ANNUAL REPORT

PROGRAM ACTIVITIES

NATIONAL INSTITUTES OF HEALTH

NATIONAL INSTITUTE OF
RTHRITIS AW METABOLIC DISEASES

NATIONAL INSTITUTES OF HEALTH

PUBLIC HEALTH SERVICE

U. S. 'DEPARTMENT OF HEALTH, EDUCATION,. AND WELFARE

(J,£- NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES
ft c Pert a V p fo (^ *►<?/>( flc^»'^«i.

Annual Project Report

Calendar Year 19

is

19S7

IATIONaL INSTITUTE OF ARTERITIS AMD METABOLIC DISEASES

Amraal Project Baporfc.

Calendar Year 1$$1

Summary Sheet

Laboratory of Nutrit-ioa sod Ersdocriaology

Estimated Obligations for FY 0958

Direct* $5ii5sOO0

Reimbursements: $20?/;800

Projects xsmaber 1 thru Xl* included <>

PHS-MIH

Individual Prefect Report

Calendar fear 19 Si

Serial No? NI&HD I

lo Nutrition & End@crin@Iogy

2o Nutrition

3o Bethe^da

par* A =

Project Titles Studies on obese and gera-free animal®

Principal Investigators Drs0 pc So Dafts 0- Miekels©n8 Rc So

Tamaraot®8 and Mr* E« 0-, McDaniel

Other Investigators s Messrs* Ao Anderson and H* Bakeraasip
Drso Eo Silverstein and Lo S©tol@£f

Cooperating Unites Section @n Fractionation and Isolation^,
LNE9 NIAMD « Project No, 12
Section on Pathologi© Anatomy a LPHS MAW =■•
proje©t KOfl 66
firginia Polytechnic Institute (Bla©ksburg)

Man fears (calendar year 191?) 8
Totals If
Professionals h
Others 8

Project Descriptions

Objectives % T© determine the biochemical and physiological al-
terations that ©ccur in animals during the develops
meat of obesity and the subsequent loss of weight by obese aiaimalsg to
study the nutrition and biochemistry of gena-free aniraalsj to detemin©
the nutritional and biochemical changes resulting from esjposure of diets
to ethylene oxide o

Methods Employed? Obesity wm produced in rats by feeding a
high fat diet? Obese and control rats were
injected with propylthiouracil;; followed by I1^, The rati© of I1^1 in
the thyroid to that in the bleed isag determined in order to secure some
indication of thyroid activity in the rats? Pituitary glands from ©b©§@
and control rats were injected into sexually immature bypophygectomised
ratso

The nutritional requirements of gem=free ratss guinea pigss etco^
is being determined fey feeding them ©teril®& conventional ration® o Th@
effect of antibiotic^ large amounts of vitamin Gs and other nutrient® s
as substitutes for certain nutrients required by ge»=>£r@e animals^, is
being studied.. Special techniques are being developed for the car© and
studjr of the gem- free animals maintained in the ge»=free tanks 0

Pag® t Serial M@= RIftMD 1

Th© r@a©tion between nia©in and ethylene ©s&d© has b©@n studied
and the r®a@tioa ©ompound resulting therefrom isolated in pur© form.-
fly means of ehFometography and a variety of physical methods* the other
©ompoisnds fo rased in this r©a©tiea are being studied*

Major Findings 8 The earlier observations of abnormalities in

the thyroid and adrenal gland® of the ©fees© rat®
have been studied in more detail c The ©bese rat show© suggestive indi-
©atiosis ©f a disturbed thyroid metabolism at least as £f|; &s &h® ^/g
ratio is ©onoeraedo In the e©ntr©l rat* the uptake ®f X^l by the
thyroid ©ompared t© the ©onoentration in the serum shows a marked in@r®ase
as the animal becomes older >> Regardless ©f the age ©f the obese rat* the
rati© ©f thyr©i& t® serum l^Jl grains at the lota level of the young
©@ntr@l rat. The interpretation ©f th<ss@ observation© are still a Miter
of ©ontroversy but they do suggest s@a® differen©© in the thyroids of the
©bese and ©ontrol rata* The aetivity ©f the adrenal glands was determined
by noting th® ©©sinophil response to the injeetion of ACTHo There 'was sso
dif feren©® in eosinophil levels between the ©ontrol and ©bese ratSc When
the pituitary glands from obese rats wer© injeeted into sexually immature
hypophysegtomized rats* the adrenal gland was the only organ whi@h showed
a marked in©r@as@ in weight ©ompared to th® ©antral? Again* the ©viden©e
suggests en in©reased horaenal aetivity -in s®m® ©rgans of the obese rats

All ©f th® pre@®ding work was don® with male rats* Obesity ©an b®
grodueed in female rats by the same method with a high percentage @f thm
reaching one kg*, in weight* These rats have more proteinuria than the
nojffiial' weight ©ontr©ls* In males the high level of proteinuria nosmlly
present obsoares any difference that might exist between th® obese and
©ontrol rats*

The level© of total lipids* phospholipid* and total ©holesterol
are slightly higher in th® blood @f th© obese than in the ©ontrol rats*
Fasting for lb hnm^& or longer redu©ed the difference in blood lipid
level® o Earlier work showed that «v@n when the ©bese rats had bean on
the high fat diet (80$ ®f ©al@rie@ from fat) for a year or more* there
were no histol@gi©al differences between the ©ontrol and ©bese r&ts as
far as the ©ororary arteries* aorta* and peripheral bleed vessels were
concerned^

■--■ The problems asso©iat©d with the maintenane© and study of g@ra~
free rats have been solved (blood samples ©an b© secured from the tail)*
Purified diets containing su©ros® as the source of ©arbohydrat© ©an be
used if the ingredients ar© sterilised separately with the sucrose being
sterilised a® a ©oncentrated (1@Q%) aqueous solution* The su©ros© and
fat ar© "gramed" in the g©gm=>fre® tank* after whi©h th© other ingredi=
©nts ar® addedc Th© resulting diet is readily eaten by the rats?
Studies are under way designed to clarify the role of intestinal flora
in the pro dust ion of f@H© acid* Sulfonamides have in the past te®en fed
t® conventional rats* and have been reported to aid ia th© development
of signs of foil© acid defioiency* Such studies ar® now being rep&ated
in gem-frs© animals <> Additional s^p©rt f©r the faportane© of th©

Page 3 Serial H@o BIAMD 1

gastrointestinal flog® in nutrition ©omes from the studies with peni©illin
and vitamin Co la eonventional animals these ©ompouadSg when added t® the
6±®t9 redu©® very markedly the animal0 § requirement; for a variety of nutri-
ents o They are both iaeffe@tive in redueing the gera°free rat "8 require-
ment for panto thoni© aeido

Collaborative work with the Virginia Polyteshni© Institute resulted
in the isolation and eharasterisation of the ©orapound formed when ethylene
oxide rea©ts with nia©in0 This ©orapound is W =>®th0xynia@inara±de ©hlorid®,.
It has none of the biol@gi©al a©tivity of the vitamiBo The above ©orapound
is famed in good yield as long m th® pK of the H@diw is kept below ?<,
During th® ©oisrsg of th® r@a©tionp th® pH in©rea@e® and when allowed to
g© up to lip a yellow ©oaporad is fossaedo Th© latter appears t@ be an
mekm formed as a result of th© rupture of th® pyridine ringc

gignifi©an©e to MAMB Researgms Obesity ©ontinues to be a major

health hasard in the United States
and has been implieated in the etiology of a variety of ©hroni© disease©,,
Th® obesity in the rats more nearly resemble® that in human being® sin©®
it is prodneed entirely by dietary mean©- Researeh leads ar© being seeded
whieh may be of value in ©linioal studies.!

The germ=>fre® work will offer ©onelusive eviden©® for the role
played by th® gastrointestinal flora in nutrition Qsr regulating th®
baeteria used in the ino©ulati©n ©f th® intestine of the germ«f rsa aniraale
it may be possible to determine the organisms that ar© Mbenefi@ialm and
those that ar® ""harmful00 as far as nutritional w@ll=b®ing is ©an©eraed 0

The work on ethylene oxide has been ©ontinued sinse this ©©©pound
is a g©od example of a substan©® that lias been proposed for use by the
food industry whi@hs although it leases noV. residue in the food after
treatment ^ yet produ©es marked nutritional ©hanges? These ©hanges w@ ■
have ©ailed mnutritional imprints80 a

Proposed Course of the Proje©tsg Th® work on th® obese rats will
• ;'•'■ b® dir@©t@d toward an evaluation

of th® ©hanges in body ©oraposition whish ©©our during th® development . of
obesity o In the germ- free areas th© studies on th® nutritional require-
meats of the rat and the fa©tors that influen©® these requirements will
be ©ontinuedo- ©a® ©ssapounds found from the intera©tion of ethylene ©s&d®
and :other vitamin® will be studied „

part B in©lud@d fas £J No £7

Pag® 4 Serial too MIAMD 1

Part Bg Heasrsg Awagds^ and Publl@at.ioa8

Publications ©they than abstraets fress this pxojset?

X* Mi@k@ls©ng Ooj Supplementation of dietasy proteins with wain® a@ids-=
Statement by th® Feed and Mutrition Board. Pubn Health R©pts? 72 §
k&Z-h&% 191? o

2o Mi@k©ls©n^ ®o and lamffiaot®^ Ho Sen Methods for th® determination of
tteiawin®o Method® ©f Bioehemieal Analysis (Do Gli@ks editor) 6g #

1958 (in pyess)o

Honors and Awards s^lating to this psojeets
Nona

-ffiB
Individual Projeet Report
Calendar fear 195?

Serial No » HIAMD 2

5L Nutrition & End©@rin©I@gy

2, Nutrition

Jo Bslhesda

Part A,

Projeet Titles Metaboli© funetion® ©f nutrients In experimental

Principal Investigators? Brs<> Go Mo Brlggs^, J.-> Go Bieri^, Mo Rn

Spivey F©x^ and M« E* H@id

Other Investigators? Co Jo P©llardg LP 0a 0rtis8 Drn 0* Mi©kels@%
Dro Mo Siivesnaan, Br0 K* S©hwarg

Cooperating Units \

Laboratory of Infectious BiseaseSpNIAXD (Dro ¥-
Ho Haas)5 Project tec 102

Man Years (©alendar yeas* 1957}
Totals 9
Prsfessionals 3
Others 6

Project Descriptions

QbJ eetives s T© study (1) the biochemistry ff physiology s and nutri-=
tiosa ©f certain essential nutrients (particularly
fat=solubl@ nutrients^, lipotropic factors^ f©li@ acid? and certain amino
acids) in experimental animals (chlckp guinea pig» rat^ and raous©)B (2)
the interrelationships of these essential nutrients* (3) unidentified
dietary factors affecting growth of animals,, and (h) the relationship of
nutrition t© health and disease P Information on these objectives is
sought in order that it may eventually b© applied to man for improved
health and nutrition.*)

Methods Baployeds Rat8s mi©®g chicks g and guinea pigs are fed
specially prepared highly purified diets eea=
posed of all known nutrients (except the nutrient^, or nutrients^ being
studied) under carefully controlled conditions o The effect of dietary
variation is studied by measurement of @linioal9 physiological £ biochemical^
and other ©hanges in the animal and its tissues.-, New chemical procedures
are developed for biochemical measurements when necessary. In ©©operation
with members ©f other laboratories the relationship of nutrition to other

Paga 2 Serial No. MMD 2

fields of science (neurology, pathology, virology, etc.) are studied..

Major. Findings; Hesults of studies on this project have contri-
buted to our knowledge of basic nutrition,
pb/jiology, and biochemistry of the intact experimental animal. In
tfri past year some of the significant findings in our Major research
fields have included;

A. Metabolism of fat°soluble nutrients s

1. Yitarain A. The new ensyme system in rat and rabbit
blood (discovered in this laboratory), which destroyed vitamin A, was
found to be in the reticulocyte and not in the mature erythrocyte.
Inhibition studies revealed that the system is different from any
known oxidases. Studies of the ensymatic conversion of carotene to
vitamin A showed that the known coenzymes associated with biological
oxidations are probably not involved in this system.

2. Vitamin E. Continued vitamin E studies have shown that
very low levels of the trace mineral selenium, known to protect against
liver necrosis in the rat, will also replace some of the functions of
vitamin E in the chick. Thus, selenium prevented exudative diathesis
and muscular degeneration but did not affect the lesion in the central
nervous system (encephalomalacia) due to vitamin E deficiency. The
production of vitamin E deficiency in the chick hj means of adding high
levels of Torula yeast to the diet is aggravated by the inorganic
fraction of the yeast. Studies on the identification of the active agent,
or agents, are being conducted.

3. Fats and fatty acids. In order to learn more about vita=
mins E and A in the guinea pig it has been necessary to continue our
studies on fat. It has been found that the metabolism of essential fatty
acids in guinea pigs is markedly different from that in other species
which have been studied. In the guinea pig one-third to one=half of

the serum fatty acid was found to be linoleic acid. In the erythrocytes
one-fourth of the fatty acid was present as arachidonic acid. The defi-
cient guinea pig does not produce as much trienoic acid as does the rat.
In the fat=defieient guinea pig the feeding of 2$ tall oil (high in free
fatty acids) resulted in poor growth and anemia; whereas 2% of corn oil
or safflower oil gave good growth and no anemia. The effect of dietary
unsaturated fatty acids on the rate of breakdown of the red blood cells
is being studied.

B. Studies on lipotropic factors in the chick (vitamin B]_2s> cho~
line, methionine, and fat)*

1. Vitamin Bi2- Of major interest in the continuance of "
studies ©n vitamin B12 is the development of a highly satisfactory puri-
fied diet for the production of a Marked vitamin B3.2 deficiency in
the chick. Using this diet, it has been found that high dietary fat
(24$) increased the severity of the vitamin B12 deficiency! under these
conditions methionine could replace vitamin B12. D^saethionine was as
effective for this purpose as DL=®ethioniae . With no fat in the diet,
the vitamin B12 deficiency was less sever® and additional methionine

e©uld not replace th© vitamin?

io Choline sad ra@thi©nin@o It is ves?y difficult to produc®
fatty livers ia chicks* However p a purified diet has been develop©d8
lew ia both eholia® and t&% which when fed to ehieks consistently
produces fatty livers,. The fatty infiltration disappears after an 18
hour fast.? Supplementation of choline deficient diets with 0*2$ inositol
has led to aa increase of the severity of perosis (slipped tendon) but
not ©f growth ©r fatty livers Q

C« Folic a<sid and related factors? Continued studies on folic
aeid in the mous© (ia cooperation with Drfl V,, Ho Haas) have shown that*
in th© absence of this vitamin^ the virus lymphocytic choriomeningitis
lost its virulence in spite ©f the fast that is was present ia the mouse
and ©ontinued to reproduce in nosmal ambers « When folio aeid was present
in th© &i®t. th® virus killed the mouse in seven days* Similar results
wor® obtaimd with histidin® deficient diets* Other studies with folic
aeid ia tt® mouse showed that dietary para^amiaobensoig aeidg a moiety
of folio aoidj, completely replaced the dietary need for folio aeid (and
behaved like folio acid in the virus studies) o Dietary peaioillin also
had a marked sparing effect on a folio aeid def ieieney* Folio aeid
defioient mio© showed an iaorease in foraiminoglutami® aoid @S£@reti©%
as do defioient rats (a® found by Dsp? Silverman) <?

In th© guinea piga in a ©ontiauatioa of earlier studies^, folinie
aeid (eitrovorura factor) appeared to hav® almost twice th© aetivity of
folio aoid la th® diet- Th© reason for this is being ©tudiedg it is
likely that th® guinea pig oan utilize th© B-form of folinie aeid^, (perhaps
th© intestinal bacteria convert* it to the L^fosm) <■

B? Studies ©a amino acids and proteins s

1<, Niacin^ tryptophan relationships and ey® changes <? In th©
guinea pig fed th© minimum requirement of tryptophan,, th® niaoin require-
ment was between 10 and 1$ mgo/kg.-, of diets With ^evy high Isvels of
tryptophan (5 gc/kg*): th® guinea pig required a© dietary niaeisse This
is similar to previous results with other animals.,- Ia the presence . of .. .
ample niaoin th© tryptophan requirement for maximum growth ranges from'
lot to lo5 g a/kg * The guinea pig leas is ©sttremely sensitive to an
inadequaaey of dietary tryptophan s For protection of the crystalline
lens against cataracts (determiaed by Dsr, Von SaHmaan) as rniieh a® 2*0
go/kg? of tryptophan is r©quiredc- However 9 unlik© the rat9 corneal
vascularization did not occur* ^tryptophan was not utilised by th®
guinea pigc.

_

2„ Amino aoid diets and unidentified factors,. In a continu-
ation @f our studies on unidentified factors ^ an amino aeid misstur© has
been formulated for us© in synthetic diet©,? This permits good growth of
eMeks up to 2 weeks ©f age with a subsequent retardation of th® nomal
growth rater, Injection of growth hormone did not prevent or alter this
retarded growth rate.? Evidence for a still unidentified growth factor is
clearly indicated by th® growth promoting effect of intact proteins fchen

Page 4 Serial Ho. JXiljD 2_

added to the amino acid diets. The factor does not appear to be an
amino acid nor any of the known essential vitamins or minerals.

3- Histidine. In connection with studies on the role of
folic acid in nucleic acid and virus formation in- the mouse a faistidiae
deficiency was developed. It was found that the faistidiae requirement
appears to be approximately 0.2$ of the diet.

Sj.jgii£iejiBiee $q> $W®).: %@M@$g£kt Additional basic information

on the nutrition, biochemistry ,
and metabolism of essential amino acids a vitamins 9 minerals 9 fatty acids ,
and unidentified factors in different animals should contribute signifl^
cantly'to our knowledge of the role of these essential nutrients in sassu
It is becoming increasingly evident that nutrition of man plays a role
in the etiology of certain degenerative and metabolic diseases;, such
as arthritisj, atherosclerosis s muscular degeneration, chronic &bor~
tiohj, dental defects^ diabetes^ certain infectious and neurological
diseases^ and certain types of cancer. Basic studies in nutrition
may lead to clues to the prevention of some of these diseases.

flSBBgftcl .Qfflffiae of Pgojectes In the following year special em=>

pfaasis will be given to sobs of
the more promising areas of these projectSj, such as studies on the
metabolism of vitamins A and E„ vitamin B12 interrelationships p amino
acid diets s choline and methionine interrelationships 9 uaidentif led
growth factor requirements p and folic acid studies.

pss>-jk H -■ > - -

.. m n

Serial Mo. fflAMD 2
PjBgt Bs Honors , Awards, and Publications
Publications other than abstracts from this projects

1. Bieri, J. G„, Pollard , C. J., and Reid, M. E. Poljene fatty acids
in guinea pig tissues. Biochim. Biophys. Acta 23s 650-651, 1957.

2. Bieri, J. G. A simplified method for administering vitamin A to
chicks fed purified diets. Poultry Sci. 36s 918=919, 1957.

3. Sehwarz, K., Bieri, J. G., Briggs, G. M., and Scott, M. L. Prevent
tion of exudative diathesis in chicks by Factor 3 and selenium.
Proc. Soc. Exp. Biol. Med. 95s 621=625, 1957.

4. Bieri, J. G., Briggs, G. M., and Pollard, C. J. The acceleration
of vitamin E deficiency in the chick by Torula yeast. J. Nutr.
(in press)

5. Fox, M. R. Spivey, Briggs, G. M., and Ortiz, L. 0. Nutrients af-
fecting the vitamin B12 requirements of the chick. J. Nutr. 62s
539=549, 1957.

6. Fox, M. R. Spivey, Briggs, G. M., and Ortiz, L. 0. Activity of eer=
tain vitamin B^2 analogues in the chick. Proc. Soc. Exp. Biol.
Med. 95s 4-98=500, 1957.

7. Fox, M. R. Spivey, Briggs, G. M., and Ortiz, L. 0. Studies of
amino acid diets for the chick. J. Nutr. (in press)

8. Reid, M. E. Guinea pig nutrition. Proc. Eighth Annual Meeting
Animal Car© Panel, San Francisco, Nov. 7=9, 1957. (in press)

9. Haas, Y. H., Briggs, G. M., and Stewart, S. E. Inapparent lympho-
cytic choriomeningitis infection in folic acid deficient mice.
Science 126s 405=406, 1957.

10. Briggs, G. M., and Fox, M. S. Spivey Thioctic acid in complete
and deficient diets for chicks. Poultry Sci. 36s 657=662, 1957.

11. Briggs, G. M. Estrogen residues in meat«=Public health aspects. •
J. Am. Med. Assoc. I642 14-73=1476, 1957.

Honors and awards relating to this projects

Br. G. M. Briggs elected Secretary of the American Institute of
Nutrition for a three<=>year term.

Dr. G. M. Briggs appointed Recording Secretary of the Organizing
Committee for the Fifth International Congress on Nutrition.

■ ■ ■ .

st Titles Diab@t©s=*=Eff©©t ©f h©Kaon®8 cm metabolism

Principal Investigators? Brs£-. Et> 0« Sesw and So So
Other Investigators
Cooperating Units?

its (calendar yeas' 1!

Others Hi
projeet Dess'adption:

Qbjeetiv©®? to muag- %h® xmmsm wnones ©:

■="''' " "•' ' " aad oa the sietabolism of carbohydrate,, fata and
prot©iB| the fa©torss in addition to fc it regulate fat isobil-

isatioHj, fat t i ms and ketone e1®!© of tt1©

(rior pituitary and adrenal glands in.va mena whieh ©©sur

in experimental diabet

Methods BTiploggd; Experimental anisaals deprived of ok© or ssor©
c====='==" ~ endocrine glands are treated Kith various

hormones o Th© effeet of the extirpations and tr@ate.aat are studied
with is vivo and in vitro methods* using eonventional and isotopie fc©eh=

Ma jo ■ to Ingsg te ©lassigal signs of diabe psrgljeessiap

" -'-^-— -"" hyperk©toHuriae hyp©rlipesaiafl and hyperasoteia I

develop within a £m hows after reaoval of the panoreas in the ratn It
It hours there is a large aeomalatioia of fat in the liver and I
Administration of siaali amounts of insBlisi aill prevent the
of these signs « injeotion of insulin 1' after panoreatest

duees an immediate deoreas© ©f bl@od glu<gos®j, ketone bodies s and noa=
phospholipid fates, At ®m hour after insulin {1 unit given subeutans
both gluseose and ketone body 1. Uf * -"^ at

three hours the lipid® are redueed -;::-. ...- . its .njeetion of

fruetos® lowered the blood ketone body level as fast as did intravenous
insulin but the effect of fruetosa raas not as prolonged* This would

Pagg i Serial Ife, MIAMP 3

suggest that insulin is not needed for the suppression of ketone b@dy
fotmstion in the diabetic?

When the panereat«rctomiiB@d rat© are fed. and given 2-, 5 unite of
insulin/gas 0 ©f foedj, all ©f the ehaaieal signs of diabetes^, with the
exception ©f hyperglycemia,, disappear within .three day®,:- the glycosuria
under this regimen is less' than 0ol gno/dayfor S$ of the carbohydrate
fed,- Withdrawal of insulin at any time results in the development @f
diabetic. e©ma8 with hjp©rket©aurias hyperlipemia;, aeewulation of fat
in the liver and kidneys8 and death within 2-3 days-; Either hvpophys-
ectomy or adrenalectomy at the time of insulin withfeg®al prevents th®
development ©f the above* Hewe,ser9 the sesdnd' operation does not hare
an immediate effect on the hyperglycemia,

Sim®, tostogeneai® is largely confined to the liv@rs this tissna©
is being studied in vitror Slice© ©f livers, @f untreated diabetic -rats
produced 3 times monT Jrofosn todies than did those of fasted aonsalsa
Accelerated ketogenesls was associated with increased liver lipid c©n=
t&itjso If adequate amounts of fatty acid© (acatate9 teutyrat®j, and
©etahoate) are added t© the medium s the increase' in ketone body synthesis
is the sarnie' for both groups r, Addition of 'substrates such as fructose,
succinate;, or malata inhibited ketogeneeis by liver sli©es« At similar
cosjcentrationii? glucose^, glutamate^ and aspartate were relatively
.ineffectual In this regard* The conditions in the liver cell that promote
and ' inhibit the formation ©f ketone bodies are zander further investigation ;

la order to determine whether ketone bodies cross the placenta,,
pregnant rats were pancreatectomized and 1? hours later the bleed level®
of ketone bodies^ as well as of glucose and fat5 were determined in the
mother and the fetuses r. It was observed that the levels of ketone bodies
and glucose were the same in the maternal and fetal blood whereas the fat
levels were much higher in the mother o However iP it was als© discovered
that pregnant rat® fasted during the last week of pregnancy als©
developed pronounced ketosis and hyperlipemia n. The blood glucose levels
were loWj> the same as in the nonpregnant animals,-. Presumably the flssd=
ings that the ketone body levels are the same in the mother and the fetus
and that hyperlipemia with fat accumulating in the liver occurs only in
the mother would suggest that the origin of the ketone bodies is in the
mother and transfer of ketone bodies across the placenta occurs 0 This
preparation provides us with pronounced ketosi® in an animal with a
pancreas and low blood glucose levels „ Studies will also be made to
find if the placenta can support the ketosie in the absence of the pitmi=
tary gland*

The effect of growth hormone and insulin on growth was studied
in hypophysectosaized^pancreateetomized ratSc These rats maintained their
body weight constant fl retained no nitr©g©ns and excreted less than 2% of
the carbohydrate fed when given 3 units ©f insulin and 7 gm° ©f food '
daily P Esstra insulin was not needed for the marked growth response of
these rats when given growth hora©ne? These findings g similar to those-
in panereateetomlzed rats with intact pituitariesj, <to not support the
suggestion of Fc, Go loung and others that growth hormone exerts som®^ if

Page : Serial &©-> Hi

not slip of its growth promoting effeet .by stimulating the seeretion
of insulifflo Th© latter studies w@r® mad® in animals (th© dog and eat)
whieh ay© sensitive t© the diabetogenie aetlen of growth hormon®g th®
increased insulin requirement in these animals wh@n treated with growth
howon© is undoubtedly neeessary for the eontrol of th® ©xaeerbation of
diabetes „

Preliminary studies in Argentina indicated that tub®°f©d hype*
physeetomised rats had nomal oral and intravenous glucose toleranees Q
These findings were ©ontrary to those reported in other speeies* Further
investigation ha© demonstrated that th© rat© of removal of axeass blood
giueos©,, in th® intravenous gluaeos© test,,, is noimal in hypophys©@t®mized
rats when tusfoe fed and subnormal when fed ad libitum* This different©
se©a®d to b® a function pf th© duration of fasting* Similar effeets w©r©
not seen in normal rats.?" Another finding in th© hypophyseot ©raised rats
was" an aeee.leration of th© reaoval rat® dteing the ©ours® of th© test
whieh was hot seen in th© nosanal-, In view of the overwhelming body of
eviden©® pointing to increased uptake of glueose by the psripheral ^©swes
of - typopnysectomised rats (Russell,, Renold .©t aloj, Krahlft and Villa}.
would seem that the %"3©r@ased removal, rates* observed in th© ad libitum
fed hypophyseotemiaei. rats during th© intravenous glug©s;e toleranee test
is the' result of giueos© being added t@ the' blood at. a faster rat® than
in' normal am&aals instead of impaired removal of glueose-,. These studio
point up the great diffieulty in interpreting intravenous giueos© t©ler=-
an©©'' test's.,-, whish are, -not eaOy dependent on the glueose removal but also
on the rate of gluieipsse addition to blood*..

Si;gni£iyan©®v.ltd KIAM) Resaarehs Th$.'$fti©T 'problem® eonf ranting

the . diabeti© patient today s
even when .'treated with insuli% are atherosclerosis and Kimm®lstl®l-
Wilson disease, -; Many -observations have indicated that tkes© ©linieal
©enditions may' be intimately sssoeiated with ' disturbanees in f at" met&bo=
iism> However j> the .nature of th@s® disturbahees8 as well as other aspects
of fat metabolism^ are poorly understood.; In th® present pr@je©tP basie ■
information '.eone'ernih'g fat metabolism is being obtained in diabeti© ,m&
nossnal rats? The interrelation between insulin and adrenal and pituitary •
hormones/in the ©ontrol of fat- utilisation arid ketosis ar© being studied:,,

Proggsed Course of th® Projeetss The in _jig© and in vitro studies -

of fa¥ metabolism in diabeti©
and normal rats will be continued* FurJh®y i2?i?estigatioa ©f th® relation^
ship-,. between insulin and growth hosmon© in growth is neeessary and will be
resumedp Studies will h® mad® ©f the signifieane© of th® fat in the renal
tubules of uneontroilsd diabeti© rats and it® possible relationship t®
the changes seen in the diabeti© patient with Kimmelstiel-Wilson diseases
Sine© th® above observation indieates th© important role of the pituitary
gland in th© development ©f ket©sis ia diabetes^, studies will be taad© of
the effeet of injeoting insulin and otter substanees direetly into th®
hypophyseal fossa on the ©ours© of diabetes in panereateetomized rats-

Part B inelud@d las /S? 13© £7

m 3

ds, and FvfoiigafcU .

Publications other than abstracts trow, thi ets

lo Seow,g> R0 0oS Wagner p E* M^ and Cas>desa A>.8 Ef£e@t ©f hypoph]
oa ^i® insella requirement and responsa to festisig ©f Kt©tailym
panereateotomized ratso Endocrinology 61 s ^©"Sflj, 195? o

to Wagner^ E° Mo and Se©ws Ro QvS Effect of insulin ©a growth in for
fed hypophyseetemized rats4 Endocrinology 61 g 2tl9=ttt0s 195? o

3-.- Se©w9 Ro Qo9 Effect ©f growth hormone ©n growth in hyp©physe©teiQlsed»
pamereateetemiasd ratgc Endocrinology 6ls $82=>586B 19$1?

kn Seoal, Re 0o» Wagner^, E-? Moa and R©n©w8 E.?p Effect ©f growth hormone
and insulin ©a body Height, and aitrogen retention in paacreateestoraiged
rats? End© ©sinology (in press)?

5^ Gheraickj, So So and Se@^s R<> 0O5 Intravenous glucose tolerance test
isa hypophysectomisesl rats* Am* $* Fhysiolo (in pr©g©)„

Honors and Awards relating to this projects
None

Individual Project Report
Calendar Tear 195?

Serial &o0 NIAMD- _4

1 » Nutrition & Endocrinology
2„ Esperiisental Liver Diseases
3c Bethesda

Part A.

Project Title; Factor 3 Against Necrotic Degenerations and its
Biochemical and Medical Significance

Principal Jhvesti gators Klaus Schwarz

Other Investigators! Calvin Mo Foltss and ffignon &c Malm (Guest Worker)

Cooperating Units;

Depto of Poultry Husbandry s Cornell University
Biochemistry Dept* Distillation Products, Inec
Analytical Service Laboratory* LC» NIAMD* Pro jo Bo,.

Man Years (calendar year 1907) :
Totals 5
Professionals 2-1/3 (1 Guest Worker)

Other? 2-1/2

Project Descriptions

Objectives ; To isolate and chemically characterize Factor 3$ to

delineate its chemical specific ity, to clarify its
significance in nutritions to develop in vitro test systems for
active compounds? and to devise net* methodTTor -the preparation
of Factor 3 active substances „

Methods Employed; Fractionation and isolation techniques s worked out

step hy step for Factor 3> are used for the con-
centration of the active substance from natural sources <> Chemical
group reactions* various physical and chemical analytical methods*
paper and column chromatography and spot tests are employed for de-
termination of its characteristics.) Highly sensitive spot and colori=
metric methods for the estimation of selenium compounds are developed
using the catalytic effect of selenium on the reduction of methylene
blue by electron donors such as JfegS and other reactions 0 Paper
electrophoresis (analytical and preparative) as well as paper strip
chromatography and new column techniques are developed and used for^
preparation and analyses of Factor 3 active compounds o Biological
effects of Factor 3 are tested in the prophylactic rat assay against
liver necrosis o Other deficiencies such as multiple necrotic

Part B includ@d (£/ No /"/

NIAMD-4

Page 2

degeneration (heart, liver, kidney and muscle necrosis) in mice,
muscular dystrophy in rabbits, conventional vitamin E deficiencies
(resorption sterility) , etc0 are used*

Major Findings; Factor 3 has been recognized to be an organic selenium

compound 0 a^Factor 3 is anionic? soluble in waters
insoluble in nonpolar organic solvents and soluble in polar organic
solvents c Its activity is not destroyed or diminished by oxidation c
In highly refined preparations decomposition with loss of activity
sometimes occurs on overheating 0 Methods of isolation have hem.
refined further so that a highly active preparation suitable for
chromatography can be obtained routinely in four steps from the end-
product of the pilot plant operation 0 Screening of a total of more
than fifty selenium compounds of known composition showed that
elementary selenium, as well as a number of organic selenium com~
pounds , are practically inactive o Other selenium compounds of divers©
constitution, as well as the selenium analogues of sulfur amino acids
are quite effective in preventing dietary liver necrosis in the rat
as well as exudative diathesis in chicks <> The !>Q$ effective dose
(EEVq) of many compounds is found at a level of 2 = 3 jig of the
element per 100 g of dietc. abactor 3 was ca 3 - 5 times as potent
as these compounds (SD^q * °® ^ * Preliminary indications are that
P-Fastor 3 is 10 times as potent as the a. form0 Inorganic selenium
compounds such as selenat®, selenite and also selenocyanate ar©
effective at the EDsJq 2 ° 3 W level*

The quantitative comparison of the biopotency of Factor 3 to
that of vitamin E or cystine shows that Factor 3 is at least 1,000
times as aetiv© as tocopherol and mora than 25*0,000 times, as active
as L<=cystinso The latter has been known since 1935 to protect against
dietary liver necrosis o However activation analyses revealed ca 2 |ig
of selenium per g of L~cystine$ and comparison of the selenium contents
and the biopotencies of cystine preparations of varying degrees of
purity has shown conclusively that the protective effect of cystine
against liver necrosis is due to the trace contamination with selenium^

Thus far<> screening of various types of deficiencies for the

effects of Factor 3 and other selenium compounds has led to the
following conclusions s Factor 3s and also selenite, prevent liver
necrosis in the rat and the mouse, heart muscle necrosis in the mouse,
peripheral muscle degeneration in the rat and the mouses kidney d©°
generation in both species, and also a severe pancreatic dystrophy
in the itous@c Factor 3* as well as other selenium compounds, have"

3

been shown to be strongly growth<=promoting„ vitamin E and Factor 3
do not replace each other but have additive effects 0 Factor 3 as
Hell as other selenium compounds do not influence testicular de=>
generation and do not prevent the classical vitamin E deficiency
syndrome (resorption sterility) 0 In the chick, Factor 3 and other
selenium compounds prevent exudative diathesis but do not influence
encephalomalaciao These results show clearly that of the Hide variety
of' deficiency diseases described hitherto solely to vitamin S a great
part is actually caused hj the simultaneous lack of Factor 3 as Hello
In rats on amino acid diets? using highly purified cystines Factor 3
Has found to have a pronounced growth stimulating effecto This tends
to indicate that selenium is essential „

The nature of the organic portion of the Factor 3 molecule remains
to be established o With the aid of newly developed spot techniques and
column chromatography on cellulose, as well as with paper electrophor-ef
refined methods for the fractionation of selenium compounds have been
developed o Preliminary evidence indicates that the organic part of the
molecule has a strong tendency to bind selenium supplied in "Hie selenite
formo

Significance to NIAMD Research: Factor 3 has the characteristics of a

very potent biochemical agent o It is
active in preventing marked and fatal anatomical lesions in several
species and has a demonstrated role in maintaining normal tissue respi=
ration o It can be anticipated to have an important role in biochemical
processes and be of significance in human diseases,,

Proposed Course of the^Froject5 The main task is presently the identic

fication of the organic part of the
Factor 3 molecule as well as the clarification of the relation between
abactor 3 ^nd p<=Factor 3° Attempts are under nay to delineate the
biological and also medical effects of Factor 3 further, for example
by the screening of its action on muscle dystrophy in the rabbit* on
the serum protein pattern in various experimental animals 9 etc0 Th©
synthetic preparation of certain selenium compounds of particular interest
regarding chemical 9 physical and biochemical properties has been initiated o
A program to compare the values measured for Factor 3 activity t^- the
necrotic liver degeneration bioassay with selenium contents determined
by activation analyses for various substances with Factor 3 activity is
under way0 Attempts to clarify whether the organic part of the Factor 3
molecule has the characteristic of an essential dietary agent are
currently being made with the aid of amino acid diets 0 Other sush-
studies are scheduled 0

Part Bs Honors* Awards $ and Publications
Publications other than abstracts frois this projects

#arzp Ko, and Foltz* Cr, M<>» Selenium as an
Part of a^Factor 3 Against Necrotic liver Degeneration .
J, Amo Chera„ Soc, 79* 3292 (195?).

2r. Schwarss K*<> Bieris Jc Go» Briggs, G0 Mcs and Seott* Mo Lcj
Prevention of Exudative Diathesis in Chicks by Factor 3
and Selenium o Proc<> Soc* Esp, Biol, and Medo 95* 621* (1907)

3» DeWitts V/o Bo » and Schwars* K„* Multiple Dietary Necrotic
Degeneration in Mice0 Heart* Liver* Muscle and Kidney-
Lesions on a Factor 3 vitamin E~Fre© Torula Yeast Ration »
Esperientia* in press.

Honors and Awards relating to this project:
None

?HS =
Individual Project

serial rfen NTAMD=5

I. Nutrition & Endocrinology
2c Experimental Liver Diseases
3o Bethesda

Project Titles Metabolic Functions of Factor 3 and fitamin E* and
the Metabolic Events in the Development of Dietary
Liver Necrosis.

Principal Investigator: Laurence Mo Corwin

Other Investigators s Klaus Schwara* Marie Green (resigned)

Cooperating Units; Wone

Man Years (calendar year 1957) .
Totals 2
Prof essional s l~l/3
Others 2/3

Project Descriptions

To elucidate the mode of action of Factor 3» and also
of vitamin E9 in intermediary metabolism o To clarify
the chain of events in the development of dietary necrosis(,

Methods Employed; The metabolism of livers and also of other organs

from rate on diets with and without Factor 3 or
vitamin E is studied using metabolic techniques such as the Warburg
respiroraeter o Tissue slices as well as various cell constituents
are used.-. The latter s particularly mitochondria* are prepared by
differential centrifugation (method of Schneider and Hoogebcom) e
P/0 ratios are measured and the effect of selenium compounds such
as Factor 3 and also of vitamin E and its derii?atives is studied c
Ensyrae systems being affected are studied o

m site of action of Factor 3* two
approaches have thus far hean used? (1) The effect
of various selenium compounds on mitochondria of normal liver and of
mitochondria poisoned by specific inhibitors was studied «, (2) The
oxidative behavior of cell components of animals on diets deficient^
in the two main protecting agents $ Factor 3 and/or vitamin Is was

Part B included Yes ffl Mo fj

HEAMD-5

Page 2

analysed 0 It was found that selenite itself at levels of
Q<& <=> loO y.g per 3 ail medium stimulates oxidation by normal
liver Mitochondria slightly*. Since arssnite is known to react
with sulhydryl groups in proteins and to inhibit oxidation in
mitochondria* and since selenium is analogous to sulfur* the
effect of selenium compounds on arssnite inhibition was analyzed 0
In preliminary studies supplementation of very small levels of
selenite produced a reversal of arssnite inhibition o The finding
could not be reproduced consistently o The cause for the variability
of "fee results is under invest! ga

Liver mitochondria from animals on the unsupplemented basal?
liver necrosia=>producing diet show normal O2 consumption with
succinate as substrate „ Greatly reduced O2 consumption is seen
with the addition of DPNo Under these conditions a large decline*
amounting to more than double that of the vitamin E supplemented
animals d is observed o Supplementation of selenite in the diet
does not protect against this portion of the metabolic derangement ,7
Oxidation of pyruvate and various other acids of the Krebs cycle
does not undergo this change „ There were no consistent differences
in the P/0 ratios of animals on supplemented or unsupplemented
rations 0 Comparison of the difference spectra of mitochondria
did not reveal a major discrepancy in the saounts of flavoprotsins
or cytochromes presents Th© alterations detected -feus far indicate
that the liver may possess a potential to shift its oxidative
functions* depending on the lack of either vitamin E or Factor 3<=

The findings ar© consistent with the concept that Factor 3
and vitamin E participate in alternate pathways of intermediary
metabolisms possibly in electron transfer systems a

Significance to NTAMD Research; Elucidation of the mode of action

of vitamin E and of Factor 3 would
be of significance 0 It would be of basic importance for the under-
standing of normal oxidative pathways * the main source of energy
for fee life of animals 0 Clarification of the primary metabolic
lesion^ and the chain of events leading from dietary deficiency of
Factor 3 and vitamin E to acute necrosis and deaths Kay produce a
more general understanding of necrosis * i0e0s death of tissue^ and
may lead to new approaches in the therapy of such lesions o

The current investigations ar©
scheduled to be extended to other
cell components such as microsomes s and to other substrates o 3@° "
lineation of the area of metabolic disfunction will be followed by
attempts to locate the defect on the enzymatic level o The extension
of the present studies to other systems 9 such as yeast cells , with
the aim to identify the catalytic ally active form of Factor 3 after
tagging the latter with radioactive seleniums is plannedc.

Part Bs Honors * Awards* and Publications
Publications other than abstracts from this projects

lo Sehw-ars* Ko* Dietary Necrotic Liver Degeneration; An
Approach to the Concept of the Biochemical Lesion.
"Liver Function* A Symposium on Approaches to the
Quantitative Description of Liver Functions'5 Ralph
Wo Brauer* Editors American Institute of Biological
Sciences a Washington, Do Go* in press.

20 Piceardo* Mo G,» The Spatial Relations of the
Endoplasmic Reticulum to Mitochondria in Liver
Cells o "Liver Functions A Synsposium on Approaches
to the Quantitative Description of Liver Function ->"
Ralph Wo Brauer* Editors, Mexican Institute of
Biological Sciences * Washington* Do Co* in press e

3o Piccardog Mo G0* and Schwarz, Ko* The Electron

Microscopy of Dietary Necrotic Liver Degeneration 0
"Liver Function* A Symposium on Approaches to the
Quantitative Description of Liver Function ow Ralph
Wo Brauer* Editor* American Institute of Biological
Sciences* Washington, Do Co* in press „

Honors and Awards relating to this project;
None

Individual Project Report
Calendar Year 195?

Serial Mo0 HI AMD =6

1« Nutrition & Endocrinology
2o Experimental Liver Diseases
3c Bethesda

Part A<

Project Title; Identification and Mod© of Action of the Dietary
Glucose Tolerance Factor (GTF)

Principal Investigators Walter Mertz

Other Investigators; Klaus Schwarzj Edith Cheney (Guest)

Cooperating Units: Won©

Man Years (calendar year 195?) :
Total s 3 ■ •

Professional: l<=i/3 (1/3 Guest Worker)

Other: 1-2/3

Project Description:

Objectives: To isolate and identify the glucose tolerance factor

(GTF) » a dietary principle required for the main-
tenance of normal glucose tolerance in rats D To delineate the foio=
chemical and medical significance of GTF and to elucidate its mode
of action o To study the mechanism by which the liver regulates the
uptake of glucose* and to elucidate the function of a newly discovered
humoral agent from liver which accelerates glucose removal by the
peripheral tissues.

Methods Employed: Glucose removal rates are measured after intra=
venous glucose injection into fasted rats 0 Male
rats are maintained on a GTF deficients but otherwise complete diets
GTF activity is estimated curativeiy by measurement of glucose re-
moval rates before and after supplementation of GTF preparations to
deficient rats- Ghemical and physical fractionation procedures are
developed to concentrate GTF from natural sources »

Protein=free water extracts are prepared from rat organs »
especially liver = For the study of the liver agent, GTF deficient
rats.8 as well as the hemldlaphragm technique in the Warburg apparatus^
and eviscerated rats are us®d<-. Tissue culture techniques are applied
to the study of the action of GTF and of the liver agent -

Part B included les /«?7

Page 2

Major Findings; Experiments with a -variety of diets have
strengthened the concept that GTF is not
identical with any known dietary agent such as amino acids* con-
ventional vitamins or known trace elements „ GIF has been differ=
entiated definitely from Factor 3 against dietary liver necrosis
after the latter was recognized to be a selenium compound 0 GTF
is water soluble and apparently of low molecular weightj it has
acidic properties and is sensitive against oxidation 0 Purified
fractions of GTF undergo inactivation by autoxidation 0 Activity
is j at least in part* recovered by treatment with reducing agents.
A method for the purification of the GTF was developed using a
by-product of the Factor 3 purification as starting material o
After six steps, preparations are obtained in substantial quantities
which reconstitute normal glucose removal rates at a dose of S mg
per 100 g of body weight 0 The best preparations of GTF prepared
so far cured the defect at dose levels of 50-100 jag per 100 g
body weights

Addition of GTF preparations to tissue cultures was found to
increase glucose uptake from the medium by 30~9$% ever that of
unsupplemented controls a Amounts of CQ03 }Ag of a highly purified
GTF preparation per cc of medium xf ere sufficient to give this effect o

GTF preparations * highly effective in the intact rat* do not
increase glucose tolerance in eviscerated animals* and also do not
increase glucose uptake of isolated diaphragm in vitro n The GTF
effect seems to be mediated through 1&e liver o This "organ contains
an agent which can be extracted under suitable conditions and which
enhances glucose uptake in eviscerated rats* as well as in vitro <>
Addition of 5 to 20 pg of crude liver extracts from normal" animal s
to hemidiaphragms increased glucose uptake from the medium by an
average of liO$ as compared to the unsupplemented controls <• The
effect is specific for liver* i-@2* extracts prepared from other
organs are ineffective * The increased glucose uptake is not accounted
for by an increase of glycogen* nor was any increase of oxidation
observed-- The molar ratio between Og consumed and glucose taken up
shifts from ca 10 in controls to ea 6 when liver extract is added 0
This resembles the effect of insulin 0 However $ the liver principle
is not Identical with insulin o It is water soluble* non=protsinaceous;
relatively stable against boiling* and effective at very 3mall dose
levels o

Significance to NIAMD Research; GTF has the characteristics of a-

new essential* dietary agent for
the maintenance of normal glucose removal rates » It can be expected
to be of fundamental biochemical and physiological importance o It
may be of clinical interest in several human diseases* especially in

md« 6

Page 3

diabetes s where dietary therapy is essential 0

These studies show that the liver produces an agent which
enhances peripheral glucose uptake 0 This opens new prospects
for the understanding of the homeostatic mechanism of blood
sugar regulation? It is of basic importance in physiology , and
it may be of significance in disease processes, such as diabetes ,
liver injury, the terminal phase of cancer, etc „

Proposed Course of the Project; Continuation and further develops

ssent of the fractionation with the
aim of obtaining the GTF in pure form*: to clarify its chemical and
physical characteristics, and to make it available for clinical
research <> It is attempted to utilise' the effect of GTF on glucos©
uptake in tiss\i.e cultures as an assay system » Currently, various
methods for the stabilisation of purified GTF preparations against
autcxldation are analyzed o

It is planned to devise colorimetric methods for the estimation
of GTF concentrates, using the oxidation-reduction behavior of GTF
as a potential* The natural distribution of GTF in dietary ingredient*;
is studied further o The effect of GTF on experimental metabolic
diseases* such as diabetes, will be analyzed 0 Work on the humoural
agent from liver will be aimed at the development of improved methods
for the preparation and concentration of this huraoural agent, and
the further investigation of its effects in tissue culture, the
diaphragm system in the Warburg* isolated organs, as well as in the
whole animal-.

Part Bs Honors t Awards.,; and Publications
Publications other than abstracts from this projects

Schwarzn Ko» and Herts, W,» A Note on the Glucose Tolerance
Factor (QTF)S a New Dietary Agent* ''Liver Function* A
Symposium on Approaches to the Quantitative Description of
Liver Function,," Ralph Wo Brauer-> Editor^ American Institute
of Biological Sciences * Washington* Do Go* in press o

Sehwaraj Kos and Pferts* Wo* The Glucose Tolerance Factor
and its Differentiation from Factor 3° Archives of Biochenio
and BiophySoj in press.

Schwarzj Ko* and Hertz? WOJ The Terminal Phase of Dietary
Necrotic Liver Degeneration in the Rate The Journal of
Clinical Investigation* (submitted) e

Honors and Awards relating to iiiis project:
None

1. ISuftrltion & Endo<

2. Endocrinology

3. Bethgsfia

Bart A-

Srojeet Titles Insulin StsaSies

Briaeipal Itesstigators: Br- Efrelpi .toderson and St., Robert Wo Bates

Otter Eesrostigatort JSSrs. Fra&ees E« $berry

&a Tears (Calendar Year 1957)
SSotals 3

ETofesssioaals 2/3
&fehers 1 1/3

Project Inscription:

CtojectiTOt So isrestigats insulin in bleed*

fethods and Major Findings; Jfsthods are beisg developed.
for tfes eaSEaetiem of insulin from p&aesa* She pl&sasa fractions are
being assayed for i&sulia activity by two different atetbods: one.
aathod using tte I^pepshyseetcsaissd^ aHeasaaisedj, diabetic B»©use?
tjbleb bas a&Baady be®n publishsdo tootter aietfesd ■sbieh is amsscffasfc
siailar uses t&e hypopb^eeteraised, @H®saaised5 diabetic rat*

Fraa vary prelisaln&ry f toalngs it appears that tfes aiaount
of ins'olin in nomal :8asting baa&n plasm is of the order of 50 ffller©~
units per stL* pXassao SMs is not significantly different from a
derived value obtained by Berson et al« and is in contrast to a
value obtained by Handle^ which is about 300 times the value -we
have obtaiaedo

Sigaif ieanee to B2MD research; Studies of this nature shauld
■be of trasendoos value S& investigating diabetes*

Proposed (BwErse of Projects Ste assay of lasulia in various
clinical elSlSiiis -will be ecradu&ted.

Part B included* Yea £X/ No /_/

Bart Bo

Publications ssther than abstracts f5?oai this project:

Anfiersoa., EoP fJtexry, F.0 Bates, Bo ¥o? and Cornfield^ Jo
A Method fcac Assay of Insulin Using AllcBan Mabetie ijypcyia^seetsiBized
B?©80 Seeo Exgyfeio Biol. & Ms&* Jgk, 321-325* 1957.

Calea 3-957

AMD _ _ 8_ _
1„ Eutrltlon and lnd<
2. 32adoer inology
3 » Bethesda

Bart Ac

Project Title; leureendoeriae studies - Jfesescephslie-iagrpo-
thalamic mechanisms influencing the
anterior pituitary.

Principal Investigators: Dr. Anderson, 33r. Wilson, Br. Bates

and Dr. Knewlton.

Other Investigators: Br. Spence (Georgetstai), Br. iSauta

Br. Baymateer (AFIP), Mr. Eedda, Mr. Byan
and Ma?. Koger.

Cooperating IBiits: Georgetown University School of Jfedieine,
Walter leed Army Institute of Research,
and Armed farces Institute of Pathology.

Man Years (Calendar Year 1957):

Professional: 2
Other: 2

Project description:

Objectives: So establish the hypothesis which this group
has proposed, Basely, that there is a mesencephalic hypothalsadc^pituitsry
activating system.

Methods Employed: Destruction of areas in the midbrain in the
dog by neurosurgical techniques and electrolytic lesions in the midbrain
of the cat by stereotaxic techniques.

AC3S release frm the anterior pituitary is studied by quantitative
assay of adrenal steroids in the urine.

lew methods for the accurate analysis of adrenocortical steroid
excretion in the urine of dogs have been developed. Shese pesmit repeated
experiments with serial detezmlBatiens over intervals as small as 2 hours,
an on the same aniaal. She procedures have recently been simplified and
speeded up. Moreover, the method has also proved a very sensitive index
of adrenocortical activity in eats. Shis constitutes a new approach to

the study of factors affecting AC-HE release in these anSjsals.

fhe -to?© my or corticosteroid components of dog urine have been
isolated and presuarotively identified. -She variations in excretion
levels of one ©f these shear much, sharper increases following stress
than does the entire corticosteroid moiety. Bevised procedures for.
the specific assay of this esse ccsaponsnt are no® being put into use .

5SS release is determined by the radio-iodine release rate fraa .
the thyroid. Seradresalia and adrenalin in the urine 'Will be measured
by the von Eulsr method.

Major Findings: transection of the midbrain in the dog
interrupts the ljyp©tbaj.affiie»pit\3itary saeehanism *rhich neemily triggers
the release of A02H in response to stress . It also disturbs the *2S&
release 0 la sense preliminary studies on eats, the placing of stereo-
taxic lesions in the midbrain has shown in soess cases as much as a
10 fold increase in adrenal steroids, -while in other cases no alter<»
ation in steroid excretion has occurred,, She anatomical studies
locating the lesions are under way* Striking changes in 2SK release
have also been observed.

^Significance to the program of EI&BSDs Central nervous
system influences on zsetabolic and endocriae activity are of tremendous
importance in th© study of all asetabatie diseases.

Proposed course of project: ¥©rfe is under ^ay on the location
of the B»senph^ie»l3cyparasBlaaic©"pituitsiry activating system. Bogs
with electrolytic destruction of the dorsal and antral nuclei of
Sudden are being studied.

2art B included. Yea IV no / /

Bart Bo

Publieatioss ©tas:r than abstracts frcsa this project;

Anderses, So, Bates, B. ¥., BnrtfcaraS', 22., Eaya&lser, ¥o,
3Joo«itoa, K», Bieeh, Bo MsE., Spenee, ¥. T»9 m& ¥ilson<> E»
ffea Effects of Midbrain and Sgiml Cord Sr&nseetioa ©a Endocrine
and Metabolic Fraootioaa© with PostuLatloa of a Midbrain %p©thaieBSico«»
Pitv&tary Activating System. Beeent degress in EbEmoBe Beseareh
nn, 1957,. Aeadesde Bsess, Saw York.

:

Isolation, -i.. >s,

PriE: ; -rs: Bz% Kobe

Ofclsez -oa-st Mr* SuXaae

and .

Coc . : Shite:

7):

'■,.1: 1*6
i .ay 2

Sro&eet Base:

Ob^c y testmem&B (t

nd prolaet:
especially

mouse from 'blood.;

eitien of ■ms%f±z& liessacme pz-v sources

ec-ffip&Ts t&eir 'd 'biological las,

; pitwitar

native el.
&M e

j© electee
and oi

:i?e es'sfcsslicai ©f Ufee sasii
are ajs&e by & ■ i©& excia

tte study of o^gaMe reaotioas a&s used to p2©p&£6 suitable
of the lasmeaes fca? structural a&alysS

■
: suitable

eloped* - we prow

fractions having a potency of 1 U/aig. ~ : ©f the

Laal 5SS. Shis is slightly better than is obtained by prev:
solvent fractionation procedures. Ouly prelirjiisary tests have 1
isafie concerning the location of the fi
in the percolation system.

!Ehe 3T^* ehiefe depletion assay for SEE is not sensitive enough
to detect the hoxmone in 6 blL* of plas- -lurae

that can be injected :lnto the birds. TJhen : :ed blood is sub-

jected to the above percolation procedure, concentrates are. secured
which permit the accurate estimation of 92SH in iaasan plassaa (hQ imi/100 si

Using old lyqpMlized human pl&ssia (from Bharpe and 3ohme)? m:
or all of the fS3 activity can be obtained in a fraction containing
about 2 per cent of the starting dry Tssight. Wcee biosssay thic
the injection of solids fros 50 to ICC xLa&ssa into oce test

animal. With such a concentrated plat .'.on* a dose-response

curve f or #31 depletion with. !ISE was obtained using the equivaler;
of 6f 12 , and 2k- ml9 of plasma/day in nine chicks. Shi3 curve was
not different in properties fraa that obtained with SSH from pi
extracts. A plasm 5^ concentration of 2-5-0 mu/lOQ a?.* -was foot
Shis saethod of extraction sakes it possible to deteasniaa quantitati--
the 3SH levels in human plasm, but requires 50 ial. ©f plassa. Oth
pituitary harnesses and also insulin, are probably present ia the
fraction, but Moassays have not yet been done for aH these other

25se 33SE potency of three strains of transplantable pituitary
tumors has Taoen followed through four transfer generations in atbyroid
mice. Ha all three strains the concentration of 3ESH in early gener-
ations ©f the tumor was about that in the nerssi souse pituita:
3n one strain the 32SE' eoncentraticai decreased with each trans:?
until it was only ©ne^-tenth the original potency, but the coneentea-
tion -was ssaintafeed in the other two strains. 33® 3SB concentration
in the blood senm of mice with these large tuiaors was 2000 t:l:
norasal concentration and it decreased Slightly with each generation.

2SK has been concentrated, from bor.Usg pituitary glands, to the
extent of 1000 times. *&& highest potency that has been obtained is
between 18 &n& 2k U.S.P. units per sag, purification has "been

accomplished by the ssse of anion and cation exchange ehraaaatography
on the cellulose derivatives diathylasiiaesthyl cellulose (SEAE-C^and
carbct^^thyl cellulose. In the course of this work the concentration

■ '
3Jhe bovine Its g hormone removed E can "be receserefi

tzsa sad appear a basic protein

contrary to reports in the literature. 3© S^H fraction consist:

ut 80$ of a jasper co^sassnt ->?hen essmiised tyr free electrophoresis.
Assessing this ia&;)er component to "be t&e X-iorKESO;, the uppes? liait ©f
potsney would "be 25 ^T.S.P* units per og* in tSae case of beef 2SS
Additional asotmts of the purified 3BH as© now being prepared c:.
jUarger seal© so that the questions of liwageaeity and teller parifi
can be studied.

chrtaatogr&pMe ssethods devised for the isolation of bovir
have bsen applied to the studs'- of 5!SE in other species, in p&rtleul/
the mouse pituitary timers described in easSLier repsrts, asad to. the
of the aaimls bearing these traaory,. Anion eseh&nge dsressatogsaphy on
K3AE-C has led to the preparation of mouse traasr 5SH -STith a potency of
between 8-3.0 U.S. P. units per Bg» Ms mouse 2SH, while similar in
respects to beef SBE, had a different partition coefficient from beef
ESE as shotm by ; .rent position in elutien ckreEtatograpbyj,

probably reflecting differences in sesse ehcsaieal groupings in the m
5SS fresa the plasm of times" bearing sice -was s&Bilar to the horaone
isolated frosa the tasso

Significance^ to ...SlfiMD rei^aroh; She ssaehanisa of action of
pituitary hormones is net, as yet, taa&erstoodi, Brogress ^rill depend
upon availability of pure horaones, a Jaawrledge of their structure,
the accuracy and sftsplieity of the bioassay procedures. Srogress in
this area has been achieved by cur Sxop?ev9d sjetbods for preparation
and bioassay of the hesmenes. It is hoped that these Eethods will
perait detesaination of blood levels of 2^S 2a patients.

Eropoged course of project; SSE is one of the few pituitary
hormones wMehTaas'lxyt been~isolated is& poire f ©cm. Efforts to prepare
pure ESH froa various* saw aaterials wiH eoa&tee and, lahan successful,
full csbsaaicai and physical eberaeterlzatiesi will follow. She physieo«
chemical homogeneity ©f bovine 2SB wiH be assessed,, its amino acid
composition -Bill be determined together with, the terainal saine acids*
For this purpose kinetic studies on ensgssatic degradation and on the
preparation of chemical derivatives xdJX be carried out. Studies ©a
the effect of SSE on thyroid physiology in the cfciefe ^111 continue*
S$jae course s'Srj^ies Mil be setfe en the blc<^. levels of 3BH in radio» -
tbyroideetcffiized jaioe SS^>Ianted with 3!SE producing tssaors t© deterMae
vhen the blood levels of 2SS rise. Studies are planned to deteraiiae
the S^ level to. blood ©f patients using our nev extraction prosedure.

?art B included. Yes /x/ No

Publications otbsr than abstracts frca this project:

Bates, Robert W. aM Cornfield, JercBis. A& Saproved Assay I&thed for
ftoyrotropMn Using Bep&stiea of 1^31 from the thyroid of Bay-Old CM
^oerinology 60, 2S5<»238, 1957,

Cendliffe, Bster fi. and Bates, Eoterb ¥0 Otonatograplsy of ST%r©tr©pMn
gel BietliyiamiaQet&yl Cellulose <> Arelu Bioebem. and Biophysics 68,
229-230, 1957.

Bates, Robert ¥«, AjaSersen, Evelyn, and Fiffitb., Jacob, ^yrotrcphia
Potency of SmnsplB&tab&a Pituitary 3hss©rs of Mice through Fofur~!£s©nsfers.
Endoerkaology jaL, 5%9»55k, 1957*

Lai Bo a EXAM) 10

1. Sutrition & End©

2, ]«&doerin©logy
3 o Bethesda

Barb &•

Erojeet Sitle: She influence of the central nervous system on
jaetabollc functions - Creatine and creatinine
jset&beHsm.

Principal Xswestlgator: Br. Eatbrya Khowlton.

Other IDxrestigator: Mr. Leonard H. Bedda

Bfen years;

Sbtal: 1 1/3

Professional: 1

Other: l/3

Project Description:

Objective; So distinguish between extrusion of intracellular
creatine and failure ©f creatine incorporation by tissues as causes of
ereatlauria f ollcrejing transection of the spinal cord or other (Sausage to
the central nervous system.

Bfethods earoloyed: SSse body creatine of a dog vias labeled
fa #5 by feeding creatine synthesized to contain 60 atoms $ excess
in the glycine moietyo 2Sas spinal cord was then transected at
Qj level . "From. the urines collected starting at the time of T^
labeling, tbs excreted creatine and creatinine are being isolated,,
degraded to sarcosine-, and purified as the toluenesuLfonyl derivative.
53ae #5 content of these products •sill be determined by mass speetrci!setry=

Jfejor findings: After study and modification of conditions
required for the various steps, creatine v&s synthesized freaa glycine
containing 60 atoms $ excess W$ "by toluene sulfonation., sethylatica.,
removal ©f tfee toluenesulfocp. radical to release sare©sine? and sub-
sequent condensation with metbylisothlourea hydriodlde*

Procedures for degradation of the isolated creatinine products
and for purification of their derivatives have been set up# the resulting
preparations will be analyzed for isotope content.

mmsp- 10

Significance to Sj^iDjgg^agjga; Since creatia& phosphate
occupies a place" cf "sosse 15^^^Seir'IE^atx«ellt£Lar energy interchange
of the sssanEalian body^ hn©y?lsdge of various phases of its Esstabollc
raeehanisias wiH contribute to tmsferstanding of diseased states. In
tl^ ^particular studies^ factors of ijsporfeanee in nexareendocrine
and aeurcBBet&bolic problems should "be mere eosrpletely delineated.

Proposed coarse of prejeet; T<&®»- data fresa analysis for W~^
abundance have been acc^u^teeQ" interpretations n&LL be rosde *srhich
dictate the course of farther iia^stigations. If 7 the postoperatively
excreted ere&tine contains the preponderance of F**, the ereatinaria
must res-nit frc® release by disintegrating muscle cells, or frcm
failure ©f the biochemical mechanisms tJhieh nsssially retain creatine
in. the cell for its part la supporting energy transfer systems. If
lr' does not appaar in the postoperative creatine ,, the creatinuria
must be due to failure- of ©ndogenously f erraed creatine to be incor-
porated into Bsuscls cellsy and Its censeouent excretion in the urine.

A similar study is planned for the cxeatinuria following midbrain
transection. It is also hoped that light \ri2X be shed on the reason
for 1&© abrupt drop in creatinine excretion seen -with this operation.

Bart B included. Yes /X/ No / /

a Ko. SXc^CO r j-0
Page 3

Bart B»

Bstolloaticas other than abstracts fsm this project:

KheffiXtosi; Xfefeya, S^eaee^, William S^ Ricoh.* Xtearid SSeK.,
Sasmalsez'., W&bb; Lasls&y, Alice, Eefida, JjeeasEra, Bahn., Bobert,
anct An<3e?scsa.> Eroly&o Metabolic Changes FclloEfing Transection of the
Spinal C©r& in Xtegs. Acta Sfe-urosyego 3?, 37^-*^>3* 1957 »

J'57

Serial Bo. HIAMD-

1. Nutrition & Endoc

2. Endocrinology

3 . Bethesda

Fart A.

Project Title: Studies on the secretion and metabolism of
adrenocortical steroids in man and animals.

Principal Investigator: Br- Fildegard Wilson,

Other Investigators: Mr. John Eorris (Until 7/1/57) > Dr. Lillian

Butler (Georgetown), Mr. David W. Ryan (George-
town).

Cooperating Unit: Georgetown University Sehool of Medicine.

fen Years (calendar year 1957):
Total: 2.0
Professional: 1.2
Other: 0.8

Project Description:
Objectives:

I. Studies on Human Subjects.

In persons with endocrine abnormalities related to adreno-
cortical function, it is of first importance to ascertain what steroids
heing secreted hy the adrenal cortex,, and to estimate their amounts. She
usual routine assay procedures applied to the urine of patients give at
best an incomplete picture of this situation. During the last few years
this laboratory has developed a simple, comprehensive technique, by which
the entire pattern of urinary steroids can readily be surveyed. Beginn:
with the prepared urine extract one person can complete 5 patterns in as
many working days. Experiments here and elsewhere have shown how such
urine analyses can be interpreted in assessing adrenocortical seere-'
activity, and to detect abnormalities in adrenal biosynthetie reactions.

H. Studies on Dogs.

To identify the major steroid hormone metabolites in
urine of dogs, in order to validate analyses based on colorimetric. dete:
minatlons.

Employed: The urines are first treated so as to obtain

the most complete hydrolysis of steroid conjugates and complete extra
of free steroids possible . This requires ineubation with 0 -glueurc

.

followed bj continuous ether extraction at pH 0.8, followed by a second
extraction at pE 0« !<?he combined neutz bions are chromatographed
on a standardised partition column •?rhich. separates steroid metabolites
according to their physiological significance. A minimum, of 7 fraet:
is collected, containing in turn (l) metabolites of androgenic pre.;
(2) dehy&rc^pian&resterone and pregnanediol, (3) androstanediol,
similar substances, (h) C3.0O9 and CojO, metabolites, such as H»3seto~
etioeholanolone and pregnanetriol, 15)' metabolites of 11-deoxyeortisol
(Cpd. S), (6). major metabolites of Cortisol, and (7) highly polar saste,
of Cortisol.

Three or four such columns can be completed in one day.

A series of 6 or more micro assays, each, specific for certain
groupings, is then applied to the eluates. OJhese are designed .to provide
a furtber differentiation of the types of compounds present in each fra
tion. Frem the results of these determinations, the excretion of most
the important classes of adrenosteroid metabolites, both normal and
abnormal, can be calculated.

For more detailed characterization of individual steroids, standard
methods of chromatography, both on partition columns and on paper, are
applied to pooled urine extracts. Ebe chief fractions are eluted,
re~ehremategraphed in several solvent systems to insure homogeneity,
subjected to color tests for specific groupings, and re-crystallized,
if possible . Identification from mobility, melting point, and absorpti
spectra in ethanol and sulfuric acid is then attempted. In addition,
derivatives such as oxidation and reduction products and acetates are
prepared, chromategraphed and characterized in the same manner for
more positive identification.

Major Findings^

I. Studies on Human Subjects: She technique for determii
patterns of urinary steroids in human subjects has so far been applied
to single patients ■with different abnormalities, so that no universally
applicable conclusions can as yet be drawn* It has been proved, howsv*.'.
•'chat important information can readily be obtained. For example:

(l) A vasmi .with hirsutism and aexie was found to have a selective
overproduction of androgen metabolites, Without any other abnormality.
Shis ra,s, therefore, a different syndroms' from classical-- adrenal hyper-
plasia in -which several abnormal metabolites are excreted in large amounts =

(2) la male stib.jeets with choriocarcinomas it has been suggested
'■■ the high levels of gonadotrophin secretion might cause the format:

of progesterone « Tsro such subject -tadied, bus neither excreted

any ©f the chief metabolite of progesterone, viz: pregnanedlol. One
thesa had an elevated excretion of oehydroepie^ydrcsterone.

Both these metabolites are easily sad accurately determined by our
procedure .

(3) A woiaan TTith an adrenocortical carcinoma and resulting Cushi:..
syndrom excreted elevated amounts of ^etoaL steroids, but Toy far the

-.most abundant metabolisms of a new -type, most likely a 21-deo:Ky '
eoxticoida Qis indica^s an abnormal biosjathetic ssehaaisai, in tsh
the adrenal tumor tissue,, presi-mably in the absence of pituitary ACE
falls to complete the 2l~hy&rcxylati©hl process.

H » Studies . on Dogs:

The chief urinary corticosteroids of dogs, which are
unquestionably metabolites of Cortisol,,, ax's 3 highly polar C-17, 20,21
triols with 03 hydreagl groups. 33heir total basal excretion level has
varied in different dogs,, between 15 and 75 ug» per £k hours. A less
polar metabolite of similar stricture was present in slightly ssaHer
amounts.

Tbs aiost abundant JL=»hetol has been identified as compound tetra-
hydroeortisol, (pregnane-3^ >11$17<A j21»tetrol»20»one). It is acccmps
fry a 30 -hydroxy isomer. . 'She combined basal excretion levels of these
metabolites *ias about 9 'ag* ^^ day in 3 normal dogs, and was dramatics
increased by at least 6 fold and probably Kiuch snore, during several days
immediately after imposing a stress.

Saaller amouats both of more polar and less polar 17,21-dihydroxy-
20-lssstones, metabolites of Cortisol, were .also present.

Shis pattern differs from that of humans in the preponderance of t
polar triols over A-lsetpls, and in the absence of any appreciable amor
of compound tetrahyclrocorfcisone .

Sigaif icance to 11A1D research:

I* She ecsaprehensive svstwqj teehaisgue we have developed
requires a mininasa of effort, yet reveals the nature of the adrenoeort:?
i^tabolites in detail o By detecting and determining metabolites ce:
©verloolssd, it has shown that the adrenocortical secretion may be quite
©sapless, and is capable of many variations from one individual to anoti :
It is potentially useful in enlarging ©w understanding ©f adrenocortical
activity and its relation to disease states.

H. The analyses of dog to?:'.; lish -fete presence

fencsao, nsasurabls steroid substances for the first t:kae. Shas the
•validity of tectei ieying colorSjjsetrie -bests fop urinary etes

as indices of adrenocortical function in dogs is cccefismed.

Ktereover, the sesje experiments demonstrated that a purified
consisting chiefly of eeaapound tetrahydroeortlsolj, affords sa exftremely
delicate criterion of changes in adrenocortical activity in dogs*
Bsnee, the effect of various procedures on adrenocortical activity can
'be determined again and again in the same dog? obviating the necessity
for sacrificing the animal in order to obtain adrenal venous blood*
appropriate asethoda of extraction ; purification, and assay^zoany erp:
can be performed am. dogs which cannot be done -with human subjects.

Proposed course of project: ffihree types of study are mate.-

1, a. Identification of the presumptive 21-deozy eorticoid found i:
urine of the adrenal carcinoma patient.

b. Application ©f a method for the detection and quantitative
determination of this type of compound to urine extracts from
selected subjects. This method has already been developed.
Results xaay shar differences between adrenocortical hyperactivity
due to earGinesaa (■where AC5H trould be suppressed) froa that due
overproduction of ACS35L

2. A study of the steroid pattern of the urine of hypophyseetcaaized
human patients during ■withdra'Kal of steroid therapy. Be tailed analyses
in such patients have act previously been performed. Shey t?311 show
nature of the lammx adrenocortical secretion in the cosaplete absence of
ACIEH stimulation* Tts change in urinary steroid pattern produced "by
exogenous AC2H is being followed as a function of tfeas.

3« Positive identification of one or two additional Major steroid
metabolites in the urine ©f dogs will be ua&rtelaen. A seaBswhat siaiilar
analysis on extracts of "She urine of cats asay also be performed.

Bart B included. Yes /X/ No / /

Fart B:

Wils©% H.j, Bala% R* C, aad IteaftSj, J. J. Stsroids is
Blood e&& T&ria© <?£ Female Mce Eea^iag aa. AGEE B^ocLueiKg ©saw.
Eadocriaology * izi garess.

Serial lo. BIAMD 12

1. Hatrition & Endo

2. Fractionation and

Isolatl
3 » Bethesda

Part Ao

Project Title: Large-scale processing of MoiogiGal mate-

Principal Investigator: John C. Keresztesy

Other Investigators: lone

Cooperating I&its: EHI - Katural Products Labo

Man Years (Calendar Year 1957):
Total: 1=5
Professional: 0»50
Other: loOO

Project Description:

Objectives: Many problems of importance in the biochemistry
of disease require the isolation and identification of substances which
are present in only trace amounts in the natural product. It becomes
necessary, in order to obtain sufficient quantities of the desired
compound, to process large amounts of biological materials, such as
liver, brains, excretory products, plant materials, etc.

Methods employed: The laboratory is equipped with large«c;
apparatus, such as stills, filters, reaction and extraction kettles, etc.
In most isolation problems the original small-scale process has to be
modified and developed, so that it can be carried out efficiently on the
larger scale. This adaptation or process development is an important
function of the laboratory. The degree of participation of the laboratory
varies according to the needs of the specific problems.

!fejor_ findings : The laboratory facilities have been used in
a variety of investigations carried on at HIE. Its main use has been
in the preparation of concentrates from horse liver for prefolic acid
and the extraction of a great -variety of plant materials for the
Hatural Products Laboratory (MI).

12

Significance to EXAM) research; Ordinary laboratories lack
facilities to effectively carry on studies ^hieh require the extraction
and processing ©f large quantities of biological materials. Soaever,
with the equipment and trained personnel of the large-scale laboratory,
sush investigations. as. the isolation of. the prafolic acids can be
undertaken and successfully completed *

Proposed course of project: So continue to offer assistance in
large-scale operations as required by such projects as prefolie acid
and the alkaloid problem (SET).

Part B not includedo Yes /X/ Mo /__/

Ca3 957

Serial Mo. MMm _ _13

lo ISutrition and"™"

Endocrinology
2= Fractionation and

Isolation
3 * Bethesda

Part A.

Project Sitlet Microbiological assays for vitamins and amino acids .

Principal Investigator: Milton Silverman

Other Investigators: E. Ba&erman, M. Romine

Cooperating Units:

Man Years (Calendar Year 1957):
Total: 3 «5

Professional: 1»5
Other: 2

Project Description:

Objective: She determination of various vitamins and amino
acids in foodstuffs, body tissues and excretory products ean be carried
out by the use of microbiological assays. Samples submitted by investi-
gators on nu'fcritional and allied projects are analyzed for their cor
of the specific vitamin or amino acid under study.

Mathods employed: The use of microbiological assays continues
to be of major iErportance in the studies on folic aeid. For example,,
with suitable test conditions one can distinguish between folic acid and
its reduced derivatives. Further, the concentrations of 5- and XO-forayi
derivatives of te-fcrahydrofolic acid in miHi-raierogram, amounts can be
determined in the presence of each other.

Major bindings: In collaboration vita- 25rs. Arthur Ship and
Donald Watkwas of the Rational Cancer Institute f a study was made of
influence of a pantothenic acid deficiency on the ability of patients
to foaa antibodies when challenged with various antigens* She laboratory
assayed urine and stool samples from the subjects for pantothenic acid.
The study was undertaken to see if various types of eaneer might be
controlled by the action of speeif ie virus infections - some of them
appeared to have a lytic effect on cancerous tissue as long as their
multiplication was not inhibited by antibodies produced by the host.

In collaboration with Br. Mickelsea, investigated the reaction
between niacinamide and ethylene oxide, including isolation and
characterisation ©f the reaction products.

i 13

She above are only eseaaples of collaborative studies which
utilize the facilities of ths laboratory*

Significance to lg£AMI3 research: 'Ehese raicrobiologieal assay
procedures'" are a very important tool for the asasurenient ©f the concen-
trations of various cell constituents,, i.e., amino acids and vitamins.

Proposed Course of gro^eet: So continue currant micro-
biological problems and collaborate vitesn called upon with other groups
for projects requiring microbiological assays for- vitamins and amino acid

Bart B included. Yes /x/ Ho / /

Page 3

E&rfc B:

Publications otter than abstracts fxm. this project:

Fields Jasass B.^ Stecterman, Daniel D.s MeDan&el, Ernest, aafl
Batannasij Howard A, 1/rinary Excretion Mteras of Some B
Vitamins in Diabetes* A Study in Patients vrith. and without
IDsgensraiive Cessnlieations« J. toe Diabetes Assoc 69 508-514., 1957*

Ship, Arthur, Sefcatten, William, K&tklns, Doasld Mo, and Koaine, £:'
Effects of Btntotbenic Acid Bef icient Diets in Bstients with

Carcincaffi.So Cancer (3n Kress) e

2. ¥z"
3» Bethesda
Fart A.

Sro^eet !Uitles Studies on Folic Acid

2*ein«i3jsl Ba^stig&tors: M. SfJLv&xmas. aofi J. Go Keresztesy

Other Investigators; R. Gardiner* 2» Esaaeldson and J". Szulraa;"

Cooperating Utalts: tass

a leafs (Calendar Yeas? 1957)%
Hotel: 5

Professionals 3
Other: 2

Beo^eet Inscription:

Studies cm folie acid.

(a) S&fcural3^MMKS«rriag fea» of (prof ©lie acid),

(b ) Enzymatic coswersien of prefolic acid t© knosm fossae*
ye) Sazysatie reduction of folic aeid»

(d) Urinary compcpjnds associated s?ith folic acid ssetabol:
St&i&es ©a the bacterial degradation of ereatin:!:;

Objectives: 2o deteraiiss the- role of folic acid and its various
derivatives* natural and synthetic in the biochemistry ©f metabolism
and nutrition* So isolate and identify prefolie acid* which s?ay have
neater biological significance in heasopoesls aad the therapy of
leuketaia than those now Known. 33© Identify the end-products associated
with folic acid Esetaboliss. So study creatinine metabolism in the
single carbon ds;aor problem.,

Hsthods exsnloyed:

"°*~|ay~Bia^"'',ffi, prefolie acids have unusual lability* fraetloaa
proeefiureg to gsrify extracts ssust be devised whieh win not cause siga
cant decomposition during processing.

(b) fh© enzyijiatie conversion of prefolie acid to the micro-
biologically active fern involves a ©abound of unlsaown structure* an
unidentified enzpse and an ead«-5a?©duot jseasured by its microbiological
growth properties . A search is being is&de for a physical property
that can be utilised for the quantitation of prefolie acid»

(e) Rirific&tisox ef the easjpes frost liver which participate
in the synthesis of eitrevoraa factor.

(d) l&entif ieation of tulnary products associated with folic
acid saetaoolisffl and their smsuresent.

(e) She iseehex&sa a? the ener^r yielding reactions of the
metabolism, ©f creatinine.

Bfejerjf&a&inggt Sao ferns of pxef olio acid can he separated
from, each other 'by selective elation frcsa ehareoal adsorbates, "by
ohrcmtographic teolmigpes and by the solubilities of their asmonte,
and torim salts . Beef ©lie A acts as the weaker acid. She soltMlity
csf the ssssoniraa salt of prefclie A in saethanol has been utilised 'bo
prepare fractions more than 5<$ pure.

as enssjpsatic reactions associated with the conversion of folic
acid to citrsvonm factor have been elucidated. When folic acid is
incubated wiSk a chicken liver preparation in the presence of reduced
triphosphepy^idine nucleotide (TM)f it is converted to tetsahydrcfolic
acid. "Rhea, the latter is incubated -with fosaiasioo glutamic acid sad
an enzyme preparation, it is cosarerted to 1-10 forsyl tetrahydrof ©lie
acid which on heating in the presence of ascorbic acid goes to eitre«
vonas, factor (U-5 fcsasgrl tetrahydrefolie acid). In the first ens^ssatie
reaction, amisiopteria prevents the redaction of folic acid to tetm~
hydrof ©lie acid but the inhibition did not irarolve the reduced feaaa of

She above reactions have been adapted to the estSmtion of the
fcasafcsin© glutamic acid present in uriBe* She urisary excretion of the

f CBsa.1iain© glutamic acid is increased isi a deficiency of folic aeid«
Shis technics has been used in a study of Alaskan Eskimos where a
fairly high incidence of ansaia exists. A significant percentage of
these people excreted large asKouats of fesaaiaino gbj&ajnie acid.
Sherapeutie trials are under 'way t© detessaine whether folic acid
supplements will cure the anemia In these people.

It has been established ("by isolation and characterisation) that
the folio acid deficient rat whose diet is mtpplsssented with several
eaaine acids and l^SSC^ excretes a large aaacmnt of A ^toglufesrate.
She latter was detected hj the ancraX©ns growth raspm&e of L. arabinosus
in a ssediisa in which the glutamic acid was replaced by rat wrasse extracts*
She excretion of the excessive assists of ^ Betoglutamte was due
prtearily to the alkaliaization of the tissues by the MSJO3 in the
suppteaent. She phen«wen@n also occurs in the aosssal rat.

An enayate system An ohielxen liver that reduces folic acid to
tetmhydrofolie acid has bees partially reified. Be&uced trlphosphe-
pyri&tee ('3SS), nucleotide was required for the eduction. Asalnerpfse:
ted no effect caa the gsaemtios ©f reduced WS but affectively blocked
its utilisation, for the x^dactioa of folic acid. She syathesis of
citrovorua factor frcss folic acid sad fcassyl glutamic acid was established

A sensitive bioXogioal procedure for the detection of fomlmiaogli: ;
acid In urine has been described- It has "been applied to the detection
folic acid deficiency in maa and the laboratory aaftasl. It has also be
applied ©a a surrey scale to several native Alastem villages, where jb©&
anemia is eadMtlc, Preliminary results sug^sst that a significant per-
centage of this population excretes fc&^aiao^xteaie acid. Xa connection
with tba Alaelssa stai&es, the hypothesis is being tested that la habitual-
abortion the subjects suffer from a fmctisasal (and presumably nutritii':
folic acid deficiency*

fflbse bacterial ensgroe which converts creatinine to asethyl hydaatoin
has been purified 4000-fold.

Significance to SfflMffi research: Ste isolation and identification of
the various metabolites of' folic acid will result ia a better uuaeretair
of the biochemistry of such diseases as sesse of tbe anemias and leuteaie
She aeclsaniems by which folic acid derivatives influence the f cra&tion
fundsiraeatal components of all living cells, the purines, are being widely
studied. Ms project is directed at discovering how the various ferns
of folic acid es&st ia the body aad hew they are changed froa inactive to
active forms .

Bfoposed course of project:

(1) Ste isolate the two fossss of prof ©lie acid and establish
their cheaaieal structure.

(2) So study the reactions concanssd Is the transformation ©f pre™
folic acid to eitrovortaa factor and to purify the eaayae.

(3) So correlate the excretion of fonatoiaegluSamic aeld to faulty
folic aeld xeetaboliess in hassans.

Page 4

^rt B:

Publications st&er tlsaa abstracts fzcm this projects

Fufctemaa., Si&aey &a& SHveaaaa, ffiltffia» Shs "Saactitjatlasa" ©f Folic
Acid by Liver". J* Bid, Ctea. S&, 31-^0; 1957.

SV&&£RazDif. JS&H&m, Eferssstesy, Jofta.C.j goval; Ge©3?gs J*3 aaad S&ediserj,
Bita C. Ci-towosfom Facto? and the Sy&t&ssiB ©f FatwylgLutass&c Acid.
J. Biol. Gbea.'jgg, 83-9^ 1957.

Fufctessaaa, Sidney. Erasjimtie Seduction, ©f Folic Acid and Bihy&rofoli©
Acid to SetEeOgtfvSfelie Acid. ^T. Siol. Gtem. 228, 1031-1038, 1957 •

HATXONAL INSTITUTE OF ARTHRITIS AMD METABOLIC DISEASES

Annual Project Report

Calendar Tear 1$S1

Sxmmxy Sheet

Laboratory of Bloahearf-stsy and Metabolism

Estimated Obligations for FY IggB,

Direst* $385*000
Reimbursements! $1U7»COO

Pro jests number 15 thru 29 inoludedc

PHS-NIH

Individual Project Report
Calendar Yeas? 1957

Serial no,. NIAMD^ 15_

1= Biochemistry & Metabolism

2o Enzymes & Cellular Biochemistry

3o Bethesda

Part A:

Project Titles The Enzymatic Trans formation of Carbohydrates
and Their Intermediary Metabolism

Principal Investigators Drc Bo L<? Horecker

Other investigators! R , Ann Ginsburg

Cooperating Units: Dr,-. Go Domagkp Postdoctoral Fellowp Damon

Runyon Memorial Furadp until September 30n
1957

Dr,-, Dr. P0 Burma^ Postdoctoral Fellow,, Jane

Coffin Childs Memorial Fund# until August 33
1957

Man Years (calendar year 1957);
Totals 4
Professionals 2
Others 2

Project Description:

Objectives; To study the pathways of carbohydrate metabolism

and their relation to the synthesis of cellular
components and to sell growth and function^

Methods Employed; The engymati© mechanisms are studied in

detail in isolated ©ell^free systems,. New
intermediates are identified,; synthesized by chemical or enzymatic
means and used for further studies,.- Isotope tracer methods ar©
used to study eltemlcal mechanisms and the role of the several
pathways in cellular metabolism Kinetic methods are used for
the study of cellular transport meehanismso

Major Findings; The major pathways of pentose fermentation in

Lactobacillus plantarum (pentoses) have now bsm
established-.- Phosphoketolase^ the key enzyme which catalyses
the cleavage of xylulose 5~phosphate to yield the energy-rlch
product^ acetyl phosphate^ was further purified and is now
available in homogeneous form,, The reaction has hem shown to
be irreversible but the mechanism of this complex process remains
unknown.

Part B included Yes fx] NoF

Page 2

Ma jog^F 1 wd ings^jgont r.

In the utilization of L-^arabinose the following steps
have been shown to occurs

lo Isomerisation to L~ribulos©

2o Esterifieation of L°rlbu3.os© by ATP to form

L=rlbulose S^phosphat©
3-; Conversion of L°ribuios© 5=phosphate to D<=Kyiulos©

5°phosphat@
4* Conversion of D^&ylulos® 5=phosphat© to acetic acid

and lactic acldo

Steps 1 and 4 were discussed In the annual report for the
preceding year* Th© :'\tikinas© which catalyses step 2 has
now been purified and used for the enayraatlc synthesis of
L^ribulose 5-=phosphat@£, making this new ester available for
further studies o With this subs tame© a n©w ©ngymep L-rlbuiose
phosphate 4«©plraeras@p was found which catalyses st©p 3? Th©
equilibrium constant for this reaction was found to be lo2P
with a slight excess of D°stylulose S^phosphat© over L^ribuiose
^"phosphate present at equilibrium*

This en&ym© constitutes th© second known 4°epimeras©o Th© first
catalyses the conversion of UDP galactose to UDP glucose,.-.
However the sans® cofactors are not present and th© mechanism
appears to be different..,

A study of deoxyribose metabolism was initiatedj- made possible
by the preparation of quantities of this substance by Drc Fletcher
and his groupo y, j^ntarua "*as shown to ferment 2"de©s$yrlbose
to produce lactic acid and acetaldehyde? This is th© first
instance in which the latter product has been shown to be a
major fermentation product,-, Th© steps in the fermentation were
shown to be

io Esterification of deosjyribose to deoxyrifoose 5-phosphate
2o Cleavage of deor/ribose 5=phosphate to acetaldehyde

and triose phosphate
3c Conversion of triose phosphate to lactic acid.-..

The enzymes which catalyse steps 1 and 2 are adaptive and
appear only in ceils grown on deoxyribos@c Both of these are
now und®% investigation,-, Reaction 20 which is reversible^ has
been employed for the synthesis of deosjyribose 5<=phosphate and
this valuable substrate can now be prepared from cheap and
readily available precursors c

El is

Page 3

Ma jog Findings? ©oat.:

The new fermentation proeess also provides a ©onvenient
method for the study of biosynthetl© pathways for deoxyriboseo
In ©oliaboration with Dr* H., H* Hlatt (Beth Israel Hospital^
Boston) ev&denee has hsm obtained in the intaet rat whleh
indieates that deoxyribose Is formed direst ly from a ribose
derivative^, and mot frora aeetaldehyde*

Preliminary studies at the Pasteur Institute have extended
existing knowledge of the glueose transport raeehanisra* New
methods have been developed for the study of this system and
it has now been shown to be sensitive to energv^uneoupling
agents su©h as dinitrophenolc- With glu©osec=C^ several
unidentified ©orapoundsp possibly intermediates^ have h@®n
ensountered*

Slgniflean©e to NI AMD | research; The new pathway for pentose

utilization in baeterlal
systems provides a prototype for other studies* It has singe
been shown in another laboratory that a similar pathway for
hessoses exists* The general signifi©sn©e of this rae©hanism
of agetyi phosphate formation requires evaluation*

The pathway of deoxyribose metabolism provides further
knowledge ©oneerniag the metabolism of this important substanee
and also new methods for its study*

Proposed Course of Projects After Dr* Horeeker0© return the

e3sperien©e gained in this field
will be applied to an investigation of permeability me©hanisras
in mammalian ©ells,?

NIH

Individual Project Report
Calendar Year 1957

Serial No*. NIAMD- 15

Part Bo

Publications jdurine^ 1957s

Takagip Yo8 and Horecker^ B,. LoP Purification and Properties
of a Bacterial Riboside Hydrolase5 Jo Biol? Chem*,, 2250
77"860 1957*

Horeekers Bo Loy Hurwitap Jop and Heppelfi Lo A<,9 The Synthesis
of Ribose 5=Pyrophcsphate and Ribose 5~Triphosphatep
£o to** Chemo So£P£) 79s 701 =702 e 1957r.

Heppelp Lc Aop Hurwitep Joj, md Horesker.p Bo Lo0 Adenine

Deaminase of Azotobagteg yinelandiip Jo Am,, Chens* Soeop
79s 630-633^ 1957c-

Kuwitgp Jo 9 Heppelp Lo kop and Horeckerfl Bo Lop The Enzymatic

Cleavage of Adenylic Acid to Adenine and Ribose diphosphate p
J* Biol, Chemo,0 226p 525=540* 1957c

Buymaj, Do Po9 and H@reskerP Bo Lo,9 L=Ribulokinase and the

Formation of D-=xyiulose Phosphate in Lactobacillus pentosusu
Bioehlm, Blophyso Aeta» 24s 660-661^ 1957.,

Vishniaep WoP Horeekerp Bo LoP and Oehoap SoS Ensyraatl© Aspects
of Photosynthesis s, in Advances in Ensyraologyp in press o

Horeek©r.9 Bo Loj, and Hiattp Ho Ho,» Pathways of Carbohydrate

Metabolism in Normal and Neoplastic CelIslP The New England
Jo of Medog in press o

Heathp Eo Cop Hurwit^p Jop Horeckero B.3 Lop and Ginsburgp Ao0
Pentose Fermentation by Lactobacillus plantarum I a The
Cleavage of Xylulose 5°ph6sjpHate0 JV BToXTclesop in press .•=

Heathy Eo Cop Horeeker^ Bo LcS SmyrniotiSp Po Zo9 and TakagiP

Pentose Fermentation by Lo altotarum II 0 L~arabinose isomerase,r,
Jo BiojU Cheffiop in press 0

Burma 0 Do Pop and Hore<sk@rp Bo LoP Pentose Fermentation by 1L>
pjjntarum lllo Ribulokinase^ Jo Biol* Chgmop in pressT

Burma p Do Pop and Horeekern Bo Lop Pentose Fermentation by Lc

pi ant arum IV „ L=ribulose diphosphate 4=eplmerasei? Jo M©JU Chem.;
in press o

Honors and Awards* Rockefeller Publi© Service Award

Individual Project Report
Calendar Year 195?

Serial NOo NIAMD» 16

lo Biochemistry & Metabolism

2g Enzymes & Cellular Biochemistry

3o Bethesda

Part Ac

Project Titles Studies on the structure^, biosynthesis ami

intermediary metabolism of nucleic acids and
nucleotides.-,

Principal Investigators Dro Leon A0 Heppel

Other Investigators? Dr..-. Russell Jo Hilraoe

Cooperating Units? Dr., So Oehoa9 New York University College of

Medicine^ New York City
Dro Maxine Singer^ PHS Post-doctoral Fellow
Dro EP Po Anderson^ National Cancer institute,,

NCI #42716 Laboratory of Biology
Dro Ac Mo Miehelson^ Ac P.-.. Guiness 8. SonP Ltdc r
Dublinp Ireland

Man Years (calendar year 1957) %
Totals 3
Professionals 2
Others 1

Project Descriptions

Objeetiyess Th© objective of this project is to discover

pathways for th® biosynthesis and breakdown of
nucleic acids and smaller polynucleotides „

Methods Employed; Kith enzymes purified from bacterial,, plant
and animal sources various foiosynthetie and
degradative reactions are carried outp The products are studied
by means of paper chromatography^, paper electrophoresis,?? chemical
analysis^ enzymatic analysis,, ion~esschange column chromatography
and isotope tracer methods 0 Enzymes are used as specific
anaiytieal reagents for study of polynucleotide struetur@0

¥£iS?..„ Z^JJlBSBl Bacterial polynucleotide phosphorylase catalyses
the reversible synthesis of polymers with the

structural features of ribonucleic acid (RMA),, Thusp with adenosine

diphosphate (ADP)s

n ADP ^==rz=»(5°"AMP)" * n po| (1)

In the reverse direction,, this enzyme catalyzes th® phosphorolysis

Part B included Yes jjxjj N© f~l

MIAMD° | | 16
Pag® 2

Major Findings^, ©ont-

of th© polymers thems@Iv@sp of "natural™ RNA and of certain
types of small polynucleotides? Dse0 Sing©? found that small
polynucleotides with a 5°=phQsphomono@ster endgroup undergo
phosphorolysis faster than th© polymers th®ms@iv©Sn However?
the ©hain ©ould b© phosphorolyied only, down to a two unit
compound r. such as a dinusleotid©-. This<> if A s adenosine^
and P = phosphate groupP and PO^ = inorganic phosphates

POl (trinucleotide) PO- (dinueleotide)

pApApApA .— „ 7. .^pApApA ^^--^^^^ -.pApA Action (2)

-j- * Stops

ADP ADP

More recently p it has been shown by 0©hoa°s group and in this
laboratory that highly purified polynucleotide phosphorylase
from Ajotobaeteg vjnelandii cannot synthesis© polynucleotide
polymsrs except in the presence of a ""primes"0.* The polymers
themselves can act as primers* W@ have shownp in addition^,
that small polynucleotides of the kind shown above are primers
and act as a nucleus for starting new chains o Ptenonucieotides
cannot act as primers a But a dinueleotide,) as pApA„ can act as
a primer p even though it cannot undergo phosphorolysiso By this
is meant that it stimulates polymerization^, which otherwise do©s
not occur until after a lag of several hoursp and it serves as
the "starter™ for a ©haino Thus with pApA and uridine diphosphate
(UDP)s

pApA + n UDP— «=->pApApUpUpUpU<,ooo00pUpUpU (3)

By specific enzymatic degradation procedures the isolated polymer
was shown to have th© structure indicated in equation (3)p with
pApA stuck to th© m front endTOo

Reversible polynucleotide synthesis thus resembles th® action of
starch phosphorylase in some respects c, The enzyme n©@ds a primer
(at least 2 units long) to begin its action^ and in th© reverse
direction of phosphorolysis th© ©niym© can shorten th© chain
only down to a length of two units o Polynucleotides with
3 " "phosphomonoester end^groups ar® inactive as primers and as
substrates for phosphorolysis,,

An important new finding is thats in th© presence of primerc
polynucleotide synthesis is ©f f©etive at very low concentrations
of ADP and UDP such as exist in cells.. This increases the
probability that this en^ym© is really concerned with in vivo
RNA synthesis o Preliminary evidence B from this laboratory and
that of Oehoap indicates that the polymers have a certain
specificity with r@sp©et to what nucleoside diphosphate they will
stimulate,, in a polymer izat ion reaction.

Pag© 3

Ma jog Findings o eont.:

The first evidence for an animal polynucleotide phosphorylas©
has b®@n obtainedo With Dr<, Anderson^ polyps* (utilisation
by extracts of ieueeraia cell lin@s.B grown as ascites tumorsp
is being studied? The bacterial polymers are mush more
rapidly split than isolated RM by these fractionso

Ensyaatie studies were carried out on ribopolynueleotides
chemically synthesized by Dr« A* MQ Miehelsono These interesting
materials resemble RMS but have mixed 2°s5° and 3°s5° linkages.
Studies were also don® on cyeilc=di~adenylic acids the material
which activates liver glycogen ph©sphorylas@o

Significance _to HIA^_R@g^rehs These studies s thenp elucidate

the mechanism of action of
polynucleotide phosphorylas® and give us a clearer insight into
polynucleotide synthesis^ in generalo it is expected that the ■'
principles being discovered hereP such as need for priiaer^
phosphorolysis to a '"limit polynucleotide1"^ and tighter binding
by the enzyme as the chain grows p will be general prineiplesc
We expect that DMA synthesis and various animal RMA systems will
show the same governing principles Q

Proposed Course of | Projects Further studies ©n mechanisms of

RMA biosynthesis are contemplated.?
W® hope to study the content of polynucleotide phosphoryias© in
the cell during different phases of activity.^ as in eniyme
induction^ when RNA turnover shows variation It is expected
that the level of ensyme should change with the rate of RMA
turnover? More studies on animal systems and nuclease specificities
are planned Q

PHS°1«H

Individual P?oj@et Report

Calmer Y©ar 1957

Serial No* MIAMD- 16
Page 4

Publieations during lg7»

0©hoap So and Mepp@l,? Lo Ar.p Polynucleotide Synth@sisp
in Ch@ffli©al Basis of H®s@dityp Baitimog-ej Johns
Hopkins Pr©ssp 615°638p 1957,,'

Heppelr, Lo Ac 9 Huraitsp JOJ> and Hog-eekex*., Bo LoP Adenine
Deaminase of Azotobaeteg vinelandli^ Jo Ara0 Chera0 S o©? fl
79p 630=633 9 1957 „

Ho?@@k@?p Bo I.op Huswitz^ JoP and H@pp©lp Lo AoP Th© Synthesis
of Ribos© S^Pyrophosphat© and Ribos® 5"Tsiphosphat@P
Jo Aoso Chenu So©, e 79p 701°702p 19570

Hepp@lp Lo AoP Kuffwitzp JoP and Hos'sek©^ Bo LoP Th@ Enzyraatle
Cleavage of Adenylic Asid to Adenine and Ribos© 5~Phosphat©g
Jo Biolo Ch@moP 226p 52§°540P 1957Q

Hilmo@p Ro JoP and Keppelp L.; Aop Polynucleotide Phosphosylai.®
in Liver Nuclei^ Jo ta,;. Ch©m,;, Soeop 79s 4810p 1957-

Singes^ Mo Fop Heppslp Lo Aop and Hilmo©p R, J9p Oligonucleotides
as Primers for Polynucleotide PhosphosylasSp Bipehlmo ft
ll£ghvs; aetao 26,, B 1957 p in psesso

Heppelp Lo Aop 02"tisp P« Jop and Ochoap SQp Studies on Polynucleotides
Synthesized by Polynucleotide Phosphoseylas© I„ Stguete© ©f Poly-
nucleotides with on© Typ© of Staeleotid© Unit and Ho Stswte©
of Polymers Containing a Mlrta® of Basesa £<■.■ Bjtojlo gfaejao P 229£
p 1957p in pgess*

PHS-N1H
Individual Project Report
Calendar Year 1957

Serial NOo N1AMD- 17

lo Biochemistry & Metabolism

2o Enzymes & Cellular Biochemistry
3» Bethesda

Project Titles Carbohydrate Metabolism in Mammalian Tissues

Principal Invest igators Dr., Gilbert Ashwell

Other Investigators? Jean Hickman

Cooperating Units? Drr, A„ Wahbae Postdoctoral Fellow (Damon Runyon)
DTo Jo Jo Burnse National Heart Institute 0

Laboratory of Chemical Pharmacology

Man Years (calendar year 1957) s
Totals 3
Professionals 2
Others 1

Project Descriptions

Objectives: The previously described findings of D^xyiulose

phosphate as an intermediate in mammalian pentose

metabolism has prompted further investigation into the biochemical
reactions of this compound and a study of the metabolic pathways
mediated by itp with particular reference to pentosuria and uronic
acid metabolism?

Methods Employed s In general^, the methods employed involve an

enzymatic or solorimetrie determination of
substrate utilisation or product formation? This is usually

followed by purification of the enzyme system involv®d.;> Isolation
of the reaction products is attempted by use of paper and columnar
chromatography and final identification made by preparation of the
appropriate crystalline derivatives*

Major Findings s Work has been recently completed on a new

enzymatic assay method for L° and D°2«yluloseo
alone or in mixtures p and a manuscript is being currently prepared.-. ^
An investigation of the purification and properties of D°8ylulokinasef

an enzyme which specifically phosphorylates D=^yiuloser:. has bssn
completed- This report establishes a basis for the understanding
of the metabolism of L~5cyluiose,; an intermediate accumulating in
the urine of pentosuric patients,-. In addition^, it provides evidence
for a key step in a new cyclic pathway for glucose oxidation in-
volving the formation and degradation of glucuronic aside

B inelud Yes (Tl f~]

Page 2

Major Findings, o

An enayai© system in guinea pig lives' capable of metabolizing
D=galaeturonie acid has been purified twenty=-fold and the
product identified as L«galaeton£e acid? To date» attempts
to demonstrate further utilisation of this compound in
mammalian tissues have been unsuccessful c In conjunction '
with Dro Albert Wahbaa cultures of Eo coli have been adapted
to i>=galacturonie acid and eell=>free preparations have been
obtained which are capable of degrading this compound to
unknown products by a mechanism vuhieh does not involve the
formation of free pentoses.^ The synthesis of C14 labelled
D-^galacturonic acid is in progress in an attempt to determine
the reaction products and to gain insight into possibly new
mechanises for uronic acid eatabolism,

In collaboration with Dro John Burns and Mr0 Julian KanferP
of the Heart Institute* a detailed study of the formation of
l>xylulos© from L=guloni© acid has been undertaken* The enzyme
system from rat and hog kidney has been partially purified (15
times) and evidence has bmn obtained which indicates that at
least two steps are involved s A DPK dependent oxidation step
has been successfully separated from the decarboxylation reaction
and presumptive evidence is now available to indicate that the
intermediate is a p*«=keto aeido Since from structural considerations,-
this compound might be expected to form ascorbic acidp serious
consideration is being given to the possibility that this compound
may represent a point of bifurcation of two divers© metabolic
pathways* Lsoj to ascorbic acid and/or to L-xyiuios® and ultimately
glucose o

Significance to WIAMD Research? It is expected that a study of

"" these compounds and the pathways

by which they are utilised will contribute to cur knowledge of
the overfall njetabolic proeess and thus lead to a more rational
approach to the understanding and treatment of the pathological
alterations in metabolism^

Proposed Course of Projects A study of the reaction products of

D-gaiacturonie acid produced by
mammalian tissues and by adapted E* ejpJ4 is in progress,- It is
hoped that information obtained in these systems may provide
insight into new mechanisms for uronic acid metabolism^ In
addition* considerable emphasis is being placed on the identification
of intermediates and a -study of the reaction mechanism in rat
kidney preparations whereby l<=gulon£e acid is converted to
L=xylulos©o It is expected that these studies will lead to new
information on the biosynthesis of ascorbic acid and the etiology
of essential pentosur.-

PHS-MH
ividual Pffojset Report
Calgndas* Yeas' 1957

Sesrial Ho,. NIAMD- 17
"Page 3
Part B.,

Publications duglng 1957s

Ash«llfl GoS CoiQ5?£met2?ic Determination of Sugas-Sp in
Methods in Ensymology,, N®w Yorkj> Aeaderaie Press0
73°i05p 19570

Ashmllp Ga{) and Hiekmanj, Joe Enzymatic Formation of

Xylulos0°5~Phosphate fs-om Ribose~5<»Phosphate in Spieenp
Jo Biolo Chea^f, 226p 65p 19570

PHS-NIH
Individual Project Report
Calendar Yeas 1957

Serial Noo NIAMD° 18

lo Biochemistry & Metabolism

2o Enzymes S Cellular Biochemistry

3o Bethesda

Part A<

Project Titles Studios on the Biosynthesis and Degradation
of Purines

Principal Investigators Dr0 Jess© d> Rabinowitz

Other Investigators s w<> Eq Pricer0 Jr0

Cooperating Units? NlMiD9 Laboratory of Pharmacology a
Toxicology p Section on Biochemical
Pharmacology

Man Years (calendar year 1957) s
Totals 3
Professional: 2
Others 1

Project Descriptions

^©etiyess The objective of this project is to determine

biological mechanisms involved in the metabolism
of purines o

Methods Employed s Enzymes are purified from suitable sources.
The products of the enzymatic reaction are
isolated using ion exchange chromatography or paper chromato-
graphy and are identified from their spectral characteristics s
by specific chemical and enzymatic assays^ and by comparison to
compounds prepared by synthetic means* These products g prepared
enzymatically or synthetically are employed in studying further
enzymatic reactions-

Major | Findings % Enzymes have h%®n purified from bacterial cells

which catalyze the breakdown of xanthine to
4 -amino-Imidazole c, The further degradation of 4°amino~imidazole
by an enzyme purified from extracts of Clostridium, .cylindrosgp
yielded a product which was isolated and identified as formimino-
glycine (FlG)o It was then shown that the formation of glycine s v
formate and NH3 from FIG required a folic acid derivative and was
accompanied by the esterifisatlon of phosphates

FIG * ADP * Pj_ ^_JM. s^ glycine * NH3 * HCOOH * ATP,,

This enzyme has recently been crystallized by Mro Pricer- (Th© one
catalyzing reaction 4e below)*

Part B included Yes m f°~l

MAMD- 18_

Pag© 2

Major Findingsg_eont.;

The folic acid cofaetor was sho» to b© tetrahydrofolfe
acid (TKF) and the detailed mechanism of formimino transfer
was found to consist of the following steps s
formimino transferase

(1) FIG + THF s — — i .iasasBSSSJsasa 5=-formimlno-THF * glycine

cy el ©deaminase

(2) 5~foMsiralno"THF — — _—£► 5P 10~aethenyl ° THF ♦ m3

eyelohydrolase

(3) 5910°methenyl<=THF * HgO < ' : ■ > lO-formyl-THF

(4) 10-formyl°THF * ADP + Pj Y ' ' ; ' "^ H COOK * THF ♦ ATP

These findings have ledc in collaboration with Dro Ho Tabor s
to the elucidation of a similar series of reactions in the
metabolism of forraiminoglutamic acid in mammalian tissues and
account for the earlier observation of Silverman and his
collaborators that formiminoglutamis acid was excreted in the
urine of folic=defieient animals o This suggests a physiological
role for formimino transfer in animals as well as in microorganisms <.

Dr? Rabinowitz has now accomplished the synthesis and isolation
of 5=formimino THF using the formimino transferase purified from
Clostridium eylindrospprumo This has led to the large-scale
preparation of the biologically active diastereoisomer of this
substance as well as of the 5^10-methenyl THFn A second enayme
(eyelodeaminase) has been purified from the same organism? this
converts formimino THF to the cyclic 5pl(Haethenyl THF.-, This
is the first demonstration of a biological function for this
compound which had been previously synthesized as the raeemie
form (anhydrocitrovorum factor) 0 The third enzyme (eyelohydroiase)
catalyzes the hydrolysis of 5P10"methenyl TKF to form 10-formyl TKFc

With DrG Taborp it has been shown that formate excretion rises
in folic acid-deficient rats.-. This rise occurs earlier in the
course of the deficiency than does the appearance of formimino
glutamic aside

Significance to MIAMD Researehs A number of observations in the

literature^, particularly from
nutritional studies with microorganisms^ suggest a relation
between histidine and purine biosynthesis and the discovery of
formisaino transfer and the role of THF ragy help to clarify this
relatione

MIAMD" _ 18 ^^

Page 3

ProposedCou£s@ of Psoje-etg Dsr0 Rabinowitz has left the

— — National Institutes of Health

and gone to the University of California o D?o William Jakoby
has joined this seetiono He also Is ©arsylng on a program of
mierobiologieal reseas-ehp designed to elueidat© the general
meehanisass involved in the metabolism of arainoc hydroxy^ easrboxyl
and sulfhydryl groups.? The teehniqu© is to isolate^, by erayiehmerat
culture teehniquep organises which ©an grow on simple organic
compounds which bear such reactive groups <>

phs;
Individual Projeet Report
Calendar Yeas 1957

Serial NOo NXAMD° 18

Part B Page 4

Publieations during 1957s

Rablnowitzp Jo CoP and Priee^ Wo So*, Js??*, An Ensyraati©

Method for the Determination of Forrai© Aeidp £.-> Blolo Chea^
2290 p 1957 J, in press*

Pro j eet Title:

Principal investigate: . . .

bigs

Coop;:. :ne

Man Ye

Total; 3-2/3
Pro! . 1: 2

Others 1

am

Objeg objective of ::- is to elucidate the

s of forraatien and utilisation of glucuronic acid in

si ,-.

-^M^^-iiiW'- ■-• " Glucuronate°C^ ;ed into nei
ratso Expire as collected and assayed for CF-^o

C^-3 and O-^4 labeled D^glucurono] L°»gulonol; i sre

injected into iver glycogen was isolated

graded and the . assayed for C-

Glucose<=C^* was incubated with rat seminal ves
inositol was isolated and

Major Findi. : - rate of

combustion of glueuronaie observed prevl
oxidation in 1
in the ra1 .
variable combu3tion0

Glycogen isolated after the of D=glucuronolactone>

6~&W to rats and L~gulonolaeti -' gs ^ I

contained only 1% of the administer

These results rule out the direct conversion of eith : sound to gl
About 20 times as much C^-3 was found in Gi as in Gg ose0

Together with s evidence for the Individual steps the!
consistent with th«

Serial' Mo 0 »BMD^_ 19
Page 2

D^glueuronolaetone —=.=§1 L=gulonolactone — — ^ L~:xylulQse + GOg

vie jsylitol ^ _ „ , via sedoheptulose ,. _ _

— —a* > ~^> B-xylulose — — — — —£-- ---> D-glucoseo

The presence of C-*-*4 in inositol isolated from seminal vesicle slice s
following incubation with uniformly labeled glucose suggests a pathway of
synthesis of inositol from glucose , and therefore an alternate route of
glucuronic aeid sjmthesis from glucose since inositol can serve as a pre-
cursor of glucuronic acid in rat kidney „

Significance to NIAMD Research => In view of the wide distribution
of glucuronic acid in connective tissue^ a defect in the metabolism of
this compound may be associated with arthritis and other diseases of that
tissue o In order to recognise any derangement at the biochemical level
in the diseased state it is essential to study the normal course of
metabolism c.

Proposed Cqurse_of Project <= Whether inositol arises directly from
glucose cannot be inferred from the experiments with uniformly labeled
glucose o Experiments are in progress in which glucose^S^C-"4 was incubated
with rat seminal vesicleo The isolated inositol will be degraded to de=>
termine the distribution of the label » Other substrates will be studied
in the same way and from these studies it should be possible to deduce a
pathway of biosynthesis 0

Part B included* Yes

NgLMD° 19
Page 3

Part B; Honors, Awards , and Publications

Publications other than abstracts from this projects

Eisenberg, F0, Jr0, Isselbaeher, K0 and Kalckar, He Studies on
Metabolism of Carbon=lij Labeled Galactose in a Galactosemia
Individual, Science, 125, 116 (1957) o

Burns, Jo Jo, Dayton, P„ G0 and Eisenberg, F0, Jr* Metabolism of
L=gulonolactone in Rats via Pentose Formation, Biochimo et Diophys,
Acta, ?SS 6li7 (1957),

Honors and Awards relating to this project; None

Individual Projee

Calendar lear 1957

erial N©0 NIAMD- 20

lo Biochemistry & Me tat
2o Intermediary Metafcolis
3= Bethesda

Part Ac

Project Titles Metabolism of Glycogen

Principal Investigators DrD M0 Ro Stetten

Other Investigators s Dr» Dn Stetten, Jr8 and Mr, H0 M0 Katzen

Cooperating Units s None

Man years (calendar year 190?):
Totals 3-1/3
Professionals 1-1/3
Others 2

Project Descriptions

Objectives = To gain insight into the normal structure, synthesis
and metabolic turnover of the glycogen of liver and muscle*

Methods Employed <= Glycogen is isolated from the livers and muscles
of rats and rabbits by methods involving extraction with acid or alkali „
Samples are separated into fractions of varying si^e by differential
centrifugation or precipitation and molecular weight determinations carried
out by light scattering* Properties of glycogen samples are also studied
by sedimentation methods and by electrophoresis on glass paper c

Radioactive glucose is administered to rats or rabbits and the
radioactivity of ihe isolated glycogen studied as a function of solubility s
sise, and anatomical distribution^

Glucose=l"POj=Sr^ has been prepared from radioactive starch and is
being used in in vitro studies of the mechanism ©f glycogen synthesis as
catalyzed by muscle and liver phosphorylase 0

Major Findings « It has hesn found that incorporation of glucose
in vivo proceeds more rapidly into the larger molecules of glycogen in
muscle and into the smaller molecules of glycogen in liver.,-. In an attempt
to understand these differences , studies have been carried out in vitro
with liver phosphorylase and muscle phosphoryla se and liver and muscle
glycogens of varying average molecular siseQ The results indicate that
each ensyme has a preference for polysaccharide molecules of a specific
sizar, Muscle phosphorylase reacts preferentially with larger glycogen
molecules than does liver phosphorylase ?

Glycogen can be isolated, in a pure state , using milder conditions
than are used in the conventional Pfluger hot alkali extraction, so that
it has a far larger molecular size than previously reported o

20

Preliminary studies of crude preparations of a mammalian
transglucosidation enzyme from liver indicate that animals are able
to utilise free glucose for the synthesis of oligosaccharides from maltose c

Significance to NI&MD Research = Alterations and defects in the
way the body metabolizes various carbohydrates have been found to be
characteristic of certain nutritional states s drug actions and metabolic
diseases o Any additional knowledge as to how carbohydrates are normally
handled may be escpected to contribute to a better understanding of the
nature of these conditions and diseases o

Proposed Course of Project = The project as originally envisioned
is nearly complete „ Hew milder methods of isolation will be sought in
an attempt to isolate glycogen in a state more nearly like "native" glycogen!

Purification and properties of a mammalian transglucosidation
enzyme which is capable of synthesizing malto oligosaccharides will be
studied „ This enzyme may possibly be responsible for the synthesis of
"seeds" for glycogen production in animals q

Part B included; Yes

3

Honors e Awards

Publications other than abstracts from this project:

Stetten, Mc R0. Katsen, H. H0 and Stetten, B0J Jrc A Comparison
of Glycogens Isolated by Acid and Alkaline Procedures, Jo Biolo
Chemo, in press 0

Stetten, M0 R0 and Stetten, Do, Jr0 Influence of Molecular Size
of Glycogen on the Phosphorylase Reaction, Jo Biolo Chem0, in press „

Stetten, M0 R0 and Stetten, D.? Jr„ Homopolysaccharides9 Conference
on Polysaccharides in Biology, Macy Foundation <= in press o

Stetten, D0, JrD Certain Aspects of the Metabolism of Glycogen,
Diabetes, 6, 391 (1957) »

Stetten, Do, Jr0 The Pathogenesis of Gout, Metabolism, 6, 88 (I95»7)c

Stetten, D0, Jr« Metabolic and Clinical Aspects of Gout, Am. Jo Med0,
22, 807 (1957) o

Honors and Awards relating to this projects

The Banting Medal of the American Diabetes Association - D, Stetten, Jrc
June 1957 &

dual ••- ro,; .
Calendar Tear 195?

Serial too NIAMD° 21
lo Biochemistry & Mel-
go Intermediary Metabolism
3* Bethesda

Part A,

Project Titles The Glycogen Metabolism of Tumor Tissues
Principal Investigators? Dr, Me W„ Nireriberg and Dr. D0 Stetten,,

Other Investigators! None

i

Cooperating Units; None

Man years ( calendar year 1907) :
Totals 1-2/3
Professionals 1-1/3
Other; 1/3

Project Descriptions

Objectives « The objective of this project is to compare "glycogen"
and phosphoryiase levels in a variety of ascites tumors and tumors in tissue
culture with normal tissues in order to determine whether a relative absence
of phosphoryiase is a distinguishing characteristic of the neoplastic tissues

Methods Employed «= The following tumors are being studied? Hepatoma
ascites j masst cell ascites , sarcoma 3? ascites s lymphocytic leukemia ascites ?
Ehrlich carcinoma ascites « Krebs-2 carcinoma ascites s melanoma S~91 sol
plasma cell ascites , and the HeLa carcinoma in tissue culture 0 Glycogen
is determined by the method of Good9 Kramer and Somogyi.- Phosphoryiase
activity is determined both by the method of Sutherland and by measuring
the progressive disappearance of glycogen in homogenateso

Major Findings = Preliminary results indicate that the different
types of tumor cells studied are unable to either synthesise or utilise
glycogen whereas the normal tissues thus far tested can do both,-

Significance to NIAMD Research - The storage of carbohydrates in an
easily accessible form is a characteristic of normal, adult, mammalian
tissues o Although tumor tissues utilize carbohydrates at extremely rapid
rates compared with normal t is sues s the ease with which neoplastic tissues
can store sugars has never been systematically investigated <, A survey of
this type might therefore point out differences in ensymic pathways between
normal and neoplastic cells ands further 9 might help to clarify the unknown
factors which control the extraordinarily high carbohydrate utilization
of tumor cells o.

Proposed Course of Project - The preliminary findings must be validated
by further control experiments ,. A more complete study using additional
normal j embryonic and neoplastic tissues will be initiated,.

Part B included 8 No

Individual Project Report
Calendar Tear 195?

Serial N0o WIAMD° 22
lo Biochemistry & Metabolis
2o Intermediary Metabolism
3o Bethesda

Part Ao

Project Titles Insulin =■ Its Structure and Fate in Continuously
Perfused Mammalian Tissues <,

Principal Investigators: Dro F0 Tietse, Dra G„ E„ Mortimore and

Dr» D„ Stetten, Jr0

Other Investigators! Dro Jo E0 Folk and Dro J, A> Gladner

Cooperating Units; Laboratory of Oral and Biological Chemistry, HIDE
Laboratory of Physical Biology, NIAKD 95

Man years ( calendar year 1907) s \
Totals 3-1/3 V

Professionals 2
Others 1=1/3

Project Description;

Objectives = It has been shown that homogenates of mammalian liver g
kidney , and to a much lesser extent,, muscle are capable of degrading insulin,
The specific fate of insulin in these tissues will be studied.

Recent work has demonstrated that the structural variations among
insulins isolated from different species are limited to the amino acids
occupying positions 8~10 of the "A" chain of the molecule „ Attempts to
develop a simple method for the characterization of this sequence will be mac

Methods Employed - The liver and hind limbs of albino rats are
continuously perfused with whole, heparinised rat blood containing trace
amounts of insulin=I^-31 ., Samples of the perfusing blood are withdrawn
at intervals during a one hour period 0 Aliquots from each sample are
assayed for l) TCA -precipitated radioactivity, an estimate of undegraded
insulin, and 2) TCA=soluble radioactivity, an estimate of degraded insulinc.

A combination of chemical and enzymatic reactions are being developed
to liberate amino acids 8-10 of the A-ehain of insulin.-.

Major Findings <=> In liver it has been observed that rapid initial
binding of insulin~I^31 occurs with a disappearance of 60=>7Q$ of undegraded
hormone in one hour* The initial rate of appearance of degraded insulin
is much slower, but at the end of one hour IjO^ of the total radioactivity
was present as TCA=soluble producto In contrast, hind limb preparations
degraded insulin to a barely detectable extent despite a significant degree
of hormone binding r>

rial Hoo NIAMD~ 22
Page 2

The intra-A chain disulfide bridge of insulin has been reduced
with thioglycolate and the -SH groups so released have been coupled with
8<=bromoethylarainen The successive action of trypsin and carboxypeptidase~B
on the product resulted in the liberation of a mixture of amino acids^
one component of which was identified as S~( S=aminoethyi) -cysteine 0

Significance to NlAMD Research = The finding of rapid binding and
degradation of iodoinsulin by intact rat liver emphasizes a role of liver
in the metabolism of insulin which until recently has been obscure n It
is conceivable that the diminution of these functions is related to the
known amelioration of diabetes in liver disease^ Furthermore^ it may be
possible to explain clinically different diabetic states by differences
in insulin degradation and bindingo

The elucidation of the sequence of amino acids 8=10 of the "A"
chain of human insulin would be of great value in assessing the role of
antigenicity in the formation of humoral antagonists to commercial beef-
pork insulin0

Proposed Course of Project <= Using the perfusion technique^ the
effects of various compounds and altered physiologic states on insulin
metabolism will be studied^ It is also hoped that the character of the
degradation product(s) might be elueidatedo

With respect to insulin structure determination it is hoped that
further proteolysis by carboxypeptidase=A of the protein residue will
result in the stepwise liberation of amino acids 8=10 of the A=chain.o

Part B included? Yes

;~ 22

Page' 3

Part Bo Honors, Awards, and Publications

Publications other than abstracts from this oroject;

Stettens DOJ Jr„ The Hyooglueemie Sulfonylurea Drugs - An Interim
Evaluation,, Ann0 Into Medo, lj6, 1005 (195?) o

Field j Jo BOJ1 Tietses Fc and Stetten} DOJ Jr0 Further Characterization.
of an Insulin Antagonist in the Serum of Patients in Diabetic Acidosis.,
J. Clinc Invest,., 36 , 1588 (1957).,

Tietses ¥.3 Gladner, J0 A, and Folk,, Jo E0 Release of C=Terminal
S~(p=aBiinoethyl) -cysteine Residues by Carboxypeptidase=Bs Biochim&
et Biophyso Acta, to be published in January s 19580

Honors and Awards relating to this project; None

Individual Project Report
Calendar Tear 195?

Serial Moo HIftMD° 23__
lo Biochemistry & Metabol
2o Intermediary Metabolism
3° Bsthesda

Part Ao

Project Titles Biosynthesis and Gatabolism of HexoseSo

Principal Investigators? Dr<> I. J0 Topper, Br* LD A0 Pesch and

Drn D0 Stettens «Jr^

Other Investirators : Dr., S0 Segal and Drc E0 R, Simon

Cooperating Units: None

Han Tears (calendar year 1957)?
Total: 3-2/3
Professional: 2-2/3
Others 1

Project Description:

Objectives = The objectives of this project are to study the
bacterial biosynthesis of L°fucoses the mammalian biosynthesis of
I.=iduronic acid and the effect of various steroids on the metabolism
of B-galactoseo

Methods Employed => In the biosynthetie studies isotopie precursors
are administered to appropriate biological systems and after suitable in-
cubation periods L=fueose and L~iduronie acid are isolated „ Chemical
degradations are performed on these sugars in order to determine the
labeling pattern c.

The effect of hormones on D-galaetose metabolism is studied in vitro
in the presence of a variety of steroids by measuring C^Og production from
isotopie galactose-

Hajor Findings <= The labeling pattern in L»fucose isolated from
cultures of Aerobacter aerogenes after incubation with isotopie glucose
indicates that the methylpentose arises from the intact carbon=skeleton of
glucose* This conversion thus involves epimerisations at C=2, C=3 and
C<=5 and reduction at C=60

The oxidation of B°galaetose to CQ^ by rabbit liver slices is
profoundly increased by methyl testosterone and progesterone s but is
little affected by either hydrocortisone or cortisone o The effect of these
steroids on glucose and fructose oxidation was found to be small and non-
specific with respect to hormone- These observations suggest that methyl
testosterone and progesterone exert their effect at a level between the
entry of galactose into the cell and the conversion of this sugar into
glucose°6«phosphaten

Serial No„ KIAMD^^g^
Page 2

Significance to NIA?4D Research - The elucidation of biosynthetie
pathways with respect to carbohydrates 9 and studies on hormonal control
of sugar catabolism might be expected to shed some light on certain
metabolic diseases o

Proposed Course of Pro.ject => An attempt will be made to delineate
the pathway by which L=iduronie aeid5 a major constituent of chondroitin
sulfate Bj is synthesized by rabbit tissue Q

The precise site of action of progesterone and methyl testosterone
on galactose oxidation by mammalian liver will be further investigated,-:
Other tissues, including red blood cells s will also be studied in this
respect o

Part B included; Xes

Serial No„ NI&MD* 23
■Page 3

Part Bo Honors, Awards, and Publications

Publications other than abstracts from this project:

Segal, So and Topper, L Jc On the Biosynthesis of L-Fucose by
Aerobaeter aerogenes, Biochim0 et Biophyso Actaj, 2J>, Iil9 (l95?)o

Simon, E0 Ro and Topper, Y0 J„«, Fractionation of Human Erythrocytes
on the Basis of Their Age, Mature, in press 0

Honors and Awards relating to this project; None

195?

too VIAND- 24

lo Biochemistry"!: Metabolis
2o Intermediary Metabo
3o Bethesda

Part Ao

Project Title: The Mechanism of Action of Ensyms3 of the
Glycolytic Cycle

Principal Investigators; Dr = Yo Ja Topper and Dro B0 Bloom

Other Investigators Miss JQ D„ Benedict

Cooperating Units: Hone

Man years (calendar year 1957} s
Total: h
Professional: 3
Other: 1

Project Description:

Objectives =■ The objective of this project is to gain a deeper ins:
into the mechanism by which enzymes s particularly those involved in gly
operate as catalysts in biochemical transformations o

Methods Employed - l) Non-isotopie substrates are exposed to the
biological catalysts in the presence of TgO and the distribution of isotope
in the products is determined 0 2) Tritiated substrates are incubated with
the enzymes and the redistribution of the isotope is then studiedo 3) Com°
plex formation between substrate and ensyme is followed spectrophotometr

Major Findings ■= Since the stereospecif ic activation of hydrogen by
aldolase and phosphotriose isomerase was reported last year it has been
found that aldolase forms a stable complex with dihydroxyaeetone phosphate
(DHA.P) which absorbs in the neighborhood of 230 rap.0 This reaction is
specific with respect to both protein and substrate,.-, The ultraviolet
absorbing material can be destroyed by the addition of aldehyde.? Further-
more, absolute identification of the hydrogen of DHA.P which is labilized
by the two enzymes has been accomplished,*

Significance to NIAMD Research ~ The enzymes studied mediate
important steps in carbohydrate metabolism*, It is felt that the elucidation
of these and other enzymatic reactions will be of aid in the understanding
and treatment of various metabolic diseases.;.

Proposed Course of Project ~ We will attempt to derive a small fragment
from the aldolase molecule which is still capable of ccmplexiijg with Dl-IiP.-.
The determination of the structure of such a fragment would facilitate furthe
analysis of the mechanism of action of the enzyme* Alsos the possibility
that complex formation between the ensyme and DHftP results in a change
the shape of the protein will be investigated c

Part B included s .Yes

31oom9 Bo

^Aldolase Compj

in Spectrophotometry Caused b

Bloom, Bo 5 Galactose Metabolism in Rat
in press o

snors and Awards relating to this pi None<

ivid.ual Project Report
Calendar Tear 191

Serial No» M3AMP° 25
lo Biochemistry & Met
2o Intermediary Metabolism
3o Bethasda

Part Ao

Project Title: The Biosynthesis of Thiamine

Principal Investigator: Dr>0 I0 G„ Leder

Other Investigators: None

Cooperating Units? None

Man years (calendar year 1907):
Totals 1-2/j
Professional; 1
Other: 2/3

Project Description:

Objectives <= T© study the mechanism of synthesis of the vitamin
thiamine j in particular t the synthesis of the thiazole ring?

Methods Employed - Experiments based upon the isotope dilution
technique will be used to establish precursor relationships with sulfur
amino acids and their derivativeso In addition specifically labeled com-
pounds will be studied as precursorSo These studies will be performed in
various wild and mutant strains of yeast and neurosporao The mutant strains
will also be cultured under conditions which may give rise to the accumula-
tion of possible intermediates as a result of genetic blocks c The nature of
possible intermediates will be investigated by paper and column chroiaato-
graphy and electrophoresis and by spectrophotometri© studies*

Major Findings <= Experiments with Torulopsis utilis suggest that
like certain strains of bakers0 yeasty these cells can synthesize the
thiazole ring in response to relatively high levels of thiamine pyramid:..
If high levels of thiamine can be obtained it would be particularly useful
in these studies because ©f the ability of this organism to utilise a
large variety of compounds as sources of sulfur 0

Significance to NIflMD Research « Cyclic compounds containing sulfur are
represented by such relatively diverse compounds as biotin and thiamine ;)
which are vitamins p and penicillin^ an antibiotic -> It is hoped that this
study will contribute to our understanding of the synthesis and raetabol:..
compounds of nutritional and medicinal importance in man and in miereorganisn

Proposed Course of Project <= S^5 sulfate will be incubated with yeast
cells in order to label the thiamine o Subsequently,, the effect upon sulfate
ineorporation of !!cold" methionine 9 homocysteine^, cysteine and similar a
pounds will be studied 0 A number of thiamine mutants of bakers0 yeast will
be examined to establish the nature of the genetic bloek and the possible
accumulation of important intermediates o

PartJBo Honors. Awards , and Publications

Publications other than abstracts from this project:

Bryant, Jc Ho, Leder3 ID G,,, and Stettens D09 Jr« The Release c
Ghondroitin Sulfate from Rabbit Cartilage Following Intravenous
Injection of Crude Papain, Areho Biochemo and Biophyso, in pres

Honors and Awards relating to this project; None

PHS
Individual Project Report:
Calendar Year 1957

Serial. Nou gIAMD°_ 2ft.

I. Biochemistry & Metaholij

2o Metabolic Enzymes

3o Bethesda

Part Ac

Project Title s

Ac. Studies on the Mechanism of the Conversion of UDPGalactose
to Glucose r

B0 Study of the Adaptation Pattern of Formation of Enzymes of
Galactose Metabolises

Co Immunological Study of Uridine Diphosphate Galactose»4-
Epimerasec

Dc Development of Methods for the Study of UDPG Metabolism
as a Function of Differentiation and Heredity.,

Eo Study of the Enzymes of Galactose Metabolism in Hereditary
Blocks in Microorganisms „

Fa Studies on Congenital Galactosemia*

Principal Investigators Herman M. Kalckar

Other Investigators? Former Participants j

Huguette de Robichon Szulmajster Kurt Isselbacher

Kiyoshi Kurahashi Elizabeth Anderson

Albert Wahba
Henry Nt Kirkman
Elizabeth Maxwell

Cooperating Units?

Drc Be Eddy? National Institutes of Allergy & Infectious Diseases .
Dr„ Frank E£senbergs Section on Intermediary Metabolism.,

Man Years (calendar year 1957) f
Total 6
Professional 3=1/2
Other 2=1/2

Part B included <= Yes

Serial No„

Pag<§ 2

Project Descriptions

Ao Studies on the Mechanism of the Conversion of UBP Galactose

to Glucose.-

OBJECTIVES - Brain lipids and lactose contain galactose* The

mammalian organism can convert glucose to galactose
and vice versa. The enzymatic inversion process by which the
hydroxyl in carbon-4 of galactose is turned so as to make glucose
is one of great interest for students of "biology and of structural
chemistry. Many suggestions have been proposed regarding the
mechanism of this process which 3eems to.be closely related to
a s£ereoisomeri2ation„ It will be recalled that DPN was found to
be a coenzyme of UDPGal-4°epiiaerase (Seeialso Dr* E. Maxwell es
report),. It was, therefore, of Interest to see whether protein^
bound DPN or DPNH could be detected in the purified enzyme „

METHOD ■» A prerequisite for studying the enzymatic inversion of

uridine diphosphogalactose (UDPGal) to UDP glucose is a
purification of the specific enzyme which catalyzes this process
and which we call UfDPGal-^epiixjerase (formerly called Galacto=>
Wal.denase)o This has been accomplished by Dr* Elizabeth Maxwell
in our group (See Dr« Maxwell's report) <, Studies on the fluorescence
spectrum of the purified UDPGal=4~epimerase (in amounts of 3=5 rage
protein per mlo) were performed by using the scanning spectrophot©=
fluorometer. The exciting wavelength used was 360 m^ and the
fluorescence spectra were recorded.

RESULTS - A highly fractionated UDPGal=4°epimerase studied in the

scanning spectrofluorometer gave a distinct fluorescence
spectrum with maximum at 410 m>uu The most effective wavelength with
respect to the exciting light was found to be 360 m(i„ The fluorescence
spectrum, as well as the maximum of the exciting light, correspond
to the optical properties described by other authors for protein=
bound reduced DPNH»

FUTURE PLANS - This same technique should be applied to fractionated

4»epimerase from yeast induced with galactose as
well as for proteins in same fractions from non~ induced yeast « This
might yield a particularly effective demonstration that the bound
DPNH belongs to the DDPGai~4°=epimerase„ As will appear from
Dr. Szulmajster^s work, described elsewhere in this report, BDPGal«4~
epimerase in yeast is only synthesized after induction with galactoses

Serial

Study of the Adaptation Pa&tern of Formation of En;
Galactose Metabolism,

OBJECTIVES - Study of the adaptive pattern of the enzymes responsible
for galactose metabolism is very important o In general,,
though the adaptive nature of the "galacto^ymase" complex is knowna
nothing has been done on the individual enzyme members of this
complex o Knowledge of the kinetics of adaptive formation of these
enzymes, specificity toward the inducer, etc„, can bring about
additional facts in the fields of adaptation and of metabolic
aberrations in mutants from microorganisms and men (galactosemia),
and also furnish fresh ideas towards the problem of biochemical
differentiation during embryonic development.

METHODS ■» Diploid yeast was grown on acetate or succinate atssaonia

medium,. Galactose was present or absent*, Aliquots
were taken either for assay in the Warburg manometer or for
enzymatic assay of lysateso The enzymatic assay of galactokinase,
Gal-l-P uridyltransferase and UDPGal~4~epimerase were performed
as described for the assay of the hemo ly sates »

Studies on

RESULTS

A yeast mutant supplied by Drc Hawthorne 8 University
of Washington, and described as unable to ferment galactose, was
identified by Dr0 Huguette de Robichon Szulmajster in our group as
being galactokinaseless. In this mutant, Dr* Szulmajster found
that the enzymes succeeding the galactokinase reaction, namely
Gal°l~P feransuridylase and uDPGal~4"epimerase, which are absent in
unadapted yeast, were found to be strikingly induced in response
to galactose * Hence free galactose appears to be a multi" inducer
of the three enzymes of the galactose pathway, following a pattern
of simultaneous rather than sequential adaptation* Apparently the
pattern of sequential adaptation by which a substrate 1 (or an
analogue) induces an enzyme which converts substrate 1 into substrate
2, etc 9 does not apply here*

2« Study of the system responsible for permeability, to
galactose in yeasts .

Non»adap£ed cells have been found to be rather
impermeable to galactose „ Increase of permeability to galactose
in diploid yeast can be shown to be related to the time of contact
of the cells with galactose.. This system, when completely developed

(after 3" 5 hours), is able to increase the intracellular concentration

Serial Mo. HIMD- 26

to 8~XQ times the external concentrations. This has been detected
by C galactose uptake by the cells in a very short incubation
period. The properties of this system are being studied Preliminary
experiments on tryptophan requiring yeast suggest that de novo
protein synthesis (free amino acids being required for its formation)
is associated with the active transport of galactose into the cello
Glucose acts as an antagonist of galactose in this system,,

i'jJECXlVES - The role of UDPGal~4= epimerase in mammals is partially
known but many questions deserve to be looked into. Is
this enzyme s for instance , essential for the normal development of
mananals, especially under conditions of a diet devoid of galactose?
The myelination of the central nervous system takes place exclusively
after birth,, Hence, at that time substantial amounts of galactolipids
are being synthesized* The conversion of glucose to galactose depends
on the effectiveness of UDPGal=4=epimerase, What would happen if
this enzyme were inhibited by analogs of galactose or more specifically
by an antibody? If anti~4-epimerase could be produced,, would it only
give cross reaction like8 for instance, the anti=p«galactosidases or
would it be & real antienzyme, i<.e,s inhibiting enzyme activity like,
for instance, anti~alcohol~dehydrogenase? If anti~4~epimerase could
be made, it may also turn out to be a valuable tool in a study of the
mechanism of reaction catalyzed by UDPGal'=4~epimerase or for a study
of the induction of this enzyme in yeast , If a formation of brain
anti=4-epiiaerase could be promoted, for instance, by autoinraunization,
how would myelination in the young animal be affected?

METHODS » The adjuvants technique by Freund was used in the immunization
procedure; our thanks are due to Dr, B, Eddy and her staff, National
Institute of Allergy and Infectious Diseases, for help and advice during
the initiation of this immunization program* Purified calf liver TOPGal-4"
epimerase constituted the antigen, A quantitative assay for anti=-4>=epimerase
was worked based on the techniques described by Dr. Maxwell for the study
of the kinetics of 4=epiaserase, Globulins were harvested from guinea pig
blood plasma or serum by half° saturation with ammonium sulfate and
subsequently dialysed, .

RESULTS - Regarding the study of antienzyme formation, it turned out

that of seven immunized guinea pigs of the age of two months,
six developed striking ant i~UDPGal~4- epimerase (against the corresponding
enzyme from calf liver) , whereas the immune globulins did not inhibit
another enzyme, BDPG dehydrogenase, from same source. Four controls
(three of which were two months old animals) which were not injected at
all or injected only with adjuvants, did not develop detectable amounts
of anti°4~epimerase. Three guinea pigs of three months, which were
receiving booster shots, kept their snti~4-epiinerase level, (This latter

2<o

group included two iactating animals)., In three older animals of
8<=12 months, no formation of an£i*°4~epimerase could be observedo
The inhibition brought about by addition of 3 mg, immunization
globulin to the 4-epimerase ranged from 50 to 80 percent.
Globulin from non~iEE3unized animals tends to give some stimulation
(40 to 90 percent) of the 4~epiBaeraset The true inhibition mays
therefore, be more intense than observed directly „

FUTURE PLANS - 'She production of anti"=4-epimerases may be valuable

tools in a study of the mechanism of enzyme synthesis s
especially in microorganisms which synthesis© 4~epimerase on an
adaptive basis. It may also be possible to resolve UDPGal-4-
epimerase into more than one step„ It is quite conceivable that
at least two enzymes might be involved in the catalysis of this
complicated process <, If an antienzyme were promo teds which pre<=
ferentially inhibited one of the components „ intermediary products
might accumulate o Studies on the possible inhibition of 4~epimerase
in vivo by means of ant i~ immunization and by analogues of gala . fcose
are also under consideration,

D.. Development of Methods for the Study ofUDPG Metabolism as a
Function of Differentiation, and Heredity.,

OBJECTIVES - Recent studies from two different groups have shown

that glucuronide coupling is a process which develops
gradually after birth.. This explains, probably, phenomena like
the physiological jaundice as well as the fatal jaundice which
develops frequently in cases of blood group incompatibility such
as erythoblastosis faetalis„ Bilirubin is a glucuronide acceptor
but if the rate of formation of the active glucuronic acid donor
(UDP-glucuronic acid) is slowed down^ non=coupled bilirubin which
is toxic will accumulate in the central nervous system.. Various
groups have found that in man there is a hereditary disease in
which the enzymatic transfer of glucuronic acid is blocked,, More~
over, suspicion is mounting that the foetus and the newborn infant
is lagging seriously behind in their ability to form and transfer
active glucuronic acid* The latter suspicion is based on experie®?n£s
on guinea pig embryos,' foetuses and newborn animals <, A specific
dehydrogenase, UDPG-dehydrogenases which is responsible for the
formation of active glucuronic acid (UDP glucuronic acid) was described
2 to 3 years ago by our group,. This enzyme has now been found to be
extremely low in the newborn guinea pig (Brown & Zuelzer s Department
of Pediatrics t University of Michigan) „ What is the basis for the
lack of this dehydrogenase in embryonic livers? What is the UDP
glucose content in embryonic livers compared with livers from animals
at various ages aftet birth? When do the enzymes of galactose
metabolism appear „ It has been known for a long time that the
galactose tolerance of the newborn is relatively low*

Besides these problems which have to do with the quantitative
biochemistry of differentiations, it would also be of great value to

?ag«s &

initiate corresponding studies on hereditary abnormalities and
especially on the more subtle changes in hereditary carriers (trait's)
of enzymic molecular diseases of haxose metabolism, in order to
institute a study of the. enzymes of galactose and glucose metabolism
in man with special reference to traits (either spontaneous or
occurring in population previously exposed to high energy radiation) s
precise and reproducible methods for blood eazymology must be
developed,, In order to be used in field stuidiesj, the methods must
be rugged and not tea tedious »

For the purpose of studying UDP glucose concentration
on the various stages of developments the Indicator
enzyme mostly used is the specific UDPG dehydrogenase « The tissue
filtrates are subjected to a fractionation with norite; the
nucleotides are adsorbed and can be eluted with ammoniacal 50 per~
cent alcohol c Internal Standards are always usedo

For a population st'^dy in man8 urine, blood plasma and red cells
or hemolysates are the only available sources . A study of urinary
excretion pattern or the application of tolerance tests have so far
yielded valuable information. However s both methods are tedious ,
inaccurate and variations are greatly influenced by hormonal factors .
The red blood cells and their heraolysates contain a large number of
metabolic enzymes s the rate of which are relatively little influenced
by hormonal factors . Dr. Henry H=, Kirteian of our section is develop-
ing promising techniques in this direction, The oxygen consumption
brought about by addition of methylene blue is used as a measure for
the activity of three enzymes „ Gal-i-P uridyl transferase, phospho=>
glucomutase, and glucose«6-phosphat:e dehydrogenase. The red cells are
carefully washed before lysis in order to suppress the endogenous
respiration and subsequently hemolysates are made by means of distilled
water. Warburg, manometry is used for the determination of oxygen
consumption.

RESULTS - As regards the first problem, the variations in UDPG

metabolism during the prenatal and the postnatal stages
of developments, preliminary observations by Drs» KIrkmans, Mr„ E.
Bynum and myself indicate that embryonic livers from guinea pigs
are extremely low in UDP glucose;, whereas livers from adult animals
contain appreciable amounts »

FUTURE ELANS « As regards UDPG metabolism in the newborn* it is

felt that an understanding of the mechanism of the
initiation of enzymes in the mammal might be of paramount ii*terest
in developing a rational approach toward the therapy of erythro~
blastosis0 To take an examples If UDPG injection in the newborn
were able to raise the level of liverP UDPG (and most likely 5 to
10 percent of the circulating UDPG would penetrate into the liver
cells) would this mean that the rate of glucuronide coupling would
increase faster? $ueh a speed=up might presumably break the

NLfiMD- 26

vicious cycle- The problem might, well be readily approachable.,
Of great theoretical interest is the question whether RKA synthesis 9
which obviously takes place more vividly in the embryo than in the
newborn;, consumes so much TOP that there is very little left for TOP

and UTP synthesis,

Concerning the question of hereditary carriers, the «ype of
methods developed could be used to check up on some existing views
on the pattern of inheritance of congenital galactosemia. They can
also be used in a study on primaquine hypersensitivity or on traits affecting
nucleoside and pentose metabolism- etc- They may also be useful in
trying to find radiation mutations in human populations which have
been exposed to high energy radiation- A study of spermatozoa
metabolism in traits carrying defect which affects the maintenance
or specific function of spermatozoa might reveal that the genotype
In shese cases have a phenotypic expression. Such an approach
should be considered in the case of male carriers of phenolpyruvate
oligophrenia c

E o Study of the JEr^zgrnes^of Galactose Metabolism in Hereditary
Blp cks in M ijLrporganigms «

OBJECTIVES » The study which our group undertook during Che year

of 1956 showed that the hereditary disease, congenital
galactosemia 8 is due to a defect in an esssyme Gal-l°P uridyltransferasej,
an enzyme which we first found in yeast adapted to galactose., Since
Dr„ Jo Lederberg at the University of Wisconsin has described a number
of bacterial mutants (mutated by a virus) unable to use galactose as a
carbon source for growth (galactose negative mutants),, Dr« K- Kurahashi
in our group undertook a study of those galactose deficient mutants -
The following objectives were set: 10 To identify the enzyme defect
in the various galactose deficient mutants and correlate it with the
existing genetic information„ 2, To further elucidate the nature of
the galactose toxicity which is a striking feature of the human
disease congenital galactosemia,

METHODS - Enzymatic spectrophotometry techniques along the lines
previously described in the investigation of cell lysates from humans
was employed ira bacterial lysates. The indicator enzymes used were
the specific TOPG dehydrogenase and the usual enzymatic analysis of
glucose^ 1 "phosphate (or glucose-S^phosphate)- Use was also made of
C*^ (Carbon**!) labeled galactose obtained through the courtesy of
Dr- Horace Isbell3 National Bureau of Standards- Growth curves of
the various mutants were obtained by means of the conventional
turbidimetrlc technique-

Pag® 8

MAJOR FINDINGS? Among seven single mutants of |. coli which we

obtained from Dr. Lederberg, three were identified
as blocked in the galactokinase and four were found to be blocked
In the Gal~l"P uridyl transferase. According to Dr. Lederberg the
various galactose negative mutants are closely coupled but never the*»
less genetically distinctly different. These results confirmed
recent findings on other microorganisms which clearly indicate that
the so-called gene is composed of numerous sub-units and that the
production of one ensyme can be interfered with by a mutation in
one or the other of the sub-units within this particular gene.
After the various mutants had been identified^ Drs. K.Kurahashi
and Albert Wahba undertook a study of the effect of the extra
addition of galactose on the growth rate of the wild type (galactose
plus) as well as of the galactose negative mutants. It was found
that the growth rate of the wild type and of galactokinaseless
mutants was the same on the glucose ammonium medium with or without
the extra addition of galactose. However , the growth rate of the
mutants blocked in the Gal~l-P uridyltransferase was markedly slowed
down by the extra addition of galactose. These observations furnish
a strong indication that galaetose-1-pb.ospbate rather than free
galactose is to be held responsible for the suppression of growth
in these organisms. The inhibition could be due to galactose™!*-
phosphate per se or to the phosphorylation of galactose which, when
acting as a blind alley, would tend to trap high-energy phosphate.
Ginsburg in our group has found that gslaetose^l-phosphate pjr se
is indeed an inhibitor of &n enzyme in glucose metabolism, namely
phosphoglucomutase. {See Br. Gin3burg8s report). This finding may
have relevance to the suppression of growth by addition of galactose
in the bacterial mutants blocked in Gal=l~P uridyltransferase. It
may also have bearings on the galactose toxicity in congenital
galactosemia since this disease, as mentioned, also constitutes an
example of a block in the Gal=l-P uridyltransferase.

FUTURE FLANS - Concerning hereditary problems, the galactose

mutants may offer opportunity to study the relation

between mutations and changes in gene products,. Lederberg has
found the existence of interesting double mutants with strong
coupling. Dr. Kurahashi has found that in these double mutants two
ensymes have become defect. Is it possible to detect an "error3
in the amino acid pattern? If so, can it be located in relation to
the terminal amino acid?

As regard to the toxic effect of galactose°l-phosphate on the
growth of the bacteria, one may wonder how the growth of the virus
carrying the defect gene is affected.

Serial No. BXftMD- ,__. 26_
Psag© 9

Fo Studies on Congenital Galactosemia

OBJECTIVES ■= Our group found two years ago that red cell hemolysates

Irons subjects afflicted with congenital galactosemia
show a striking defect of the enzyme Gal~l°P uridyl transferase. It

remained, first of all, to establish that other cells show the same
enzyme defect „ In other words , that it is a general enzyme defect ,
Since early diagnosis of congenital galactosemia is of the utaaost
importance, it was also felt desirable to develop the enzyme test
for Gal~l~P uridyl transferase in such a way that it could be used
as a reliable test in the early diagnosis of the disease,. Subsequently;,
it would be of interest <:o use more sensitive tools such as, for
instance, radioactive galactose in order to differentiate between
various cases of congenital galactosemia. Is the incorporation of
Gal~l»P completely blocked, or are there cases where residual activity
persists?

14

METHODS - The use of G ^labeled galactose and the enzymatic

preparation of C^-Gal-l-P proved particulary useful in
micro assays on liver biopsies from galactosemia subjects,.
Fractionation with norite was performed and the radioactivity which
is incorporated in the uridinenucleotide (fraction adsorbed on
Norite and eluted with 50% ammoniac®! alcohol) is a sensitive
measure of the presence or absence (or of a striking defect of the
enzyme Gal~l=P uridyltransferase, i„e,s

* *

Gal=l=P + OTPG ^ssst G-l-P + UBPGal

UBPGal ^sssii UDPG

For the in vivo project, a 24~year old male subject who is mentally
retarded on account of congenital galactosemia was selected „
Galactose 5 pG G^ was infused intravenously and oral administratioa
of menthol was instituted in order to secure any glucosidonic acid
which might be formed from the labeled galactose. The menthol
glucosidonic was crystallised from uric acid and determined for
radioactivity. This project was performed with Br, F, Eisenberg
as the chemist and Br, Kurt Isselbacher as the clinician,,

The ensyoatie assay as used in the service of diagnosis takes
advantage of the following facts - 1) Hemolysates of human red blood
cells do not have 4=epimarase activity; addition of BPM is required.
Hence, if Gal-l-P is incubated with UBPG, UDPGal is formed and G-l-P
is released. The stoichiometry has been found to be strictly
quantitative in more than fifty different specimens of hemolysates
from various subjects, 2) Heither WBQ nor UBPGal undergo significant

Serial No„ NXAMD^ 26
Page 10

side reactions. Hence, the extent of UDPG disappearance as determined

by DPN reduction at 340 mp, brought about by the specific UDPG

dehydrogenase agree quantitatively with the formation of UDPGal

as determined by the renewed reduction of DPN upon further addition

of the second assay enzyme which is fractionated calf liver UDPGal=4»

epimerase*

FINDINGS - The defect in Gal">l~P uridyltransferase in congenital

galactosemia which wa3 first detected in red blood cells
was also found in liver and in red blood cells of the umbilical cord.
These findings , therefore,, establish that the defect in Gal~l~P
uridyltransferase is general and is congenital and hence* presumably,
constitute the basis for the abnormality,. In the case of an adult,,
the incorporation of Gal~l-P into nucleotide was not completely
blocked although markedly lowered.. The remaining reaction may, however;
be due to an alternative Iwt related pathway „ Dr. L Isselbacher
(now at Massachusetts G-neral Hospital) has found evidence for the
existence of such an alternative pathway in mammals , although the
capacity of this alternative reaction is smalls

It has been possible to develop a highly reproducible diagnostic
method using hemolysates and the above described enzyme assay c Ey
this method it was possible to diagnose a feeble-minded 12~year old
male subject as galactosemic, who had been misdiagnosed as a victim
of another disease and hence overlooked as being galactosemic t Ws-j&t
became particularly important in this case was the fact that a
three month old sibling of the above subject was revealed as being
galactosemic as wello In the latter case,, Drs, Ga Donnell and
W„ Bergren., Los Angeles Children 8s Hospital, instituted immediate
galactose^ free diet and the young sibling recovered and should
presumably be protected from later brain damage.,

FUTURE SUGGESTIONS - Screening of institutions for feebleminded for

congenital galactosemia by a simple screening
technique. First screening should be galactose excretion urine
(reduction,, test paper) upon administration of galactose in suitable
amounts c Second screenings enzymatic test for Gal-l-P uridyl"
transferase in hemolystatesc

Studies of the disease congenital galactosemia by immunological
techniques might be of value in trying to resolve the seemingly
homogenous biochemical disease into various groups „

Serial No. NXAMD- 26,
Page 11

Studies on the enzymatic formation of uridine diphospho-
glucuronic acid, description and isolation of TOP glucose
dehydrogenase, as well as identification of UDP glucuronic acids
have been described in the annual report for previous year.

Studies on galaeturonic acid metabolism in microorganisms
were conducted by 0r, A„ Wahba under the supervision of
Dr<, Go Ashwell who has developed a program on the study of 6h.«
metabolism of this compound in the mammalian organismo The report
of Drc Wahba8s work will therefore be found in Dr„ Ashwell 8s
report «

PHS~NIH

Individual Project Report

Calendar Year 1957

Serial No, HIAMD- £6

Pag© n

Part Bt Honors, Awards, and Publications

Publications other than abstracts from this projects

Kalckar, H. M< Biochemical Mutations in Man and Microorganisms.,
Science, 125, 105 (195?) .

Andersons Elizabeth P., Kalckar, HL M» , and Isselbaeher, Kurt J.,
Defect in_the_incorporation of falactose-l-pftgjBJgtg^Jntto
Nucleotides in LJver Tissue from Patients with Congenital

Galactosemia, Science, 125, 113 (1957)..

Eisenberg, Frank Jr., Isselbaeher „ Kurt J., and Kalckar8 H. M. ,

Studies on theJietab^Msj^^^gj^labeled Galactose in ajgajactose^c
Individual, Science, 125, 116 (1957).

B3§&

57) =

Science, 125, 115 (1957

Stromingers J. L», Maxwell, E. S,, Aselrod Jo, and Kalckar s BL Mu ,
Enzymatic Formation of Uridine Diphosphqg]^curonie_Asid; J, Biol.
Chem., 224, 79 (1957).

Kalckar, H. M. , Anderson E.a and Munch»Petersen, Enzymatic Synthesis
and Metabolism of Uridinedipb^s^hoj^cojyj^CompoundS;, Pubblicazioni
della Stasione Zoologiea di Napoli, xms 119-125 (1957).

Kalckar, H. M. and E« P. Anderson, Enzymatic Determination of BTP,
Methods of Enzymology, Vol. 3, 976 (1957).

Strominger, J. L„, Maxwell, E. S. and Kalckar, H. M. , Determination
Vol. 3, 974 (19

ofJBPG^and UTP by means ofjg dehydrogenase, Methods of Enzymology

Anderson, E. P., Kalckar „ H, M<, Kurahashi, K., and Isselbaeher, Kurt J.s.
A Speclficjngymatlc Assay for the Diagnosis of Congenital Galactosemia
I. The Consumption Test, J. Lab. Clin. Med., 50, 469 (1957).

Maxwell, E. S., Kalckar, H. M. , and Bynum, E„, A Specific Enzymatic
Assay for Diagnosis of Congenital Galactosemia II. The Combined
Test with 4-enimerase. J. Lab. Clin. Med., 50, 478 (1957).

Kalckar, H. M„, Saulmajster, H. , and Kurahashi, K., Galactosejggtabolism
in Mutants of Man and Microorganisms,, International Symposium on

Enzyme Chemistry in Tokyo, Japan, October 1957, in press.

Serial Ho- NIAMD-
Pag® 13

Pare Bs (continued)

Kalckar, He M= s Some fomaideratipn* Regarding Biochemical Genetics
in Man, Perspectives in Biology & Medicine, Vol, X, Koc I, 3-16,
Autumn 1957n

Kalckar, EL Mc , and Maxwell, Biosynthesis and Metabolic Function of
Uridine Diphosphoglucose in Mammalian Organisms and its Relevance
to Certain Inborn Errors, Physiological Reviews,, in press,

Kalckar, H< HM Uridinediphpsphogalactpse? Metabolism, Enzymology
and Biology. Advances in Enzymology , in press..

Bergren, William, Donnell, George and Kalckar, H, M. , Congenital
Galactosemia and Mental Health, Lancet, in press .

Szulmajster, Huguette de Robichon, Galactose,, a multi- inducer of the
enzymes of the galactose pathway in Saccharomyces cerevisiae,,
Science, in press e

Kalckar, Herman M„, Discussion, on Observations on Galactose Defect

Mutants in Transduced Microorganisms, in McElroy, Wo Eo, and Glass, B«,s

edso, Chemical Basis of Heredity^ Johns Hopkins Press, Baltimore, 19S7<

Honors

Guest Lecturer, Government of India, Atomic Energy Department,
Calcutta and Bombay, October-November, 1957.

Guest Lecturer, Max Planck Gesselschaft, Munchen, November 1957*

Guest Lecturer, Denmark" s Naturvidanskabelige Samfund, Copenhagen,
November 1957.

Scientific representative of Section on Metabolic Enzymes, NIAMD;
Japan, India, Israel, German, Denmark „

Foreign Msmbar? 9eg Kongelige Saoeke Fidensksbernes Selsksb

Awards

Presidential Award International Poliomyelitis Congress, Geneva 1957

Medaille Biochimie Societe de Chimie Biologique Paris 1957.

BES-H1H
Individual Project Report
Calendar Year 195?

Serial No, EUm- 27

PART A.

Project Titles

Ao Galactose~Glucoee Interconversion

Bo Role of UBPGal as a Galactosyl Donor

Principal Investigators Elisabeth So Maxwell

Other Investigators s

Cooperating Units;

Man Years (Calendar year 1957)
. Totals 1=1/3

Professionals 1
Others 1/3

Project Description;

A= Galactose-Glucoae Interconversion

OBJECTIVES - The mechanism of the galaetose°glucose interconversion

has been the subject of many investigations „ Since
LeloirBs discovery that the inversion of configuration takes place
while the monosaccharide is in glycoside linkage to uridine diphos-
phate s interest has centered around the more detailed mechanism ©f
the inversion itself 0 Our own investigations were undertaken to
study this mechanistic Since our discovery that BPN is necessary
for the reaetions experiments have been designed in an attempt
to show unequivocally whether or not the reaction proceeds by
oxidation-reduction as is suggested by the requirement for DP$0

METHODS AHB MAJOR FIMDIKGSs Both DPK and BPM labeled in the para

positions with tritium were prepared,.
No tritium could be detected in either UOPGal or OTPG after running
the reaction in the presence of either labeled BPN or labeled WW.
for periods of time well in excess of that required to reach

Fart B included Yes

Serial No. NIAMD» 27

NIAMD-

equilibrium. Thus, if the reaction does proceed by oxidation-
reduction with BPN functioning as the hydrogen transport agent,
the lack of transfer of tritium from the para position of BPN to
the hexose mast indicate either thaV the hydrogen originally
removed from the hexose is stereospecifically reintroduced into
the isomeric hexose in the second step, or that the para position
is not the active site in this reaction,, Although the latter
possibility seems unlikely, it would serve also to explain the lack
of incorporation of tritium from BPNH labeled in both hydrogens
of the para position,, Another explanation might be that
enzymatically formed BPNH is tightly bound during the reaction
and does not exchange with RPMH in the medium. Added BPNH was
found to be a strong inhibitor of the reaction. It seems
unlikely therefore that it is unable to react with the enzyme.
Further studies will be necessary to show unequivocally whether
or not oxidation-reduction is the mechanism of this reaction*

The purification of the enzyme UDPGal°4°epimerase from calf

liver acetone powder has been improved and a method for storing
it for long periods of tise without significant loss of activity
has been found. Many of the properties of the purified enzyme,
including Ks for TOPG, UBPGal and DPN, pH optimum, inhibition by
mercury 9 etc., have been studied. The purified enzyme can now
be used in a routine enzymatic assay for the detection of con-
genital galactosemia (See section on galactosemia).

Bo Role of UBPGal as a galactoayljgSBSS

OBJECTIVES - There has recently been a great deal of interest

in the role of uridine diphosphoglycosyl compounds
as glyeosyl donors . Studies from other laboratories have indicated
that UDPGal can serve in such a capacity in mammalian systems
forming lactose and gal&etolipids. Other studies indicate that
UBPG is involved in mucopolysaccharide synthesis. The blood group
substances, mucoid compounds which contain galactose, occur in
large amounts in certain mammalian tissue. Virtually nothing is
known about this biosynthesis. Our own studies have been under-
taken in an attempt to see whether or not UBPGal is involved in
their enzymatic formation. It is hoped to eventually extend these
studies to a more detailed study of the enzymology of these interest-
ing mucoid substances.

METHODS AN® MAJOR FINDINGS s The mucoid blood group substances,

which are present in large amounts in
secretions of gastric mucosa, contain about equal amounts of
B galactose, L fucose, N acetyl glucosamine and N acetyl galactose
amine. GastrTc mucosa has therefore been selected as the first
tissue for investigation. The !8foifid«s factors" (di- and tetra-
saccharides containing galactose and N acetyl glucosamine) are
also present in this tissue.

Serial Ho. MIAMD- 27
Page 3

An enzymatic method for preparing UBPGal labeled in the
galactose moiety with C^ has been devised. Preliminary results
using the labeled UDFGal in an enzymatic system prepared from
guinea pig gastric mucosa have been encouraging. Radioactivity
is incorporated from UDFGal into an alcohol insoluble material.
Labeled glucose, glucose^6»P galactose and galactose~l»P are
inactive in the system. The nature of the product is as yet
completely unknown.

SUMMARY = The mechanism of the enzymatic galactose glucose inter**
conversion has been investigated further., particularly

with tritium- labeled pyridine nucleotides. The purification of
the enzyme8 UDPGal=4~epimerase has been improved and its properties
have been studied*

A project has been initiated for the investigation of the

possible role of uridine diphosphogalactose as a galactosyl
donor in polysaccharide synthesis in mammals. Since large amounts
of the mucoid blood group substances containing galactose are
present in gastric mucosas this tissue has been selected for the

initial experiments. Preliminary results using UDPGal labeled
in the galactose moiety with &*•** are encouraging.

Serial Mo, HIAMD- 27
Fag© 4

Publications other than abstracts from this projects

Maxwell, Elizabeth S.t Kalckar, Herman Mo, and Bynum, Elward,
A Specific Enzymatic Assay for the Diagnosis of Congenital
Galactosemia, II. The Combined Test with 4-Bpimerase,
J, Lab, & Clin, Med,, 50, 478-481, (1957),

Maxwell, Elizabeth So, The Enzymatic Interconnexion of Uridine-
diphosphogalactose and Uridinediphosphoglucose. J. Biol, Chem,
in press o

fes -ran

Individual project Report
Calendar Year 1957

Serial H©„ MIAMB- 28

1. Biochemistry & Metabolism

2. Metabolic Enzymes
3c Bethesda

Project Titles

A„ The Metabolism of myoinositol and other cyelitols
in living organisms 0

Bo The study of Pentose Phosphate Metabolism,,

Principal Investigators Arthur Weissbach
Other Investigators?

Cooperating Units?

Man Years (calendar year 1957);
Totals 2
Professional; i

Others i

Project Descriptions

OBJECTIVES - To study the metabolism of myoinositol and other
cyelitols in living organisms.

METHODS AW MAJOR FINDINGS » The use of the enzyme ;, inositol

dehydrogenase for the enzymatic
determination of myo~ inositol has been further studied,. This
assay is specific for myoinositol and no other cyclitol
tested reacts in this system. This is the first rapids sensitive;,
and specific enzymatic assay for myoinositol, This system has
al3© been adapted to the enzymatic determination of scyllo^myo

Part B included Yes

Serial Ho, MIAMP- 28

Pag® 2

inosose which is the product formed from myo-inositol in this
o&id&tiorao

myo-inositol Seyllo=i&yo= inosose

By altering conditions , the enzyme also provides a means
for the estimation of the inosose by utilising the reverse
reaction and measuring DPSSH disappearance „

Scyllitol j, another of the hexahydroxy cyclitol isomers 8 has
been prepared in large quantities in preparation for studies
concerning its possible metabolism^ During the preparation of
scyllitol from scyllo-myo inosose and sodium borohydride2 a new

complexj scyllitol diborate (I) postulated structure was isolated*

$ ^ H

off

(x> Cxi) cm)

This compound is of more than routine interest since it
indicates that scyllitol may exist under certain conditions with
its hydroxyl groups in an all-axial conformation III* rather than
the all -equatorial conformation II „ The all-equatorial conformation
is the one generally accepted as the probable ones since the all-
axial conformation has never been observed or considered as a
possible structure because of energetic considerations 0 Another
possibility is that the ring of the cyclitol is so deformed by
the attack of the borohydride ion in the reduction process that
it becomes capable of binding two borate molecules,

OBJECTIVES - The study of pentose phosphate metabolism,*

METHODS AM) MAJOR FIJBINGS - In the continuing study of pentose

phosphate metabolism in cell-free
bacterial systems of L coIi„ it has been found that a desoxy
sugar acid is formed from ribose-5-P©^, This compounds, which is
accumulated in amounts up to 20% from the substrate, has been

\

isolated avid identified. It is an Oketo acid containing a
2-keto8 3=deoxy grouping. Degradation studies and synthetic
preparation have indicated it has the structure

(I) ,

&c;a°5

CT2OH

Biosynthesis of this compound in |0 coli extracts requires
ribose-5~pho£,phate» phosphoenol "pyruvate, WN, and & sulfhydrvl
source such as glutathione. The compound first formed is
probably the phosphorylated form of I which has previously been
described by Srinivasara, Sprinson and Davis „ The phosphorylated
compounds 2~keto8 3-deoxy 7»phosphoglucoheptonlc aeid3 is known
to be the precursor of shikimic acid and aromatic rings in Ec coli
mutants . This is the first report of the dephosphorylated form""
occurring naturally and it Is of interest to note that it occurs
in wild typi! E. coli, 2~-Ke£o, 3-deoxy hexonic acid phosphates
have b^en reported to be formed in other microorganisms
<E< saccharj^ilia) but the heptonic acid has not. During the
course of this work, the color test of Waravdekar & Saslaw, which
was developed for desoxy sugars and discovered by these workers
while at the National Institutes of Health; has been adapted for
the assay for 2»ke,;o, 3-deoxy sugar acids. This modification of
the test is of great sensitivity and very valuable for assaying
compounds of this type. Much of the later work on the identification
and enzymatic synthesis of 2<°ke£os 3«deoxy heptonic acid v&s don§ in
collaboration with Br, J. Hurwitz of the Department of Microbiology s
Washington University ichool of Medicine,

SUMMARY MB SIGMIPICAMCE TO THE INSTITUTE - A rapid and sensitive

enzymatic assay for
myoinositol which will be of aid in studying the metabolism of

this compound has been further studied.

In the course of preparing scyllitol (one of the isomers of
myoinositol) 8 a new complex^ scyllitol diborate8 has been isolated.
The structure of this compound may shed further light on the
stereochemistry of these ring compounds.

The. metabolism of ribose~5~phosphate in E. coli leads to the
accumulation of 2~ketos 3=desoxy heptonic acid. This compound
has not been reported befores though Its phosphorylated derivative
is an important precursor of aromatic rings in bacterial metabolism,
A previously reported color test has been modified aad affords an
excellent assay for sugar acids containing the 2™keto8 3=dnoxy structure-

Serial Ko„ HIAM0- 28
Fag© 4

Part: Bs

Publications other than abstracts front this project;

Weissbach, Arthur , The Ensyaatie Determination of Myo-inositolj
Biochimc et BiophySo Actas in press »

Individual Project Report
Calendar Year 1957

Serial Ho0 HjAjg>- 29

Project Title °

Ao Cause of Galactose Tonicity is Galactosemia,.

Be Purification ©f UDF6 Pyrophosphorylase from Mung
Bean Seedlings 0

Principal Investigators Victor Ginsburg
Other Investigators;
Cooperating Unless

Man Years (calendar year 1957) s
Total; 1-1/3
Professionals i
Others 1/3

Project Descriptions

Ao Cause ©f Galactose Toxicity in Galactosemia „

OBJECTI¥ES » The demonstrated absence of the ensyme Gal»l~P traas=

uridylase in galactosemias still leaves unexplained
the toxic effects ©f galactose 0 Work carried out in this laboratory
(Kurahashi & Wahba)s as well as in other laboratories 9 indicates
that abnonaal Gal°l»P accumulation is eoneoaitant with the appearance
of naetabolie disorders „ It was hoped that a molecular reason for
galactose toxicity might be f©und„

Part B included K@

Serial fe, 1XAMP- 89

Pagg 2

METHODS fflffl RESULTS - Ttfhile attempting to deteraine, on a
biochemical level , Meeker abnormal
concentrations of Gal-l=P might be harmful to cellular metabolism
it was found that this ester strongly inhibits the important
glycolysis easy®® phosphoglueomutase (PGM) in vigrqo The mechanism
of this inhibition was studied in detail because of its possible
importance as the actual cause of galaetoeeraie symptoms,, Routiae
biochemical methods were used in this studjc It was found that
the formation of galactose diphosphate (Gal DP) at the expense
of glucose diphosphate (GDP) was the cause of inhibition as shorn
in the following reactions

6-1=9 + GDP ~£s5 GDP -5- G-6-P

/ Gai~l»P

pgm

(

"5

f *3al DP

G-

6«P

Hence, GDP, which can be considered as a coenzyme in the overall
reaction is being slowly depleted by the formation of relatively
inert GalBPo The interconversion of G=l-P and G"S~P by PGM can
be inhibited 90 percent by the same levels of Gal<=l=P that occur
in the tissues ©f galaetosemies s Whether or not PGM inhibition
is physiologically important remains to be seen* All attempts to
assess this question in experimental animals have thus far been
unsuccessful 0

B° Purification of TOPG ovrophosphorylase from Maag Bean Seedlings.

OBJEGTlfE = In d£g<sussions of galactosemia, the possibility of

direct incorporation of Oal<ji=F into uridine nucleotides
by reaction with UfP has mainly been ignored although such a
reaction lias been demonstrated in yeast and in higher plants « How~
ever, it has recently been shown that liver extracts are also able
to catalyse this reaction,, This finding would have a bearing ia
studies on galactosemias as an alternate pathway for galactose
metabolism would thus be available in bypassing the deficient enzyme
Gal-l»P transuridyla@e0 In the ease of higher pleats, it was
unknown whether the activity towards TOPGal was due to an unspecifis
UBPG pyrophosphorylase or to a different ensyme0 A purification of
HSP6 pyrophosphorylase was therefore undertaken, partly to settle
this question and partly t© obtain experience in the chromatography
of proteins on Sober^Petersen columns , as this technique was utilised
in the purification procedure „

Serial No„ M1AMD- 1®

eg*

METHODS MSB RESULTS - The methods used in Che purification were

classical with the exception of the Sober»
Petersen column,, The enzyme was purified over 800-fold and its
properties were studied*, It was found to be specific for UDPSc

FUTURE HESEMCH ~ With experience gained in the use of Sober~
Petersen columns during the purification of
WBPG pyrophosphorylase, it appears feasible to apply this method
to general problems in the study of protein synthesis,. Preliminary
experiments are sow In progress on the incorporation of radioactive
amino acids into proteins ©f microorganisms during adaptive enssyme
formation and during phage infection0

Further experiments on the reason for galactose toxicity
in vivo are also planned 0

NATIONAL INSTITUTE OF ARTHRITIS AM) METABOLIC DISEASES

Aramal Proj«xfc Report

C&lestiar Xear W$7

Suaaaaiy Sheot

Laboratory of Ch©misfergr

Estimated Obl^ateona for.FX 2°$8

Total: $?5O88O0
Direct? #01*3*000

Rsimburacittsatss $201^800

Projects muabfflr JO thru 61i inoXudedo

PHS-HIH
Individual Project Report
Calendar Year 1957

HIM©- SO

Bart At

1. Chemistry
2= Carbohydrates

3. Bstheeda

Project Title: Studies on tte Synthesis of Carbohydrate Derivatives
for Biochssaieal and Medical Research

principal Investigator: Hewitt G. Eleteher, Jr.

Other Investigator©: Ho w. Otehl, D. L. MaeDonald (V.S. from 8/15/57),

R„ K. Hess, E. W. Tracy, B. Vis. (Fellow), H. B.
Wood, Jr.

Cooperating Unite: Hon©

Man Years:
Total: 7

Professional: U V3
Other: 2 2/3

Project Description:

General Objectives:

Ao To investigate new synthetic pathways for the synthesis of
carbohydrate derivatives of biochemical and medical
importance «

B0 To provide cooperating biochfKists and clinicians with
otherwise inaccessible carbohydrate substanees!0

Co To extend knowledge of the eheaiieal properties of bio-
ehQEically important carbohydrates.

Specific Objeetiires s

Ao To study the cheMstry of D-ribose in order t© evolve sew
or improved synthetic pathways to the ribofuranosides and
particularly selectively substituted derivatives thereof 6

Bo To explore the chemistry . of 2«-d©Qxy~B=>ribosie6 specifically
• methods of synthesizing 2-»deo2y=D»ribofuraiK)sidf8 and those
phosphates of 2-deqky-D-ribose which have besrn demonstrated
to be (or suspected of being) saetaboHe intermediates) 0

Co To synthesis© i&teraediatas of tiie metabolism of carbo-
hydrates other than 2-d@oxy=B=ribos®0

Do To devise new syntheses for ket©ge@0

Progress during 1J957?

ihamb- m

Peg© 2

A, Ki® first knowa9 crystalline D=ribofurano8yl halidea
Z^^di^benzoyl-D-ribofuranoayl chloride., found in the
earlier"course of this project^ was studied further be-
cause of its unique utility in the synthesis of both
a~B~ribofuranosid@s and B-D-ribofuranosides „ From this
halide was obtained 5~0~benzoyl~1j2c3~0«b8nzylidene-a~B<=
ribofuranoesj, a novel substance whose utility for the
synthesis of D~ribofuranosides under exceedingly mild
.conditions was demonstrated.

Bo A new and general synthesis for th© synthesis of B^arabino-
furanosides was developed* Both anomeric J^-di-O-
bsnssoyl^B-arabinofuranosyl bromides were ©btaineoTin
crystalline for$a„ Aside from the synthetic possibilities
thus opened,, these two substances are of theoretical
interest as the first anomeric pair of furanosyl halidss
to be made available.

The hydrolysis of 3p5»di~a=fcenzQyl~a™B«arabimofuranosyl
bromid® is accompanied by°an aeyl migration to give H s>3B$^
tri~0~benzoyl°8=B~arabinofuranose. This observation con-
firms a similar one made earlier in the B-ribofuranose
series and is of particular interest since H s3 ,5-tri-O-
benaoyl=»2-0»methylsulfonyl'=-B-B=arabinose was shown to"
afford ribofuranosides and ribopyranosides under alkaline
conditions,, Earlier synthetic methods available for the
synthesis of such substances hatre required acid or neutral
conditions o

Go Methyl 2-deoxy=3s5"di«0-tolylsulfonyl°J>-ribofurano8id©
was obtained — apparently the first synthetic crystalline
2»deoxy-D=ribofuran©side derivative, Both ancaserie
benzyl 2-deosy-D=ribopyranosides were obtained in crystal"
line for® and characterized through various crystalline
derivatives,

D, The occurrence in nature of nucleosides containing B=xylo=
furanose residues has led to the re-examination of methods
for the synthesis of such compounds. It was found that

2 ,3 flU«5"di^)°beazylidene^aldehydo°D°xylog® dimethyl aeetal
(easily prepayable from D°xyios9~in nearly quantitative
yield) may be used for the synthesis of B=xylofuranosides0 v

E, Treatment of ^6^di-0=msthylsulfonyl°29li:3s5"=,di'=2=methyleRe==
L°iditol with a sodium alkoxide has been shown to. give th®
doubly unsaturated derivative B~threo~Ui)8°dim8thyl3&3°
1^39597ranaphthodioxane0 This facile reaction holds promis®
of providing a new synthesis of 2«ketoses and dlkete-sugarso

HIAMD-

Pass 3

Significance of the project to the program of the Institute?
The synthetic methods and the materials produced in the
course of this project have been and will be of utility to
various research groups in NXAHDo

Proposed course of projects

Item Bp C, and Ba lasted above under "Specific Objectives"
will be eisphasizsd furing H?580

Part B included? Yes

NIAMD-

Part Bs Honors , Awards , and Publications

Publications other than abstracts fros* this project;

Vis, E. and Fletcher, II, G., Jr., Mts5»Anhydro-f>=D-ribofupaaoee and
the «Monoac©tone Anhydroribcses 9 of hsvam and Stiller, " J. Am. Chesa,
Soe„s 2£„ 1182-1185 0957)»

Wood, H. B,, Jr., Diehl, H. W. and Fletcher, IL G., Jr., "1,2*1^6°
Bi-0-benzylidem-a-3>glucopyranos© and ImproveBssate in th© Prspara*
tion of lifl6-0=Benzylidene™D=glucopyranose,e' J. Am. Chem. Soe., 79,
1986-1988 (1957)* "*"

Vis g E„ and Fletcher e IU Go, Jr., ""Conversion of 1 ,6-Di-O-methyl-
sulfonyl-2,U:3,5-di-Q-K®thyle3^~L-iditol to IVthreo°Ufl8-Dimethylena»°
t ,3ff507-=aaphthodioxane,s' J, Org, Chem., 22, 7123tTI 0957).

Wood, H„ Ba, Jr., and Fletcher , H. 0., Jr., n1 -0-B®nzoyl-a-D-
talopyranose," J, Am. Chem. Soc, 79, 323i.°3236~7l957)<.

Wood, H, Bo, Jr., Biehl, H. W. and Fletcher, H. G,, Jr., B1,2;3,5~
Di-0-benzylid9Re-a«>D-glucosean J. Am. Chem. Soc.. 79* 3862-386U
0957), —

Ness, R, K, and Fletcher, II. G., Jr., "Ribofuranose Derivatives from
3 ,5-=Di°0-benzoyl-l>ribosyl Chloride. I, 1,3f,5-Tri-0-benzoyl'=|3=D-
ribose and 5-O-Benzoyl-1,2,3-0-bQasylidene=a-D-ribose,ls J. Org. Chem,
22, 11i65»1ii69~(1957).

R. K. and Fletcher, H. G., Jr., ^Ribofuranoee BsrivatLves from
3,5-Di-0~ben3oyl=B"ribQsyl Chloride. II. Further Reactions of 5-0-
BQmQyl»102,3~0"ben3ylidene«a»B-ribose,B J. Org., Chem,, 22, 1U70- °"
11*73 (1957)<. "

Individual Project Report
Calendar Year 195T

HIAM)- 31

lo Chemistry
2o Carbohydrates
3, Bethesda

Bart As

Project !H.tle: Mjcroanalytlcal Laboratory

Principal Investigators William c. Alford

Other Investigators s Paula M» Parisius, Evelyn G, Peake,
3yron Baer, Rosemary L» CBrien (to Jferch 8, 1957),
Charles 6. Remsburg (to February 28, 1957), Elizabeth H, E&th,
(from May 19, 1957), William R, Jones^ Victor E. Hart.

Cooperating Units: Section on Exp, Liver Diseases a INES NIflMD 4

s"*Mn" «n Pnly««eeharid«B . LCP. NCI #261
M&n Years:

Total; 7

Professional: 3

Other; 1*

Project Description:

She MLcroanalytical Laboratory is a service organisation
which provides necessary analytical services for research person-
nel of the National Institutes of Health, and to a limited ex-
tent, for persons of other governmental agencies . Bie scope of
this work is summarized as follows:

lo About 9000 elemental, functional group and instrumental
analyses were performed during the past year, 23iis number is
below last year because of the retirement of Mr, Charles Remsburg
early in the year, and the subsequent conversion of that position
to a research category, nkese, with the approximate number ©f
each, include: carbon and hydrogen (2,500), nitrogen by Dumas,
KJeldahl and Nessler methods (2,&X>), reducing sugar (&>0), halogens
(250), phosphorus (400 ), lactic acid (200), functional groups such
as methoxyl, acetyl, benzoyl, carboxyl, active hydrogen (150),
weight loss-moisture-ash etc, (600), metals such as sc&itsa
potassltm, calcium, barium, iron, zinc etc, (100), infrared and
ultraviolet spectra (1,650), optical rotations (250), miscellaneous
(350), Recipients of this service include about 125 research
workers of the HIE staff. In addition, analyses are performed for
governmental agencies outside the HH, insofar as they can be
handled without interfering with the progress of 3SIH research «
Agencies which have received such services at various times ln-»
elude the Hav&l Medical Research Institute, Bethesda, Ml., the

HIAMD 31

Tuberculosis Research laboratory, U0S<>P<,H,S», Sew York, the
National Bareau of Standards, lise Steval Research Laboratory,
Anacostls, amd the Food and Drug Administration, Washington,
D. Cc

2, During the year, a Gary Recording Ultraviolet
Spectrophotometer has been added to the equipment of the
laboratory operated by Mr, Jones, Infrared and ultraviolet
spectra are routinely obtained for members of the Carbohydrate
Section of the laboratory of Chessigtry and also for persons in
other Institutes and to a limited extent for other government®!
agencies, Such additional services are provided only to the
extent permitted by the work load from the laboratory of
Chemistry,

3° During the year the staff of the Microanalytical
laboratory has attested to provide services of a special
nature to two separate research teams, namely, that of Br,
Klaus Schaarz (HIAMD) and of Dr, Peter ISora (NCI), The former
involves a study of dietary importance of selenium vhile the
latter is concerned with the anti-carcinogenic properties of
various polysaccharides. In both cases it has been necessary
to devote a large amount of time to the adaptation of ultra-
micro methods to the analytical problems involved. This work
is continuing.

Bart B incltdeds Ho

Individual 'Project Report
Calendar Year 1957

NIAMD- 32

1 . Chemistry

2. Carbohydrates
3„ Bethesda

Part A;

Project Titles Higher-Carbon Sugars s Anhydro Sugars s Sugar Alcohols
and Their Derivatives .

Principal Investigators Nelson K, Riehtmyer

Other Investigators s Jamas W. Pratts Laura C, Stewart, Emmanuel
Zissisfi Edward' W„ Tracy

Cooperating Units : None

Men Years s

Totals 6 1/3
Professional! ^

Others 2 1/3

Project Descriptions

Objectives s To evolve generalizations relating the physical
and cneHcSTp^operties of the groups of substances named in the
project title to their configurations and conformations.

Methods Employed and Major Findings s In continuation of our
studies on the formation of raonomeric noaredueing anhydro sugars by
the action of acids on hexoses^ heptoses^ and heptuloses^ research
in 195? was directed toward two sugars with the galacto configuration^
preliminary experiments with D*»galactose have led~to the isolation
of 1s6-anhydro»a-D-galactofuranose3 and from L°galacto--haptulos® we
have isolated two anhydrides^ one of which is most certainly 2g7»
anhydro- P-»L°galacto-heptulopyranose and the other is currently
believed to be anh3^0"a"L^galacto-»heptuIofuranoseg although there
ar® two other possible structures that can be written to explain its
failure to be oxidized by periodate„ A considerable amount of 3^
deoxy-D-ribo-hexose was prepared for use in clinical investigations*
and work on the preparation of 3°deoxy°I>»xylO"'hexo3® s B-deoxyD-
lyxo-hexosea and their anhydrides was continued, A study of & novel
trans formation of a mannose anhydride to a talose anhydride is in
progress^ and the preliminary results have been encouraging. In
concluding our study of tri^O^methyleKevolsmitol^ the postulated
intermediate a l^sUflO-di-C^methyleisevolemitolj, was Isolated! the
methylenation of p-sedoheptitol has yielded a tri=0«ffiethy3jene
derivative whose structure we are now attempting to establish*

Proposed Course of Projects Continuation of these and closely
related topics,

Part B included? Yes

HIAMD- 32
Fag® 2

Part B: Honors, Awards^, and Publications
Publications other than abstracts from this project:

ZissiSp E,9 Stewart, La Co and Kichtffiysr, No K., "sXhe Formation of
a^T-Anhyiiro^-D-^Mmo-hsptulopyranos® by the Action of Acid on D-
nannoHfeptttloBe and of Alkali on 3Etaayl a-D-D^mo-BsptuLopyrano-
sidej also l,6-Anhydro-0-*D*>n»n3SQpyranose from B-Mannos© in Acid
Solution/* Jo Amo Chem. Soce, 22, 2593°2597 (1957).

Pratt, J«Wo ©Ed Richtayer, Uo K*5 "Crystalline 3-Deoxy-a>=D»rib£-
hexosSs Preparation and Properties of l,6"=Anhydro~3"d®03£y':°P'=3"'
arabino°bjexoTOrrfflnose „ 1 B 6"Anhydro°3°d@Q2gy-g~D-ribo Hbaapayranose
and Related Ccsapounds," J, Am„ Chem, Soc., 22, 2597-2600 (1957).

Ziasis, Eo aad RLchtmyer, No YL<,S "Structure of Tri°0°E@thyl®3S=
volemitol," J, Orgo Chemo, 22., 1528-1532 (1957).

PHS»NJH

Individual Project Report
Calendar Year 1957

KXAMD 33

Laboratory of Chemistry

Analgesics

Bethesda

Part Ao

Project titles Chemical structure and analgesic action..
Principal investigator: Nathan B. Eddy
Han Tears:

Total?

3o?2*

Professionals

Oo33

Othar:

3.iil

Project descriptions

Objective; Accumulation of data on variations in analgesic
activity and toxicity with variations in chemical structure,
whether a new basic structure or systematic modification of a
known analgesic structure „

Methods and results s Mice are used for both analgesic and toxi-
city studies. For the farmer we employ our modification of a
well~known hot plate method, a dose-effect relationship design of
experimental procedure based on standard bioassay methods and
probit analysis of the data<>

Approximately 13$ compounds have been received to date during
19$7« About 35 have been made by the chemists of this section as
described in their projects % the others have come from outside
sources-, as part of the cooperative program of the section with
other laboratories in our objective. The greatest number have
come from Merck Sharp & Dohme (substances related to meperidine)
and from Hoffmann=La Roche of Basel, Switzerland (substances
related to levorphanol) = Each of these large groups will be the
subject of a joint publication on chemical structure and action
in the respective series- Compounds have been received from
seven other industrial firms. Compounds are now known in both
the meperidine and morphinan series which experimentally have
many times the activity of morphine. Also as part of our coopera-
tive program many of the compounds tested here are processed for
screening for addiction liability. All of this data contributes
to the consideration given prior to selection of specific
compounds for clinical trial.

KIAMD 33

Peg© 2

Approximately one=third of the time of the Chief of the Section
goes to supervision of the research program^ coordination of the
results and consultation with the cooperating agencieso

Approximately ten percent of the time is devoted to adminis-
tration of the sectiono Balance of time is devoted to advisory
functions to other government agencies 9 and industry as secretary
of the Committee on Drug Addiction and Narcotics of the Kational
Research Council and to United Nations and World Health Organiza-
tion on problems related to addiction.

Part B included

KIAMD 33

Part Bo

Publications?

Eddy, No Bo "Addict! outproducing versus 8 habit ~ forming'"*
Guest editorials J. Amer» Medo Ass<>, 163> l622 (1957).

Eddy, No B. "The history of the development of narcotics".
Law and Contemporary Problems, 22, 3 (1957) «

Eddy, Murphy & May. "Structures related to morphine. IX.
Extension of the Grew® sporphinan synthesis in the benzmorphan
series and pharmacology of some benzmorphans'U Jo Org0 Chem,,
22, 1370 (1957) o

Eddy, Ko Bo, Halbach, Ho and Braenden, 0. J„ "Synthetic sub-
stances with morphine=like effect » Clinical experiences
Potency, side effects, addiction liability"« Bullo VJorld
Health Orgo, (in press) o

Honors and awards:

In April two lectures were given at the Bahamas Medical Con-
ferences (1) New developments in analgesics*, and (2) Addiction
the present situation..

From May 5 to May 31, attended twelfth session, United Nations
Commission on Narcotic Drugs as member of Uo S» delegation*

June 10 to lha participated in conference on current research
at Addiction Research Center (NIMH), Lexington, Kentucky0

October l!i to 19, participated as Chairman of eighth session of
Expert Committee on Addiction Producing Drugs, World Health
Organization, Geneva, Switzerland.

JPHS-HIH

Individual Project Report
Calendar Year 1957

NIAMB

Chemistry

Analgesics

Bethesda

Part A°

Project title;

1) A Position Isomer of Racemorphano

2) H-Substituted Derivatives of 2'-Hydroxy~2,5,9-Trtraethyl-&;7-

Benzmorphan*

3) Synthesis of C -Nuclear Labeled 3-Hydroxyanthranilie Aeid0

Principal investigator; g„ L, Kay

Technical Assistance: Js Harrison Agcr

Man years;

Total % 1,6

Professional; 1*0

Other ; 0*6

Project description:

Objectives; To continue the study toward solution of the problem
of drug addiction, toward discovery of improved analgesic and
psychotherapeutic drugs and toward possible parallelism between
analgesic and conditioned- response- Slacking action.

Methods: 1) By an 8-step reaction sequence, an isomer (I) of
3-hydroxy-N-methylmorphinan (racesoorphara) has been totally
synthesized and its structure proved., Noteworthy is the ease with
which a saturated dinitrile intermediate (II) undergoes ring
closure- hydro lysis to a ketophenanthrene derivative (albeit in
only 301 yield) by the use of refluating 30% hydrochloric acid, A
jg-situated electron-donating mathosyl and favorable sterie factors
are explanations. The hydrogenation reaction to form II is
apparently stereospecifie but not chemically selective; maximum
yields of II were 50% with the hydrogen entering from the imino-
ethano (cage) side of the molecule, predictable from models and
proved by Hoffmann elimination of the methiodide of the methyl
ether of II if one assumes (reasonably) that the H at position 10a
does not participate in this elimination; racemorphan and I lead
to the same phenanthrene derivative , The compound designated as I
is 3-hydroxy-N-methylheteromorphinan ©r 1,2,3,9, 10, lOa-hexahydro-
6-hydroxy-ll-methyl-l,4a(4H)-imino«thanophenanthrene while IS is
9-dicyamomethyl-5-Cs-JDethoxyphenyl)-2-methylmorphano

NIAMD

2) The asabicyelo compound 2 '-hydroxy 2,5,9-trimethyl 6;7-benzmorphan
(III) (prepared by us) has bean shown to have analgesic action and
addiction liability comparable to morphine (animal studies) and
conditioned-response-blocking action in mice and rats (SKF program)
superior to chloropromazine. In another series, g-dl-methadol (a
derivative of methadone), also prepared in this laboratory end shoan
to be devoid of addiction potential in both man and the monkey is
about half as potent analgesically as IXI (more so than meperidine)
but only a twentieth as effective as III in blocking a conditioned
response. To study further a possible parallelism between or
dissociation of these two pharmacologic actions N-substituted analogs
of III have been synthesized by either of two methods,

3) From maleic anhydride labeled with C at the ethylenic carbons
C^-nuclear labeled 3-hydroxy ankhranilic acid has been synthesized
for DTo Mehler's studies in tryptophan metabolism.

Major findings ; 1) A change in the position of N- closure from 10 to
1 ia the 3-hydroxy N methylmorphinea series decreases analgesic
potency SO fold,

2) 2'-Hydroxy 2,5,9-fcrimethyl-6;7-bengmorphan comparable to morphine
in analgesic effectiveness and addiction potential is superior to
chloropromaeine in blocking a conditioned response in mice and rats.
Replacement of methyl on the nitrogen by pheaethyl in this totally
synthetic compound produces a very potent analgesic, one 10 times as
potent as morphine,

3) Several phenanthrylamino alcohols previously prepared under the
antimalarial program at NIH have been found by the Smith, Kline and
French laboratories to have a strong antibacterial effect against a
number of organisms,

4) Novel synthetic routes have been discovered for simple and com-
plicated naphthalene and phenanthrene derivatives.

Cooperating Laboratories; Smith, Kline & French laboratories have pre
vided facilities and personnel for pharmacological studies. Also, com-
plementary to pharmacological investigations in this section.

Part B included; Yes

8IAMD 34

?ag« 3

Part Bo

Publications s

Bo Lo May0 Structures related to morphine, VII. Piperidine
derivatives and examples ©f failure in the Knoeveaagel reaction,
J. Org, Chem„s 22, 593 (1957) .

Bo Lo May and E» Mo Pry« Structures related to morphine. VIII »
Further syntheses in the benzmorphan serieso Jo Org. Chenio, 22,
1366 (1957), —

No Bo Eddy, J0 Go Murphy & Eo Lo May. Structures related to
morphine. IXo Extension of the Grave morphinan synthesis in
the benzmorphan series and pharmacology of seme benzmorphanSo
Jo Org. Chem., 22, 1370 (1957).

Chapter in Burger's Medicinal Chemistry (2nd Bdo) entitled
"Analgesics" (in press) „

Honors i

Presented three (hour- long) lectures on "Alkaloids" to the
Research Associate group of NXH.

PHS-NIH

Individual Project Report

Calendar Year 195?

KIAMD 35

Chemistry

Analgesics

Bethesda

Part A.

Project title: Studies on the metabolism and enzymatic degradation
of narcotic drugs and changes in the degradation
brought about by chronic administration of these
drugs o

Principal investigators Julius Axelrod.

Other investigators s Joseph Cochin

Cooperating Units: M~CS=Ph»l<>

Man years*

Total s 0*66
Professionals O066
Other s Fone

Project descriptions

ITIH black rats treated chronically with morphine, nalorphine,
raorphine°nalorphine mixtures and norraorphine for periods ranging from
10 days to four weeks were tested for analgesic response to a given
dose of morphine before and after chronic administration of the above
drugs and liver preparations from the above groups were assayed for
N~dem©thylating and N=dealkylating activity and compared to untreated
animals o It was found that the depression of enzyme activity paralleled
in all cases the depression of analgesic response.. Cross tolerance to
an analgesic test dose of morphine was noted in the nalorphine and nor~
morphines-treated animals and this was accompanied by a depression in
the ability of liver preparations from these animals to dealkylate
either morphine or nalorphine o The animals receiving a mixture of
morphine and nalorphine showed an analgesic response and a depression
of N-dealkylation which were somewhere in between the responses given
by untreated animals and morphine-treated animals* depending on the
ratio of agonist^antagonist usedo

During the course of these experiments, it was necessary, because
of the inadequacy of the chromatophic acid procedure, to modify a
method for the determination of formaldehyde to make it applicable to
biological fluids and this modification will be included in a report
on these studies now in preparation..

Ml'AMD 55

Page 2

Because of certain limitations of the rat tail-flick method as
a measure of analgesic response, we adapted the mouse hot-plate
technique of Eddy for use with the rat and found it quite useful,
especially in studies where repeated measurements of analgesic
response are required,.

A preliminary report of these studies appears in the Jo Pharmacol „
119s 139 (19*7 )o

Proposed course of study « 1) A comprehensive study of the effect
of stereoisomerism of analgesic drugs on enzymatic N~demethylation
and of the ensyme kinetics of N-dealkylation<>

2) Chronic administration to the rat of a drug which K»deraethy-
lates but does not produce analgesia or cross°tolerance to analgesic
drugs (eogo cocaine and/or dextrorphan) to see the effect on the
N-=demethylation of narcotic substrates by liver preparations from
these chronically treated animals «

3) It has been reported by Qrahovats and co-workers that quinine
potentiates the effect of morphine when given in conjunction with it
acutely or chronically and we propose, therefore, study of the effects
of quinine on the enzymatic N=dealkylation of narcotic drug substrates
when given acutely or chronically in conjunction with narcotics o

Part B included: Teso

NIMD J3

Page 3

Part B„

Publications s

Axelrods Je and Cochins Je The inhibitory action of N»allyl-
norraorphine and the enzymatic R^demethylation of narcotic
drugSo Jo Pharmacol-, and Exper„ Therap. (in press) »

PHS-HIH

individual Project: Report
Calendar fear 195?

NIAMD 36

Chemistry

Analgesics
Bethesda

Part Ao

Project titles Asainohydropheaanthreass as synthetic analgesieso

Principal investigators James 6c Murphy

Man years;

Total s 1.0
Professionals 1„0

Project descriptions

Objectives Continuation ©f research on certain aminehydrophen-
anthrenes ^ith similarities to morphine structure.

Methods and results % Completing the work on the hosiolog series
(nitrogen in homologous position) ©f the aminohydrophenaBthreneso
it was found possible to conduct kydrogesaolysis ©f I O-cis-J.,2,,
3,4,4a,9, 10, 10a-oetahydro-l@-dimsthyimjin©Bfflethyl-9-pheaarathr©l5
ME 7316) sad get a 721 yi&ld of the deoxy eesapouad II <ei©-i82D
3,484®,9el©,10a-oetahydro-10-disiethylaminoa>ethyl-phenanthreae8
NIH 7480) .

The UoVo spectrum of II in both location of absorption maxima
and their intensity supports the assigned structure rather thaai
that of an isomer which could arise by catalyst promoted migration
of hydrogen o

For the purpose ©f obtaining oore exact ssorphine analogs ©f
the aminahydropheasathrenes, resort was had t® -the isoaitrosation
of XII (6is-l„2,394D4ao10m-h@xahydr©-9(l©H) phenanthr©®©) <shich
led to & fifty-three percent yield of I? (eis-1, 2, 3,4,4a, 10a-
h«3«ahydr©-10-is©nitro8©-9-phenaathr©ne) sjfaieh in the presence ®£
acid tmb eatalyticaliy reduced,, enabling an isolated yield of
75% of the amiws-ketone 7 (10-®!^!^-eis-l82B3,4p4a9iQa-h@xahydr©-
9£10H) phenanthrone, MM 7548) »

As was observed in the "hemolog series" , the reduction of ¥
to®k a different ecsurse according to the method of reduction
chosen* Catalytic reduction gave a© 84% yield of a 0-alcohoi,
711 (9-10-«dno-cls-l,2,3,4,4a,9, i0&10a-oetahydr®-9-pheaanthr©l)
&hila the us® of lithiwa alumims© hydride gav© 71, the d-alcohol,
in 56% yield,

MIAMD° 36
Page 2

Projected wosk includes the es^lstioa of this series of
aMadfc^3x>^2eEt®Btto@m©? especially with regard to the eon-
figusutlon of tfa© functional groups, and tevelopsmt of infer-
ffiatiaa on eosspcwnd© 2mlat@a to the little investigate 1,2,3 tk-
tetrahyteonephthylBjsine-g T*hieh are of interest in ssr/egsO. ws$®,
io©0, ag a siagple analog of ras&'phiae still retaining the amino
function and the arassatia yingj in the surprislagly high activity
that ha® bean dCTsaostsuted for the closely related substance,
3>^ihydTO-2^tagt&yIaiMna^

Part B included: Yes

niamd 36 _

Pag@ 3

Part Bo

Publications;

Everetts L„ May, James Go Murphy s Nathsa B, Eddy, "Structures
related to taorphiise,, VII", J„ 0rgo Chem., (in pr«ss)<>

PHS~NIH
Individual Project Report

Calender Year 1957

Serial § NIAMD_ _ 37_
Chemistry

Bethesda

Part Ac

Project title « Studies on the development of tolerance to drug©
as raeasured by various behavioral responses*

Principal Investigators Joseph Cochin

Other investigators? Gonan Kornetsky

Cooperating units* Serial Wo„ M-CS (C)~3»

Man years?

Total t 0o66
Professional j 0.33
Other s 0„33

Project descriptions

Rats were given morphine chronically and their response to a
test dose of morphine was measured before, during and after chronic
administration of morphine. The responses measured were the anal-
gesic response on the hot plate and a behavioral response,, namely,
swimming a fixed distance in an alley. It was found that, although
the two parameters tested paralleled each other, tolerance to the
analgesic effect of a test dose of morphine developed more rapidly
and disappeared more rapidly on withdrawal than did tolerance to the
effect on swimming. A preliminary report of this work was given at
the 1957 fall meetings of the American Society for Pharmacology and
Experimental Therapeutics by Dr. Cochin»

The swimming technique was also applied to the investigation of
drugs to which tolerance supposedly develops, but which are not
analgesics, such as LSD* Preliminary results indicate that tolerance
to LSD develops much more slowly, in the VJH black rat, at least,
than was heretofore supposed, and that 2o$<=3°0 mg/kg/day is the maxi-
mum dose of LSD to which tolerance can b© demonstrated.. Atropine, in
doses which in themselves have no measurable effect on swimming or
behavior, potentiates the effect of LSD in both the acute and tolerant
animal.

Proposed studies; Work will continue on the use of swimming as a
measure of drug action and as & tool in studies on tolerance to drugs.
It is also proposed that a comprehensive study in animals of respond-
ers and non°*r®sponders to drugs be undertaken. W® hope in this way
to find a clue to the problem of the placebo reaction and the hypo
responders in mana

Part B included s I®.

PHS-NIH

Individual Project Report

Calendar Year 1957

Chemistry

Analgesics

Betheada

Part A,

Project titles Clinical evaluation of analgesic ces&pounds.
Principal investigators Joseph Cochin
Other investigators Nathan B. Sddy

Man years s

Total s 0,43
Professionals 0,43

Project descriptions

Objectives An effective analgesic,, relatively free of aids
effects, suitable for chronic administration, because relatively
free of tendencies towards tolerance end physical dependence
(addiction).

Methods and findings s Usisag a double-blind technique and

of the regular nursing staff as observers under the supervision
®£ the investigator ©ne of a series of coded medications is
administered daily to patients requiring continuous pain relief
by morphine or related substances. The series includes £w d@ses
of morphine as standard,, one the optimal dose of 10 mg,, the other
half that amount, and tw© doses of each of tw© new analgesic drugs ,
again one dose twice the sis© of the other. Thus the patient is
his own control and the smaller dose of morphine being at or belcs
the level of effectiveness introduces evaluation of placebo effect,

a) A brief series ©f observations were made on tw© morphine
mixtures, ©ne of which m@rphine -J- polyphloretin phosphate, was
designed to extend the duration ©f analgesic action without
materially modifying effectiveness, 21© significant difference
with the new mixture frcm standard morphine could be demonstrated.

b) Since it has been shows that nalorphine can produce In man
an analgesic effect equivalent t© that of morphine,, and sine® nal-
orphine is nan- addicting but has a high incidence of side effects,
other substances related to nalorphine at least in that they have
experimentally the ability to antagonise t® ®«sae extent ©srphiae-
like effects but may have more analgesic effect than nalerphis&a in

D 38

Pag@ 2

experimental animals should be tried clinically., Tss© such sub-
stances have been incorporated into a series ©£ coded medications
as described above and are undergoing trial in the Clinical
Center c, One of these, K-propylnoraorphiae, is apparently to©
strong an antagonist,, Its administration precipitates disturbing
abstinence phenomena in individuals somewhat dependent upon their
regular aarcotiCo It® use must be abandoned.. The ©ther,
N-mefchallyl aonuorphine, a weaker antagonist and a better analgesic
in animals, is working out well and its study will be coatinuedo

IMs work proceeds very slowly because of the small number of
patients with pain requiring narcotic administration which become
available t© us in the Clinical Center .

2?art B included? Mo

PRS-NIH

Individual project Report

Calendar Year 1957

nam 39

Chemistry

Analgesics
Bethesda

Part Ao

Project title:

a. Relationship of structure to analgesic activity in the
morphine group of alkaloids.

b„ The antiviral activity of certain acridine derivatives.

Principal investigator: Lewis J« Sargent.

Technical assistances Jo Harrison Agar.

Man years;

Total • 1.25

Professional: 0.85

Other : 0.40

Project description:

Objectives: 1(a) Efforts are being continued to develop thera-
peutically useful analgesics from the opium alkaloid thebaime.

1(b) An attempt is being made to complete the project on
basic research in thebaine chemistry which the late Dr. Small
had brought nearly to completion.

2. There have been reports of variable antiviral activity
of several acridine derivatives in the recent literature. Some
of these substances were found to interfere with the growth of
influenza type viruses A and B in tissue culture,, Because of the
availability of certain appropriate intermediates prepared in
another connection it was deemed pertinent to make a preliminary
exploration in this areas

Methods and findings; 1(a) The original expectation (referred
to in the previous report) e£ transforming 7-aeetoxydihydre-
codeinone into the unknown dihydroee-deine derivative with the
alcoholic function at C-7„ has as yet not been realised. An
unexpected fission of the 4,5-oxygen bridge during Raney nickel
desulfurization of an intermediate dithioketai led to the forma-
tion of an isomer of 14-hydroxytetrahydrodesoxyeodeiae. The
phenolic character of the latter was demonstrated through aethyl-
atioa with diasomethane. Other means of eircu&wenting this

^8T;i 2

obstacle are being examined,, because it would be highly desir-
able to know something of the analgetic activity of this codeine
isomer o

1(b) In regard to the product of unknot structure obtained by
the action of methyl hypobromite on thebaine (by Dr„ Lo F« Small),,
it was recently found possible to abolish the carbonyl group in
this substance through reduction of the newly prepared thioketal*
This simplification of the molecule should be of value in arriving
at the ultimate structure of the thebaine produeto

2o The synthesis of three new carbinol amines derived from
N-acetyloctahydroacridine along with the proof of structure of
these substances have been achieved. It is proposed to have
these screened for antiviral activity.,

Significance of research activities;

lo Because the potentialities of transforming the medicinally
useless morphine alkaloid thebaine into pharmacologically
beneficial drugs are by no means exhausted, research in this
direction will be continued.,

2* In order that a maximum of information be obtained in
regard to the chemotherapeutic spectrum of substances prepared
in the course of our research, selected intermediates will be
screened for possible CHS, antiviral end carcinolytic action..

Part B included: Yes

Part Bo

Publications;

The Structure of X-w-broraoacetyl-H-acetyl^lQ-dihydroaeridiae
by Levis Jo Sargento Jo Org» Chem0 , 22, 1494 (1957) »

Honors;

Presented three one-hour lectures on the chemistry of the
alkaloids in connection with the NIH Research Associate
progress

PHS=MH

Individual Project Seposrt

Calendar Year 1957

HXAMD 40

Chemistry

Analgesics

Bethesda

Fart A.

Project titles Synthesis of Structures Related to Morphine.

Principal Investigators Bdward Mo Fry.

Han years s

Total s 1.0

Professionals 1.0
Other s 0.3

Project descriptions

Objectives The synthesis of compounds to be tested for analgesic
activity. These substances are to be designed to add to the
present knowledge of the effect ©f structure on activity.

a) The investigation of a synthesis alternate to that of Grew
for elaborating substituted bensoraorph&ns was successfully
completed, and the work may be extended to compounds with a
5-mesabered B-ring.

b) In view of reports that the methyl group on nitrogen under-
goes oxidative degradation in vivo„ the N-methyi group of
morphine was replaced by formyl and carboethoscy group® for

testing.

e) A simplified means of attaching the 3-phenyl-3-©x®-n-propyl
residue to the secondary riag nitrogen was developed.
Attached to the ring nitrogen of other analgesic compounds
this grouping had greatly augmented activity. Hence investi-
gation of its effect when added to the morphine structure.

Part B included s Yes.

MIAMD 40

Saga 'I

Pert B,

Publications:

"Structures Related to Morphias* VIII » Further Syntheses in the
Bensomorphan Series'^ Everett© L, May and Edward M» Fry, J„ Org*
Cheifflo, 22» 1366 (1957)o

"Gyeiizatien of an Unsaturated Bera&yl Ether t© a Hydrofuran"p
E= Mo Fry, Jo Org» Ch@m0j (in press) a

Individual Project leport
Calendar Yeas? 1957

HI/MD- 41

1. Ohgiaistsy

2. Analgesics

3. Bathesda

Part A;

Project Title: Editorial Service

Principal Investigator: Charles I. Wright

Other Investigators: lone

Cooperating Units: Hone

Man Years:
Total: 1
Professional: 1
Other:

Project Description:

As Chairiaan of the Editorial Cessaittee of this Institute, I
received and recorded 454 Kanuseript© (ranging frosa hooks to
abstracts) since Moveaiber 1, 1956. Each manuscript was usually
assigned to two or saor© cambers of the Ccmnittee, including the
Chainaaa, for review. When no board member qualified the Chairman
sought and received aid from members of the other Institutes or
occasionally from persons in the national Bureau of Standards and
the Haval Hospital Research laboratories. She evaluation of each
reviewer's comments was made by "the Chairman, especially when a
manuscript was not reecsssended for publication or severly criticised,
A few such manuscripts required consultation with the author and a
second or third reading before final approval or rejection.

Part B included: [Bo]

PHS— NXH

Individual Project Report
Calendar Year 1957

Serial Ho. HIAMD- 42
1. Chemistry
2.. Steroids
3. Bethesda
Part A

Project Title: The Structure of Cholegenin and Isocholegenin

Principal Investigator: Erich Mosettig

Other Investigators: Malcolm J. Thompson

Cooperating Units: lone

Man Years (calendar year 1957):
Total: 1.67
Professional : . 33
Other: 1.33

Project Description:

Objective. —Structural studies on cholegenin and isocholegenin
were continued.

Methods Employed. ■"-Since a number of .oxidative and reductive
degradations gave no strictly unambiguous results a total ten- step
synthesis of a dihydrocholegenln (ring F opened) has been nearly
completed.

Major Findings. --At the present^, it cannot be stated definitely
that the formula proposed by Spring et al. for cholegenin is
correct.

Significance. —The occurrence of a steroidal metabolism of
sapogenin type in the animal organism would be unique from a
biogenetic point of view.

Serial Ho, HMMDr__4gs

Page 2

Part B: Honors, Awards, and Publications

Publications other than abstracts from this project:

Scheer,, I., Thompson, M. J. and Mosettig, S. : "C-22
Isomeric Dihyarosapogenins from Kryptogenin, "
Jc Amo Chem. Soc, 79, 3218 (1957) •

Publication concerning report for 1955* but not reported there:

PATENT: Mosettig, B., Sato, Y. and Kata, A.: United.
States Patent Office, 2,684,365, "Method of Converting
TOmatidine into Ai6_Allopregnenolone, " patented
July 20, 19?4c

-SIH
Individua :t Report
Calendar Year 1957

Part A

Serial Ho. HIAMD- 43
1. Chemistry
2o Steroids
3> Bethesda

Project Title: The Partial Synthesis of Sulfhydryl-analogs
(positions 11 and/or 17 J of Hydroxylated
Corticoids and Other Steroidal Hormones

Principal Investigator: Erich Mosettig

Other Investigators: Toshio Kawasaki

Cooperating Units: Cancer Chemotherapy lational Service Center,
BCZ.

Man Years (calendar year 1957)°
Total: I.67
Professional : 1 . 33
Other: .33

Project Description:

Objective o --This study has been commenced with the objective of
finding antimetabolites of corticoids and steroidal sex hormones »
Such antimetabolites may shed light upon the mechanism of action
of antirheumatoid and cancerchemotherapeutic steroidal agents.

Methods Employed. °-The method employed thus far consist
principally (a) in converting steroidal epoxides to the corresponding
episulfidesj, and subsequent opening of the episulfide; (b) in the
adding of thloacetic acid to the appropriate (isolated) double
bonds* and subsequent hydrolysis.

The epoxides and double bonds thus far involved are the
93 lip-epoxide, the 11^ 12p=>epoxide, the 9?il-double bond,,) and the
llj, 12~doubl@ bond. As was to be expected steric hindrance posed
great difficulties, but it appears that the opening of the
lla 123= epoxide ring has been achieved (30-40$)..

Major Findings.— Largely of negative nature 9*11" and 11^, 12-
eposides resist conversion by conventional methods to corres-
ponding episulfides.

Part B included No gSTf

PHS—NIH

Individual Project Report
Calendar Year 1957

Serial No. NX-AMD- 44

1. Chemistry

2. Steroids
3» Bethesda

Part A

Project Title: Isolation and Characterization of Cholesterol
from Trypanosoma cruzi

Principal Investigator: Erich Mosettig

Other Investigators: Malcolm J. Thompson

Cooperating Units: Section of Physiology (Br1. Theodor von Brand)5
Laboratory of Tropical Diseases,, NIAID,

Man Years (calendar year 1957):
Total: .67
Professional: -
Other: .67

Project Description:

Objective. °~This is part of a program to Investigate the steroidal
content of a number of parasites^ protozoa and bacteria.

Methods Employed. — Standard methods^, chromatographic allusion^.
U.V. and I.R. spectrophotometry.

Major Findings. ■"-From the unsaponifiable fraction of the extracts
°f Trypanosoma crusi (supplied by Dr. von Brand) cholesterol has
been isolated and characterised unambiguously. The U.V. spectra
of impure fractions indicate the presence ©f a steroidal 5s7"diene.

Significance. "-Presence of cholesterol in 5ES£5£§2E§ cruzi.

Part B included No [x]

i/l&ual Project Report
Calendar lear 1957

Serial No. NIAMT?° 45

1. Chemistry
2* Steroids
3. Bethesda

Part^A

Project Title: Infrared and Ultraviolet Spectroscopic Studies

Principal Investigator: Harold K. Miller

Other Investigators: Mrs. Anne H. Wright

Cooperating Units: None

Man Years (calendar year 1957):
Total: 2
Professional: 1
Other: 1

Project Description:

a. Approximately thirteen hundred infrared spectra were made
to support investigations in progress in the Steroid Section.
Attempts to correlate infrared spectra of morphine and related com-
pounds are being continued. Investigation in the infrared spectral
region from 15 to 40 microns indicates that some important data
relating to heterocyclic structure might be found in this region
(Cs Br prism). A micro illuminator ("infrared microscope") has
been fabricated.? and has allowed spectra to be taken on fifteen
micrograms of substance and less. This instrument is being
evaluated. A synchronous recorder has been constructed which
permits the transfer of spectra to a punch card system simul-
taneously with the production of the large scale spectrum.

b. Mrs. Anne H» Wright is in charge of the Cary Machine
and has during the past year carried out determinations for
members of this section and for members of the other sections of
the Laboratory of Chemistry, in particular., of the Analgesics and
Metabolite Section®. Occasionally there are also determinations
to be done for scientists outside of this laboratory. The
number of determinations is approximately 2-10 per day, depending
largely on the amount of information available in regard to
physical and structural properties of the compounds to be
determined.

Part S ins

:t Report
Calendar fear 1957

Serial Mo. SIAMD- 46

1. Chemistry

2. Steroids
3« Bethesda

Part A

Project Title: Excretion of Adrenocortical Hormones in Urine

Principal Investigator: David F. Johnson

Other Investigators: Erich Heftmann

Cooperating Units: Walter Reed Army Medical Center supplies
pregnancy urines . Dr. Bunizn has supplied
urine from subjects treated with 2-methyl-
cortisol (Clin. Invest., A&R),, B'lAMD. 110c

Man Years (calendar year 1957):
Total: 2.67
Professional: 1.33
Other: 1.33

Project Description:

Objective. «•-(!) Study of corticosteroid excretion in pregnancy.
(2) Metabolism of synthetic analogs of cortical

hormones .

Methods Employed. -°The urine extracts are fractionated on
partition columns with water as the stationary phase. Gradient
elution with petroleum ether containing increasing amounts of
dichloromethane gives fractions of the individual adrenocortical
hormones. The fractions are analyzed by ultraviolet absorption
and reduction of blue tetrazolium. Their identity is established
by paper chromatography.

Major Findings.— The patterns of corticosteroid excretion in
women show significant changes in the course of pregnancy as well
as some individual variations. Complications such as toxemia^
diabetes, and rheumatoid arthritis appear to have an effect on the
excretory patterns^ but more data will have to be accumulated
before definite trends can be established. While the A*-derivatives
of cortisone and Cortisol ar@ interconvertible in the body, no
conversion of 2-methylcortiscl to 2=methylcortisone was found to
occur. Reduction of this analog in C-20 has been demonstrated.

Serial Ho. HIAMD- 46
Page 2

Significance. --An increase in corticosteroid excretion in
pregnancy may be expected on th© basis of clinical evidence,
especially in toxemia cases. The nature and amount of adrenal
steroids produced in the course of normal and abnormal pregnancies
may increase our understanding of the causes and effects of increased
hormone production.

Part B included Yes [x]

Serial Bo. KIAMD- 46

Part B: Honors p Awards, and Publications

Publications other than abstracts from this project:

Johnson, D. F. , Heftmann9 E. and Bunim, J* J. ;
"Intercon version of A^-Cortisone and A^—Hydro-
cortisone in Man," Proc. Soc Exptl. Biol, & Med.
9j£, 291 (1957)- '

Publication pertaining to last year's reports

Johnson* D. F., Heftinann<, E« and Hayden, A.:
"Determination of Individual Adrenocoi'tical Steroids
in Urine," Acta Endocrinologica 2% 3^1-357* 1956-

Individual Project Report
Calendar Year 1957

Serial Ho. HIAMD- 47
1° Chemistry
2. Steroids
3° Bethesda

Part A

Project Title: Study of Fecal Steroids

Principal Investigator: Erich Heftmann

Other Investigators: Ekkehard Weiss and Erich Mosettig

Cooperating Units: lone

Man Years (calendar year 195?) •
Total: 2

Professional : 1 . 6j
Other: . 33

Project Description:

Objective. --To identify steroids in feces.

Methods Employed. --In addition to adsorption and partition
chromatographic methods, the preparation of derivatives has been
found valuable in the isolation of substances from stool. So
far^ six different substances have been isolated in crystalline
form and their identity is being investigated. Pilot experiments
on the non-ketonic fraction have also yielded a number of
crystalline fractions.

Major Findings.— The present investigation is the first
indication that 17-ketosteroids may be excreted by way of the
feces.

Significance. —For an understanding of steroid metabolism in man
it is of fundamental importance to know which steroids are
excreted by way of the feces.

Part B included

8®rL®l MOc Mm3^ »7

Pag® 2

Bart B; Honors,, Awards,, aad RabXieatioaa

Fsblieatioas otter tiaaa abstract® fro® this projects

E®ftean% S*s "A Simplified Matfaissatieal bwtnnfc
of tba f&eory of Parti-tie® ChrsssaSography/'
GbrosatograuMe Methods* 2^ 5 Cl957)«

PHS--NIH

Individual Project Report
Calendar Year 1957

Serial Ho. MIAMD- 48

1. Chemistry

2. Steroids
3 » Bethesda

Part A

Project Title: A Study of the Steroidal Alkaloids

Principal Investigator: Yoshio Sato

Other Investigators: George Latham and Erich Mosettig

Cooperating Units: Laboratory of Chemical Pharmacology,

(Dr. Marion Cotton) » HEX and Laboratory
of Chemical Pharmacology, (Dr. Murray J.
Shear ), NCI.

Man Years (calendar year 1957):
Total: 2.67
Professional: 2
Other: .67

Project Description:

Objective. —With the objective of finding new., rare and fruitful
sources for the production of biologically active steroids the
examination of the degradation possibilities of various steroidal
alkaloids has been continued.

In view of the widespread occurrence of Solanum species? th«
plant source for solasodine, efforts were made to increase the
yield of 5* l6~pregnadien~3f3-ol-20»one in the degradation of
solasodine.

Methods Employed. — * An approach to this objective has been our
finding of a novel and remarkable conversion of the steroidal
alkaloids, solasodine and tomatidine^ by a one~step deamination
process to the steroidal sapogenins, diosgenin and neotigogenin
in about 10$ yield. The major isomer is the novel steroidal
sapogenin having a modified 5-membered furano (F) ring. The
various reactions of this new isomer are being investigated^
particularly that of its conversion into a pseudo derivative (as
in the regular sapogenins) which would be a useful intermediate
for conversion into progesterone.

Serial No. BIAMD-
Page 2

Major Findings.— a. A new steroidal sapogenin isomer 5-=E2smbered
furano (F) ring is formed by a one- step deamination process of
solasodine and tomatidine.

b.. A number of nitrogen- function containing
steroids derived from tomatidine5 solasodine and veratrine have
been supplied for testing for hypotensic and antiaccelerator
action.

c. A number of steroidal alkaloids and of
steroidal sapogenins were supplied for carcinolytic testing to
the Laboratory of Chemical Pharmacology (Dr. Murray Shear and
Dr. Morris Belkin)* NCI. The major part of these compounds
produced considerable tumor damage. From the tests it became
obvious that most of these compounds are fairly toxic. Never-
theless certain ones may produce tumor damage without too great
a cost to the host.

Significance. "—Possibility of the utilization of solanum plants
as commercial steroidal source.

Serial Id. MiiMD;
Page 3

Part B; Honors , Awards, and Publications

Publications other than abstracts from this project:

Sato, Y. and Latham., E. G., Jr.: "Conversion of
Steroidal Alkaloids,, Tomatidine and Solasodine into
Bihydrosapcgenins, " J. Org. Chem., 22, 981 (1957).

Sato, Y., Latham, H. G. , Jr. and Mosettig, E. : "Some
Reactions of Solasodine, " J. Org. Chem., 22, 1^96

(1957)=

Sato, ¥., Latham, H. G., Jr., Briggs, L. H. and
Seelye, R. N. : "Conversion of Tomati&ine and
Solasodine into Heotigogenin and Diosgenin and into
& Common Constituent 5(2-22, 25-epo2cyfurostan-3P-ol, "
J. Am. Chem. Soc, 79* 6089 (1957).

Publication pertaining to last year0© report:

Sato, Y. and Latham, H. G.* Jr.: "Chemistry of
Dihydrotomatidines, " J. Am. Chem- Soc.^ 78, 3146
£1956).

EHS—HIH

Individual Project Report
Calendar Year 1957

Serial Ho. KIAMD-
1„ Chemistry
2. Steroids
3» Bethesda

Part A

Project Titles The Anthrasteroid Rearrangement and Carcinogenesis

Principal Investigator; William R. Bfes

Other Investigators: John Steele and Erich Mosettig

Cooperating Units: Endocrinology Branch (Dr. Roy Hertz)-, NGI-800-C
Laboratory of Chemical Pharmacology (Dr.
Murray Shear), and Cancer Chemotherapy
National Service Center, SfCI

Man Years (calendar year 1957):
Total: 3

Professional : 1. 33
Other: I.67

Project Description:

Objective. -"This study was undertaken partly for elucidating the
factors Involved in the anthrasteroid rearrangement and partly for
investigating the synthetic approaches to compounds to be
submitted for pharmacological and biological studies.

Methods Employed.— Chemical and physicochemical methods were used
to investigate the mechanism of the anthrasteroid rearrangement.
Yarlous compounds which were prepared by synthetic procedures were
isolated hy chromatography.

Major Findings. — -(1) The first step in the conversion of poly-
unsaturated steroids to anthrasteroids was found to be an allylic
reaction. This allowed the postulation of intermediates which
were independently verified.

(2) The conversion of pregnenolone to 5j>7s9*1^~
anthrapregnatetraen-20-one was successfully carried out.

(3) Preliminary steps necessary for conversion
of dehydroeplandrosterone to 5, 7* 9s l4-anthraandrostatetraen°17^=ol
were developed.

Serial Ho. HIAMD- 49

Page 2

Significance. -Bie anthrasteroid rearrangement is considered as a
possible route for the metabolic conversion of steroids to
carcinogens or other pathogenic agents (e.g. steroids to methyl-
benzsnthracenes). She present investigation showed that in the
series of reactions which lead from 9(ll)-dehydroprovitamin=D to
anthrasteroids the first step is a bond migration reaction. Shis
study also elucidated the course of the i-steroid rearrangement of
provitamin-=D and its 9(ll)-dehydro analog and showed that the 9(H)-
double bond has a strong influence on this reaction. She synthesis
of 5» 7* 9a l4-anthrapregnatetraen~20~one made available the anthra-
steroidal analog of progesterone for biological, examinations -

Fart B included Xes [x]

Serial No. H1AMD° 49
Page 3

Part Bs Honors, Awards, and Publications

Publications other than abstracts from this project:

Nes, W» R. and Steele, J. A.: "Influence of a 9(H)-
Bouble Bond on the Course and Mechanism of Alkyl-
oxygen Cleavage Reactions of Unsaturated Steroids,"
J. Org. Cliem., 22, 1^57

Publication pertaining to last year's report:

Nes, W. R. and Shoppee, C. W. : "She 3>5-Cyclo-
steroid Rearrangement," J. Chem. Soc, 93 (1957) •

Individual Project Beport
Calendar Year 19^7

Serial Kb. HXAMD;
1» Chemistry
2. Steroids
3« Bethesda

Part A

Project Title: The Structure of the Aglycone of Stevioside

Principal Investigator; Heinz F. Lichti

Other Investigators: Erich Mosettig and Inder Sen Gupta (Fulbright
Fellow)

Cooperating Units: None

Man Years (calendar year 1957) •
Total: 2.67
Professional: 2
Other: .67

Project Description: This is a continuation of the project given
under the same heading in the report of December 195*h> December
1955 and December 195&.

Objective: To determine the fine structure of the aglycone.

Methods Employed: The investigation is concentrated on
establishing the structure of ring C and S in stevioside^ steviol.,
and ieosteviol. It is now practically definite that steviol and
isosteviol possess structures:

steviol ieosteviol

Major Findings: In agreement with these structure formulas,, the
rearrangement of steviol to isosteviol as well as some other
reactions can be mechanistically explained. In accordance to IB
spectra and chemical experiments* steviol and isosteviol seem to
possess a gem-dimethyl group in the ^-position* while C-5 is th-
most probable location for the carboxyl group.

Significance: The steviol-isosteviol structure has become of r^srj
acute interest since it could be tied up with the garrya alkaloids
andj, more recently^ also with cafestol.

Part B included

PHS—ISIH

Individual Project Report
Calendar Year 1957

Serial Ho. ELAMD- 51

1. Chemistry

2. Steroids
3 • Betkesda

Part A

Project Title: Partial Synthesis of 21-Glucosiduronates of
Corticoids

Principal Investigator: William W. Zorbach

Other Investigators: Erich Mosettig

Cooperating Units: Clinical Investigations, MR (Dr.
Ralph Peterson)* SIAMD 110-G

Man Years (calendar year 1957):
Total: 1
Professional: 1
Other: -

Project Description:

Objective. --To partially synthesize 21«glucosiduronat@s of
corticoids in an effort to secure active, water-soluble derivatives,
and to study their biological behavior.

Methods Employed. —The method of Sonigs and Khorr for the
preparation of glycosides was investigated first. This involves
treatment of an alcohol with an appropriate acylglycosyl halide
in the presence of silver carbonate which reacts with the hydrogen
halide formed during the reaction. Carbon dioxide and water are
liberated, the latter of which may be removed by azeotropic distilla-
tion with carbon tetrachloride. This method has several disad-
vantages, i.e. a large excess of the costly acylglycosyl halide is
required, heating is necessary, and several hours are required for
completion of reaction.

An apparent improvement is a novel oligosaccharide synthesis
recently described by Bredereck which was next investigated. This
involves treatment of a tritylated hydroxy! group with an

Serial Ho. MlfiMD- SI

Paps 2

acylglycosyl halide in the presence of silver perchlorate to give

a blocked glyeosid®. Heagents are employed in ssolar proportions ,
the reaction is rapid, and takes place at low temperatures.

Major Findings. --"Methyl a-1-broiaotriacetyl glueuronate when
allowed to react with H~deoxyeorti©osterone, in the presence of
silver carbonate gave approxteately a 50$ yield of a blocked 81-
glucosidurcnat® of 11 -DOC. The free 21»gLueosiduronate could
not be obtained in crystalline form. The blocked 2X-glucosiduronate
was also obtained Toy treating the 21-trityl ether of 11-BOS with
methyl a-1-broriotriacetylglucuronate in the presence of silver
perebXorate.

Significance. -°She above method psxmitB the synthesis of 21°
glucosiduronates of corbieoids that have been neither isolated
from natural sources nor synthesized before in the laboratory.

Part 3 included Ho [x]

Individual Project Beport
Calendar Year 1957

KEAID- 52

X. Chemistry
2* Metabolites

3p Bethesda

part .'V;

Project Title: l. Cfoordination of a prs^aia of synthesis and tee',
of Metabolites and antimetabolite®., 9- New
inhibitors anfi substrate© of cholinesterase.

Principal Investigator: Bsrahard Witkop

Other Investigators:

Cooperating Units: Bg». geyssour Fries? , Hasral Msdieal Center j Msrek,
Sharp & Bohatej fte, Upjohn Co.; Sterling-Winthrop
Besear<8h laboratories; Squibb Institute, for
Medieal Research; J. H, Geigy, Easel; 'and Ghas.
Pfiser & Co*

Jfcn lears:
Total; 2/3
Professionals 1/3
Others 1/3

Project Description:

Pjfo^egt: 1. Utilisation of industrial connections to obtain
ecragouad® of value or interest in the general program on E&stabolites
and anttagtabolite®. 2« Hew inhibitors and substrates of eholin«
esterases

Cfejeetives: To save tisae, labor and money at the BJH for fun-
mental research by letting eoiEsereial laboratories furnish, test or
synthesize new metabolites, precursors, iates«di&t®!3, etc

l^-QILJiP^P^® '• 3.° threo-m-Sydroxyphenylserine (obtained
through Hoechster FarbwerkeTliEowed pharmacological activity in rati
different froa 0= and m- tyrosine. This led 2r« S» Udenfriend to a
study of possible decarbossylases. Besolution of ssst&nephrine was
achieved through the cooperation of the Sterling-Winthrop Beseareh ^
Institute, who also provided basic pharaacologieal data for these
eosipcunds. The JOecspsund a© well as B,L=noK8atanephria® were placed
at the disposal of various groups: the Vancouver and Osaka group®
studying aroasaturia in schizophrenics, X&*. Charles Winter, JSsrck,
Sharp and Bohms, testing th® new metabolites on trained rats, in
which they do not retard, but accelerate the discing tisse (sasphet-
amine effect) . BS". A. Sjoerdssaa, HHI, was able to locate norsssta-
nephrine and 3~Mth©3$ytyraffiin® in phaeochrcssoeytcBa tumor. S.

NIAMD $2

Pag© 2

Leeper and H. Weissbach, MS* found that norsstanephrin® Is a
better substrate fop MO than setanephrine. Fffisar recently supplied
alkaloids from Peruvian plant© which vere tested by Dr. H. ¥©issbaeh,
MHI, and found to b© powerful inhibitors of MO Indicating a rational©
for their psychotropic effects.

2. J. H. Geigy, Basel, furnished eis- and trms°diasainoeycLo°
hexane" derivatives which were tested hj Dr. S. L. Eriess as inhibitors
in the system eholine©terase°aeetyl choline. Their inhibition was of
a new kind and depended in a characteristic way on the concentration
of the substrate. She Squibb Institute for Medical Research furnished
streptsmine -which started the investigation on a new type of estsr
lablXimbipn (see separate project sheet). The Upjohn Cosspany
donated a large amount of neasaine (from neomycine), the starting
material for deo^ystreptaffiine, a dlaaainocyclitol of great interest
in our labile ester and cholinesterase studies » Eli Lilly furnished
neo-inosajBin®"2 for these studies.

part B included! J,¥esJ

NIMD° $a_

Page 3
Fart B: Honors, Avards, ami Publications

Publications other than abstract© from thia project:

Goodwin-,-*. , and Witkop, B. , "Quinol Intermediates in the Oxidation

of Phenols and Their Baarrangasents. Oxidation Jfeehanissas XVIIJ/'
J. Aa. Shea. Soc, Jj), 179 (3-957).

Witkop, Bo , and Folts, G. M. , "Studies on the Stereochemistry of

Ephedriae and pfseudo-lphe&rine, " J. Am. Cass. Sac, Jj?, 197 (1957).

Foltz, 0. H. , and Witkop;, B. , "She Stereochemistry of the 1-Fhaoyl-
1,2-propanediol® aa& of a-Xse^phe&rine," J. Jto. Chea. Soc., 79, SOI

(1957).

Patchett, A. A., and Witkop, B., "Studies on Hyaroxyproline," J. Mm*
Chem. Soc, 79_, 185 (1957).

Witkop, Bo , and Felt's, 0. M. , "Configuration of 5-Hydroxypipeeeilie
Acid from Bates," J. to. Chem. Soc, 79, 192 {2.957).

Fries©, S. L., Patehett, A. A., and Witkop, B., "She Acetylcholin-
esterase Surface. VII. Interferons© with Surface Binding as Re-
fleeted hy fiajsj/Eatie Response to Turiein©, Batonleine and Belated
HeterOeyeles, '' J. Am. Gtem. Soe«> Jj?, 459 (1957).

Freter, K« , Aselrod, J. , and Witkop, B. , "Studies on the Ghezaieal
and Enzymatic Gssidation of Lysergic Acid Diethylamide," J. Am* C
Soc, J2, 3191 (1957).

Witkop, Bo, "Quefcrachanlne I." J. Am* Saea. Soc, TO_, 3193 (1957).

Freter, K.j Weisshaeh, H., Udenfriend, So, and Witkop, B., "Bio-
chemical and Pharmacologic&L Studies with D= and L»5°I
phan," Proc of the Soc for ESqaeriisental Biol, and Medicine,

725 (1957).

Honor® and Awards relating to this project;

Appointed a s^afcer of the Mitorial Board of the J®umal of Organic

Chemistry.

PBS-MH
Individual Project Report
Calendar Yeas- 1957

NIAM&- S3

1. Ohealstry

2. Metabolites
3- Bethesda

Pari; A,

Project Title; Polyacetates of Quaternary and tertiary Jfesao- aad
M-aralnoeyelitols as Substrates and Inhibitors of
Cb^linesterase,, as Model© for a New Type of Ester
Ls&ilisation and a® Heuroffiu@cular Blocking Agents .

Principal Investigator: B. Witkop

Other Investigators: G» Holland

Cooperating Units: S« Sriess, Haval Medical Center

Man Years:
Totals 1

Professional : 1
Other:

Project Description:

Objectives; To establish a role and possibly a use for deriva-
tives of those mono- and diaminohaacitols that occur as 'building stones
of antibiotics; to explore the sterie limitations and requirements
for (polyfunctions!) substrates of eholinesterasej to find neuro-
muscular blocking activity for cyclic analogs of dimethylaaainoethenolo

Major Findings; Significant differences have been observed in
the spontaneous and ensyme-» catalyzed hydrolysis of diastereoiso&.
quaternary peata-Q~aeetates in the mvo inositol sad seillitol series »
Labilisation of several ester groups has been noticed -with 0 -tetra-
acetates of ditertiary and naonoquaternary streptamine derivatives. .
These findings have been written up as a Cojsamieation to the fflitor
of the Journal of the American Chemical Society,

Further Course of Project: The nature of the labilisation of
ester groups in these molecules seems to be a result of the over-
conformation of the hera-substituted cyclohexan© ring. This is being
studied now in the 2<=deoxystreptamine series, obtainable from neamin^
a building stone of the widely used antibiotic neomycine A.

Part B included; Ho

Individual Project 'Report
Calendar Year 1957

JJLAMD- 34

Part A,

1 . Chemistry

2. Metabolites
3 • Bethesda

Project Title: 1. Synthesis of Polymeric 2- and 5~Hydroxytryptophan8*
2. Selective OKidatlon and Degradation Method® for
Tryptophan in Peptide® and. Proteins.

Principal Investigator: B. Witkop

Other Investigator®: A, Patchomlk (V.S., started June 1957).

Cooperating Units:

Mam Years:
Total: I-1/3

Professional s 1
Other: 1/3

Project Description:

Obj_eetive£: l) To synthesize polymers of 2-hydroxy~ and 5~
hydroxytryptophan in order to observe changes in their suitabil;-
a® substrates or inhibitors for the enzymes involved in their for-
mation or destruction. 2) Ta> investigate methods for the selective
attack of tryptophan in peptides and proteins in older to split
selectively peptide bonds next to suitably- changed tryptophan Molecules,

Methods Etenlqyed: l) Polymerisation is being achieved via
water- catalyzed opening of N-earbobenzyloxy anhydrides. 2) Selective
oxidation of tryptophan is being accomplished by the use of H-brorao-
succinimide under special conditions «

Major Findings: 1} The polymers of 2»hydroxy-£- and of 5-
benzyloxy~D,L- tryptophan have been prepared aad submitted for enzymi
testing. lj By ultraviolet differentia]!, absorption spectrophotometry
the selective axsxk smooth oxidation of H-csrbobenzoxytryptophan, N-
earbobenzoxytryptophylglycine, gramicidin- A and lysosyme to (broadr^
oxindole derivatives has been demonstrated. Proteins containing no
tryptophan are not significantly altered or inactivated by small
quantities of IBS as was found out with rlbcnuelease.

Further Course of Project: It is hoped that either the oxindole
carbonyl or' the hydroxyl of a diosdJidole, obtained by further oxida-
tion of the precursor oxindole, will interact with the adjacent pep'
bond and labilize it sufficiently for preferential opening. This
method, if successful, will provide an approach to the structural
elucidation of tryptophan peptides of unknown structure, such as grami-
cidin, and will permit controlled stepwise asedification and cleavage
of tryptopnan-rida enzymes, such as lysozyme.

Part B ineluded: Ho

-ioh

Individual Project Eeport
Calendar Year 1957

NXAS®-__ J|

1. Chemistry

2. Sfet&bolites
3o Betbssda

pai:<t A.

Project title: Synthesis and Enzymatic Degradation of ^(5H)~
Xmidaaolone-5(^)-acetic acid, a New Metabolite
of Histamine.

Principal Investigator: B. Witkop

Other Investigators: H. Hhy (V.S., started Sept. 1957)

Cooperating Units: 0. Hayaishi, Serial Ho. Ml/tap 88

Man Years:

Total: 1/3

Professional: 1/3
Other:

Protest Description:

Objectives: In the bacterial degradation of histamine to fom-
iminoaspartle acid one missing intermediate has been postulated to be
4(5E)-imidaaolone-»5( k) -acetic acid. This compound must either be
extremely labile or mast be rapidly aseta'faoliaad, since its existence
could not be shown.

Major Findings: By cautious liberation of the ce-ethyl ester of
L-formamidinoagspartie acid hydrochloride UV speetrophotossetric evidence
was obtained for the rapid formation of the highly unstable imidazolone
which was degraded, ssaoothly by a purified enzyme preparation frost
hiBtajnlne-degrading psewdomonaa. Boiled ©nsyme was without effecto

Further Coarse of Project: Th& requiressents and inhibitors of
this particular enzyme will be studied and compared with those of
the other enzymes that open up 4(5H) -imidazolone and 4(5H)«imidaso"
lone-5(k) -propionic seid, whose synthesis is being carried out. Shere
is considerable interest in the type of cyclic aeylamidine bond in-
volved and in the mechanism of its cleavage.

Part 3- included: Ho

ES3-NIH

Individual Project Report
Calendar Year 1957

HXAMD-

1 . Chemistry
2. tfetabolites
3- Bethesda

Part A:

Project Title: Labile Metabolites. V. The Biogenesis of Form-
iminogiycine.

Principal Investigator: Berahard Witkop

Other Investigators: Kurt Freter (V.S., left July 1957)

Cooperating Units: Dr. J. C. Babinowitz, UM} Serial Mo. ®mm 18 .,

Man Years:
Total: 1/3
Prof ess ional : l/3
Other:

Project Description:

Project: Synthesis of missing ssetabolis intermediate in the
bacterial degradation of xanthine to foraiminoglycine (FES).

Objectives: To study the behavior of an unusually reactive
molecule, the properties and the requirements of the ensyajes involved
in its formation and destruction -and the course of its autoxidatioa
and condensation reactions.

Method® ESspLoyeds After the failure of all conventional ring-
synthetic methods for the preparation of unsubstituted 4C5H)~imid°
azolones, the ssaooth cyclisation of the ^phenylethyl ester of Fid
led to imidasolone.

Major Findings: ^(5H)-Tmide,solon@ turned out to be stable only
in dilute solutions and* at room temperature, for a few hours.
Enzyme extracts of Clostridium gylindrosporuan smoothly degraded
imidasoloae to FIG. Boiled ensyi&a had no effect. These findings
rule out formiminoglyein© amid© as intermediate.

Proposed Course of Project: Two further asissing metabolites "
having imidasolcne structures will now be synthesized, one is a
bacterial breakdown product of histamine (Hayaishi), the other an
important normal metabolite of histidine (H. Tabor).

Part B included: Jen

HlfiMD- 56

Pag© g
Part B; Honors, Awards, and Publications
Publications other than sfostraota fsrosn this project:

Ureter, Ko, Rabinewits, J. 0,, aM Witkop, B., "Labile Stofffweeheel-
pro&ukte. V. Zur Biogeneee &®s FoMsi&dnoglyeins aus M?H)~
Imidazolon," Jubilee Isma® of Annslea &@r Gheaie honoring Ho
Wi©land's 80. Birthday, Vol. 60?, 17^-187 (1957) •

Honors and Avarfts relating to this project:

-BOH

Individual Project Beport
Calendar Year 1957

WXAMD- S?

Bart A.

X • Chsia&stry

2. Metabolites

3. Bethesda

Project Title: Mechanism of O&i&ation of Catechol to els^eis-
Muconie Acid (pyroeateehase Iteaetion).

Principal Investigator: Bemhard Witkop

Otter Investigators: A, A. Batehett (left April 1, 1957)

Cooperating Units: 0. Hayaishi^ Serial Mo. ®um 86

Man Years:
Total: 1/3
Professional: 1/3

Other:

Project Inscription:

Project: To clarify the Essehanlsni of the (enzymatic) cleavage
of catecfiils to dlearbo&ylic acids*

Objectives: 2© find and synthesis© intesssediary labile hydro-
peroxides a® the first reaction products between catechol substrates
and molecular oxygen as jsediated by the action of oxygenases such as
pyroe&te chase .

Methods Bsoloyed: Bather than starting with catechols and
oxygen, a synthetically imre feasible s»thod, i.e. , addition of
hydrogen parotide, under anhydrous conditions, "to o=quinones was
used. °"

Ifejor Findings: o-Qainone was ruled out as an intermediate in
the conversion ©f catechol to eis , eig-»BH«^ie acid. Its non^enssyiBatie
disproportionation in aqueous buffer to catechol aM huxaie acid eissi™
lated a 50$ conversion of ©-quinone to saiconie acid on standing, She
in vitro conversieo of o^qulnaae to smconie acid was achieved and
pointed to the existence ©f an intermediate unstable hydroperoxide
whose diserie analog could be isolates and subjected to rearraagessst
and transformation reactions offering weleesas analogies to the
useehanisia of the ensyaiatle "©s^genolysis" of catechol.

Shis project will not b© continued for the tlsse being.

Bart 3 included: Yes

Serial Ho. NIMB-^^^L^
Pag® %
Part B: Honors, Avaafds, and Publications

Rablieations other than abstracts trtm this project:

Hayaishi, 0., Patehett, A.. A., and Witkop, B., "fiber den teehanisssus

der BrenseateeMn-Ossyda®® (Pyr©cat©ehase)~B©aktien. Oxidation
Mechanises. XI." Jubilee Issue of Liebigs Annaien tor Otaiie^
honoring H. Wielaad's 80. birthday, Vol. 608^ 158-167 (1957) .

Patchett, A. A. , and Witkop, B. , "Oxidation Mechanism®. ZXX.

On the Maehaaism of Oxidation of o-Qninon© By Hydrogen Bsread.de,"

lyndon p. Bmll Stesorial issue of J. Org. Chem., 22. 1U77 (1957)-

Eenors and iterd® relating to this project:

Invitation by 0. Eayaishi to b© a lecturer sad diseussant at hi&
Symposium on the feehaaisa of Oxygenizing Ensyass, IV. 3nter=
national Congress of Bioehtnu, Vienna, Sept. 1=6, 1958.

EHS-H2H

Individual Project fteport
tolendar Year 1957

NZAMD- 5©

1. Ohersi&try

2. Mstaboiites

3. Bethesda
Part A;

Project Title: Inhibitors of the Biosynthesis and Breakdown of 5-
Hydroxytryptasaine

Principal Investigator: Bsrnhard Witkop

Other Investigators: K. Foster (V.S. , left July 1957)

Cooperating Units: S. Udenfriend, E. Weissbaeh and fi. Bedfieia,
Lab. of CSLinical Bioehesu, HE, Serial' Ho*

Man Years:
Total: 2/3
Professional: 1/3
Other: 1/3

Project Description:

PrOjjeet; Synthesis of cxsspounds which inhibit 5-hydroxytrypto-
phan decarboxylase and/©r (serotonin) mansasia© oxidase (MAD).

Objectives: The inhibition of the eas^s© which decarboxylates
5~GH=tryp1xsphan would prevent the forasation of serotonin, and have
practical applicability in diseases characterised by overproduction
of serotonin, such as liver carcinoid syndrome. The inhibition of
^® i£ ZiZ£ aQ^- ^ eentral locations is known to result in narked
psychic effects. The control of both of these processes is highly
desirable.

Methods SSmaloysd: A new approach to compounds with "antisero-
tonin" activity was patterned after the observation that the hailueln©=>
genie agent lysergic acid diethylamide (USD) is setabolisaUy inac-
tivated by the introduction of a hydroasyi group in the «- indole
position., Such conversion® were carried out with tryptas&ne, trypto-
phan, 5-benaylosytryptaiaine, serotonin and 5-ben2ylosytryptophan»

Iflajer Findings: The corresponding oxindole derivatives revealed,
in the asdne series, interesting neighboring group participation of
the priissary asino groups with the aside group of the osindole ring,
and turned out to be good in vitro inhibitors of purified 5-OH-trypto-
phan decarboxylase as well as MAO.

Proposed Gourde of Project: Another type of even better MAO
inhibitor was discovered in (B-iaetbylJtetrahydrohas'iasa derivatives.

Pag© 2

Two alkaloids oceu£?ing natoally in plants from Steij I.e.,
haOTalia® and tetyahyds-ohasmine, showed the highest inhibitory
action of MAD o"b@©yv®& so faff. For la vivo action "loading"
experiments with intact anisals requiring snore material will
have to be carried out.

Part B included: Yes

HlflMP- 58

Fags 3

Fart B; Honors, Awards, and Publications

Bablie&tions other than abstract© from this protest:

Witkop, B. , "Bioehesnistry of Msntal Illness, " University of
British Coluisbia Rxblieationj, Biological Sciences Series Ho. kf
p. 25 (195T5»

Honor® and Awards relating to this project:

Invitation by the Upjohn Co., Balaasaseo, to be present as
lecturer and panel discussant at Symposium on the Bioenaaistry
of Sfental Msaases, Kov. 14, 1957.

Individual Project Report
Oals&n&ar Year 1957

MXAMD- 39

1. Chemistry

2. Metabolites
3» Bethesda

Bart A.

Project Title: Bggulation of Growth of AnimL aM Plant Cells by
derivatives of Natural HydroxyaaBino Acids.

Principal investigator: Beraharfi Witkop

Other Investigators: ■ Arfehur A. B&tahstt

Coopea-ating Waits: Br. F. 0. Steward, Cornell Unlvez-gsity
Dr. 0. Mitcraa, KEK, Serial Mb.

Man "fears:
Total: 1/3

Professional: 1/3
Other:

Project Description:

Fro J set: To determine the influence of hydroag/'apln© acids and,
their analogs a® possible regulators or inhibitors of cellular growth .

Objectives: So accomplish a shot® direst control of protein
tissue regeneration, formation of collagenous sear tis
by direct and local application of eyto~aetive agents than was
hitherto possible by rsaaote and hoiraonal control »

Msthotig Etegloyed: A plant tissue culture system has been used
to detect the growth inhibitory effect ©f a nusaber of nitrogenous
compounds and to determine, where possible, the metabolic Bite at
which the subataaee in question may act. The tissue culture system,
consisted of expLsnt© from carrot root stissalated to grow by cell
division. This was suggested because the cells which grow in thi®
way synthesize a protein in which proline is incorporated and which
is unusually rich In hydrossyprolin® for a pla&t protein*

Major Findings: Hydro3£y-L-proline strongly inhibits the growth
of the carrot tissue under the stimulus of the coconut ©ilk. This-
effect is reversed by casein hydrolysate but only by proline of the
aaain© acids which are present in the hydrolysate. (Slight ability
Of other amino acid© to reverse the hydrossyproline effect ussy be
attributed to their ability to give rise to praline). The foss&tion
of hy&roxyproline from prolin© in the protein of the growing cells
is postulated, and it follows that hydrosyproline is a specific in-
hibitor for the utilisation of proline in this form of protsin syn-
thesis. Various eoBpounds that contain a hydrssy-L-proline jroiety

Page 2

possess growth inhibitory properties? in this system,, and their effect
is similarly reversible by proline. Presumably they act at the sasa
site as hydro2£yproliae» Tb& L compounds of hydroxyproline are more
inhibitory than the D and ^"substitutions of hydroay-L-proline,
especially those on the nitrogen atcsa tend to reduce its growth in-
hibitory effect due to its role as a proline anti-metabolite.

In chicken embryos 5 mg. per egg of |r^»ketcproline leads to
the accumulation of higher amounts of f&se hy&ro3Q>proline, about
100°300<p more, than in controls, thereby the conversion of Jj-lj-keto-
proline to hydroxyproline could be @xelud®d»

She presence of 4-ketoproline protects hy&roxyproline to a
considerable extent from attack by the powerful hydroxyproline-
degrading ensyi&es present in liver mitochondria.

Part B included; Xes

Peg® ;": '

Bar-t B: Honors, Awards, . and Biblieatio&s

S'ublieation® other than abstract® frcza 'dais- project:

Stewssdj, F» C, Follara, J. K«, Batehett, A. A., and Witkop, B=,
"The Effect of Selected Nitrogen Ccsspounds on the Growth of ELant
Tissue Cultures," Biochixaiea et Biophysics Acta, in press-

Honor© and At?ar&© relating to this project:

Appointed a -neniber of the Editorial Board of the Journal of
Organic Chemistry*

■HEI

2Mivid.ua! Project Report
Calendar Year 1957

numd- so

1. Chemistry

2. Metabolites

3. Bsthesda
Part A.

Project Title: Mstabolism and Biosynthesis of Catechol toine©
Principal Investigator; Bernhard Witkop
Other Investigators: Siro Senoh

Cooperating Unit©: So Udenfriend, HHI, Serial Mo.

J. Aselrcd, NIMH, Serial Kbo^M^^Efa-g.
Dr. Sydney Archer, Sterling-Winthrop Research
Institute

Man Years: "
Total: 1
Professional: g/3
Other: 1/3

Project Description:

Synthesis of Novel Metabolites of Dopamine, Sorepingphrine,
Adrenaline and Other Catechols of Physiological Bsportanes. Slagsi-
fication of Biosynthesis of norepinephrine.

Objectives: To establish metabolic parameters for important
endogenous horsaones, to eharaeteriae new catechol zsetsbolites, to
find labile precursors of norepinephrine by elucidating the mechanism
of its formation.

S&thods Employed: Development of synthetic methods for partially
O-aethylated catechol amines, of analytical ssethodg for their separa-
tion. Application of tracer technique using tritium, ^G and ^"0 to
follow the fate of (N-acylated) dopaisine on oxidation in vitro and in
vivo, ®->g«j in adrenal slices, in phaeochroasocytoBB tumor, etc.

Major Findings: By synthesis there have been obtained the fol-
lowing new catechol amine metabolites: 1. m~0~Methyldopsjaine (3-
laethosytyramins); 2'. m-0=!fethyldihydroa^phenylalanine, isolable froa
rat urine after administration of dopa; 3. Nornssstanephrine (m»0-
methylnorepinepbxine); 4. ISetanephrine (m-G»H©thylepinephrine) t all
identical with the natural metabolites found in urine, brain, adrenals
etc.

A novel type of ^arrangement of H-acylated dopaquineaes has
baen observed. These o-quinones are apparently in equilibrium with
their p_-quinoid quincBsithines which easily add the elessents of water
or alcohol© to yield norepinephrine or transformation products still
under investigation.

KIAMD 60 ...
fegs 2

-_ Course., of ggo^eet; Work is in progress to elucidate
the struetare of a large maaber of transformation pro&uet® of H=
earbobeaByloj^dopaquiaoae end to establish the possible rdle of
qjainonemethinee In the biosynthesis of noradrenaline.

Part B included: Ye©

HIAMD- 60

Part B; Honors, Awards, and Publications

Publications other than abstracts from this project:

Axelrod, J., Senoh, S», and Witkop, B«, "Synthesis and Identifica-
tion of New Catechol Amine Metabolites," J. Pharmacol.. & Esptl.
!3Dherap» , in press.

Axelrod, J., Iniseoe, J<» K. , Senoh, S., and Witkop, B. . "0»Methyia=
tion the Principal Pathway for the JfetabolisBsi of Epinephrine and
Horspinephrine in the Eat," Biochimo et Biophysics Acta, in press.

Honors and Awards relating to this project:

SBS-SXB

Individual Project Report
Calsad&r Year 1957

H3AMD- 61

Bart A:

1. Chsmistry

2. Metabolites

3. Betheada

Project Title: Concerted Acid-Base Catalysis of Decarboxylation:
A Possible Enzyme Model.

Principal Investigator: Louis A, Cohen

Other Investigators: None

Cooperating Units: None

Man Years:
Total: 1/3
Professional: 1/3
Other:

Project Description:

To study the mechanism of decarboxylation of ^-substituted eln~
naasic acids and related compounds under relatively"mild condition®
of pH.

Objectives: To show that concerted acid-base catalysed decar-
boxylation can serve as a model for enzyme systems utilizing the
same principle of action.

Methods Employed: The use of ultraviolet spectroscopy to measure
the kinetics of a series of reactions*

Ma;) or Findings: Only j3»hydroxy and £=dimethylaminocinnamie acid,
of all compounds tested, undergo facile decarboxylation in the pH
range 2«10. The pH optimum appears at pH 3-4.

Significance: The system under study is structurally similar to
pyruvic acid, fumarie acid, urocanic acid and, particularly, to the
pyridoxal-ajaino acid completes. The information gained in this study
may further an understanding of the ensyzeatic decarboxylation of thee®
systems.

Future Course of Project: To complete the kinetic study and to
test the catalytic activity of internal "push-pull" catalysts such
as S-hydrosOTy^dtog .

Calendar Year 19 57

HIAMJ> 62

1. Chemistry

2. Metabolites

3. Bethesda
Part A.

Project Title; Transannular Interaction of Peptide and Ester Bonds

Principal Investigator: Louis A. Cohen

Other Investigators: None

Man Years:
Total: 1/3
Professional: 1/3
Other:

Project Description:

A study of possible covalent interaction hetveen peptide and
ester bonds.

Objectives: To demonstrate the ability of peptide bonds to
react with esters to fom "reversible covalencles" when offered a
favorable geometrical configuration.

Methods Employed: The use of continuous infrared spectroscopy
to follow the course of a reaction and to detect relatively unstable
intermediates; the use of nuclear magnetic resonance spectroscopy
(planned) to assist in structure determination.

Major Findings: Synthesis of the first compound essential to
the study has been achieved.

Significance: The study is of Importance in determining the
ability of functional group® of proteins to contribute to geometry
and stability by other than hydrogen bonds.. Such information i® of
relevance in protein structure studies and in the mechanism of
proteolytic ensyme action.

Future Course of Project: To determine the stability and
chemical properties of such interactions; to extend the study to
more complex systems.

Part B included: Ho

Individual Project Report
Calendar leap I957

HMMD-____63_

I.. Chemistry

2. Metabolites

3, Betbeada

Part A:

Project Title; Stabilized SVe© BadieaX Analogs of Arasatis ^aim
Acids.

Principal Investigator; Lewis Ao Cohen

Other Investigators: Bone

Cooperating Units: Ife®

Man Year®:
Total: 1/3
Prof sessional : 1/3

Other;

Project Description:

Th@ synthesis and enzymatic study of certain analogs of tyrosine
and thyroxine containing tertiary butyl groups on the aromtie riago

Objectives; To desaonstrate the possibility of inhibiting un-
stable free re&leal intosssadiates on the ensysas surface rather than
inhibiting the primry substrate.

Major Findings; The synthesis of d5..»tertiary-butyltyro8ine is
near eosipletio&o

Signifi^nce: A study of the amino aside and of their fre©
radical derivative® in ensyxnatic systems may provide information on
the isaschanissas of enzyme action and on the role of the phenolic group
on the ensynss surface .

Proposed Pours® of Project: To eoasplete the synthesis and p@r~
form the enzymatic testing.

part B included; Yes

HIAMP- 63

mm 2

FartJ3: Honors, Awards, and Fuhlieations

Publications other than abstracts from this project:

Cohen , Lo A., "Electronic Control of Sterie Hindrance in Hindered
Phenols/' J. Org. Ghem., 22, 1333 (1957) .

Honors and Awards relating to this project:

PHS-NXB
Individual Project Rei

Calendar Year 1957

HIAMD- 64

1. Chemistry

2. Metabolites

3. BetheMs

Pert A:

Project Title; The Synthesis and Biochemical Activity of Oerta&a
Sequences of Insulin «

Principal Investigator: Louis A. Cohen

Other Investigators: Gerald P. Holland

Cooperating Units; Uone

hlsax Years:
Total: 1/3

Professions!;. 1/3
Other:

Project ©ascription:

To synthesize and study the enseymatio properties of certain
cyclic disulfide fragments of insulin.,

Oye^ives: To determine the relative importance of geometrical
factoid va„' chemical constitution in the hormonal action of insulin
and in its sensitivity to insulinaseo

_F3ndings: A new sssthod for the synthesis of sulfur-
peptides has been developed which permits the preparation

of peptides with several cysteine residues and the specific manipu=

lation of one sulfhydryl at a tine*

Significance: Since the variation in certain aiaino acids in
the insulin molecule does not alter the horasonal activity, it is
essential, to know whsther the over-all geometry alone is sufficient
to i&part biological activity. In the hoxie ©f contributing to this
problem as well as to an understanding of the jaeehanism of insulin
action, this project has been undertaken.

Future Course- of Project: To eoanplete the synthesis of the
desired cyclic peptides and to study their behavior in enzymatic
and physiological systems.

Part B included s No

NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES

Annual Project Report

Calendar Year 2957

Summary Sheet

Laboratory of Pathology and Histochemistry

Estimated. Obligations for FY 1958

Total* $1i99#1GO
Direott $361^,000
Reimbursements! $138^100

Pro jests number 65 thru 77 inoludedo

Serial Np. EIMD 6$^

PHS»HIH 2, Histochemistry

Individual Project Heporfc 3. Betheada
Calendar Year 195?

Part A.

Project Titles Adaptation of chemical analytic procedures to
histoehemical use and their application to
pathologic histology for th|3 further identic
fication of chemically eharaeterisable laorpho-
logic entities in tissues.

Principal Investigator: R. D. LiHie

;ors G. G. Gleaner*

3 & Ps Dr. H. M

MJHsLCBs Dr. H. Weis&ach et alJIIAMD 28

KXAMDsLKfls Dr. G. W„ Brom

MCIsEs Dr. H. J. Levlne

Man Years (calendar year 1957) s
Total? 3-1/2
Professionals 2«l/2
Others 1

* Dr. G. G. Gleaners 6 months at N2HS last 6 months at Johns
Hopkins on advanced residency training program.

Project Descriptions

On completion of the recently published" series on the melanin segment
of the pigments., attention ima focused on the so-called "yellow eellsf of
the gastrointestinal mucosa.

The recently reported indole reactions appear to controvert the
Erspamer identification of enteroehromaffin as J-hydroxytr^ptairaine.. The
resoreinol hypothesis of Goiaori is controverted by the failure ©£ carbonyl
reactions dther to demonstrate" or to block demonstration by other reactions
and by the demonstration of a reversible oxidative blockade of the as©
coupling reaction ^hich suggests a p^diphenol or aadaophenol structure,. The
reduction product does not rapidly reduce aeid silver nitrate as catechol'
groupings would There appears to be at least two varieties^ one "ahich

Serial Ko. .KEBP),,, 6jL,

"Page 2 """*""■

couples readily with diazaaiu© salts, the other not.

A netf procedure fop bi.stiocheiii2.o3Ll deasonstratioa of tyrosine
residues in protein xaas introduced s based on the protein dis&otis&tioE
of the wool and silk chemists.

A procedure for localisation of monoamine oxidases has been
studied testoehexaically and in vitro.

The Kile blue procedure for fatty acids and lipofuscins lias been
extended to such sites of relatively high earbosgrl concenimtion as
pepsinogen granules. Methylation prevents the reaction? and a histo*
logically new technic of dessethylation "Kith alkali has been introduced £
■anion restores it.

Part B included Yes

Serial Ho. HIAMD 6$_

Part jh Honors^ Awards, and Publications

Publications other than abstracts from this project:

G. G. Gleaners Simultaneous demonstration of bilirubin, hemosiderin,
and lipofascin pigments in tissue sections. The Am. J. Clin. Path.
2£*l-5, 1957.

H. M, Fullmer (DsH & P) and R. D. Lillies The staining of collagen tfLtb
elastic tissue stains. J. Histoehem. Cytoehem. Jjll^llj, 1957,

R. D. Lillie and G. G. Glenners Histochesiieal aldehyde blockade by
aniline in glacial acetic acid. J. Histoehem. Cytoehem. 5sl67~X69,
1957.

R. D. Lillie 5 The ssanthydrcl reaction for pyrroles and indoles in

histochemistry* Zymogen granules, Xensss enteroehrcssaffin and me2anins.
J. Histoehem. Cytoehem. 5sl88~195, 1957,

G. G. Gleaner and R. B. Lillie 5 The histoehemical demonstration of
indole derimtires by the post coupled p-dimethylasdBohensylidene
reaction. J. Eistochem. Gybochem. 5:279-296* 1957.

G. G. Glenner: The histoehemieal demonstration of indole derivatives
by the rosiadole reaction of E„ Fischer. J. Histochem. Gyboehem, 5 s
297-3Gifc 1957.

R. D. Lillie, J. P. Greco Henson and H. 0. Jacquier Burtners Metal
reduction reactions of the iseXasaiass silver and ferric feOTieyanide
reductions by various reagents in vitro. J. Histoehem. Gytoehexa. 5s
311-32)4* 1957.

G. G. Gleaner and R. D. Lillie s A Ehodoeyaa technics for staining the
anterior pituitary. 9&dn Technology J2tl87«190, 1957.

R. D„ Lillies Metal reduction reactions of the melasdnss Histochemie&l
studies. J. Histoehem. Qytocham. $}%2%™333s 1957*

R. B. Lillies Trichosanthin, the yellow granular pigment ©f guinea pig
hair follicles and hairs. J. Histochem. Gyfeoeham, £i3li6-3S3> 1953. .

R. D. Lillies ferrous ion uptakes A specific reaction of the melanins.
A.M.A. Arch. Path. 6Usl00-103, 1957.

R. B. Lillies Adaptation of the Morel-Sisley proteia diaaotisation

procedure t© the hisbochemieal demonstration of protein^borad
tyrosine. J. Histoehem. Cyfeochem. 5t528-&2, X9S7»

Serial SJo. MBMD 60

Tap"3

G. G. Gleaner, H. J. Bortaer and G. W. Browi (A:LM) t The faistochesBieal
demonstration of monoamine os&dase activity by tetrasolitia salts.
J. Histoehem. Cytoehem. 0s091~6O6, 1957.

H. Weissbach (HsLCB)s B. G. Redfield (HsLCB)s G. G. Gleaner and C.
Mitoma (HiLCB) z TetrasQlium redaction as a measure of monoamine
os&dase activity la vitro. J. Histoehera. Gyfcoehem. 0s6Ol«6O0, 196'?.

H. J. Levine (GsE) and G. G. Gleaners Observations on tryptophan
staining of the pancreatic alpha cells. J. lat. Cancer Inst. »» in
galley —11.57.

R. B. Li31i.es The Kile blue reaction of peptic gland symogen grannies?
The effect of methylation and alkali demethylation. J. Risfeachem.

Cytoehem. 6: (March 1908?)

Honors and Awards relating t© this projects

Dr. R. D. Lillie @s elected President of the Histoehemical

Society for 1907-58.

Serial Ho. MM1D66 |
1. PathologyTTS^c.
FH&JDH 2, Histochemistry

Individual Project PwGport 3. Bethesda
Calendar Year 195?

Part; A.

Project Titles Studies on the histochemieal reactions
of mueoproteina.

Principal Investigator: S. S„ Spieer
Other Investigators: None

Qoopemting Units: None

Han Tears (calendar year 19$7)
Totals 3
Professionals!
Others 2

Project Description*

Reports oa the histochemieal changes following injection of papain
in mishits and on the distribution of glycogen in the kidney and lung™
projects are not-? fin press. Current investigations follow two general
lines of endeavor, namely the application of histochemieal procedures
to pathological conditions and development of tmv histochemieal methods*

In the first category, studies en iebred strains of mice susceptible
to amyloidosis haw shosaa that amyloid localises en collagenous structures;
primarily in vein wills and basement membranes* It has not been possible
here to establish, a correlation between amyloid deposition and plasaia
cell proliferation, which is a relationship frequently referred to in
toman cases of amyloidosis* However, histochesical methods applied to
these animals do reveal several possibly related or unrelated lesions
including % roast cell proliferation, hemosiderosis, ceroid deposition
and accumulation of lipofuscin pigments in endocrine glands and Giaceio
positive insoluble lipids in abdominal lymph nodes. One aspect of this
investigation concerns the histoehesdcal ehameterisation of mouse and
human amyloid, and the evidence thus far disagrees -Kith the prevalent
concept of a sulfated polysaccharide.

Serial Mo. HHHD 66

Newly observed sells fa the periphery of the thesis lobbies of
mice ha^e attracted seme attention. These large cells tahich increase
in sise sad number raith ages contain granules apparently composed of
carbohydrate and insoluble lipid material.

By a sequential methylation (effecting desulfation) and
demethylation procedure carried cut at several pH lewlSj it has been
possible to deraoastmte that the metachrczaasia of nozraal cartilage
depends on sulfate groups. The orthochromasia of eheaidroitin free
cartilage my then be attributed to non sulfated polysaccharide in
the cartilage isatrix.

Attempts are 5a progress to develop differentiating ssethods
for detection of tissue aldehydes and ketone groups including the
naturally occurSng constituents as well as those fosssed from carbo-
hydrate and nucleic acids by oxidation and hydrolysis respectively.

Part B included

Serial Ho. 12MD 66

PageTT"*"5

Fa.rt._Bs Honors, km,rdss and Publications

Publications other than abstracts from this projeerfcx

S. S„ Spicar and J* H. Bryant* GarbiXage changes in papain treated
rabbits — in Press — Am, J. Pathology.

S. S. Spicer and J, E. Bryant: Systemic changes in papain and in

chondroitin sulfate injected mbbits •— in Press — Am. J. Pathology,

S. S, Spieers Histological localisation of glycogen in the urinary
tract and long «— in Press »» J. Histoefe®. Gyfeoehem.

PES
Individual Project Report 3.
Calendar. Tear 195?

Part A.

Project Title* Studies on renal structure and function through
the use of higtocheaieal techniques.

Principal Investigators J. B, Longley

Other Investigator: lone

Cooperating Units: HIBR-H & P: Dr. Burstone

St. Elizabeth's Hospital *Dr. Lassen
Georgetown Unlv.Hospital:Br. Lillienfield
G. Washington Med. ISch. zDr, Still

Man Years (calendar year 195>7)
Total: 2»l/2
Professional: 2
Other 1/2

Project descriptions

1) Application of a new histoehemieal method for esterase to the
kidney of various species has revealed a xsssber of new sites of
specialised metabolic activity. In the rat these results have b
particularly interesting because they shed light on the hemodynamics
of the renal medulla, a subject in turn closely related to the renal
urinary concentrating mechanism. In this species the endothelium of
the efferent arterioles of the glomeruli are stained, as is the blood
plasma itself in certain areas. The blood supply of the medulla is
delivered exclusively through the efferents of the juxfca»msdullary
glomeruli lanich fosm bundles descending into the deep parts of i:
Their staining by this method distinguishes them from the return ves
"Bhich occur in the bundles, and the relations revealed between the t^o
emphasizes the perfect suitability of the bundles to fraction as causa
current exchangers, Within the papilla of the kidney, hj virtue of
staining of the plasroa, it is considered that plasma proteins must be
concentrated. Bed cells are relatively mre in these sites* and it is
evident that an effective medullary mechanise for separating red eells
from piasssa exists^, probably also in the bundles.

2) Confirmation of the foregoing conclusions and quantitation
of the various shifts teplied in medialXary blood composition has been

Serial No.

trades-taken using Evans blue tagged proteins, and ieotopically tagged
albumin and red cells. The "hematocrit" of the "blood" in the renal
papilla of the rat has been found to be about one fifth that of
large vessel blood. The papillary plasma protein concentration
relative to large vessel blood is still being determined. The
identification of the esterase staining material of the papilla as a
plasma has been confirmed by its electrophoretic isolation from rat
plasma. It moves separately from the albumin or any of the globulin
fractions (with Lassen, St. Elizabeth, and LiHienfield, Georgetown).

3) As part of a summer training program for medical students
attempts were made, from June through September, to apply fluorescent
and radioautogmphic techniques to the estimation of turnover times for
some of the soluble constituents of the renal ajedulla. Satisfactory
progress was aade, and it is hoped to resume the program nest year.
One incidental finding of particular interest was that inulia
administered to rats is concentrated in the epithelium of the prcc&mal
convoluted tubule (with Still, Hastings, aad Abdu, George Washington U. )

k) Work continued on a review relating to functional and
morphologic sites in the vertebrate kidney.

Part B included

Serial Sic. HIMD 61

Part 3s Honors, AT&trds, and Publications

Publications other than absts&cts frcsa this project?

H. J. Bortner, R. 0. Mm, and J. B. Longley? Observations on the
redaction and quantitation of neotetmzolium. J. Histoehem.
Cyfcocbem. £*I27-13U, 19$1»

R. C, Balm, and J„ B. Longleys Quantitative effects of a mercnrial
diuretic on the distribution of renal succinic dehydrogenase in
the rat. J. Fbaxm. Esp. Then 1183365~367, 1956 - Received 1.25.5?.

Honors and Amrds relating to this projects
None.

Individual Project Report
Calendar Year 1937"

Serial No, NLJM^

1. Pathology & Histochemistj

2. Pathologic Anatomy

3. Bethesda

Project Title: Study of pathologic lesions induced by nutritional
deficiencies, vaccines, antibiotic and spermine,
and those oe@urr5.ng naturally in man.

Principal Investigator: L. L. Ashburn

Other Investigators: Ernest G. MeDaniel, and Floyd S. Daft;
George A. Hottle; Chester W. Emmons;
Celia W. Tabor.

Cooperating Units: LNE & LPT •- NIAMD; D.B.S.; LID - NIAOT*

niamd 1
Man Years (calendar year 1957):
Total: 2
Professional: 1
Other: Secretary 1

Project Description:

Study of a new drug (with conventional methods) which has shown
considerable promise in therapy of a specific fungus infection -
experimentally inf eeted mice. A wide dose range was used. No
systemic pathologic lesions were recognized in therapeutic dose
range. At lethal and sublethal levels, changes include fatty
metamorphosis and focal necrosis of liver, lipoid depletion of
adrenal and minor renal damage. There is some evidence that the
antibiotic stimulates liver cell proliferation. Further effort
will be mad© to evaluate this point. This study is with Dr. Chester-
Emmons, NIAID.

Studying the effect of certain nutritional deficiencies, in germ-
free rats. Preliminary data indicates that folic acid deficiency
produces bone marrow depression at least as severe in garm-free
as in conventional, rats. Effect of possible substitutes for folic
acid will be evaluated. Even though some germ-free rats die in a
poorly-nourished state, there is no pulmonary infiltrate, and peri-
bronchial lymphoid tissue is notably absent. Studies will continue
and broadened to include patothenic acid, choline and methionine
deficiencies. This study is with Mr. E. G. MeDaniel and Dr. Floyd
S. Daft, NIAMD,

NIflMP *3

Fage 2

Spermine studies are being reactivated to attempt evaluation of
possible role of vascular lesion in pathogenesis of renal lesion..
Mechanism of focal myocardial necrosis in some spermi® rabbits
will be studied - this with Dr. Gelia Tabor.

Study of allergic encephalitis has been started to evaluate some
vaccines as passible allergens j only preliminary data at present -
this with Dr. George A. Hottle, DBS.

With staff of our laboratory . study continues on human tissue
specimens obtained from PHS Indian and Bureau of Prison Hospitals.
Such study serves diagnostic and research purposes. In latter
category, Indian tumors, collected for 20 years, have been classifi*
and charted b^ organs and systems for comparison with other racial
group - this study is with Br. Huth L. Kirschstein.

Part B included! No.

PHS-NIH

Individual Project Report

Calendar Year 1957

Serial No. N1AMD

1. Pathology & Histochemistry

2. Pathologic Anatomy
3« Bethesda

Project Title: Preparation of stained tissue sections for investigation
and diagnostic purpose

Principal Investigator: Mr. Roy Reed - Head, tissue preparation

laboratory o

Other Invest igator: None

Cooperating Units: None

Man Years (calendar year 195?) s
Total: 8*
Professional: 0
Other: 8

Project Description:

The statistical report of this unit is shown below. The work load
increased over last year by about 6,500 prepared slides. In addition
to preparing material for projects conducted in the laboratory of
Pathology and Histochemistry, many other investigators received
advice and service. These include: Drs* Despopoulos, Duncan, Hyatt,
Ross and Saunders of NHI; Drs. Crawford, Kilham and Li of DBS;
Drs. Dewitt, Jacobs, Oliver and Remington - Lab. of Trop, Dis.;
Drs. Brodsky, Craighead, Hesselbach and Rowe - Lab. Inf. Dis»; Dr.
Freund - Lab. of Jinmunologyi Dr. Baer - Dental Institute.

Tissues from a small number of animals were prepared for ten other ^

Polio vaccine control work continued; tissue from 3*920 monkeys
were processed for histopathologic study. Stains of tissue cultures
on glass surfaces were made by giemsa technique. A stain for
dextrain was added to our procedure list.

s Including 1 full time technician furnished by DBS to work on
polio vaccine work.

NIAMD *9

Page 2

Specimens
Accessioned

Stained slides
Routine Special

Animals

7,906*

9,087^\

Surgicals

2,151

7,709 > 19,4M

Autopsies

125

1,814. )

Slides Total .

21,912

59,9

* Source of animal material:

DBS (mainly monkeys) 3,920

NIAMD 2,167

Other Institutes 1,819

Part B included! No

PHS-NIH

Individual Project Report
Calendar Year 1957

Serial No. NIAMP- 10

1. Pathology & Histochemistr

2. Pathologic Anatomy

3. Bethesda

£SCk •

Project Titles Studies on experimental endocarditis and glomerulo™

nephritis, high altitude, iodates, and catechol amines.

Principal Investigator: Benjamin Highman

Other Investigators: Drs. P. D. Altland* H. M. Maling, J. Roshe,
S. E. Webster and Mr. E0 G. Thompson.

Cooperating Units: LPB, NIAMD (Dr. Altland and Mr. Thompson ).HiAMD 9k
LCP, NHI (Dr. Maling).
Surgery Branch, NHI (Dr. Roshe)
LPT, NIAMD (Dr. Webster). NIAMD 88

Man Years (calendar year 1957):
Total: 1
Professional: 1
Other: None

Project Description:

Aortic insufficiency and an associated marlced susceptibility to
endocarditis has been induced in dogs by perforating an aortic
leaflet. Highman, Roshe and Altland have shown that an infection
resembling acute and subacute bacterial endocarditis is induced
consistently in such dogs by either single or multiple intravenous
injections of cultures of Staphylococcus aureus, and Streptococcus
mitis respectively. Many dogs also develop visceral infarcts, a
diffuse proliferative glomerulonephritis, and occasionally focal
glomerular lesions similar to those associated with human endo-
carditis. To determine whether or not the increase in cardiac
work load resulting from the aortic insufficiency was the major
factor contributing to the susceptibility, a Hufnagel valve was
inserted in the thoracic aorta before bacteria wore inject edj this
valve did not prevent either endocarditis or glomerulonephritis.
Administration of cortisone did not prevent the development of the
diffuse proliferative glomerulonephritis suggesting that the latter
is not due to an antigen-antibody reaction. The development of
infarcts in the spleen and kidney could be determined generally
during life by a sharp rise in serum alkaline phosphatase. Studies
by Highman, Thompson, Roshe and Altland on dogs with infarcts pro-
duced by vascular ligation indicated that the rise is due to ab-
sorption of alkaline phosphatase from the infarcts and that the

MIAMD _?0,

Page 2

serum alkaline phosphatase reaches a peak in about 24. hotels and
retains to normal in about a week after infarction. Studies are
continuing on the effects of early and delayed treatment with
antibiotics, on the sequellae of the disease, and on the deve-
lopment and regression of the histopathologic changes .

In a study with Dr. Altland, it was shown that dogs given bacteria
after prolonged discontinuous exposures to 25,000 feet simulated.
altitude develop moderate susceptibility to endocarditis and
glomerulonephritis . The susceptibility to endocarditis is less
than that reported previously in similarly exposed rats and less
than in dogs with aortic insuff ieiency . In obese rats , altitude
tolerance is reduced and visceral changes attributable to high
altitude tend to be more severe » The effects of exposures to
altitudes above 25,000 feet on normal dogs and dogs with various
cardiac abnormalities has been under study. Studies are also being
made to determine the effects of high altitude exposures on the
development of atherosclerosis and other lesions in rabbits given
high cholesterol diets.

A study with Dr. Webster on the toxicity of iodides and iodatas
is nearing completion. Mo major findings to report.

A seareh for new effective antiarrhythmic drugs has been handi-
capped because the mechanisms underlying such arrhythmias has not
been clearly understood. In a study with Dr. Maling, we have
demonstrated severe fatty changes in the myocardium of dogs sacri-
ficed S hours to several days after intravenous infusion of large
doses of either norepinephrine or epinephrine. During the period -
of 2 to 5 days following large infusions of these catechol amines,
marked cardiac arrhythmias and ventricular tachycardia can be
induced in such dogs by small test doses of epinephrine or noreplne~
phrine. The severity of the induced arrhythmias roughly parallelled
the severity of the fatty changes in the myocardium. Studies ss^q
in progress to determine with the aid of various drugs and biochemical
procedures a possible causal relationship between the fatty changes
and the cardiac arrhythmias.

Part B included: Yes

Serial ]

Page J
Honors j, Awards, and. Publications

Publications other than abstracts .from this project:

Alt land, Paul D., and Highman, Benjamin? Effects of High Altitude
Exposure on Bogs and on Their Susceptibility to Endocarditis. The
J. of Aviation Med. 28:253-259, 1957.

Webster, Stewart H. , Rice, Mary E., Highman, Benjamin and Von Oettingen,
Wo F.s The Toxicology of Potassium and Sodium lodates: Acute Toxicity
in Mice. J. of Pharm. & Exp. Therapeutics. 120:171-178, 1957.

Highman, Benjamin, Thompson, Edwin C, Roshe, Joseph and Altland, Paul D,
Serum Alkaline Phosphatase in Dogs with Experimental Splenic and Renal
Infarcts and with Endocarditis. Proc. Sec Exp. Biol. & Med. 95:109-312,
1957.

Roshe, Joseph, Highman, Benjamin and Altland, Paul B»: Effect of Euf-
nagel Valve on Susceptibility of Dogs to Endocarditis* A. M. A. Arch.
of Surg. 75:680-683, 1957.

Altland, Paul D. , Mlekelsen, 0. and Highman., B. : Effects of Exposure
of Obese Rats to Simulated High Altitudes.. Am. J. Physiol. 191: Dec,
1957 (In press).

Highman, Benjamin, Roshe, Joseph and Altland, Paul B.: Endocarditis
and Glomerulonephritis in Dogs with Aortic Insufficiency: Production
by Single Bacterial Inoculation and Effect of Cortisone. A. M. A.

Honors and Awards relating to this project: Served as Guest Demonstrator
for Fresh Tissue Pathology at the A.M. A. Convention in New York City,
June 4-, 1957.

NIH
Individual Project Report.
Calendar Year 1957

Serial No. NIAMD- 71

1. Pathology & Histochemistry

2. Pathologic Anatomy

3. Bethesda

Project Titles The pathogenesis of viral diseases.

Principal Investigators Ruth L. Kirsehstein

Other Investigators: Is Dr. George A. Hottle. 2: Dr. Samuel Baron.
3s Dr. C. P. Li. Ui Dr. Joel Warren. 5s Dr.
Laurence Kilham. 6s Dr. Alan S. Rabson. 7s Br.
Karl Babel. 8s Drs. R. Huebner, ¥. Row© and
I. Brodsky.

Cooperating Units: Is DBS, LVP„ 2: DBS, LVP. 3: DBS, LVP.
4s DBS, L¥P. 5: DBS, LBP. 6s NCI, LPA.
7s NIAID, LID. 8s MAID, LID.

Man Years (calendar year 1957) s
Total, s 1
Professionals 1
Others None

Project Descriptions

1. (With Dr. George A. Hottle and staff)? As outlined in the
Current Regulations for the Monkey Safety Test for -Poliomyelitis
Vaccine, histologic sections of lumbar and cervical spinal cords
of monkeys inoculated with vaccine are examined for the presence
of lesions. 300-400 monkeys a month are examined.

If suspicious lesions are encountered, study of further sections
of brain and spinal cord is .undertaken. Lesions of suspicious
viral nature are encountered occasionally. These must be dif-
ferentiated from poliomyelitis.

It is expected that this endeavor will continue as previously.

2. (With Dr. Samuel Baron}: Lesions of expartoental Types I, II
and III Poliomyelitis w& being compared in Rhesus and South
African Yervet Monkeys. Th© susceptibility to infection and
the pathologic response of the two types of monkeys seem
comparable.

Pag© 2

3. (With Dr. G. P. Li)s An attenuated live poliovirus w&s ino-
culated into monkeys as in the Vaccine Safety Test* The
pathologic lesions in these animals are minimal. However,
lack of substantial numbers of monkeys has caused cessation
of this project at the present time.

4.. (With Dr. Joel Warren): It is known that poliovirus is an
OTterle virus. In an attempt to see if multiplication of
virus takes place in the intestinal tract and where, monkey
5.1©al organ cultures in tissue culture media are inoculated
with virus. The ilea are examined histologically for lesions.
A limiting factor has \>®m the rapid autolysis of the mucosa
of the intestine. The virus, however, does appear to mul-
tiply so that continued attempts at preservation of viability
of tissue are essential. Expansion to include human intestine
is contemplated.

5,6. (With Dr. Lawrence Kilham and Dr. Alan S. Habson): Study of
the' pathology of fibroma virus in adult and young rabbits and
squirrels reveals a different pathologic process. The viruses
are apparently more virulent and of much greater malignant
potential in suckling animals. Further study of the virus in
suckling squirrels and rabbits is contemplated.

7. (With Dr. Hab®l)s The "S.A. virus" Isolated, some years ago,
apparently gives a clinical neurologic lesion in hamsters and
chicks. Histologic study of the brains and spinal cords of
these animals shows no definite lesions. Further study of
peripheral nerves and muscles will be done to explain the
apparent lack of olinico-pathologic correlation.

8. (With Drs. Huebner, Rowe and Brodsky)s A filterable agent
isolated from mouse Ehrllch's ascites cell tumors has been
passaged in mice as cell-free filtrates. These mice develop
large spleens and often die of rupture of the spleen. The
pathologic picture needs further study for classification as
a malignant process. It is proposed that further studies be
undertaken including th© giving of the "agent" to splenectomiaed
mice and to suckling animals as well.

Part B included? No

PES

Individual Project Report
Calendar Year 1957

Serial No. NIAMD 72

1. Pathology & Histochemistry

2. Pathologic Anatomy

3. Bethesda

Project Title: Study of mechanisms involved ia infectious diseases

Principal Investigator: E. M. Lesrner II

Other Investigators ? Leon Sokoloff, Robert R. Williams, Kurt J. Block ,
Victor Haas, Laurence Eilham.

Cooperating Units: LPB-NIAMD, A&R-NIAMD, LID-NIAID, LBP-DBS

Man. Years (calendar year 1957):
Totals 1^~
Profession&l: 1
Other: g-

Project Description:

Continued studies with an arthritigenic strain of S^^^gbaeJJJAjg,
monilif oralis isolated by specialised cultural techniques have pro-
duced osteoarthritis in 92-93$ of more than 100 rats injected
intravenously, At the acute stage of boa®, joint, and periarticular
lesions (5-7 days post injection), the infecting microorganism could
be recovered from the joints but extremely rarely from the blood.
Examination of animals at daily intervals has shown pathologic
changes in joints as early as 24 hours after injection. Blood
cultures are positive for 48=72 hours after injection, then become
negative in most animals. Joint cultures are positive for the same
period as blood cultures, then become negative, and become positive
again 6 days after injection. This would suggest multiplication of
the microorganism in the affected tissues. Immunological studies of
infected rats with acute osteoarthritis have shown a slight elevation
of plasma opsonins , a 10-fold increase over uninfected animals* Sera
from infected rats have reacted positively in the modified sheep cel-l"
agglutination test for rheumatoid arthritis. All sera so tested have
given positive titres, ranging from 1:4. to 1:64,' while all sera from
uninfected animals have been completely negative. The same sera have
reacted positively in the bentonite flocculation test for rheumatoid
arthritis, giving titres of 1:4 to 1:128, after these sera had been
stored for on© week at 5° C, whereas fresh sera have generally reacted
completely negatively in this test, as have sera from uninfected
animals. This immunologic response will be pursued, as well as studies
of the filterable "L" forms of S. mo£M£g^A§."

NIAMD

Study of mice infected with lymphocytic choriomeningitis virus and
treated with ainathopterin has been extended to a larger series,
confirming preliminary findings that mice so treated survive and

have either negative or extremely slight lesions , although LOM
virus is recovered in high titre from the brain . Modification of
th© course of the disease with this drug appears to be relatively
independent of the quantity of virus recoverable from the host.

Preliminary chemical studies with rabbit myxoma virus indicate that
chloroform kills the virus and destroys its transforming factor for
rabbit fibroma virus. However , treatment with diethyl ether kills
the virus but does not destroy its transforming property, paralleling
heat treatment in this respect. This would suggest a water-soluble,
heat-stable component of the virus which is responsible for the
transforming effect upon fibroma virus. The possibility of desoxy-
ribonucleic acid or some derivative is being followed, and electron
micrography is being employed in an attempt to correlate morphological
changes .

Part B included? Yes

Serial No. NIAMD 72

Part B; Honors 3 Awards, and Publications

Publications other than abstracts from this projects

LerneTj Edwin M. II and Silverstein, Emanuel s Experimental Infection
of Rats with Streptobacillus monilif ormis «. Scions© 126s 208-209, 1957.

Honors and Awards relating to this projects None.

PHS-NIH

Individual Project Report

Calendar Year- 1957

Serial JJo. NIAMD-.

1. Pathology & Histoehemistr-y

2. Pathologic Anatomy

3. Bethesda

Project Titles Comparative pathology

Principal Investigators Ladd N. Loomis

Other Investigators? Walter Newton, Paul P. Weinstein, Jack C. Jones,
Alexis I. Shelokov and A. W. Pratt.

Cooperating Units s MAID, LTD (Newton, Weinstein and Jones}.
NIAID, LID (Shelokov). NCI, LPHI (Pratt).

Man Years (calendar year 1957):
Totals 1

.: 1

Project Description:

The effect of germ-free parasites upon germ-free guinea pigs is being
studied with emphasis on the hosts tissue reaction (and the deve-
lopmental stages of the parasite) in relation to the tissue-barrier
concept of host specificity. It lias been found that some parasites
(roundworms) complete their life cycle in an abnormal host for the
first time. The work is continuing.

The comparative neuropathology of a Russian "Type four poliomyelitis
virus" was studied in Rhesus monkeys. While some of the C.N.S. lesions
were indistinguishable from those caused by true poliomyelitis virus,
other monkeys contained C.N.S. lesions that allowed a differentiation
to be made. These findings combined with extensive serological studies
identified the Russian virus as identical to Coxsaekie A7 virus. These

A virus of the gastroenteritis type is being studied in suckling mice,
rabbits and Rhesus monkeys. It causes both visceral and C.N.S. lesions.
These are being evaluated against those caused hy more well-known virus
entities. The work is In progress.

NIAMD_73

Page 2

A report on th© nature of the skeletal muscle lesion earns ©d by
the Rous tumor has been submitted for publication. The sub-
cutaneous Rous tumor regularly invades the underlying muscle
tissues in a characteristic manner. Photographs and some theories
are presented of th© muscle lesion. The project is completed.

A study of th© spontaneous diseases of domestic animals used for
research is being continued. Special attention is being given to
th© extensive demyelination seen in the brains of dogs with a
history of infection with canine distemper viras.

Part B included? Yes

Serial No. NIAMD 73
Page 3

Part B; Honors, Awards s and Publications

Publications othsr than abstracts from this projects

Habel. Karl and Loomis, Ladd N.j Goxsaeki.® A7 Virus and the Russian
"Foliovirus Type 4". Proc. Soe. Exp. Biol. & Med. 95s 597-605,

Honors and Awards relating to this project: None.

PHS-
Individttal Project Report
Calendar Year 1957

Serial No. MI AMD Ik

1. Pathology 3

2. Hematology

3. Bethesda

Part ft.

Project Titles Regulation of hemopoiesis.

Principal Investigators % Frederick Stohlman, Jr. 9 and George Brecher

Cooperating Units; Medical Department 9 Brookhaven National Laboratory

Man Years (calendar year 1957);
Total; 5.5
Prof e ssional : 1
Other ; U- 5

Project Description;

Objectives; Study of red cell, white eell9 and iron turnover with
particular reference to the physico-chemical identification
and physiology of erythropoietine 9 the erythropoietic
stimulating factor.

Methods employed; Erythropoietine (E.P.) is assayed in rats with
suppressed erythropoiesis9 irradiated9 starved or hypophys-
ectgmized. Its effect on RBC production is measured by the
Fe red cell incorporation technic and reticulocyte response.
Red cell destruction has been estimated utilizing the DFP
and Cr technics.

White cell turnover is being studied in rats and mice by
transfusion of labeled cells and in, vivo labeling 9 using
primarily tritiated 'thymidine and radioautographs.

Major findings:

1. A. hundred fold purification of erythropoietine has been
obtained utilising an acid heat denaturation and alcohol
precipitation procedure. The heat denaturation step results
in a 70-90$ loss of E.P. but does permit the assay of foreign
plasma in the rat.

2. Studies have defined the rate of release of E.P. in
response to hypoxia and bleeding (3 hrs) and disappearance
from the plasma after withdrawal of the stimulus9 half
disappearance time 8-10 hrs.

- 2 - Serial No. HI AMD
Page 2

3. Increased E.P. production is responsible for the poly-
cythemia following hypoxia 9 the magnitude of the response to
E.P. ? however , is limited by the oxygen available for the
primarily aerobic normoblastic proliferation.

A. Evaluation of DPP32 (Di-isoorophyl flouronhosohonate) in
the study of red cell survival indicates that it cannot be
used as an injritro tag but does give survival curves similar
to those observed with the Ashby technic and better than those
obtained with chromiums) t^hen used as an in vivo label,

o
5. Thymidine H is incorporated into DNA which provides a
permanent label. Heavily labeled lymphocytes persist in the
spleen for prolonged periods. Their survival in the
circulation is of the order of a day or two. Published
observations of apparent long life srians of lymphocytes can
orobably be attributed to continued release of snail numbers
of labeled cells over a prolonged period rather than to long
survival of these cells in the circulation.

Significance to the program of the Institutes Anemia is a common
complication of arthritis and in many instances may be
considered to be a metabolic disorder. Understanding of the
mechanisms of red cell regulation are not only of basic
interest but should eventually result in improved therany.

Proposed course of project; Continued efforts at isolation of E.P.
utilizing column fractionation and enzymatic digestion are
planned. Investigation into the physiology of red cell pro-
duction will include studies on the mechanism of E.P. release
and its relationship to oxygen requirements. The existence
of an inhibitor has heen suggested by both clinical and
experimental observations and studies aimed at identifying
such an inhibitor are planned.

White cell turnover studies are planned in terminal cases of
brain tumor and will be carried out in Brookhaven.

Part B included Yes jj$ No £J

Serial No. NIAMD
"Page"

Fart, B ; Honors, Awards, and Publications

Stohlman, F. Jr. , and Brechers G. ; Humoral Regulation of Erythro-
ooiesis III. Effect of Exposure to Simulated Altitude. J. Lab. &
Clin. Med. /& % 890, 1957.

Stohlman, F. Jr., and Brecher, G. % Humoral Regulation of Erythro-
poiesis IV. Relative Heat Stability of Erythropoietins. Proa. Soe,
Exp. Biol. & Med. 9J5s 797, 1957.

Gladner, J., Laki, K. , and Stohlman, F. Jr. % DIP Labeled Thrombin.
Bioehemica et Biophysiea. In press. December 1957.

Brecher, G. , and Stohlman, F. Jr. z Humoral Factors in Erytliropoiesis.
Progress in Hematology. Edited by L. M. Tocantins. In press.

Stohlman, F. Jr., Brecher, G. , Schneiderman, M. , and Cronkite, E. P. i
The Hemolytic Effect of Ionising Radiations and Its Relationship to
the Hemorrhagic Phase of Radiation Injury. Blood. In press.

Brecher, G., and Cronkite, E. P. s Gastric Lesions in Experimental
Animals Following Whole Body Irradiation. Am,, J. Path. In press.

Brecher, G., Cronkite, E. P., Bond, V. P., and Dutcher, T. F. :
Problems of Leukoeyte Transfusion. Acta Hemat. In press.

PHS-NIH

Individual Project Report

Calendar Year 1957

Serial No. fflAMD 75

1. Pathology & Histochemistry

2. Hematology

3. Bethesda

Part &.

Project Titles Studies on normal and abnormal human hemoglobin.

Principal Investigators H„ A.. Itano

Other Investigators: S. J. Singer, E. Robinson, B. Hudson

Cooperating Unitss Department of Chemistry, Yale University (Singer)

Man Years (calendar year 1957) s
Total: 2
Professionals 1
Others 1

Project Descriptions

Objectives: To study the physical chemistry, biochemical genetics,
and clinical significance of the normal and abnormal human
hemoglobins.

Methods employed s Moving boundary electrophoresis; spectro-
photometry? kinetic and equilibrium measurements with pH
meter and spectrophotometer.

Major findings:

(a) Intermediate compounds of ferrihemoglobin and ferri-
hemoglobin cyanide have been separated by moving boundary
electrophoresis. Itano and Robinson

(b) Hemoglobins A., S, and C have been found to dissociate
reversibly to their respective half -molecules in acids how-
ever, recombination of these half molecules by neutralization
of acidified mixtures does not result in the formation of
"hybrid" molecules, i.e. molecules composed of half -units
from two different forms of hemoglobin. Itano and Singer

(c) Samples of rare hemoglobins from the laboratories have
been analyzed electrophoretically. In addition to samples
from several laboratories in this country, samples from
Turkey, England, and the Belgian Congo have been studied
during the past year. Itano and Robinson

Serial No. MI AMD 75
Page 2

Significance to the program of the Institutes

(a) The inherited abnormal forms of human hemoglobin provide
useful experimental material for the study of the effect of
gene mutation on the biosynthesis of proteins and of the
structure of hemoglobin. Since differences in the physical
■properties among the various forms are small9 differences
that arise from alteration of the molecule after biosynthesis
must be distinguished from differences that are incorporated
during biosynthesis* The separation of intermediate compounds
provide a clear demonstration of one type of alteration of the
hemoglobin molecule that can be induced after completion of
synthesis of the molecule* According to the intermediate
compound hypothesis} the efficiency with which hemoglobin
transports atmospheric oxygen depends upon the presence of
four hemes in each molecule and upon the interaction of these
hemes. The separation of intermediate compounds confirms the
physical validity of this hypothesis. The failure of hybrid
molecules to form under conditions of dissociation and
reassociation implies the presence of an inherent structural
specificity in the bonds which holds the half molecules of"
the respective normal and abnormal forms together. The
examination of samples from other laboratories has been under-
taken as a contribution of this laboratory toward maintaining
order in the identification of rare inherited forms of hemo-
globin. Correct identification is essential since the location
and frequency of the different types provide valuable data for
studies in population genetics and anthropology.

Proposed course of project: The studies undertaken during the past
year will be extended. In addition9 the effect of various
small molecules on the physical properties of hemoglobin will
be studied. Studies on the terminal amino acids of hemoglobin
will be undertaken with the collaboration of Jules Gladner of
the Laboratory of Physical Biology.

Part B included Yes S No £J

Serial No. MML—TL
"Page 3

Part B; Honors, Awards, and Publications

Pauling, L„, and Itano, H. <u (editors) | "Molecular Structure and
Biological Specificity". Baltimore, Waverley Press, 1957,

Itano, H. A. 3 Specificity in the interaction of sickle cell hemo-
globin molecules. In "Molecular Structure and Biological
Specificity" vv< 166-173, 1957.

Itano, H. A, | Interpretation of electrophoretic, solubility, and
denaturation data in the study of hemoglobin differences.
Federation Proc. 16, (Sept,, 1957), in press.

Itano, H. A, | Electrophoretic analyses of the abnormal hemoglobins.
Me R„ C„ Conference of Hemoglobin. Washington, D. C. 1957 (in press)

Itano, H. A, j The human hemoglobins; their properties and genetic
control. Advances in Protein Chemistry 12, (1957) (in press).

PHS-NIH

Individual Project Report

Calendar Year 1957

Serial Koc NIAMD_

lc Pathology & Histochemistry

2, Hematology

Part A.«

Project Title; Studies of dietary factors and embryonic growths

as affecting rat leprosy and experimental amyloidosis

Principal Investigators George L„ Fite

Man Tears (calendar year 1957) s
Totals 1-1/3
Professionals 1
Others l/3

Project Descriptions

Objectives;

lo To investigate the effect of vitamin E deficiency on
rat leprosy s following the lead of Bergel and Mason in
their work on human leprosy s in which they reported
enhancement of the infection in rats fed E-deficient diets
rich in cod liver oil„ This work, if trus9 may be of great
significance, and appears of readily susceptible analysis
using rat leprosy.

2, To explore further the unreported work of H. S» N.
Greene in transplantation of lepromas to brain tissue. It
is planned to explore this field widely; by implantation of
infected embryonic tissues into non-susceptible animals9
not only into brains but testis and other organs*

3o To examine the infectivity of Mc Leprae Murium growing
in the ascites tumor of the mouse.

4.. To analyse experimental amyloidosis in animals using
several dietary procedures and rat leprosy as the immediate
causative agento

Methods employed; Animal experimentationo

Major findings; None

Serial No. WTffljD.
Page 2

Significance to the program of the institutes A direct attack
on basic problems in nutrition as related to chronic
infection.

Proposed coarse of projects See objectives.

Part B included Yes hj No

Serial No, flglAMD,
Page'"

Part, B s Honors? Award s9 and Publications

Fite9 G. s Leprosy Society s and Hansen's Disease., Int= J
Leprosy. 2^2 457-4-63? 1956.

PHS-
Individual Project Report
Calendar Year 1957

Serial No. NT AMD

1. Pathology & Histochemistry

2. Hematology

3. Bethesda

Part A.

Project Title; Pathogenesis of Experimental Arthritis and Pathology
of Rheumatism.

Principal Investigators Leon Sokoloff, M. D.

Other Investigator: Emanuel Silversteins M. D.

Cooperating Units; NIAMD, LNS? Dr. Mickelsenj RLAS Dr. Jay,
NIAMD, LPH9 Dr. Lerner N1AMD 72

Man Years (calendar year 1957) :
Total; 3.9
Professionals 1.9
Others 2.0

Project Descriptions

Objectives; Investigation of factors influencing development of
degenerative joint disease in small laboratory animals?
production of Inflammatory arthritis.

Methods employed: The role of genetic factors, thyroid function*,
early skeletal maturation and dietary obesities on the
development of osteoarthritis are being studied by anatomical
means; and? in the case of thyroid function, by several
physiological and chemical modalities.

Inflammatory joint disease has been studied by injection of
adjuvants, adrenalectomy and more recently, by collaborating
with Dr. Lerner in studies of Streptobaeillus moniliformis
infection of rats„

Major findings; Osteoarthritis has been produced in rats by a

high fat diet resulting in obesity, but similar findings were
not consistently obtained in mice. The incomplete evidence is
that high fat intake, in susceptible animals affects the joint
disease adversely. Incomplete studies indicate that obesity
ean be dissociated from the arthritis by crossing STR/lN with
A/IN mice. Genetic variation in thyroid function has been
demonstrated among several strains of mice? but this cannot
be correlated with the occurrence of arthritis.

Serial Wo. MI AMD ?7
PageTF

An interesting by-product has been the discovery of a
spontaneous^ apparently primary polydipsia and polyuria
occurring regularly in an inbred strain of mouse.

Significance to the program of the Institutes Disability due to
degenerative joint disease is of great economic importance.
The present studies were directed at discovering whether
osteoarthritis is an inevitable concomitant of aging or
whether specific etiological factors and preventive measures
could be discovered. Genetic factors have been found of
major importance but other influences studied have not been^
with the exception of obesity produced by high fat diets in
certain animals.

Proposed course of the project: Six months will be required to
complete the genetic and dietary studies noted above. A
small number of animals will be retained to complete skeletal
maturation studies. Further extension of these experiments
appears unprofitable. After that time9 learning and applying
technics of electron microscopy to problems of rheumatic
pathology are contemplated.

Part B included Yes ^ Wo £J

Serial No. HI AMD __ ]]_
Page 3

£i.£t.-B' Honors, Awards5 and Publications

Lsrner 1IS E. M.9 and Silverstein9 E. s Experimental Infection of
Rats with. Streptobacillus moniliforraiso Seiences 1265 208-209?
1957.

Sokoloff$ Lo9 and Habermann, R. T. s Idiopathic Necrosis of Bone
in Small Laboratory Animals9 A. M. A. Arch. Path. In press.

Silverstein9 E. s and Sokoloff, L. : Osteoarthritis in Guinea Pigs5
Arthritis and Rheumatism. In press.

NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES

Annual Pro jest Report

Calendar Year 195?

Summary Sheet

Laboratory of Pharmacology and Toxicology

Estimated Obligations for FY 3g$8

Totalt $U76^5QO
Direct! $3*l5*00O
Reimbursements % $131$5O0

Projects number 78 thru 91 included©

FHS-NIH

Individual Project Report
Calendar Year 1957

Serial No. NIAMD.

1, Pharmac ologjT & _!Toxic olcg y

2, Pharmacology

3, Bethesda

Part A,

Project Titles The physiological function, intermediary metabolism,

and pathological effects of spermine, spermidine, and
related amines.

Principal Investigator(s)s Drs. S. M. Rosenthal, C, W. Tabor, B, DubiiT

Other Investigator (s) % Drs, H. Tabor, E, L, Jackson.

Man Tears (calendar year 1957) s
Totals 3-2/3 yrs.
Professionals 3-1/3 yrs.
Others 1/3 yr„

Project Descriptions

Objectives^ Spermine and spermidine have been found to be potent
nephrotoxic agents. Analyses of animal tissues, and of plant and bacteria
cells, have demonstrated a wide distribution, frequently in high
concentration. Studies are conducted to elucidate its metabolism,
physiological function, its relation to renal disease, to spermatozoal
physiology, and to bacterial defense mechanisms,

I-Iethqds i ^mployeds (l) Isotopically labeled compounds (G^ N^)
were employed to further elucidate the biosynthesis of these polyaraines ,.
(2) The amine content of various plant and bacterial cells was furlher
studied, using chromatographic techniques, (3) Continued efforts were
made to antagonize the renal toxicity of spermine using a fatal
nephrotoxic dose in mice as teat object,

Maj or. Findings s In the colon bacillus it was found that spermidine
synthesis is dependent on pH, being suppressed in acid media. At pH 8
synthesis was obtained in bacterial sonicates, and the enzymes involved
have been partially purified. With these : preparations the folloi^ring
biosynthetic pathway was showas

ATP * L Methionine feigyme.I. » Compound I

Compound I Ea.zy^r U - CO2 » Compound II

Compound II I^g16 m * Spermidine,
Putrescine

Part B included [x] Yea [ ] Wo

Serial No, NIMD 78
Page 2

Compound I is Kactive methionine57 which is deearboxylated and then
transfers its propylamine group to putrescine to form spermidine. In the
systems studied this has been an important pathway of methionine metabolism

Studies of the distribution of the polyamines in vegetables and
bacteria have shown them to be in nearly all cells., but with wide variations
in concentration and in the relative amounts of each amine. In some
bacteria they constitute over 20 per cent of the nonprotein nitrogen, and
isotopic studies have shown a high turnover rats of putrescine.

Significance to HI&MD Research? Spermine and spermidine are widely
distributed in mammalian and plant tissues and no previous work has
demonstrated any physiological function or relation to diseases. It is believed
that this work will throw light on their biochemical significances and that &
causative relationship may be shown to some types of hussan renal disease,

R^ojpoced^ Course m of .Pro j ec 1 1 (l) To complete our knowledge of the
biosynthesis and fate of these polyamines^ and to relate these findings to
their physiological and pharmacological effects, (2) To elucidate the
bound state in which they exist in cells, (3) To further study the renal
toxicity, bacteriocidal and spermatoeidal effects in their relation to
physiological function and possible role in disease.

■HIH

Indi-yidual Project Report
Calendar Year 1.95?

Serial No, NIAHD 78

Page 3
Part Bi Honors s Awards, and Publications

Publications other than abstracts from this projects

Tabor 9 H», RoBontfcal, 3. Mo* m& Whor, Co Wa Rol© of patreBcim
and methionine in the tomosynthesis of spermidine,, J„ Am„ Cheau Sec*
2£s 2973, 1957 .

Green©, R. C„ Incorporation ©f the carbon ehaiB, of Ksthioaiae
into spermidine, J„ AiBer, Chess. S©cu 2223929a 1957.

Individual Project Report
Calendar Year 195?

Serial Hoo BlaMD . .79 _
1. Phsrssseoiogy &*°fffi!cS^j
2„ PteBJ&eology
3- Bethesds

Project Title; The chemotherapy of mouse leprosy.

Principal Imestigator: T„ f „ Chang

Other Inwstig&torsu None

Han Years (calendar year 1957) s

Totals 1-1/3 yr0
Professionals 1 vr„
Others 1/3 yr0 "

Project rescript ions

Ob^ectiTOas The emluation of therapeutic effectiTOsxess of drugs
in kous© leprosy. The tissue eultur© of intracellular parasites.

Methods i|^l@yedt8 Intraperitoneal iaf action of mice %sith M«J|fJ>r§e
""rSacSS of monocytes by a test tub® nsethod specialty"
in this laboratory.

J:^ Oil

.Major Findia^gg The eviration, ©f satsrine leprosy in intraperitone. .
infected Eic© "sass studied by wekly escaHidnations (np to 10 weeks}
histologies! changes in K38©nterie spreads snd the bacterial ©numerat
in spleen and li^es8,, A lineal relationship ms found between the time ®ad
the severity ©f the infection* Definite growth of leprous infection "ma
noticed as early as three weeks after inoculation. AnimLs which tssre
inoculated irlth killed bacteria or treated with Isojti&sid showed a steady
decrease of the infection,

Streptoimriein showed strong suppressive activity in s©use leprbsyj
hydnoearpic acid, hydn©3nlfenes dihyilrosulfon©, propylthiouracil, thyroid
extract, sad abal©a© infusion, only raeak actirityi and gmm, globulin,
no activity.

Asosig Esany failures in the cultivation of Hgroteeterium leprae
aaarluiB in tissu© culture of Essnoeytes, obtained from peritonea! esrad&t©
©fTeprous isd.ce, three esperiasata sherod definite Koltiplieation of the
organises o The ©rganisES showed elongation, bundle forss&tioa ®nd

Part B included [2] Yes [ ] D©

Serial No. JSTIAMD 79

Psge 2

tremendous increase in number within the monocytes in untreated
culturesj while cultures treated with isoaiaaid ©r infected with
killed bacteria revealed no growth at all,

Streptorayein, 1 ug0/5slof was found to inhibit the growth
in intracellular BCG in the tissue culture of monocytes,, Diamine^
piraelic acid *Jas not found in tissues of ssouse leprosy,,

Significance, to N3IMD Research s The leprosy studies ®re carried
out in cooperation with*T!heniiierican Leprosy Foundation (Dr. Chang is
on a Fellowship from theni)o The results are applied to their clinical-
evaluation studies *

Proposed Coursei of Pro.jeetg Continuation of evaluation of drugs
in mouse leprosy, using both lsng~ terra and short- terra techniques <,
Continuation in the study of tissue culture of BCG and Mycobacterium
leprae rourium0

Serial Mo, N1MD 79

Page 3
PHS-NIH
Individual Project Report;
Calendar Year 195?

Part B s Honors, Awards, and Publications

Publication® other than abstracts from this projects

Chang., Y» T. Chemotherapy of murine leprosy, V, The effects
of various conciliations of ljsIjB<=-diaMn©diphenyl sulfon® (DDS),
streptomycin and isoniasid (isonicotiBic acid hydr&zide) on mouse
leprosy, Xnternat, J, Leprosy 2^:30?»32ii, 1956,

Changj, Y0 T, Chemotherapy of murine leprosy, VI, The effects of
isonicotinylhydrazone of 2~carboxyTOthc^~3~^thoxybenzaldehyde (Cosspound
373) and isonicotinylhydrazone of 2=carbeacymethQ2ybenzaldehyde (Compound
377) on mouse leprosy, Internat. J, Leprosy 2J>sNo0l9 1957«

Chang j Y, T. Chemotherapy of Earin® leprosy, VII, The effect of

cycloserine (Seromyein) on mouse leprosy, Internat, J, Leprosy, In press <

FES
Individual Project Report
Calendar Year 1957

Serial Woo HM1D 80 ,,

la Fharmae ologyl&rf 52S>togir
20 Riarm&eolegy
38 Bethesda

Part A0

Project Titles Metabolic processes , drag ^©tta&s, m& physical
chemistry related to electrolyte distribution
and bioelectric®! phenomena in nerve*

Prineip&l Investigator? A« Mc Shanes

Other Investigators s N» D<, Qershfeld and G, Paul Bianchi (NIH Fellow)

Man Tears (calendar year 195>7 )s
Totali ii=2/3 yrso
Professionals h yrs0
Others 2/3 y?<,

Project Descriptions

Object^res^ Ionic moveisant and the bieeleetrical characteristics oi
unexelR^ ("resting" ) nerve have been found to be susceptible of
modification by alterations in the rate of isatabolism and by phys-
changes (eage, in menforane permeability such as induced by drugs) ^hiek
leave metabolism unaltered,. The present studies have been directed
towards establishing in greater detail ~ chiefly in nerve^ but to _
extent also in Euscle - the interrelationships between jsstabellssj, ion
distribution rate of ion aoven®nt9 and resting and action potentials
as affected hy ionsf; drugs , etc<,

Methods Employed? Desheathed sciatic nerves of the toad,, ^hieh our
studies have demonstrated to be remarkably stable in vitro for long
periods of tirae^ are exposed to experimental coadiliomTTor varying
intervals of tiase, usually in the presence of radioisotopes ©r after the
radioisotopes have been taken up by the tissue „ The rate of uptake or
rate of loss of the isotopes is compared with controls t© evaluate ex-
peridental effects <, The Beelman flass spectrophotometer provides date
on the soditm and potassium c patent of the tissue. Resting and action
potentials are f ollouad potentiosstricalXy and by cathode ray oseil«
legrsphy using specially designed circulation units that saJLaiHLze
changes in potassium ceascsntratiea of the Interstitial fluid ^feieh others
vise would ©ccur under our experisseat&l e editions „ The role of
aet&boliss is evaluated by following th© ionic and electrical chaages
during a&exi&s in the presence or absence of iisdoacetata^ with glueoae

Part B included & Yes [] No

Serial No„ NI&MD _J0.
Page 2

or other metabolites present, and during th® pest^anoxie recovery in
oxygen. The influence of drugs is judged by following their actios
on the sama physiological properties of nerw before, during, and

after metabolic inhibition,

leaie movements ia single giant ax©ns of the squid during activity
have sis© been studied by th® Rv©lt&ge=clsj8p;, technique,

Ma^or Findings (1) Tins i course nof &ctlm of, ,8a anesthetic (cocaine),
at coad^Mon"'''bIockiEig conceatr&tlgas^ -i^Jsodim^d pe^gioa^A^gs
JmS^^ " Thjjnaas

IffiidleTIEmlioM "(activeT*^Sasport of ioas

present or largely bloelSdT>y"©n€sd,a or a cestjinatioa ©f anoxia and
iodoaeetate poisoning (thereby leading BpBssi,s©n transfer ),

Potassium influx is decreased hj th® alkaloid by about the same
absolute anasunt whether the influx includes both active and passim
consonants, or has been previously reduced to 1/3 <=> lA ^7 inhibition.
This effect is matet&Imsd in the continued presence of the anesthetic,

Potassium eutflux ia uninhibited nerve undergoes a transitory d®=
cline and returns almost to normal ia the continued presence of th© drug,,
The elevated ©utflux of potassium in inhibited nerve is largely
obliterated by cocaine j this is a sustained effect and is duas to
prevention of exchange of potassium for extracellular sodium.

Sodium outflux is completely unaffected by cocaine whether metabolism-"
is inhibited or not. Only the elevated sodium influx during inhibition
is eliminated* and this effect is sustained.

The depolarization of n©rv® hj mtsbolic inhibition is almost
eliminated by cocaine, Th© resting potential of uninhibited serro under?
goes a small transitory rise ia potential in cocaine.

These studies indicate (a) that ia resting nerve the low infest
cellular level of sodium ia achieved hy metabolism by rejeciiag sodium
it enters the cellular mimbmmi from tfos swrounding medium (sodium
sigjajl, ratter than by extrmdiag sodium fr« th® axoplasm m& (b)
acts predosrijsaatly en the permeability ©f tte fiber

exclus

to sodium and potassiwa iesss rather than en th® active

transp®rt proc@ss| its action largely tm th® influxes, with little ©r
«ly a transitory effect en the ©utfltscsSg suggest© that th® actio© of"
this alkaloid is restricted to -tee outermost layer of th® cellular

Serial He. HIM© 80

Page 3 r—" — ""

(2) The effect of anestjtotiGS IprmxLm* cecaing) ^.tto^lnyBrd

giant moBSo'°°°fkese""c'wrekts 'iEs^dVpTOSsedr^ThiV'Imlc^^s*'^^^^
ls«reB8®ffii""itK. persssability, wUh&ch norrally occurs first to sodium then
to potassium when the fibers are "clamped" at a sufficiently low membrane
potential, is reduced by the anesthetics*

(3) The role of carbohydrate metabolism in potassium retention and
sodium exclusion b^yertebrate 'ner?jn[Mudred^b3rRMlcl?e^th' Fellow,
"S^'filSchiJ7^'m^Lucoie slows' the rate of anaerobic K* loss from de°>
sheathed Bufo T_narinug sciatic nerve in the first h to 6 hours, There-=»
after, the beneficial effect of glucose on anaerobic I(* retention is
lost. Cessation of potassium loss, during a 2 to 3 hour pestancxic
recovery period in oxygen, is invariably h&ispered by prior anaerobic
utilization of glucose for periods as brief as h hours* Increase of
the phosphate level causes greater loss of K*, but during the oxidative
recovery phase the rate of loss is no greater in glucose than In the
absence of the sugar.

Spike potential measurements showed a decrease which paralleled
the rate of' loss of K*„ The spike potential decreased during anoxia,
the decrease being substantially slower in the presence of glucose.
When raygen was admitted, the spike potential partially recovered within
10 Minutes, the recovery being less with glucose in the ssediusa,.

Adenosine reduces potassium loss under anaerobic conditions as well as
durtag post&noxic recovery in oxygen,

(k) Interactions i among ions with_respect to n ionic | flaxes in
vertebrate"5 nerve ' an^T^cleT^o^el^ify^S' naturV°oT''^°pas8age of
ions,"into"°aa3ri)uT*oinES cell, studies are in progress on the effect
of different anions (CI, NO3, SCW) and of the concentration of sodiusa
and potassium in the msdiua on (a) the distribution of sodium and
potassium, (b) the outflux and influx of sodium and chloride, and (c)
the uptake of the radioisotope Ca*».

It has already been found for nerve that 'sodium outflux is unaltered
by removal of extracellular sodium (by replacement with choline^), by
removal of both sodium and chloride (by replacement with sucrose), ©r by
removal of potassium from the medium* As one replaces CI with -more
"pelarissable" anions (SCH > NO, > CI), the decliae of ionic gradients J
(to©Uj [K]^ decreasing, [$&3i "increasing) "is accentuated and action
potentials decrease* Also, the uptake of Ca*» ±& inscraased,

to^HIftlffiJtesaarchg These studies provide B&sthods for
two e@Sular°*,derang®Sents9 vis,, metabolic insufficiency
and increased gagisbrffln© le&kiraess, either of which say cause cells to rim
dousfoill with respect to ion distribution and bioelectric®! potentials*

Serial Ho. HMI1D §0

Page k — —

They also demonstrate that, within certain Units, these derangements
sre individually susceptible of treatment „ either by providing suitable
metabolites or by the application of agents which reduce mAnme leaki~
ness to ions* Since, as far as is known, the general features of all
other cells of the body with respect to ion distribution and resting
potential are sia&lar to those of nerve, the principles being elucidated
may prove useful in efforts to analyze the basis of cellular deterioration
in traumatic disorders or in disease and to delay or prevent such
deterioration,,

Proposed Course of Projects; (l) The action of an agent such as
veratriae, which exerts physiological effects (eog», on excitability,
asenfeane potential, and leskiness to sodium and potassium) opposite t©
those of the anesthetics, will be examined with respect to t&e ionic
fluses in inhibited and uaihibited nerve as in the case of eocaisss,,
The affect of hydrostatic pressure on the relative effectiveness of
eocaise and veratrine on ionic fluxes is also to be explored as a pos<=
sible Kesns of distinguishing differences in the mechanism of interaction
of the drugs with the cell surface *

(2) The action of pH, which can be used to control the fraction of
anesthetic' that is present as a cation or free base, ©ay be explored to
determine in what form anesthetics are effective in reducing mesribrane
permeability to ions0

(3) The f&stebolic studies are being extended to include measurements
(a) of the production of COg and laetic and pyruvic acids and (b) of the
consumption of oxygen under the sass experimental conditions previously
employed «,

Serial Mo« NIAHD iimm go

Pag« 5
FHS-NIH

Individual Project Report

Calendar Tear 1957

Part Bg Honors, Awards , and Publications

Publications other than abstracts from this projects

Shanes,, A. M. After-potentials in nerve. In "Electrophysiology of
the Heart" (II. II. Hecht, Editor), Ann, N. X. Acad. Sci. 65?9li3, 1957.

Shanes, A. M. Ionic transfer in a vertebrate nerve . In "Ifetabolic
Aspects of Transport across Cell Mesforanes" (G. R. Murphy, Editor), p. 12?,
Madison, University of Wisconsin Press, 1957 .

Aoatneik, E„, Freygang, W., Grundfest, II., Kiebel, G., and Stones,
A0 M. The effect of temperature, potassium, and sodium on the conductance
change accoapaoylng the action potential in the squid giant axon. J. Gen.
Physiol. J£:333, 1951*

Shanes, A. M. Some aspects of jsyella in nerve trunks . In
"Progress in Neurobiology" (So R. Korey wd D. B. Tower, Editors) . In
press „

Shanes, A0 M. Electrochemical aspects of physiological, and
pharmacological action in excitable cells. I. The resting cell and
its alteration by extrinsic factors. Pharmacol 0 Rev. 10s La press.

Umms md Awards 2©laMsg te fcM& preset?

Dr. Shanes participated as an invited guest speaker at the Symposium
• on Myelin held in conjunction with the rasetings of the American Neurological
Association in Atlantic City June 15, 16, 1957.

Dr. Shanes has been invited to speak to the Physiological Society
of Philadelphia on Deceiver 17, 1957.

Articles by Dr. Shanes, based on talks given at renent symposia,
have appeared in two monographs published this year (see Publications
©bows).

Dr. Shanes has be@n invited to speak ©a ien transport at the
IMversity of Philadelphia Medical School in Spring, 1958.

Dr0 Sfeanss has been iavitod to spsak at Hmdboldt Oaivwsity,
GerB&ny, at an international syspssiasa ©gs the "ffechanisa ©f ©scit®bilitgr!S
to be held March 31 thru April 2, 1958.

Individual Project Report
sar 1957

Serial Ko„ Nl^D^^JH.

1. Ph&risacology & Toxicology

20 Pharmacology

3. Bethesda, Md. and Lima, Peru

Project Titles Mechanisms and therapy of" traumatic shock*

Principal Investigators)? Ers„ R„ Carl Mlllican, Kehl Markley,
M0 Bocanegra, and S„ M. Rosenthal.

Other Investigator(s); Mr. J. Rust

Man Tears (calendar year 195?) '
Totals 2-1/2 yrs.
Professionals 2 yrs„
Others 1/2 yr.

Project Description;

5^£SM22£L ^ '^ie s^e^iveness and mechanisms of action of

certain drugs in shock,, (2) Clinical, evaluation in burn shock of
large volumes of saline solutions.

Ifeth^dj^^EmployeJ^ (1) Standardized techniques developed in this
laboratory for tine production of burn and tourniquet shock. (2) M
studies, analysis of fluid distribution, hemodynas&c changes, and e
on body temperature in experimental animals, (3) The clinical study
conducted in Lima, Peru involves comparison of saline solutions alone
with saline solutions plus plasma, on an alternate case basis „ iixt
correlary laboratory studies in hemodynamics, bacteriology ? cadoerinolcE
and renal function are carried out.

Major Findings s (l) Experimental shocks Ghlorpromazine,
chlorpromaaine sulfoxide, and promazine, given prophylacticaliy, h«
a therapeutic effect in tourniquet shock* No therapeutic effect was
observed from phenergan and pyrathiazinea Chlorpromazine produces the
following effects in mices a) marked and sustained lowering of body
temperature! b) increases blood volume, lowers plasma proteins
hematocrit, indicating & movement of extravascular fluid into the
circulation! c) the hemodynamic effects of intravenously administer
fluids are prolonged! d) swelling of the injured area of tourniquet-
shocked animals is markedly reduced, Chlerprsmszine sulfoxide had ao
demonstrable effects on body temperature and vascular system of non
mice or on the swelling of injured ma.ee a

t B included [x] Yes [ ] No

Serial No0 NI&HD _ __ 81
Page 2

(2) ^Gliiiical^ St-gdies . The first part of the project c ©sparing
large volumes of saline solutions with plasma plus minimum amounts of
saline has been completed* and demonstrated that saline was equally as
effective as plasma. The current study comparing in children large
volumes of saline alone with large volumes of saline plus plasrea is
under way. Approximately 70 cases in each group have been accumulated
and show a mortality from shock ©f 21 per cent with saline and 8 per cent
with saline plus plasma. These results suggest a benefit from plssmfts
although they are not statistically significant.

Studies have been completed on (l) the hemodynamic and renal
changes in shock treated by saline and by plasma (2) the adrenal steroid
response to bt?rns of varying severity^ tested \>y the steroid excretion
following a standardized dose of ACTH (3) the effects of large volumes
of saline loading in normal subjects (k) the ability of natiws at high
altitudes to withstand stress.

Proposed Course of ..Projects (l) Continued study of the basic
mechanisms that produce shock, and the effectiveness of various
therapeutic measures, (2) 2?ollosing the ceiapletien of the current clinical
evaluation of plasma therapy it is proposed to evaluate the effectiveness
of whole blood therapy in bum shock.

FHS-NK

Individual Project Re;
Calendar Year 1957

Serial No, NI&MJ)

Part Bs Honors, Awards, and Publications

Publications other than abstracts frosa this project

Milliean, R. C„ and Rhodes, C„ J„ Relative effectiveness of
certain phenothi&zine derivatives against tourniquet shock in mice,,
«$■„ Pharmacol, and Exp„ Therap„ In press,

Millican, R, C„ and Rhodes, C0 J. The effects of chlorpromaziae
and chlorpromazine sulfoxide in normal and tourniquet shocked mice,,
J„ Pharmacol,, and 23cp0 Iherap, la press,

Markley, K., Bocanegra, M„, Morales, G„, and Chi&ppori, N,
Oral sodium loading in nonhydrated normal individuals „ J. Clin„
Invest , 36; 303, 1957.

Navares, E. and Markley, K„ Postoperative water and
electrolyte changes in natives at high altitudes, J. Applied
Physiol o In press.

PHS-N2H

Badividuffil Fr®3©et Rsport

Calendar Tear 195?

Serial N®0 NI&MD 82

lo Phari^cel^y'^f^Se^j^^

2a Pharsseolcgy

3* B@t4ieed©a Ma„ sad Lim* Peru

Parti ,

Project Titles She rol® of gara^a glsfealia in resistance to iafectiem
fros org&misiss of Ioh Tirul©jae®0

Principal £msstig&tor(s)s Drs<> R0 G&rl Millicaap Kehl Markleys
Nicholas A„ Kef elides, ©ad S, M» Rosenthal,

Other Inv©stigalor(s)s Mr<> J„ Rust

Man Years (Calender year 1957) s
Totals 2-1/2 yr0
Professionals 2 yrD
Otters 1/2 yr0

Pro^eet Descriptions

Objectives* (l) Using the techniques developed in this laboratory,
tourniquet and burn shock is produced in mice* Delayed deaths in 1 to I;
weeks occurs in burned mice but not after tourniquet sh@ek0 The r©le of
infection in these deaths is beiag studied. (2) Production of fatal
inf eetions from PsendoBica5Bsffi Stephylefeoecus9 and ether erganissss of Issr
Tirulenee in sale® rendered stssceptibl© byOTtessi1®® bwss£ by prats
mm% with. Basste©* doses of cortlsom, ©r "fey isajeeti^g the orgauiss©
suspended in mcia0 (3) Elimination ®f gasgEa globulin ®sd astlbietie
therapy in these experimental infections and in clinical iafectio&s
following burns (Peru Project),,

Methods Enplerysds (1) Standardised trauasatie sheek prodtssed 1b
jaiee by~©ppHcaxIoa®? tourniquets to kind legs ©r dipping a&iaals in I
rater 0 Mortality studies used to steady (a) effectiveness of antibioties
and other antibacterial agesits in treatasssstt of delayed deaths f ©listing
extensile burs injury^ (b) susceptibility of tourniquet^ sad bura*»sh£v:-. .
mice to infection from ©rg&aiSBS of low virulence,,

(2) Enhanced susceptibility of Hie© to j^udgjaMas^ragjLnpsa ."
infection and to other organisms of low virulence by means of cortisone
pretreatment and hog gastric mucin,, Mortality used to study effectiveness

Part B included [x] Tes [ } No

Serial No, H3AMD 82

Page 2 — — — -

of human gamma globulin alone and cos&iaed with antibiotics against
the various infections .

M^lorJ^ndingsj^ BacperiHgntal Study, (l) Delaysd, Deaths
FoHowl^^urns/ Chloramphenicol in ssdlsklly tole^atsc! doses l©ssr®d
is9rtHi",^r3SrSg 7 to Ik days fsiis^iag burn t^ary* Other ©atibiQtieSj)
io^oa chlor tetracycline, esytetracyclin®, tetraeyelin®, Polyagai® B, and
alsandosyein, had littl© or m© effect,,

(2) Lowered resistance was dejaonst?&t©d t© Ps@udo^»gs infection
observed following ©xteasiv© burn iss^ury but sot ?©HoS!ng™tom?niqtj®t
injury,,

(3) Humn gasssa globulin was highly active in protecta&g sic©
©gainst infections froia Psettdoaaaas a©CTgia@sa (h strains ) , Stfjsho,
aureus (k strains ), E„ coli T2°str^asJ7^gfc^s_ jg3ggMj.is {2sto?ai3ss)r
andBact. aerogenes TTstrains). Gamma globulin afforded no protection
against infections from B. cell (6 strains ), Proteus morgan!,, rettgjri,
▼ulgariss and Klebsiella pneumonia©.

(h) Antibiotics combined with gamma globulin potentiated the
action of gassssa globulin in Pseudogpnas infection,,

(£) Plasma from different species of laboratory anisals protected
mice against Psgudomonas infection,. Plasmas of man, dog, rat had higher
protective titers than rabbit and guinea pig plasmas, while mouse seruja
was least active. Weaker titers were observed in young animals „

Clinical Studyg Si cooperation with Division of Research Grants,
a elinie&l project has been set up in Lima, Peru to evaluate the
therapeutic role of gassrsa globulin in PseudoKmas septicemias f oLlowlng
extensive burns. In our previous work there it has been shown that a
high per cent of burned children die from this infection. An alternate
case method is employed and extensive bacteriological, eleetrophoretic,
and immunological studies are being made. Definitive results will re-
quire two or more years.

Proposed Course of Projects (l) Further experimental and clinical
work on. causes and treatment of delayed death following extensive burns.
(2) Studies of possible role of bacterial factor in shock. (3) Studies
on the mechanism of protective action of gassraa globulin against
infections of low virulence, (k) Purification of the protective • factor,
its electrophoretic distribution in plasma fractions, and the use of
immune serum, to obtain more active material. (£) Peru Project.
Evaluation of clinical value of gamma globulin and antibiotic therapy
in "infections.

PHS-NIH
Individual Project Report
Calendar Year 195>7

Serial No, NIaMD,

Page 3
Part B? Honors, Awards, and Publications

Publication® other than abstracts frosa this project

Rosenthal, S, M„, Millican, R, C„, and Rust, J. A factor in
human gamma globulin preparations active against Pseudoaoaasii aeruginosa
infections, Proc, Soc, Esper, Biol, Med, 2ks2!li,HL9lfFr^

Millican, R, C, Rust, J,, and Rosenthal, S, M, Gamma globulin
factors protective against infections from Pseudoaionas^ and other
organisms. Science 126s $09 3 19f?7»

Markley, K„, Gurraendi, G,, Mori Chavez, P„, and Bazas, A, Fatal
Pseudjpaonss septicemias in burned patients, .Ann, Surgery XbS si?? -'..
195?»

Serial No, NIAMD- 83

1= Pharmacology & Toxicology
2„ Biochemical Pharmacology
3° Bethesda

Part Ao

Project Titles Kistidine, Histamine & Related Imidazoles & Amines

Principal Investigator; Herbert Tabor

Other Investigators; B„ Ames, H. Bauer, and L. Wyngarden

Man Years (calendar year 1957) s
Total; 5
Professionals 3
Other; 2

Project Description;

Objectives; To study the biosynthesis, intermediary metabolism,
and pharmacological activity of these compounds in order to under-
stand better their physiological and pathological role.

Major Findings; A. Work has been continued on the purification
of the following ensymes from liver concerned with the metabolism
of formiminoglutamic acid,

(1) Pormimino-glutamic acid + tetrahydrofolic aeid -*■
Formimiaotetrahydrofolic acid + glutamic acid

(2) Formiminotetrahydrofolie acid ■* 5,10«methenyl~
tetrahydrofolic acid + NH3

(3) 5,10-Methenyl-tetrahydrofolic acid -* IO-formyl~
tetrahydrofolic acid

Enzyme I and II have been purified 1000 fold, and have been
separated by differential inactivation with either chymo trypsin
or ammonlao Further work has been done on the isolation and
characterization of formimino-TKF.

Bo These ensymes have been used as the basis for a covenient
assay for formiminoglutamic acid in the urine. The assay has
been used in folic-defieient animals and confirms the previously
described high urinary formiminoglutamic acid in folic acid
deficiency (prev. papers of Silverman, Daft, Tabor, etc.). This

Part B included Yes [l] No [ ]

method has also been used, in collaboration with Dr. Howard Hiatt
of the Harvard Medical School (Beth Israel Hospital) to assay the
urines of leukemic cases given methotrexate. Very high urinary
formlmino glutamic levels were found.

C. During these studies it was found (with Dr. Jesse R&bincwitz)
that formic acid is markedly elevated in folic acid-deficient rats.
The formic acid does not corns from histidine, but appears to come
from tryptophan.

D, Further studies on histidine biosynthesis have been carried
out by Dr. Ames.

(1} Imidasoleglycerol phosphate ~* imidazoleacetol phosphate

(2) Imidazoleacetol phosphate -* histidinol phosphate

(3) Histidinol phosphate -» histidinol

U) Histidinol -* histidine

During the past year enzymes catalyzing reactions 1 and 3 have
been purified and their properties described. A chemical synthesis
of D-erythro-imidazoleglycerol phosphate from ribose-5 phosphate
and formamidine has been developed. Various genetic mutants
requiring histidine for grovth. have been shown to be lacking
different enzymes in the scheme shown above. The enzymes levels
in mutants blocked early in the pathway have been shown to be the
same as the wild-type organism, indicating that these enzymes are
constitutive, even though their substrates have not been present
at any time.

S. (See project report of Dr. S, M. Rosenthal for studies on
the role- of putrescine and methionine in the biosynthesis of
spermidine.)

Significance to NIAMD Research; Histidine is an essential amino
acid, and its products and derivatives enter into many important
metabolic relationships. The C-2 of the imidazole ring enters
into the none carbon" pool, and thus these studies are closely
related to other studies on the role of folic acid and vitamin
B-12 carried out in this laboratory and elsewhere In NIAMD.

Histamine is important because of its potent pharmacological
activity, and is most likely an important physiological agent.
Histamine studies are of relevance to such diverse study fields
as gastric secretion, neutral transmission, allergy, anaphylaxis,
and pituitary ~adrenal interrelationships.

Serial No. NIAMD-
Page 3

Proposed. Course of Projects

lo Histidine Metabolism

A, Further studies on the purification and characterization of
the various enzymes concerned with the conversion of histidine to
glutamic acid* Particular attention will be devoted to the
metabolism of formiminoglutamie acid in tissues from normal and
leukemic animals and in various microorganisms »

Bo Closely related to these studies will be studies on folic aeid-
eatalyzed transfer reactions » Experiments will also be conducted on
the relationship of folic acid and its derivatives and vitamin B-12»

C Biological syntheses and degradation of carnosine, anserine.,
ergothioneine, and other related imidazoles,.

Do Further studies on the levels of the various histidine biosynthesis
enzymes in genetic mutants with a view to tmderstanding the effect of
the genetic mutation on enzyme production

II o Histamine Metabolism

A» Enzymatic and chemical syntheses of imidazoleacetic acid
riboside o

Bo Physiological and pharmacological studies as indicated during
the development of the projecto

G, Histamine metabolism in a mouse mast-cell tumor*

957

•ial Noo NiAMD- 83
Page 4
Part Bs Honors, Awards & Publications

Publications other than abstracts from this projects

(1) Tabor, Ho and Rabinowits , J» Co Formiminoglycine, formiiaino-L-aspartic
acid, formimino-L~glutamic acido Biochenu Preparations. 5_»(l9~57)o

In press o

(2) Bauer, H„ and Tabor, Ho Cyanomethylimidaaole and imidazoleaeetic acid
hydrochloride* Biocheao Preparations £ (1957) ° In press »

(3) Ames, Bo Ho The biosynthesis of histidine s L-Histidinol phosphate
phosphataseo J. Biolo Chem*, 226, 583 (1957)7

(4.) Hayaishi, 0», Tabor, EL, and Hayaishi, To N-Formimino-L-aspar b

as an intermediate in the enzymatic conversion of imidazoleaeetic acid
to formyl-aspartic acido Jo Biolo Cherau, 227, 161 (1957) c

(5) Tabor, H», Rosenthal, So M„, and Tabor, Co W, The role of putrescine
and methionine in the enzymatic biosynthesis of spermidine in
Escherichia coli extracts, Jo Am» Chem* Soc, 7£, 2978 (1957) »

(6) Tabor, Ho Isolation and determination of histidine and related
coiapoiindso "Methods in Enzymology," edited by So P» Colowick and
No 0, Kaplan, Academic Press, New York, III, 623 (1957) «

(7) Ames, Bo No The biosynthesis of histidine s D-erythro~ImidazoIegl;
phosphate dehydrase. Jo Biolo Ghent., 228, 131 (1957) .

Awards o

Ninth Annual Arthur S„ Flemming Award, 1956

Individual Project Report
Calendar Year 1957

Serial No. NIAKD- 84
lo Pharmacology & Toxicology
2. Biochemical Pharmacology
3« Bethesda

Part A.

Project Titles Enzyme and Endocrine Studies on Tryptophan and
Nicotinic Acid Metabolism

Principal Investigators Alan EL Mahler

Other Investigators; Catherine Rhodes

Man Years (calendar year 195*7)?
Total; 2-1/3
Professionals 1
Others 1-1/3

Project Descriptions

Objectives s To isolate the individual steps in the sequence of
reactions resulting in nicotinic acid fonaations to study the
properties of the enzymes involved, and to describe the intermediate
metabolites. With the reactions availables to study the relation
of these enzymes to altered metabolic conditions.

Methods Employed; Enzymes are obtained from various sources and
purified by the variety of methods currently used in this field.
Chemical and physical* especially spectrophotometries methods are
used to measure enzyme activity and to identify products. Possible
substrates and products are synthesized by conventional organic
chemical techniques* Isotopic compounds are synthesized and radio-
activity is measured to follow the course of reactions in vivo and
in vitro. Animals are treated to produce altered metabolic states,
and enzymes from such animals are assayed.

Major Findings s The control of liver enzymes b^ hormones has
been further investigated in collaboration with Mr* McBaniel.
Picolinic carboxylase had previously been shown to be increased in
the livers of diabetic ratsf and the increase had been shown to
depend upon adrenal hormones such as cortisone. It has now been *.
found that the effect of cortisone is antagonised by administration
of a crude pituitary preparation* The effect of the pituitary
preparation is not obtained with growth hormone , prolactin* or
thyroxin.

Part E included Yes [x] No [ ]

Serial No. NIAMD- 84
Page 2

Experiments with liver slices have failed to yield conditions for
causing enzyme increases in vitro „ but have revealed an extensive
leaking of enzymes from the soluble fraction of intact cells | the
enzymes appear intact and active in various aqueous media used for
bathing the slicess and the cells retain their characteristic
appearance on histological exami nation,,

Administration of 1,2-C v 3-hydroxyanthranilic acid to rats results
in the excretion of approximately 30 per cent of the label as CO2
and the bulk of the remainder as components of urine . The urinary
components have been separated into several fractions bj chromatograph
these fractions are being further purified to permit identification.

Significance to NIAMD Re search s Two lines of inquiry are related
to NIAMD research. One is a study of the reactions that influence
niacin metabolism in order to gain more insight into the biochemistry
of this vitamin. The other is the analysis of the effect of hormones
on liver enzymes, which may give information about the nature of the
metabolic lesions in diabetes.

Proposed Gourse of Project; It is proposed to identify the
pituitary factor that influences the effect of cortisone on litfer
enzymes. It is also proposed to attempt to relate the increase in
enzyme to either accelerated synthesis or diminished destruction.

The metabolic fate of 3-hydroxyanthranilic acid will be further
investigated by attempts to identify the urinary excretory products.
The search for the enzymatic synthesis of nicotinic acid will be
continued.

Indi ,

Calendar Year 1957

Serial No. NIAMD- «&

Page 3

Part Bs Publications

Publications other than abstracts from this projects

(1) Mehler, A. H. and May, Eo L. Studies with carboxyl-labeled
3-hydroxysnthranilic and picollnic acids in vivo and in vitro „
J. Biol. Chem., 223_s 44-9 (1956) „

(2) Mehler, A, E., Bloom, B., Ahrendt, M. E., and Stetten, Dewitt, Jr.
An artifact in spectrophotometry caused by fluorescence . Science .
In press o

(3) Topper, Y. J», Bloom, Bo, and Mehler, A. Ho Spectrophotometric
evidence for formation of a dihydroxyaeetone phosphate-aldolase
complexo Science o In press.

(A) Hayaishi, 0. , Rothberg, So, Mehler, A* H,, and Saito, Y» Enzymatic
formation of kynurenine from tryptophan,, J. Biol. Chem. In press.

(5) Mehler, A. H. Introduction to Ensymology. Academic Press, New lorkc
November 1957.

PHS-NIH
Individual Project Report
Calendar Year 1957

Serial No. NIAMD- 85

lo Pharmacology & Toxicology

2. Biochemical Pharmacology

3. Bethesda

Part A.

Project Titles The Biochemistry of Secondary Sulfur-containing
Compounds

Principal Investigators Simon Black

Other Investigators; Mrs. Eileen M. Harte (previously Mrs. Phyllis
Downey) and Dr. Edith C. Wolff

Man Years (calendar year 1957) *
Totals 3-1/3
Professionals 2
Others 1-1/3

Project Descriptions

Objectives % A long term objective is discovery of enzymatic
mechanisms involved in the synthesis of constituents of living tissue
particularly of proteins .

Methods; Cell-free enzyme extracts of yeast and other organisms
are tested for their ability to convert certain sulfur-containing
compounds to new substance3o Chemical^ chromatographic 3 radio-
chemical, and radioautographic methods are used. When new substances
are found the mechanisms of their formation are elucidated by
classical enaymological methods.

Ma.jor Findings, (l) A new amino acid has been isolated and
crystallized from yeast. It has been characterized as a previously
unknown isomer of p-methyl lanthionine containing one L-amino group s
one D-amino group, and a methyl group on a third asyme^ric centers
the configuration of which is not yet assigned.

HOOC -C-C-S-C-C- COOH
L ? D

This compound could arise through a condensation of cysteine and
threonines, a possibility being considered. It is structurally

Part B included Yes [x] No [ ]

Serial No, NIAMD-_
Page 2

related to similar compounds found in certain antibiotic polypeptides
and the possibility that it enters into the formation of peptides is
also being considered,,

(2) The enzymatic formation of the methyl thiol ester of phospho-
glyeeric acid^ mentioned in the previous report, has been found to
occur in two steps involving the glycolytic enzymes phosphoglyceroMnase
and phosphoglyceraldehyde dehydrogenase. The latter enzyme catalyses
thioester f orm&tion from methyl mercaptan and the corresponding aeyl
phosphate and requires the presence of DPN„ It also catalyzes its
formation from the corresponding aldehyde plus methyl mercaptan and
DPN. Analogous reactions have previously been described for this
enzyme j, but with other thiols which did not allow use of the natural
substrate (acyl phosphate or aldehyde) and which proceeded at very
much slower rates >

Indications have also been found that aspartic p-semialdehyde
dehydrogenase, a TPN-lihked enzyme discovered in this laboratory .,
catalyzes methyl thioester formation from its substrates, p-aspartyl
phosphate and aspartic p-semialdehyde * in a manner analogous to the
reactions of the phosphoglyceraldehyde enzyme »

(3) Though the sulfoxides of methionine have never been considered
to be naturally-occizrring this possibility must be reconsidered in
view of the recent finding of optically active sulf oxides in nature 9
particularly of a lower homologue of one of the methionine deriva~
tives, (+) S-methyl L~cysteine sulfoxide. Investigation of possible
enzymatic involvements of the methionine sulfoxide isomers has
revealed a TPN-linked system in yeast which quite specifically
reduces the sulfoxide group.. This process and its implications for

a metabolic role for at least one isomer of methionine sulfoxide are
being investigated*

Significance to HIAMD Re search 8 Knowledge of the intimate chemical
transformations in living cells will serve as a basis for better
understanding of the nature of disease and its more intelligent
treatment.

Proposed Course of Projects The immediate plan is to learn the
relation of newly found substances and reactions to established
biochemical processes.

5©et Report
Calendar Tear 1957

Serial No, NIAMD- 85

Page 3

Part Bs Publications

Publications other than abstracts from this projects

(l) Dox<meyj P» F„ and Black, S» A new naturally-occurring isomer of
l^-methyllanthioninec JD Biol* Chem., 228, 171 (1957) .

,al Project Report
Calendar Year 1957

Serial No. NIAMD- 86

1. Pharmacology & Toxicology
2e Biochemical Pharmacology
3» Bethesda

Part_Ao

Project Titles Biochemical Genetics of Bacteriophage

Principal Investigators Bruce Ames

Project Descriptions

Objectives; To study the viruses «?hich attack E* coli in order to
understand the transfer of genetic information from desosyribonucleic
acid (DNA) to protein.

Proposed Course _of„ProJ[eets

A. It is planned to investigate the proteins of bacteriophage
in order to correlate the genetic mapping of the'" DNA with amino acid
sequence in protein, particularly the protein corresponding to the
tjj mutation in T/„.

B« Attention will be paid to the biochemical steps in phage
saturation in an attempt to block the maturation at various points <>

C. It is planned to investigate the effect of various compounds
on the frequency of crossing-over in phage »

Part B included Yes [ ] No [Xj

V It X ".-

Serial No, NIAMEK

1. Pharmacology & Toxic

2. Biochemical Pharmacology

3. Bethesda

Part A.

Project Titles Biosynthesis and Metabolism of Sialic Acid

investigator: L« Warren

Man Years (calendar year 1957)?
Totals 1/2
Professional: 1/2
Oth

Project Description:

Qbj^sM2S§.! ^° study the biosynthesis and intermediary Jsetabol:
of sialic acid (neuraminic aeid) in order to understand its role i
physiological and pathological states.

C-14 gluosamine and galactosamine have been synthesized by
chemical methods. Attempts are being made to determine whether
these compounds or G-14 glucose 9 fructose, glyceric acid -or a
acid can be incorporated into sialic aeid by preparations o:
submaxillary gland and hen oviduct.

Sialic acid has been found in considerable quantity in human
semen and female cervical mucus. The relationship between sialic
acid content and human fertility is being studied.

Sialic acid labeled with C--L4 has been synthesized. by organic.
chemical methods. This material will be used to investigate the
coupling of sialic acid to laetose b^ mammary gland. Neuramin-
lactose consisting of lactose and sialic. acid is, found in mills;.

Significance . . to . EI AMD- jgesearj gh s Sialic acid is found
large quantities in most mucopolysaccharides. It is found in W

hormone j semen? liver 5 and brain. The
si of sialic aeid in blood is elevated in rheumatic £ex
Virtually not] its biosynthesis and func

.terations in disease states.

F-gogo.sg,d .Course, .of .Projects To complete investigations listed
above.

Part A.

1. Pb

ect Titles :' aromatic i

ids.

Slai?

Principal Inves

':■ Inves tig a

Man Years (calendar year 1
Total; 3

Professionals 1=66
Others 1.31*

Project Descriptions

Objectives; To study the metabolism and pharmaeo]
activity of aromatic amino acids and r

M e t hod s Employed.! Through enrichms

ive enzyme technique, enzymes were isolated an

fied from various microorganisms wiv

ical transformation of biologic

c omp ound s . Ch emi < s p e c t :■: L c m e t ho :

used together >-*-" to si inti

mechanisms of .dual reactions.

Major F'i nd i ng s % I: as lb/ re;;

oxygenases 5 namely tr; an oxygenase ?.tic
oxygenase and pyrocatec"

were characterized and studied in detail,, (A)
zoleacetic acid oxygenase was shown to utilize
pheric he possible inte;

zoloneaeetic acid esized 1

Witkop, was shown to be metabolized by the enzy
preparation. These findings support our hypo:
metabolic pathway that imidazoleacetic acid is?
Pg. ejjdomonas , degrade; roxylation,

hj hydrolysis and ring opening to 1 ;ic

acid". (B) L-Kynurenine hydroxylase was obtained .
rat liver as a soluble enzyme and v/as char-act
as oxygenase. This enzyme requires certain. anior
such as Cl", Br", Cn~ or N^ for maximum activity.

genases are ge
concerned with the transforms f various r
compounds | a new type of oxygenase, lactic ox :
decarboxylases was now found to incorporate atmi
pheric oxygen into the product, acetic acid,
C-j-Cp bond of L-lactate was cleaved enzymaticall;

It appears that the other atom of the oxygen mole-
cule was reduced to water as follows?

CEU-CHOH-COOH + 0ol8— * CH, COOl8H + C0„ + Ho018

Incorporation of atmospheric oxygen into ■ <•;.
constituents of various types of microorganisi .
investigated with O.10, With strictly aerobic bac-
teria, such as Pseuaomonas, Mycobacteria, Serratia,
etc, as much as h to 9% of the oxygen of the cell
material was derived from atmospheric oxygen, whereas
with the facultative aerobes the incorporation was
less than 0.^, The effect of various growth condi-
tions on the degree of incorporation is now being
investigated o

A new enzyme, benzeneglycol dehydrogenase, is i
isolated from rabbit liver acetone powder and pu3
about 30 fold. This enzyme catalyzes the transforma-
tion of trans-- 5»6~dihydroxycycloh'exadiene to catechol
and requires TPN as a coenzyme .

Significance to the program of the Institutes Discovery
■ of a number of oxygenases and Investigations of the
mechani -. of reactions catalyzed by various oxygenases
opened up a new field in the study of biological ox:
dation processes. They play a major role in the met
olism of aromatic amino acids and related compounds
which are of great biochemical importance. It is hoped
that elucidation of their metabolism will lead to bet-
ter understanding ot their nutritional, pharmacological
and toxicological activities.

Proposed course of project^ The work will be pursued to
establish individual steps and obtain thorough under-
standing of their metabolic pathways. Studies with -
O2 an^ H^O to investigate the mechanism of enzy-
matic hydroxylation and oxygenation of various compounds
will be continued.

Part B included = Yes

ications other than abstracts from this projects

Katagiri, M. , and Hayaishi, 0., Enzymatic degradation
:.etoadipic acid, J. Biol, Chem., .226, V39=¥*8, 19

Shiiaazono, Ho, and Hayaishi, 0„, Enzymatic decar
of oxalic acid, J. Biol. Chem., 22£5 151-159 » 1957o

Hayaishi, 0. , Tabor, H., and Hayaishi, T., N»Formimino=
aspartic acid as an intermediate in the enzymatic conver-
sion of imidazoleacetic acid to formylaspartic acid, J=
Biol« Chem., 222, 161-180, 1957.

Ohmura, E. , and Hayaishi, 0», Enzymatic conversion of
formylaspartic acid to asoartic acid, J. Biol. Chem-,
22Z, 181-190, 1957 .

Hayaishi, 0= , and Sutton, tf. B., Enzymatic oxygen fixat: .
into acetate concomitant with the enzymatic desarboxylc
of L- lactate, J. Am» Chem. Soc, 23. » '+809, 1957 =

Hayaishi, 0, , Patehett, A. A,, and Wltkop, Be, Uber c
mechanismus der brenzcatechin»oxydase(pyrocatechase)=
reaktion, Liebigs Anno Chem. 608, 158-167, 1957 »

Hayaishi, 0. , Incorporation of atmospheric oxygen into
cell constituents of a growing culture of P s eudomonas^
J. Am. Chem. Soc, 22, 557&-77 ■

Hayaishi, 0.? Enzymatic synthesis and degradation of ox=
acid, Seikagaku (Japanese) 22, 3^0-3^, 1957 -

Hayaishi, 0., Rothberg, So, Mehler, A. Ho, and Saito, Y.
Studies on Oxygenases. Enzymatic formation of kynureni.
from tryptophan, Jo Biol. Chem. (in press-Dec. a 57) °

Rothberg, S. , and Hayaishi, 0., Studies on Oxygenases.
Enzymatic oxidation of imidazoleacetic acid, J. Biol. C
(in pr es s= Dee. ° 57) -

Hayaishi, 0. , Katagiri, M. and Rothberg, S., Studies on
Oxygenases. Pyrocatechase, J, Biol. Chem. (in press-Dec. J 57 i

Saito, Y., Hayaishi, 0., and Rothberg, S. , Studies on Oxy-
genases? Enzymatic formation of 3~hydroxy~L=kynurenine
from L-kynurenine, J. Biol. Chem.'. (in press-Dec. ^57) •

Honors and Awards relating to this projects

Dr. 0. Hayaishi was Chairman at the International SympOL
on Enzyme Chemistry in Tokyo, October 1957 and was invil
to organize and be a Director of the Colloquitim on Enzy-
matic Oxygenation, Vienna, Austria, September 1958.

; et Re;
Calendar Year -

Serial Ho. MIAMD-
1. Pharmacology & Tox
2= Toxicology
3<> Bethesda

Part JL°

Project Titles Biosynthesis of Gramicidin J.

Principal Investigators Dr. 0. Hayaishi, Dr. K.Ku:
and Dr. Y. Saito

Other Investigators Mr. Albert Greenfield

Cooperating Units? Dr= S. Otani, Dept. of Bioche::
Osaka City University, Osaka, Japan.

Man Years (calendar year 195?) «
Totals 3.68
Professionals 2.31*-
Other s 1.3^

Project Description;

Ob.iectivesi To elucidate the mechanism of biosynthes
intermediate metabolism and degradation of a hexa
tide, Gramicidin J.

ik

Methods Employed % (1) Incorporation of C -labeled

acids into gramicidin J was studies by using the gi
ing cells of B. brevis st. Nagano. Radioactive a
acids which "constitute gramicidin J were added to
culture of B. brevis at several different stages of
growth. After 2h hours of total incubation, the
terial cells were harvested by centrifugation and
gramicidin J was extracted by ethanol from the
The maximum incorporation was found when the rad:
active amino acids were added at the end of logarj
growth phase,, Much less incorporation was obtained
when labeled amino acids were added to the cultur
initial or stationary phases of growth. (2) Rate
incorporation of labeled amino acids were f olJ
measuring the radioactivity of gramicidin J which
obtained from the cells harvested at different ir
tion periods after addition of labeled amino acid to
the culture at the end of lagarithmie growth phase.
(3) Bacillus brevis grown in a tryptone medi
harvested at the stationary phase of its growl

Seri ■.

Page 2

disintegrated by mechanical cell disintegrator,
tact cells were removed by low speed centrif ligation
and the supernatant was used for the study of the
corporation of C-'-4"- labeled amino acids into gramici
J in a system fortified with various energy sources
and cof actors .

Major Findings; (1) From the Qo% ethanolie extracts of
cells which were harvested after 2 or h hours incuba-
tion with C -labeled ornithine at least h radioactive
substances besides radioactive gramicidin J were sepa-
rated as possible intermediates by paper chromatcgr'-
scanning method- From the cells which were incubai
with C-^*- labeled valine for 2 or h hours 2 radioact
substances were also separated- One of them was
ified as valyl-orni thine by Rff value of paper cir
raphy and hydrolysis after separation of the pure s
stance- (2) So far the production of gramicidin J
was not observed under any conditions of cell-free^n
system, but the incorporation of L- phenylalanine- 0
into two unknown compounds was observed- The prelim-
inary identification of the above compounds revealed
that one consisted of L-phenylalanine . and L~proline
and the other L=» phenylalanine, L-proline and one uni-
dentified amino acid.

If the amino acids in the above two compounds
follow the right sequence of amino acids in gramicidin
J5 it is possible that they could be intermediary pe
tides on the pathway of the biosynthesis of gramici
J- More rigorous test for identification of the above
peptides are being undertaken.

Significance to the program of the Institute? Gramicidin
J, an antibiotic produced by Bacillus brevis is a
cyclic polypeptide. Its structure has been elucidated
by S. Otani and Y. Saito as follows?

D- Ornithine.

L-Proline L~valine
/ - f
L=Phenylalanine L- Ornithine

VT . /

J> Leucine

Since there has been no other detailed work done
on polypeptide biosynthesis except that of glutathione
by J. E. Snoke et al. , the study of biosynthesis of
gramidin J has been undertaken. The objective of this

Ser:

study is to elucidate whether the biosynthesis
gramicidin J follows the pattern of step-wise
tion of polypeptide as is the case of biosynthesi
glutathione or it follows the "template mechanism"
through the activated amino acids , as has been pro-
posed for protein synthesis = The other alternative
is that these polypeptides could be a degradative
product of a large protein molecule =

Proposed course of projects Identification of the in
mediary peptides and studies on the mechanism of
matic synthesis of such peptides and gramicidin J
be carried out with intact cells and cellfree syi t

Part B included - No

PHS-NIH
Individual Project Report
Calendar Year 1957

Serial No. NIAMD- 90

1. Pharmacology & fox:

2. Toxicology
3» Bethesda

Part A.

Project Titles Toxicologic studies of iodatess, surve
of toxicological literature.

Principal Investigators? Dr. S. H. Webster and Dr. ;
F. von Oettingen

Other Investigators x - Mr. E = F. "Stohlman and Mr.K.Moa

Cooperating Units s Dr. Benj . Highman, Pathology, Ser,
NIAMD^^ and Dr. Ralph Gunk el, Retinal Examinations,
Ser. No, NIAMD_.

Man Years (calendar year 1957)°
Totals ^2/3
Professionals 3
Others 1-2/3

Project Descriptions

OMegjy^esj. The toxicology of iodates as a basis for use
in iodized salt to prevent goiter.

Methods Employed; Administration of potassium iocU

food to experimental animals and subsequent examina-
tion by hematological, ophthalmological, chemica.
and histopathological methods.

Major Findings s (a) One dog of the 19 died during the
year and three others became so ill they xirere sacri-
ficed. The remaining 15 survived the 12~1L!- month te
period. Potassium iodate was found to have pronounced
emetic action and also a marked irritant effect on the
stomach and duodenum. With few exceptions, even at"
high do ■ ingested iodate was not excreted in the
urine as iodate but as iodide. Individual suscepti-
bility varied greatly. The chief toxic effects noted
in affected animals were emesis, anorexia, loss of
weight and temporary anemia. Methemoglobin formation
was negligible. During the last half of the experiment
a marked tolerance towards the drug was acquired.
Diarrhea, hj which significant amounts of KX0-, were

eliminated by the gastro- intestinal tract, frequen"

occurred o Wight blindness could not be detected in

any of the animals at the end of the experimental
periodo

(b) A review of the literature on the relation be-
tween physical-chemical properties and the toxicity o:
aliphatic acids and their esters has been continu
Chapter I on formic acid and its esters and Chapter I]
on acetic acid and its esters are essentially fin
Additional chapters on the higher homologues of these
acids and their esters are in preparation. It
hoped that these studies will throw some light on the
mechanism of the toxic action of these compounds.

The review and abstracting of current publics
on clinical eases of poisonings ^oj chemicals and
peutic agents is being continued, covering about 100=
150 publications per month. This is essential fo:
to date information on the toxic action of such agent*
which then can be passed on in emergencies.

.Significance to the program of the Institute; The use
of potassium iodate to iodize salt is a problem of ia
ance in tropical countries, where unstable and hygro.-
properties of KI make its use impractical. The f\
of this study should enable one to judge whether KI0-, is
a safe substitute for the unstable KI at present used to
iodize cooking salt and salt-feed mixtures for cattle,.

Proposed course of projects The animal work has been ■
finished and analysis of the electroretinographic data
(with Dr. Gunk el), examination of histopathologic spe
mens (with Dr. Higbman) , and chemical and microscopic
analyses are now going forward. Preparation of a rev
covering the last three years of work on iodates and
iodides will be undertaken for the Food and Drug Admini-
stration and for Dr. Kevin S. Scrimshaw, Director of
Institute of Nutrition for Central America and Panama.

Part B included - Yes

Page 3
Part_JBj, Honors, Awards, and Publications

Pj*l?2^i^l.0i?s °fr]LQ±„J:fo-an abgjrjacts from tnis ^pro,i:-

Webster, S. H. , Rice, M. E., Highraan, J3. , and
von Oettingen, W. F.» The toxicology of Potassium and
Sodium Iodatess Acute Toxicity in Mice* J. Pharm„
Therap., Vol, 120, No. 2, 171-178, 1957"

von Oettingen, Wo F. . Carbon Tetrachloride; What the
American Standard Acceptable Concentrations Means to
The Magazine of Standards, 28, (8) 261-2, 1957 .

Individual Projee

57

Serial No. NIAMJ>_
1= Pharmacology & Toxico
2o Toxicology
3o Bethesda
Part A.

Project Title 2 Studies on the cytological locali::.

of streptococcal hyaluronidase by means of specific
fluorescent antihyaluronidase globulin.

Principal Investigators Dr. E. W. Emmart

Other Investigators? Cooperating units

Dr. R. Mo Cole (NIAID) Ser. No.

Dr. Eo L. May (Lab. of Chem. , NIAMD) Ser. No. _|3_
Dr. J. B. Longley (Lab. of Histochem. .NIAMD) Ser.
Dr. I. Klatzoj, Surg. Neurol. Branch, Clin. Nuerop

(NINDB-58-C)
Dr. B. Horvath (NINDB) Ser. No. _
Dr. Pi. A. Resnik, Lab. of Ophthalmology, Sect. Che

(NINDB) Ser. No.

Mr. W. A. Turner, Assistant, Mr. K„ Moats.

Man Years (calendar year 1957)2
Totals 3.33
Professional: 3
Other: .33

Project Description:

Objectives: (1) These studies on streptococcal hyal
onidase have been undertaken because this antigenic
.enzyme which is capable of being retained within the
body is responsible for the depolymerizing of the
ground substance of the connective tissue. One of
the changes involved in the inflammatory syndrome
rheumatic disease concerns these changes in the inter-
fibullar substance. It Is therefore the purpose of
these studies to elucidate the areas of absorption of
this enzyme.

(2) This stLidy was undertaken in order to deter-
mine the localization of myosin in normal tissue and
in pathological tissue.

Methods Employed: (a) This has been accomplished
by the isolation of high purified streptococcal h
uronidase (900 TRU per mg) from the culture medium of

= 91
Page 2

streptococcus group C, strain 7, secondly, by
duct ion of antibody in rabbits following intraven
injection of the enzyme and lastly by the study of
the absorption and localization in tissues of the
experimental animal following injection of the enzyme.
This is accomplished by the Coon"s fluorescent
body technique. By this technique injected an
ot naturally occuring antigens are tagged in tissue
sections following treatment with fluorescent anti

The study of the localisation of myosin by me;
of fluorescent tagged antimyosin globulin was made
possible by the isolation of myosin in highly pi
form by Dr. B. Horvath and the preparation of sera
from injected rabbits.

Mal2£_FiSdiS£sj. (a) Using the mouse as the experimental
animal a study has now been completed of the localiza-
tion of the injected enzyme in various tissues.
areas high in cincentration of hyaluronic acid-
eye and the knee joint— -and therefore of special
interest with relation to this enzyme—- have been set
aside for special study. The. study of the absorption
of this enzyme by various areas of the eye is now in
progress.

(b). Both teased- fibers and frozen Tissue sec-
tions of mouse, rat, rabbit and man were stained with
the fluorescent antimyosin globulin and the myosin
localized in the A & Z bands. Also teased muscle
preparations from 8 different human myopathies we
studied.

££SE2sed_course_^f_EgcsIec^ Continuation of studies on
localization of (a) hyaluronidase, (b) myosin, (c)
and of the lens proteins, employing the fluorescent
antibody technique. Localization under both normal
and pathological conditions will be employed.

Part 3 included - Yes

Serial No 91
Page 3

^art_..,B_» Honors, Awards, and Publications

Pujgliga^ions___pjto^j^_than abstrag_ts_f Irom^this pro.j c
Papers in press-

Emmart, E. W., Cole, R. M., May, E» L„, and Longle;
Studies on Streptococcal hyaluronidase and antihya
idase II - The localization of the absorbed enzyme
means of fluorescent antibody globulin. Journal of
Laboratory Investigation.

Emmart, E. W., Observations on the absorption spec
fluorescein, fluorescein derivatives and conjugates.,
Arch. Biochem. and Biophysics.

Klatzo, I., Horvath, B,, and Emmart, E„ V/», Demonstra-
tion of myosin in human striated muscle by fluorescent
antibody, Proc. Soc. Exp. Biol, and Medicine.

NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES

tenia! Project Report

Calendar Tear i$Sl

Summary Sheet

Laboratory of Physical Biology

Estimated Obligations for FY l£g8

Total* 8986,968
Directs STUCCO©

it $272,968

Projects number 92 thru 10? included <

PHS-NIH

Individual Project Report
Calendar Year 1957

Serial No* NlAHD-flg

lo Physical Biology
2o Physiology
3* Bethesda

Part A„

Project Title; Pulmonary ventilation

Principal Investigator: Heinz Specht

Other Investigators: Roscoe G0 Bartlett, Jr» , Howard
Brubach, Richard Trimble

Man Years (calendar year 1957):
Total: 5
Professional: 3
Other: 2

Project description? Studies of pulmonary ventilation and
the cost of breathing in air and under water s as well as
effects of airway resistance on breathing capacity have
been carried on with human subjects*

Objectives: The principal objective of the overfall project
is to explore new phenomena regarding breathing behavior
with regard to their physiological significance „ In order
to accomplish this various studies of physiology must be
undertaken in order to accurately control the experimental
situations, Thus, in underwater swimmers it is hoped that
ultimately breath velocity patterns at various depths may
yield information that can be compared with our studies at
high altitude. The interrelation of work of,, and resist^
ance to, breathing have been sought as limiting factors in
normal as well as abnormal conditions of breathing,, Such
data are also of interest to the Office of Naval Research*

Methods employed: Subjects are studied for oxygen consumption {
carbon dioxide production, breath velocity patterns , and
volume measurements as necessary* New devices for several
of these measurements were made, including those needed to
collect data under water*

Serial No, NIAKD-92
Page 2

Major findings? The oxygen cost of resting ventilation in
air was shown to be „5 cc/liter which is corroborative
of data in the literature,:, At 50 liters per minute the
cost rises to 08 cc/liter and at 80 liters per minute
to 200 cc/litero

Underwater ventilation was shown to double
the cost of breathing and since the closed system used
in this study is hydraulically balanced the added cost
is ascribed to inertial difference between air and water
environment,, A consequence of this increased inertial
work is a limit on rate of pulmonary performance which
was observed in earlier fin swimming experiments but
seemed to have no ready explanation at that time since
it occurred at rates of ventilation well below the
capacity in air at similar exercise loads „

Studies on maximum breathing capacity (MBC)
were initiated and carried out to establish its validity
in measuring ventilatory ability. It was early established,
that "voluntary" efforts were not necessarily true measures
and that airway resistance and rate chosen were very
critical in determining the output 0 It was shown that the
peak performance at each resistance level was at the same
tidal volume at any chosen rate. It was further shown
that since fourfold increase in airway resistance lowered
the MBC little j, the usual insistence on special low
resistance test equipment was not necessary for the
essentially normal subject „ It was pointed out that the
method used in this study could give a measure of the
magnitude of internal pulmonary airway resistance,,

Further studies on factors determining the
MBC have shown that the short cut method known as the
"fast vital capacity" (FVC) prediction suffer from a
deficit in predicted air flow due to the assumption of
instantaneous reversal between breath phases - an error
which increases with rate of breathings Means for avoid-
ing this error in ventilatory capacity measurement were
pointed out. From a three dimensional plot of airway
resistance 9 breathing rate and MBC it was possible to
define the limiting mechanisms determining the magnitude
of maximum pulmonary ventilation at various breathing
rates and resistance levels.

Serial Ho„ Ul^jJh.
Page 3

Major findings (cont'd)?

Because previously available methods have
been so laborious the literature contains little informa-
tion on the energy cost of breathing,, To span the entire
ventilatory range it was necessary to use three experimental
approaches 0 With this improved and simplified instrumenta-
tion it was observed that each breathing rate established
a different O2 uptake-ventilation curve0 Thus when tidal
volumes were varied at each breathing rate a family of
curves resulted,.

An O2 uptake=»ventilation curve produced by
"optimum" combinations of breathing rate and tidal volume
(which are close to inherently chosen combinations) was
shown to be composed of the "efficient" segments of the
02 uptake=ventilation curves 0 As the maximum effort at
any given breathing rate was approached the O2 cost per-
unit ventilation increased very rapidly producing a large
range of O2 uptake values for subjectively "equivalent
maximum" efforts 0 It was therefore suggested that when
used as a clinical test more uniform results could be
expected if the breathing effort were less than maximum0

A volume "deficit" as a result of time "loss"
in flow reversal during the breathing cycle has been intro=
duced as a newly described factor affecting the work of
breathing. It was shown that this factor results in in=
creasing nonelastic work with increasing breathing rates
at constant pulmonary ventilation,, The opposite effect of
increasing breathing rate on elastic and nonelastic work
at constant pulmonary ventilation establishes an optimal
breathing frequency for a given pulmonary ventilation0
Tiiis optimal frequency may be determined from the family
of Oo uptake-ventilation curves 0 It was also shown that
this family of curves may be adjusted so as to also indicate
the optimal breathing frequencies for various alveolar
ventilations. An explanation is presented for the wide
differences in previously reported O2 uptake-ventilation
curves c,

Significance to NIAKD research: In order to extend the under-
standing of both normal and abnormal respiratory gas ex-
change it has become necessary to explore the significance
of phenomena beyond those used classically for measuring
pulmonary f unctionc Inasmuch as physical environment
affects such phenomena the study of atmospheric density
effects on breathing will ultimately form a new set of
criteria for judging the level of normal or abnormal behavior 0

Serial No0 NIhMD~92
Page k

Proposed course of project: It is planned to continue the
line of attack carried on this year with some excursions
into measurement of internal airway resistance by new
means „ Other studies as indicated in the course of this
work may also be initiatedo

Part B included Yes [%J No £J

Serial Noe MlaMD-92

Page 5

Part Bo

Publications other than abstracts;

Bartlett, R0 G0 and H, Specht, Energy cost of breathing de-
termined with a simplified technioue, J, Applied Physiol, ,
It (1) 84-86, July, 1957,

Bartlett, Re G, and H, Specht, Maximum breathing capacity vdth
various expiratory and inspiratory resistances (single and
combined) at various breathing rates, J, Applied Physiol,,
lis (1) 79-83, July, 1957,

Bartlett, R, Go , Jr, Restraint hypothermia and I^l uptake by
the rat thyroid0 Proc, Soc, Kxptl, Biol,, 94: 654-656* 1957c

Bartlett, R, G, and P, Bernstein, Effect of clipping on body
temperature of restrained and non-restrained rats exposed to
cold, Proco Soc, Exptl. Biol, Med., 94; 639-640, 1957o

Bartlett, R, G. , Jr, , H, F, Brubach and H0 Specht, Oxygen cost
of forced breathing in the submerged resting subject. In press -
J, Applied Physiol,

Bartlett, R„ G, , H, Brubach and H, Specht„ Some factors determin-
ing the maximum breathing capacity. In press - J, Applied Physiolo

Bartlett, R, G0 , H,, F, Brubach and H0 Spechto Oxygen cost of
breathing. In press - J, Applied Physiolo

Bartlett, R, G, Evidence for "diffusion respiration8 in rhythmic
breathing, J, Applied Physiol, , 10s (2) 207-209, March, 1957c

Goff, L, Go, Roberto Frassetto, and H, Specht, Oxygen requirements
in underwater swimming, J, Applied Physiol,, 9 J (2) 219-221, Sept,.
1956,

Goff, L, Go and R, G, Bartlett, Jr0 Elevated end-tidal CO2 in the
trained underwater swimmer, J, Applied Physiol,, 10: (2) 203-206,
March, 1957,

Goff, L, G, , Ho F, Brubach, and H, Spechto Measurements on
respiratory responses and work efficiency of underwater swimmers
utilizing improved instrumentation, J, Applied Physiol,, 10; (2)
197=202, March, 1957,

Serial No<> ftIIA?>iD-92
Page 6

Publications other than abstracts (cont'd);

Specht5 H0 , L0 Go Goff, H0 FQ Brubach^, and Ro G0 Bartletto Work
efficiency and respiratory response of trained underwater swim-
mers using a modified SCUBA0 J„ Applied PhysiolOJ, 10s (3)
376-382, May, 1957.

PHS-NIH

Individual Project Report
Calendar Year 195?

Serial No0 NB££d2JL
lc Physical Biology
2„ Physiology
3o Bethesda

Part ac

Project Title; Circulatory reaction of dogs to polyvinyl-
pyrrolidone (P?P) and of rats to dextranc

Principal Investigators Louise HQ Marshall

Other Investigators: Charles H„ Hanna

Cooperating Units: None

Man Years (calendar year 1957) '
Total: 3
Professional: 1
Other: 2

Project description:

Objectives: To provide detailed knowledge of the procedures
which attenuate the response of sensitive species to these
synthetic macromoleeules0 Particular attention xvas given
to in vivo and in vitro experiments on cross refractoriness
to another histamine-releasing agents „ and in rats to other
stressing procedures which have been reported to attenuate
their shock- like reaction,.

Methods employed: Continuous recording of blood pressure has
been used to demonstrate the systemic effects In animals 9
and the gross appearance of intravascular dye to indicate
the passage of fluid across skin capillary walls „ Perfused
hind limb preparations are utilized to demonstrate in vitro
effects by flow and weight changes „

Major findings: Pretreatment with large doses or with gradually
increasing doses of the potent histamine~liberators compound
48-80, does not alter the reaction of dogs to PVP„ In pre-
treated rats vasodepres3ion after dextran did not occur s
but bluing was seen0 This resembles the reaction seen in
both species after or during ether anesthesia,,

Serial Mo0 NlaKiyjE.
Page 2

Major findings (cont'd):

No attentuation of the dextran reaction in rats
occurred after acute or chronic vagotomy , after lumbar
or cervical sympathectomy ^ after spinal cord transection ^
during insulin hypoglycemia , after electroconvulsion,
after acute exposure to a hot environment 9 or after
administration of large doses of nor~adrenalin?

In the perfusion experiments, the expected vaso-
dilation and weight increases when red ceil suspensions
containing colloid were perfused did not occur 0 Instead
there was evidence of vasoconstriction in preparations
from normal animals , while in those from refractory animals
there was relatively little effect,

Significance to NIAMD research: Detailed study of these

reactions helps clarify the role of histamine in anaphylactoid
reactions and the basic physiology of normal metabolism,,

Proposed course of the project; The effect of PVP upon the
renal excretion of dye will be studied,,

Part B included Yes (%J No £J

Serial Noc
Page 3

Part B: Publications

Marshall, Lc H0 and C0 H0 Hanna0 Circulatory reaction of tolerant
and nontolerant dogs to polyvinylpyrrolidone and of rats to dextrai
am0 Ju Physiolo, 189; (1) 209-213, April, 1957,

Marshall, L0 H0 and 0o H„ Hanna„ Effect of ether and barbiturate
anaesthesia on the reaction of rats to dextran and of dogs to poly-
vinylpyrrolidone o In press -- to appear in Am0 J« Physiol, , 191;
(2) ? , 1957o

FHS-HIH

Individual Project iteport
Calendar Year 1957

Serial No„ MIAMD^oji

1„ Physical Biology
2& Physiology
3o Bethesda

Part Ac

Project Title: Effects of hypoxia on physiologic mechanisms,,

Principal Investigator: Paul D„ Altland

Other Investigators: Edwin C„ Thompson^ Milton Parker s Edna Devlin

Cooperating Units: Pathologic studies involving animals exposed
to simulated high altitude conducted with cooperation of Dr„
Benjamin Highman, Section on Histochemistry and Pathology (see
Serial No0 NIiiKD-70 ),

Studies with obese animals conducted with
cooperation of Dr= Olaf Kichelsen of Section on Nutrition and
Endocrinology (see Serial No« NIAMD-1 )0

Man Tears (calendar year 1957);
Total; 4
Professional: 1
Other: 3

Project descriptions

Objectives: (a) To study the effect of experimental arterio-
sclerosis on survival of rabbits to acute exposures to high
altitude0 To test the hypothesis that anoxemia is a predis-
posing factor in the production of arteriosclerosis „ (b) To
determine factors affecting susceptibility of dogs to experi-
mental endocarditis including effects of altitude exposures 0

Kethods employed: Altitude exposures conducted in decompression
chambers o Physiological and bacteriological methods have been
usedo

Major findings: Rabbits with severe arteriosclerosis induced by
feeding a diet rich in cholesterol survived acute exposures
at 32s600s 35s400 and 38s600 feet as well as controls 0 Rab-
bits fed a high cholesterol diet and exposed continuously to
16^000 feet simulated altitude (p02 86 mm HgD ) showed no
increase in degree of arteriosclerosis in the aorta compared
with others not exposed to altitude 0 The altitude exposures
did induce an apparent differential cholesterol distribution
in visceral organs 0

Serial No, NIAMD- 9)i
Page 2

Major findings (cont8d)i

Dogs readily tolerated repeated daily exposures to 25„OOQ
feet simulated altitude for several months 9 but eventually died
as the result of severe polycythemia^, cerebral hemorrhages and
renal damage. No cardiac valvular vegetations or visceral infarcts
were produced, but there was some thickening of the cardiac valv©s0
Susceptibility to experimental endocarditis was only moderately in-
creased in dogs exposed to 25*000 feet long enough to produce poly=
cythemia (hematocrit 6? to 81),

Significance to N.IAI4D research! Provides basic data on the relation
of hypoxia to development of cardiovascular diseases. Results indi-
cate that hypoxia may increase susceptibility to bacterial infections ,
and may alter the pattern of distribution and deposition of fatty
substances in the body.

Proposed course of the projects Studies on the mechanisms which in-
fluence altitude tolerance will be conducted,, The work on the
effect of hypoxia on the deposition of cholesterol in animal tis-
sues will be continued. The effect of hypoxia on the physiology
of reproduction in the rat and dog will be investigated. Experi-
ments on the longevity of the erythrocytes and on factors in-
fluencing erythropoiesis in cold-blooded animals will be continued.

Part B included Yes [3 No £J

Serial No. NIAKD-9U
Page 3

Part B: Publications

Altland, P. D. and B0 Highman. Effects of high altitude ex-
posures on dogs and on their susceptibility to endocarditis0
J0 Avia. Med., 28: (3) 253-259, June, 1957o

Altland, P. D. and B0 Highman. Effects of high altitude exposure
on dogs and on their susceptibility to endocarditis . Jo Avia.
Med., 28: 253, 1957.

Altland, P. Dc, 0. M.ickelsen, and B. Highman. Effects of exposure
of obese rats to simulated high altitudes. Accepted for publica=
tion Am. J. Physiol. , 191: (1) 1957 o

Highman, B., E0 C. Thompson, Jc Roshe, and P. D. Altland. Serum
alkaline phosphatase in dogs with experimental splenic and renal
infarcts and with endocarditis. ProcD Soc. Exp. Biol. Med., 95s
109, 1957.

Highman, B0 , Jo Roshe, and P. D. Altland. Endocarditis and
glomerulonephritis in dogs with aortic insufficiency. Accepted
for publication AMA Arch. Path.

Roshe, J., B. Highman, and Paul. D0 Altland. Effect of Hufnagle
valve on susceptibility of dogs to endocarditis. AMA Arch. Surg.,
75 J 600, 1957.

json, E. C, A tray for staining frozen sections in quantity,.
Stain Tech., 32: 255, 1957*

PHS-NIH

Individual Project Report

Calendar Year 1957

Serial KoD NI^lD-ffS
1„ Physical Biology
20 Physiology

Part A,

Project title; Some biophysical and biochemical aspects of
insect respiration

Principal Investigator: John B„ Buck

Other Investigators; Stanley Friedman, Margaret Lc Keister^
Helen Dc, Parks David P0 McCarthy

Cooperating Units: None

Man Years (calendar year 1957):
Total; 6
Professional; 4
Other;

Project description:

Objectives: Our unit is pursuing several related aspects of

insect respiration^, among which are (l) carbohydrate metabolism^
with special reference to the mobilisation and utilization of
glycogen , (2) metabolism during the transition from aerobic to
anaerobic conditions and (3) physics of gas transfer 0

Methods employed: Carbohydrate metabolism is being followed by
chromatographic methods of identification and assay of various
types of blood sugars ^ chemical isolation of tissue of glycogen^
and respirometry of intact organisms and of tissuesc Enzymatic
pathways are being followed by use of radioactive tracers „
The aerobic=anaerobic transition zone is being studied by
measuring oxygen uptake before „ during and after exposure tc
various partial pressures of oxygen,, The biophysical aspects
of gas transfer involve dimensional study of the respiratory ^
system^ respirometry and computation.,

Major findings; In the adult fly glucose is converted very

rapidly into trehalose in the intact organisms, and trehalose
is split into glucose and other compounds by homogenized fly
tissueo The enzyme responsible for the hydrolysis has been
identified as a specific trehalase, not a general rl* =glucosidase;

Serial No,
Page 2

Major findings (cont'd):

A system apparently converting trehalose to other compounds
by a phosphorylative pathway is also being studied, as is
the enzynie(s) responsible for the glucose-trehalose con-
version,, In fly pupaes respiration is C^-limited at concen-
trations as high as 15$ at ages of 1, 2 and 5 days^ but not
at 0, 3 and 4 days,, Below 15% 9 respiration is limited at
all ages^ with the period of minimum limitation coming at an
earlier age the lower the ambient 02 concentration,, Two
hours of hypoxia induces little if any O2 debU In fly
larvae £ respiration also begins to be limited at 15$ ambient
concentration t but curiously enough even severe hypoxia is
not relieved by a 10~fold surgical enlargement of the valves
leading into the respiratory gas space,, An explanation of
this paradox is proposed on the basis of the physics of dif-
fusion through small pores „

Significance to NIaMD research; The studies on intermediary
metabolism are specifically oriented to contribute to the
Institute's diabetes program,, It is hoped that they may shed
light on the basic mechanism of glycogen mobilization,, The
anoxia study has already indicated that insects may show
interesting differences from mammals in regard to oxygen
debt accumulation and anaerobic metabolism,. The biophysical
study 8 it is hoped s can contribute to the theoretical know=
ledge of gas transfer mechanisms in respiratory systems „

Proposed course of project; The results so far obtained have
been so interesting and the leads for future work so promising
that no change in general objectives or procedures is contem-
plated o

Part B included lea (%] Wo [J

Serial No0 NIAKD~$>S
Page 3

Part B0 Honors ^ Awards , and Publications

Publications other than abstracts;

Buck, Jo B0 Triggering of insect spiracular valve,., Pubo in book
entitled "Physiolo Triggers" by Am„ Physiolo 3oe0 , ppc 72=79 s Aprils
1957,

Park, Ho D<, Modification of X-ray injury to Hydra Littoralis by
post irradiation treatment with magnesium sulfate and glutathione.,
Biolo Bullo, Ills (2) 240=247, 0cto.5 1956,,

Buck, Jo B0 Book review of "Annual Review of Entomology" s pub=
lished by Annual Reviews, Inco , Palo Alto, Calif „ Bulla AmD Insto
Biolo Sciences, 7% (2) 41, Aprils 1957o

Buck, Jo Bo Book review of "Hvad finder jeg pS strand en (What I
find at the seashore )"0 Bull0 Am0 Instn Biol0 Sciences, 7; (2) 40,
April, 1957 o

Friedman, S0 Determination of carnitine in biological materials,-,
In press ~ Archives of Bioehenu Biophys,,

Fraenkel, G0 S0 and S„ Friedman0 Carnitineo In press - Vitamins
and HormoneSo

Buck, John and Mc Keisterc Cyclic CO2 release in diapausing pupaec
Ho 'Further data0 In press - Jo Insect Physiolo

Buck, John and S0 Friedman0 Cyclic CO2 release in diapausing pupae,
II I o CO2 capacity of the blood; carbonic anhydrase0 In press - J0
of Insect Physiolo

Buck, John* Possible mechanism and rationale of cyclic CO2 retention
by insects. In press - Proceedings Tenth International Congress of
Entomology0

Honors and Awards relating to this project (Jo B0 Buck):

(a) Member of Executive Committee and of Policy Committee,
rticerican Society of Zoologists „

(b) Member of Affiliation Conmittee, American Association for
the Advancement of Scienee0

Serial No» NIAKD-9S
Page 4

Honors and awards relating to this project (J0 Bc Buck) (cont°d)s

(c) Representative for .American Society of Zoologists on the
American Institute of Biological Sciences'1 Council,,

(d) Member of the Public Relations Committee of the American
Institute of Biological Sciences,,

PHS-NIH
Individual Project .Report
Calendar Year 1957

Serial No„ NIAMD-96
lo Physical Biology
2e Physiology

3° Eethesda

Part A«

Project Title: Ionization studies in the upper atmosphere,,

Principal Invest igators Herman Iagoda0

Other Investigators: Mardalee B0 Dickinson.

Cooperating Units: None,,

Man Years (calendar year 1957):
Total: 2

Professional: 1
Others 1

Project description:

Objectives: (1) To secure information on the biological action of
heavy primaries on living tissue by monitoring the path of the
particles through the body of test animals0 (2) To study high
energy interactions of cosmic radiation in regions in excess of
10 Bev which are not available in the laboratory,, These studies
are chiefly of interest in the development of nuclear physics?
They might conceivably be of future importance in the field of
medicine in the same sense that the neutron discovered in 1930
led to the production of radioactive isotopes of medical and
biological interest some 20 years later0

Methods employed: Study of cosmic radiation at high altitudes „
Exposure of nuclear emulsions in balloons and rocketsG

Major findings: A small group of guinea pigs flown for 6 hours
in the stratosphere have been recovered and their brains studied
histologically at the Armed Forces Institute of Pathology, By
means of emulsions attached to the skull w@ have been able to
predict the path of individual very densely ionizing particles
through the brain,, The tissue blocks have been sliced according
to these projections thereby facilitating the tedious work of
microscopic examination,, In one animal a spherical brain lesion
was observed,, about 100 microns in diameter, located along a
predicted pathD This suggestion of biological damage must be
pursued further using animals with brains of larger volume 0

Serial No„ NIAMD-
Paee 2

Major findings (cont'd):

The manned stratosphere balloon flights by Capt0 J. Kittinger
and Major Dc Simons of the Uc S„ Air Force were monitored for heavy
primary thindown hits0 On the later flight emulsions were strapped
to Major Simon's arms and chest in an effort to correlate the graying
of individual black hairs as a result of the passage of a heavy
primary through air follicles,,

Significance to NIAMD research: Basic investigations in nuclear physics
with significance to the health of military personnel flying at
altitudes above 60,000 ftc Provides information on the potential
biological action of individual multiply charged ionizing particles
on tissue, a field of growing interest in the treatment of deep
seated tumors by heavy particle (proton) irradiation,,

Proposed course of the project: This is largely dependent on the extension
of the program by the USAF which provides the special balloon fligS
for the exposure and recovery of animals at high altitudes „ However^
material recovered from the 1957 manned flights are in study, and will
require the better part of a year for completion of the analysis „

Part B included les ffj No [j

Serial No„ MIjIjPrM.
Page 3

Part B: Honors, Awards , and Publications

Publications other than abstracts from this project:

Yagoda, H0 Book review of Heins Gartmann' s "The men behind the
space rockets" translated by Estace Wareing and Michael Glenny0
Scien0 Monthly, 83 J (6) Dec0, 1956c

Yagoda, H„ Frequency of thindo\*m hits by heavy primary nuclei in
emulsion and tissue, J„ Avia0 MecL, 27: (6) 522-532, Dec, , 1956c

Yagoda, H0 Anomalous heavy cascades recorded on ¥iking 10 Rocket
Flight0 Nuovo Cimento, 6 s (3) 559-570, 1957„

Yagoda, Hc A study of cosmic ray heavy primary thindown hits in
a phantom of a human brain0 Reports and Discussions of the 3rd
International Congress of Neuropathology , Brussels, July 1957$
pp0 171-177c

Yagoda, K6 An improved isothermal method for the processing of
thick nuclear emulsion pellicles 0 1st International Conference
on Corpuscular Photography, Strasbourg , France, July 1957 - in
pres8„

Yagoda, He Emulsion cassettes for cosmic ray study in high altitude
rocketSo 1st International Conference on Corpuscular Photography,
Strasbourg, France, July 1957 - in pressc

Yagoda, H0 Book review, "Physical Techniques in Biological Research".
?olo II, Go Oster and Ac V«„ Pollister0 Science, 126i 215, Auge 1957c

Yagoda, HQ Book review, "Isotopic Tracers in Biology" by M„ Karaen0
Science 126s 754, 0cto 1957o

Honors and Awards relating to this projects

(a) American representative at the 1st International Conference
on Corpuscular Photography,,

(b) Served as rapporteur for the session on the neuropathology
of ionizing radiation at the Brussels International Congressn

(c) Presented address on the Biological Implications of Cosmic
Radiation at High Altitudes for the Martin Astronomical Society at
Baltimore, Maryland

Serial No,, KIAKD-96

Honors and Awards relating to this project (cont°d):

(d) Served as consultant for the Hand Corporation and Cambridge
Air Research on micro-meteorite detection,, Prepared report entitled?
"Micro-meteorite Observations from the Flight of USAF Aerobee 77"
urtiich has been reproduced for distribution by the Geophysics Research
Directorate, Air Force Cambridge Research Center^ June 1957$ 10 pp +
10 illusto

(e) Elected to Fellowship in the ;toierican Physical Society,

PBS-IIH
Iadi^id«l Project Report
Calessdar I ear 1957

Serial Wo. NIAMD-97

1. Physical Biology

2o Physical Biochemistry
3. Bethesda

EirU -

Project Title: Th@ TOchamism of the orderly aggregatloa of proteins
ia boildiag up biological straetwes.

Prlaelpal Investigators: Dr. Kol

Laki and Dr. Ho A. Saroff

Other Investigators: Dr. ¥. J. Bows

Dr. H. R. Carroll

Dr. Do R. Kcsalaz

Dr. F. Irreverre

Dr. Lo B. Mannings

Dr, R. Bo Simpson

Dr. J. A, Gladner

H.

L.

Martla

M

Jo

Mo

Callanan

F.

Saad

A.

R.

Hayden

E.

F.

Wilson

R.

Lo

Evans

Eo

N.

Smith

E.

R.

Mitchell

Cooperating Units s

Dr» E. Weiabach, LTD-NJAID (Serial No*
Dr. H. Sober9 LB-NCI (Serial Ho.
Dr. J. E. Folk8 OBC-NIDR
Dr. F. Tiets©s LBM~$IAMD

(S©rial No. MDlfalj
(Serial Ho, &2

Br, F. Stohlmaaj LPH-HBMD (Serial Ho9 to,

Dro P. Promove9 6MRT-NHI (Serial No*

Dr. E. MUialyi9 LCPM-HHI (Serial Ho.

Dr. B. Horvath$ MN-HIHDB (Serial No. ___ _

Man Years (calendar year 1957) : Totals 15-2/3

Professionals U-g/3
Others 2

Project Descriptions

flUfetfoy*

The otej®<8tiw§ of 'this project I® to stusSj the mechanism of
polysaarisatloaa of proteins ia order to gala insight as to how
cell® build ?$p n@twrk steactrares. The stnsetwes involved ia
msseaLar contraction asd blood coagjil&tlea ar® the restalts of
protein p©3y»risati©m<, The isaia part of this project is,
therefore 9 d@vot©d to the study of proteiaa involved la mssstalar
contraction and blood coagwlatloa.

Pag© 2

i^thods;

The method of attack is both direct and indirect* Tii the
direct attack the proteins of muscular contraction and folo
coagulation are separated from their native milieu and ar©
studied undo? arbitrary conditions which are selected to re-
properties of interest, la the indirect attack studies are
mad© on scam© other already better known proteins to gain Sj
formation before the direct attack is made.

In these studies the procedures of biochemistry and
physical chemistry ar© employed. For examples paper and ion~
©aschange ehrcmtogr&phy, ©nsymology? ultraeentrifiagal analysis 9
osmometry, light-eeattariag measwamsats, electrophoresis s
diffusion m®&sw««sntss etc.

flavor Findings.!

The relationship and the difference between the tropomyosin
A of annelids and molluscs and the tropoaiyosia B of molluscs and
arthropods has been further clarified. The tropomyosin B is
similar to the classical tropomyosin of vertebrates 9 while the
tropomyosin A appears similar to the tropcsayosln submit isv
fro© myosin by Laki after performic &eid esdd&tion or trypsin
treatment (Kevins, Saad, Laki9 Carroll).

In urea solution an N~t«rmlnal glutamic acid was found in
rabbit skeletal tropomyosin. This finding, together with
previously reported finding of a C-terminal grcups strongly
speaks against the ey@lopeptide theory of treponyosin (&omins58
Saad).

It was found that when xmyosin is placed in an alkaline
solution, it releases subunits with molecular weight of about
189GOO amounting to almost $&%> of the protein. They are found
in myosins from both smooth and striated ssusel© (Komina9 Carroll ?
Mitchells Laki).

A method for analysing magnesium in biological materials
which contain calcium has been demised*

The ioessetrie contraction of glyeorol-extsr&etod ma©@le

fiber-bundles in certain ionic ©m>ir©»§nt8 hm® been eosapared
with the rate of feydrolysing ATP in the sam© ©ERrirossaent.,

Preliminary experiments indicate that the amount of energy
released frosa the splitting of ATP by a contracting fiber™
buadl© is immns® when compared to the work capacity of the
fiber.

The toxicity of peataohlorophenol and pentabromophenol oa

myosin ATPas© has h®m. determined and the effect of thsse

Serial No. IH2©_221_.
Page 3
Mp.1or Findings; (Cont'd.)

halopheaols on the contraction of fiber^bro&les studied. The
results indicate that this type of poison inhibits both the
splitting of AT? and the contraction of fiber (Bowen) (as
cooperation with Dr„ Eugene Weiabaeh of NIA3D.)

A procedure for the isolation without degradation of
Mixtures of the fibrlno peptides (eo=»flbyin) has been worked
out.

From amino acid analysis by the method of Moore and Stein
ffldMjayga molecular weight of peptide A was found to b© §200 and
of peptide B 30@9»

Both peptides has?© C=termlaal arginineo Peptide A appears
to have another argiaine adjacent to the G°t©rainal argiaine.
It is proposed that this arg-arg bossd is responsible for the
selective action of thrombin upon fibrinogen (Gladn®r8 Laki«
Kerning s Sober, Folk)*

The discovery that thrombin reacts with dlisopropyl-
phosphofluoridat® CDFP) has been further utilised. Radioactive
DFp3& ^3 ©ssployed to investigate the steiehias©try of the
reaction. These studies indicated that thrombin is & relatively
small molecule (Mw ~«^ 15,000). When this intact but inactive
thrombin was injected into rabbits the radioactivity rapidly
disappeared from the blood stream and was found within two hours
mainly in the lungs (Gladner* Laki? Stohlaan).

The detailed investigation of the new earbo^peptidase
(called earboaypeptidase-B) briefly reported last year? has
been undertaken. The symogen and the active e&rbo:^p©ptid&se«»B
from pancreas has been purif led 20-fold. Detailed kinetic
studies show that the new ©nsyas® hydrolases only JBfig amino
acids from the carboy! end (C-tOTminal) of polypeptide chain.

Th® significance ©f these results ar@ twofold s (1) this
is probaMy the ©nsyme responsible for the release of the all-
important dietary amino acid 3ystee to th© body following
tryptic digestion of proteins and (2) the msgm can be used
as an analytical tool for the ©nsymtie determination of basic
amino acids in th© terminal positions (Giadners Folk).

As a sidelight9 the ©asys&s has been shown to hydrelys®
S««mino (B-ethyl) cysteine from the G-terminal position of
polypeptide chains. This grouping can b® created by chsmieal
and easymati© treatment of a protein prior to its attack fof
carbo^rpeptldase-B fGladner, Tie t assy Folk) .

Serial No. JOHE-St

Page 4

Th© cowers ion of the aarf.no group of serum albumin into
the non«=polar nitroguanidlno group with S-a&sthyl isothio-

nitrcurea is under eontimssd investigation with studies
being a*ad® oa the nature of the modified albwin.

A study has been completed oa the reaction of sepper with
albumin in which it has been demonstrated that copper
binds to the SH group to bring about a dtoerisatloa of th®
protein Boleeule.

In th© course of our studies on the SH group of ©ansa
albuasinj another site (or sites) which binds Bsrcury with a
high association constant was repealed. A detailed study on
the nature of this reaction has been undertaken.

A new effect of anions on the binding of sine to sens
albualn has been discovered* It was found that th© thioeyanate

ion (compared to nitrate) increases th® strength of binding of
sine to serum albissin hj a factor of about 50. The implication
of this reaction is that there Eight be a clustering of the
isildasole groups in eerusi albwin (Saroff 8 Simpson).

A new method of preparation of th® protamine 9 ealsategg
froa th© rip® sper® of a single species of soImoss; was developed^,
aiad the product characterised to show that in this ease as wall
as in eeaaercial pr©paratieass cheaieal heterogeneity exists
even though physical aastho&s indicate hesaogeneity (Mitehell9
Callanan, Carroll).

Quantitative determination of free and bound hydros^-
proline hav® been done in the urines of various ncraal and
pathological conditions. There- is an interesting correlation
between the free and bound hydrosyprolin® in sob® types of
kidney disease as eoapared with normal and other types of
diseases so far essgrainsdo

These studies oa the excretion of hy&recsyprolin® and
hydrosyprolia© peptides in newals and In various diseases
coupled with f ©©ding ©sperlBsnts should g±m to iaf eraaaiiesi
and a better understanding of the bieehMistry of collagen..

Steadies were made on th® hydrolytic products of nitro-
arginine in 6 N HC1. Using paper chromatography mid iota^exehaag©
chromatography about Iff compounds have been identified* This
finding should give us additional basis for speculating on other
pathways of arginine s&stabolisaa.

lfelssL£iaMsga-: (Co»i

Serial No. NML23L
Page 5

A quantitative and sensitive color reaction has been
developed for y™,guanidinobiatyric acid. Tbe urines of normal
and pathological conditions examined to date showed varying
emounts of this guanidin© compound.

Last year we reported the finding of a new cyclic imino
acid in Papaya fruit by paper chromatography. We have isolated
it in crystalline form. It behaves chrcasatographically and by
paper electrophoresis as well as by color reactions and in its
behavior under the W light as 2s6-»piperidine dicarboxylie
acid . N

Continuing last year(|s work9 gaiama-guaaidinobutyric acid
from calf's brain was isolated in. crystalline form and also
prepared the flavlanate (Irreverrej Hayden).

Siffliificance s

When part of the protoplasm or the whole cell (as in, e.gc?
cell division or muscular contraction) performs mechanical w<
a network structure is built up at least temporarily 9 mainly
through an orderly polymerisation of globular proteins. This
structure then reacts with the surrounding medium and by
utilising metabolic energy (stored in ATP e.g. )' performs work
(muscular contraction s ameoboid movement). In order to under-
stand this esmechanc«ehemical coupling'8 (the interaction of
structure with the surrounding) and its disorders, ? we must know
how such structures are built up. Muscular contraction and blood
coagulation are examples of processes where structures are built
tip throingh protein polymerisation. Such knowledge eventually
will lead us to the understanding of certain diseases of muscle.
Study of blood elotting9 in addition to supplying clues for
protein polymerisation 9 gives us better understanding of the
disorders of blood clotting.

When both direct and indirect approach leads to some specific
disease (e.g. hemophilia 9 rheumatoid arthritis) the advantage
offered by studying the disease is utilised to the extent
profitable.

In the nest calendar year the project will be pursued along
the lines presented above. The following is intended to give
illustrations.

Serial No0 HB©Jg2_

Page 6
Proposed Couffae of Protect: (Coat »d. )

The splitting 155 of the Hy©sin molecule by various methods
will ha further investigated,.

Because of the similarity of the polyrasrization of actin
to the phoaphorylaa©-b«a transformation, the dephosphorylatioa
of ATP in aetia polymeriaatie® (j>vmlmsly dimerieatioa) will
also be studied*

Since the DFP inactivates thrombin 8 presumably by reacting
with or near the sit© of catalytic activity, partial degradation
of Dip32 thrombin by chemical awl enzymatic means is at present
wader way» Elucidation of the amino acid sequence around the
point of attachment of the DIP group it is hoped will give as
inkling of the molecular saeehsnism of the action of thrombin,,

The effect of thiocyaaat® ion to increase the binding cf
sin© to serum alfeaaain is being continued 8 particularly with
reference to the possibility that there is a clustering of
imidazole groups in serum albtaaiao

Studies osS salmia® protein are in progress* to determine the
effect of ©barge and changes in charge due to binding of different
ions on the ccaf iguratlonal properties of the molecul©o

Studies on the excretion of hydresyproline and its peptides
will be continued in the hope that w® gain saor© information on
the biochemistry of collagen.,

Because of the Importance of Ye«^Robutyrie a°i£ *& Beure-
chemistry studies will be continuing on the biogenesis of Y"
guani&iaobutyrie acid8 a close analogue of y^ealaobaATvle acido

Serial Ho. IiML22L«^..
Page 7
ESSUS* Honors, Awards, and Publications

Publications other than abstracts from this projects

1. Bowea, Wo J. , and Gershfeld, Miriam* The effect of monovalent

salts on the acceleration of myosin B ATPas® by magnesiums
Biochim. et Biophys. Acta 2J,, 315-323 9 May 1957.

2. Callanan, M. Joan, Carroll, William R. , and Mitchell, Ellis Ro :

Physical and chemical properties of protamine from the sperm
of salmon (Oncorhynckus Tshawytacha) I« Preparation and
characterisation. J. Biol. Chem« , in press.

% Evans, Ro L», and S&roff, Ho A.: A physiologically active

guanidinated derivative of insulin. Jo of Biol- Chem. 228_.s
295-304, Sept., 1957

4. Folk, J. E. , Gladner , Jules A* % Carbo^peptidaae-Bo lo Puri-
fication of the zymogen, and specificity of the ms&m° Jo
Biolo Chem. , is pr@ss»

§. Gl&dner, Jules A. , and Folk, Jo E. . Garbosyp®ptidase-»Bo II«
Mole of action on protein substrates and its application to
earboxyl terminal grousp analysis . 3* Biolo Chora. , in press.

6. Gladner, Jules A* , Laki, I. , and Stohlman, F. s Labeled DIP-
thrombin. Biochte. et Biophys. Acta, in press »

7o Irreverre, F. , Evans, R. L. , Hayden, A. R. , and Silber R. s Th©

occurrence of gscHSffia-guonidinobutyric acid. Nature JJ|08 704,
Oct. 5, 1957.

8. Tietse, F., Gladner, J. A., and Folk, J. E.<, Release of C^tersainal
S»(P«'>^aisoethyl)-»eyst®ine residues ^ carbo3£yp@ptidase»B. Bio-
chtoo ©t Biophys. Aeta9 ia press.

9» Tersian, L@^on A., Isnreverre, F. , and Stabler, N. . A study of

nitrogen and uric acid patterns in the excreta and body tissues
of adult Aedes aejgSDS&i. J. Insect. Physiol. , in press.

10. Komins, D. R. , Saad, F., and Laki, K. . Some chemical character"

iatlcs of annelid, Mollusc, and arthropod tropomyosins.
Transactions of Conference on th© Chemistry of Muscular
Contraction (Japan), In press.

11. Komina, D. R. , Saad, F. , and Laki, K. . Vertebrate and inverts-

brate tropomyosins. Nature 179_, 206-2(775 Jan. 26, 1957.

Serial No. HIAMD 97

PagLO* Publications (Cont'-'d.))

12. Icsains89 D. R., Saad9 F. , Glada@r8 Jo A. 9 aad Laki, K. i

Meurasaliaa tropoByosias. tosh. Biocham. and Biophys. ^9
16-28, Jialy 1957.

13, Blufflj J* 9 Keruin, T. B. 9 and Bowen, ¥. J. « Dependence of length

of rausel® fibers wpon ATP concentration. Arch. Bioetem. and
Biophys. 66, 100-13J 9 Jan. 1957.

H« Laki9 K. s and Kitainger9 C. s Heat changes dwing the dotting
of fibrinogen. Nature Jgg, 985 s Near. 1956*

15° Boweffl, W. J.: Adeaesinetriphosphate and the gharteniag of
a$us@ular Models. J, Cell. and Ccssp. Physiol. 499 Sisppl. 1,
May 1957.

16. Laki s X. t Studies on the composition of contractile sracle

proteins. J. Cell & Gcraip. Physiol. $«fo Suppl. 1, May 1957.=

17. Mihalyi, E. 9 Laki, X. , Knoller, M. I. : Nucleic acid and nueleo-

tide content of s^osin preparations. Arch. Biochsa. and Biopbysu
6£, 130-U3? May 1957.

18. Nannlnga, L. B. : Th® binding of magnesium, calcitssa and chlorine

ions to he®ry and light ffierossyosin. Arch. Blcehem. and Biophys.
7g, 3^6-366, A^ag. 1957.

19. Lewis, M. S., aad Saroff, H. A.s The binding of ions to th® rauascl©

proteins. Jfeasisresemts on the binding of potaasirea and sodium
ions to s^osin A, isyosia B, and aetin. J. of A®. Chess. Sec.
2E, 2132-2117, May 5, 1957.

20. Pie®, X. A., frwrerre-, F.9 aad Wolff, H. L. : Th® separation mid.

determination of cyclic imino acids. J. Biol. Ghem. 2jg|9 687«»
697, Dec. s 1956.

21. Lakis K. s A tropas^-osin-llke fra^aent of denatured myosin.

Transactions of Conference on the Chemistry of Jtacular Con-
traction (Japan) 9 in press.

22. Laki, K.: A simple method for the isolation and crystallisation

of tropca^osla fros the awseles of the clam, fennas Msreenaria.
Arch. Bioehesa. and Biopsy®. 6j, 2^0-242, Ear. 1957.

23. Nanninga, L. B. t Fom&tion constants ®ad hydrolysis at 100° of

e&lcim and ja&gaeslw cosjplessss of adenosine tri=>, di« and
monophosphate. J. Phys. Cbssa. &, 1M4«1H9, Sept., 1957.

Serial N©0 HIAMD

Peg® 9

Pg£tJ|s Publications ((Ge»t8d<,)

24° Zipkia8 I»s A&amlkg E. 9 sad Saroff 5, H. A. » Bomdery electron
phoresis of toman parotid aaliwa. Pros. Sec. Essp. Biol, and
Mad0 SS? 69~71s 1957.

25« Saroff, H. Ac s A theory for the biding of ©alorid® ioas to
sens® albwain feas<$d on a hydrogen bonded model. Jo Phy®.
Chem. 61, 1|64, Oet. 1957.

26. Saroff, Ho A. s The blading of ions to the missel© proteins. A

theory for K* and. Na+ blading based on a hydrogen bonded and
chelated model. Arch. Biochem. and Biophys. 7J,, 194s Sept*
1957.

27. Saroff, Ho Ao and Choate, W. L. s Reversible disser fonaation in

the reaction of copper with bovine serum albumin. Arch.
Biosbem. and Biophys . , in press.

28. Simpson, R. Bo , and Saroff , H. A. : Decrease of swlfhydryl titer

in serum albumin. J. Am. Chem. See. , in pr©ss.

Indi:

Calendar Year 1957

Serial No.JjTfiMD;

1. Physical Biology

2. Physical Biochemistry
3» Bethesda

Part A.

Project Title; Immunochemical approaches to the isolation
and characterisation of proteins.

Principal Investigators R. R. Williams

Other Investigators: S. S. Stone, J. C. Jenkinss D. Francois,
and C. Isreal

Cooperating Units: Arthritis and Rheumatism Branch of Clinical
Investigations
Drs. J. J. Bunim and K. J. Bloch
(See Serial No. KM>!D<JsO§sC_Jj

Man Years (calendar year 1957): Totals 5

Professional? 2

Project Descriptions

Others 3

aSMlSIL2 Te@ characterisation of proteins of general
particular biological interest as to their intimate structure
has been approached by many chemical physical techniques.
The immunochemical approach presents soma unique oppor-
tunities to make use of the high degree of structural
specificity which antibodies possess. This group of in~
vestigators has made use of this property to achieve a proteir
purification technique and hopes to extend its work into the
field of fine structure of proteins.

One biologically important group of proteins now under
investigation are those involved in the sensitised sheep cell
agglutination activity of rheumatoid arthritic serum. Work
is proceeding on the isolation and characterisation of one or
more of these. Its purpose is related to inquiry into the
diagnosis and pathogenesis of the disease.

Jfethods ? A variety of standard immunological methods in-
cluding precipitation titrations and sensitised sheep cell
agglutination as well as several standard methods of protein
and amino acid analysis are employed. The latter include

Serial No. HflMBagfl— _ ^.

|fe|h©Js„s C«at»do) MS® 2

sedimentation and electrophoresis aaathods and eforosi&tograpliy.

Son® new sasthods developed by th® group will be listed
in ma jo? findings and publications*

£§M@SLJMffii^ - s®® too Bunia.

Serum for fractionation has been furnished by Dps, J. Jo
Broim9 Kurt Blcchs and R» G. Black from the patients of the
Arthritis and Rheumatism Branch of Clinical Investigations.,
Also sera for diagnostic test® tee bsen furnished by th©
Clinical Center , Mi, Alto ¥®t@rans Adsaiaistr&tion Hospital 9
Walter Reed Aray Hospital , George Washington University
Hospital and Johns Hopkins Hospital,

^ajor Finding:

(1) Past work on th© use of aatig®n<°antibedy reactions
on supporting M®dia for th© purification of proteins was pub=>
lished in two paper© «

(2) A Mthod for th® quantitative estimation of th©
Sensitised Sheep Cell Agglutination Factor (SSGA) , which
measures its inhibition of th® hemolytic activity of ros®nsi<=>
tisedM Newcastle Disease Virus (NOT), has been developed and
studied* Tests on more than 1§0 sera have indicated good
correlation between NOT hemolysis inhibition and SSGA tits?®.
Fractionation of sera with high SSC titres also indicate th®
two activities appear in the sass® fractions. Correlated sedi»
mentation studies indicate both activities are associated with
the ^jaaeroglobulias™ having sedimentation coefficients of 19
or greater as proposed by luakel and associates.

(3) Quantitative data using the Ks®nsitia®d«c NOT hemolysin
inhibition test indicate that th® virus in&etiv&tioa can be
related to th® rheumatoid factor by us© of th© familiar Ion Irogh
a&sorptiea isotherm in the sasse aanasr as ecfflpl©B@»t titration.

• 3 ~

Serial No. NIAMBgj>8

J|lffi£fie§raegs Bevelopmsnt of quantitative Mtheds of assay
and tins rssultant opportunity for vigorous analysis of the data
enhances the chances of further understanding of the activities
and pathogenesis of see© of th® aaeroglobulins involved ia
rheuaatoid arthritis ©ad other diseases. Progress toward
purification of the Baeroglobullns involved will b® necessary
to ascertain their natwr® and origin g and effects on tissues,

£S^ES^id_Cowi®_ofJPTOieeis

(1) Fwther purification of present preparations .

(2) Attempts to establish ■.::.: rigorous criteria of purity
using precise specific activity data In conjunction with the
usual physical criteria*

O) Characterisation of end groups ? associated non-
protein components and asslsto acid analysis following purif lcatIon0

(4) Collaborative project with Br. E. ¥. Erasart on
possible cytotoxic effects of the proteins involved rasing cells
In culture a

Partji ; Honors, Awards, and Publicatl
Publications other than abstracts from this projects

1. R. R. Williams and S. S. Stone; Application of antigen-antil
reactions on supporting media to the purification of proteins.

I. A Model system using bovine serum albumin and bovine y*
globulin. Arch. Biochem. and Biophys. 71s 377-385, Oct. 1957.

2. S« S. Stone and R. R. Williams: Application of antigen-antibody
reactions on supporting media to the purification of proteins.

II. The removal of a subtilisin-like enzyme from carboaqy~
peptidase preparations by anti-subtilisin on cellulose. Arch.
Biochem. and Biophys. 71s 386-392, Oct. 1957.

3. R« R. Williams j D. Francois and J. J. Bunims An approach to
quantitative estimation of the sensitised sheep cell agglutination
factor in rheumatoid sera. Proceedings of a Symposium on Sero-
logical Phenomena in Rheumatoid Arthritis sponsored by the
Arthritis and Rheumatism Foundation. In press.

PHS-BTH

Individual Project Report
Calendar Year 1957

Serial No. IIAMD-j
Physics! Biology
Molecular Biophysics
Bethesda

£S2LJU

Project Titles Investigation of the macromoleeular org&ni:.
tion of living matter.

Principal Investigator: Ralph W. G. Wyckoff

Other Investigators; Dr. L. ¥. Labaw Odile Croissant

Mr. V. M. Mosley S» Goldsstaub

Persis Griffin (Visiting Scientists)
Karen Zehner

Cooperating Units ;

Madame H. Espagne ) ¥isltorg
Madame Lise Enjalbart )
Wniversite de Toulouse,,
Toulouse j, France

Man Years (calendar year 1957) s
Totals

Professionals
Others 4

Project Description;

Objectives; To gain information about the macroatolecules that
are essential constituents of living matter , to see how they
are arranged in the structures they form and to see how this
arrangement is altered by infectious and degenerative disease =
To study certain of these saeromolecules (such as viruses) in
purified form after isolation from the living material.

Methods Employed* The electron microscopy of microorganisms,,
cells and tissues in suspension or thinly sectioned; the
physicoehemlcal characterisation of macromolecular compo™
nents isolated from such material using electron microscopy,
2=ray diffraction,, and similar established techniques 5 the
development of new physical procedures ? including X-ray
Mcroscopy9 to further such characterization.

Serial Ho. ffiUft^
Page 2

Major Fi&dlnggs The work done in Bethesda daring the last
has proceeded along the following main lines t

(1) During the last year there has beea a steady extension of
the electron microscopy at very high resolution which was begun
last year for the direct photography of molecular separations ia
crystals of organic compounds of comparatively low molecular
weight. Using an old electron microscope,, modified here for
this work9 molecular spacings have been recorded down to about

9 angstroms from several chemically related indanthrene deriva-
tives. This work is being actively pushed to provide aa
adequate experimental basis for an explanation of this close
approach to the photography of atoaic detail; it also seeks to
ascertain the best way to exploit this new procedure for exs
ing organic molecules. A series of X-ray diffraction measure-
ments is now being ©ade on these sase compounds to provide data
essential to the full interpretation of what the Eaieroscope
reveals .

(2) The cooperative electron microscopy with the Pasteur
Institute designed to determine how a number of typical viruses
develop within infected tissues is continuing. During the last
year KL1©„ Croissant has again spent several weeks in Bethesda
working on this project. Especial attention has been given to
the examination of tissues diseased with vaccinia and with one
of the APC viruses; significant progress has beea made in as
lating evidence showing the steps by which the elementary infec-
tious particles of vaccinia arise. Continued study is being
made both here and ia Paris for the further improvement of the
techniques of specimen preparation. This has led to a better
understanding of the factors that now bring about some damage

to all tissues embedded for electron microscopy and Is leading
to a continuing improvement in the tissue sections needed for
these investigations. Such better techniques are essential to
the satisfactory application of the electron microscope to all
problems which deal with diseased tissues and thus for appliea-
tlon beyond that for virus studies.

(3) Progress has been Bade in the development of methods of
X=ray microscopy in directions that will extend further its
application to biological problems. During the last year an
X=ray tube has been made here which yields excellent photographs
of the thinnest tissue sections that can be cat for optical
microscopy. Its ir&Ty soft X-rays will provide adequate contrast
in sections through unstained soft tissues no more than 1 micron
thick and in these photographs th© resolution is at least as good
as the highest attainable with the optical microscope. Special

Serial Ho. Mill

Major Findings j (cont'd)

attention has been given this year to the development
microscopic methods directed towards the identification of
chemical elements present in a sample through their character-
istic X-ray absorption and the fluorescent X-rays they emit,,
These methods are being applied with increasing success to
elements of medium atomic weights and this success is leading
to their gradual extension towards those lightest elements
which are of saxiaum biological interest.

(4.) The collaborative studies with the Dental Institute
designed to apply our new physical aethods to the study of
teeth and their development is being continued. Daring the
last year this has been E&laly in the direction of essploring
the further applications to dental problems of our expanding
X-=ray Microscopy.

Significance of research to Institute:: The refinement of
electron microscopy , both as regards attainable resolution
of the instrument and delicacy of fixation and preservation
of tissues, has given this laboratory high standing in the
field. The pursuit of specific structural analyses of
viruses and their stages of development has resulted in laying
the groundwork for study of fine structure and the relation of
function to structure in other biological forms. The develop^
ment of other techniques of visualisation of fine structure
and location and assessment of specific substances is opening
new areas of study in mlerophysiology.

Proposed course of the projects It is proposed that cooperative
work on viruses with the Pasteur Institute be continued on a
reciprocal basis of use of facilities and personnel and that
the development of both electron and X^ray microscopy will be
stressed in Bethesda.

Part B in@luded

Page 4

Part Bs

Publications other than abstracts from this project;

Croissants Odile; Lepine, Pierre; and Wyckoff , Ralph W. G.,

Sur le developpement du virus vaccinal , Annales de Institut
Pasteur. In press,

Lafa&t?, Louis W. aad Wyckoff , Ralph W. G„, The electron sderoseopy
of ferritin crystals. Bioehim. et„ Biophys. Acta, 2J,s 263-266 ,■
1957 .

Labau, Louis W. and Wyckoff, Ralph W. G., Molecular striae fros
an indanthrene dye, Proc. Rati. Acad. Sei., December , 1957.

Mosley, feraon M.s Scott, David B., and Wyckoff, Ralph W. G. ,
X~r&y microradiography of tissue sections with magnesium
radiation. Bioehim. at Biophys. Acta, 2^s 235-237, 1957.

Wyckoff 9 Ralph W. G., Electron Microscopy. Yale Sei. Mag.s
Jl; (7), April, 1957.

Wyckoff , Ralph W. G., The electron microscope in crystallography,,
Horeleo Reporter, £: U) 93=94s 1957.

Wyckoff, Ralph W. G„, Crystal Structuresy'Section IV, Chapters
11 and 12. (1957)

Wyckoff, Ralph W. G., The World of the Electron Microscope. The
Yale Press. In press. (This is the first volume of a nm
series entitled ^Trends in Science" to be published by the
. Yale University Press™)

Reported last year as being = IN PRESS

Labaw, Loais W. and Wyckoff , Ralph W. G., The electron microscopy

' of miBttte crystalline detail. Proc. Koninkl. led. Akad.

Wetensehap., Amsterdam, Series B, J9j (5) 449=450 (1956).

Labaw, Louis W. Labaw and Wyckoff , Ralph W. G.„ The structure of
southern bean mosaic virus -protein crystals, Arch. Biochem.
Biophys., 6?: 225-236, 1957.

Wyckoff, Ralph W. Gfc, La Structure des Cristaux Macro^Mbleeulaires
Au Microscope Eleetroniqae „ Ball, aierosp. appl., 2s Series,
It (1) 1-8. 1957.

Serial No, NIAMD-99_
Pa&e 5

t«d).

Scott s D. Bo aad Wyekoff, R. W. G., XXI. Carbon surface replicas
for electros microscopy and electron diffraction. J. Royal
. HLeroscop. Soc, (Trans, of the Soc.), 2£s U) 217-222, 1956.
(This was not published until 3-ate in December, 1956).

Challice, 0. E., Bttllivant, S. aad Scott, D. B., The fine structure
of some cytoplasmic inclusions of oxyntie cells. In press.

PHS-NIH
Individual Project Report
Calendar Year 19^7

5a rial N0o BlAflD-100
lo Physical Biology
2o Molecular Biophysics
3o Bethesda

Part A,

Project Titles The physical chemistry of membranes and complex
membrane systems of biological interests

Principal Investigators Karl Sollner

Other Investigators: Rex Neihof, «arc Lewis, Ruth i-icGlintock0

Cooperating Units! A loose form of cooperation is maintained
with Dr,, Charles W0 Carr of the Department of Physiological
Chemistry, i.edical School, UniVo of rdnnesota and with Drc
iiugen© Grim, Deptc of Physiology, also of Univ0 of Minnesota,,

Via.n Years (calendar year 19!?7)s

Total: 3 lAi- yrso

Professionals 3 l/U yrs0

Other s None

Project Descriptions

Objectives s A physicochemical study of membranes and membrane
model systems with the purpose of providing a rational
physicochemical basis for the elucidation of numerous
phenomena in living organises, for instance, electrolyte
balance and electrolyte distribution, the accumulation of
electrolytes in living cells, cell and nerve potentials,
and electrophysiology in general 0

Methods anployeds The preparation of membranes of highly
characteristic and specific electrochemical properties
(the methods having been worked out by the principal
investigator and his collaborators), and -the investigation
of these membranes, and of membrane systems in vhich such
membranes are a functional part, by physicochemical,

Serial ;jo0 NSU-ID- 1QQ
page 2

Methods Employed (Cont "do ) s

especially electrochemical methods^, such as potential and
resistance measurements^ also by chemical analytical
procedures, including radioactive tracer methods -

Mgj or Jj^nding 3 : Theoretical considerations have led to
the prediction that the ratios of the rates of the electrical
transportation across permselective membranes of any two
species of ions of the same charge coexisting in solution^
should be predictable quantitatively from the bi~ionic
potentials arising with the same ions across the same
membrane o Likewise it can be anticipated that the ratios
of the rates of electric transfer of any two coexisting
species of Ions of the same charge should be identical
with the ratio of the rates of their exchange across the
same membrane against a third ion (a topic reported on in
the last annual report )a Experimental test of these
concepts yielded results in fair agreement with the pre*=
dictionso However significant deviations^ outside of the
range of the experimental errors,, occur regularly,, We
are tentatively inclined to believe that, contrary to the
situation prevailing in free solution^, an interaction
occurs between ions of the same charge when they are
transported electrically across fairly dense ionic membranes c

Methods have been worked out to purify protaminsin such
a manner that membranes prepared with these purified prepa=
rations regularly show extreme ionic selectivity, of the
order of 10,000:1 in o01 N solution,, The general electro^
chemical properties of these membranes were studied in
detail 0 These permselective protamine collodion membranes
are at present the only known anion selective membranes
of extreme ionic selectivity which from the electrochemical
point of view behave in a strictly regular and simple
manner 0

The work on the theory of accumulation of electrolytes— =
both of anions and cations simultaneously=~against concen=
t ration gradients was continued «, Several systematic, but
still preliminary experiments with somewhat simplified
(and therefore not fully cogent) systems were carried out
in which up to 80 fold accumulation of electrolyte was
achieved under conditions for which the theory predicts a
roughly hundredfold accumulationo

Serial No0 illA-MD-lOO
page 3

Significance to Research of the Institute; In order to under =>
stand electrolyte relationships in living cells and tissues^
it is necessary to have accurate information on membrane
model systems which3 under carefully controlled knowi condi=
tionss reproduce at least some of the major in vivo phenomena „
The work of recent years, particularly the study of polyionic
potentialss of absolute and relative rates of ionic fluxes
under various conditions^, and the construction of an in vitro
model of electrolyte accumulation have brought us significantly
nearer to an understanding and an ±n vitro reproduction of
the type of effects which ultimately mustTgovern the in vivo
osmotic behavior of cells and tissues 0 The work already
carried out indicates that even fairly complex membrane
systems j, similar to those found in living nature „ may prove
in the f areseeable future amenable to a complete and quanti=
tative physicochemical analysis 0

Proposed Course of Project; Finishing the current studies
on the relative rates of the electrical transportation
across permselective membranes of two coexisting species
of ions of the same charge 0 Continuation of the theoretical
and experimental work on electrolyte accumulationo Further
work on membrane equilibria s particularly certain peculi=
arities of membrane equilibria in ionizing solvents such as
water* Continuation of the work on absolute rates of exchange
of ions across permselective membranes from the experimental
and theoretical point of view0 Theoretical and experimental
studies on the relative ratts of flux of several coexisting
species of ions of the same charge across permselective
membranes in continuation of previous work on the electrical
potentials^, "polyionic potentials'^ arising in such systems „
A preliminary study concerning the forces involved in the
formation of regular structures by means of the exploration
of long rang© forces of attraction and repulsion between
microscopic and macroscopic particles Q (The basic experi-
mental techniques have been worked out and tested by the
senior investigator before coming to Nffi„) These latter
studies are designed to furnish an insight into the physical
forces which create organized structures of various levels
of complexity and size as those existing in living systems 0

Part B included les A7 So tj

Serial No0 NIAMD=20(
page k

jgtrt Bj

Publications other than abstracts from this projects

Nffiihofs Hex and Karl Sollner0 The physical chemistry of the
differential rates of permeation of ions across porous membranes,
Faraday Soc0 JiscussionSj, No0 215 9U~101i,, 19$6C

Contributions to the Discussions of the Faraday Society-, Mo0 21?
19$60 Ko Sollner9 pc 120* 123., 127/128,, 132/l33;, 2lU/2l£s by
Rex Iteihof^ pc 13§ and 136/137 «

Grim^ Lugene and Karl Sollner0 The contributions of normal
and anomalous osmosis to the osmotic effects arising across
charged membranes with solutions of electrolytes 0 The Journal
of General Physiology ^ lj.0* QQ7^Q99s, 195? °

Gottlieb^, M0 H0£ Hex Meihofsand Karl Sollner0 Preparation
and properties of strong base type collodion matrix membranes 0
Jo Physical Chemos &L5 l£U=l593 l?57o

Neihof s Rex and Karl Sollnero. The transitory overshooting of
final equilibrium concentrations in mmbrane systems Ttiich
drift toward the Gibbs~Donnan membrane equilibrium 0 J« Physical
Chenio, 6ls 1^9=i63r 1957 o

Calendar Ysar 1957

Serial No„ HI AMD -101

1, Physical Biology

2 c Photobiology

3 „ Beth® sda

Project Titles Research in photobiology,.
Principal Investigators ?,, S„ Bradcett
Other- Investigators* Umer Lidd«l

See PrS j@CtSS

a„ Olson

bo B©ck«r

e<> Sharpie so

d., Ghamay

Cooperating Units? Continuing cooperation with the Labora-
tory of Infectious Diseases, NIAID = Brs0 Matter^ 01*
DuBuy., etc (MIAXD Prsjeet No„ 29) - on physical ms&si"
and th® evaluation of instrument al development s0

Actively participated in th© forasation of an 0f£i«;
Mathematical Research as! th© Mathensatle-s Panel <,

Cooperated in th© study of emtral computer require^

&<gnts°

Consult ed ©n th® implications of radiation develop

Dr„ Liddel has just resumed his wrk with HIH which

inelisdes research within this s@et.ion and consultation
with th© Directors ©n high energy and nuclear research
developssat s of wide implications 0

Man Years (calendar year 195?)
Totals 3-5/6
Professionals l-l/l
Others 2-1/2

Project Descriptions

Objectives? This project undertakes to advance ©ur
understanding of biological isalecular structure in

Serial N< --101

Page 2

Objectives (Cont«d)s

its relation to- the organized function of radiant
energy exchange and utilization. It doss so by a
number of specific enterprises reported by the princi-
pal investigators as s<^>arat@ projects,, The c^itral
objective provides conuacn interest and contribution
through asetingSp discus si onss and interchange 0
Original instrumentation underlies saich of the
research ani serves the several projects, New
specific projects stem from the central objective 0
While all the projects are of a basic nature they range
from an inquiry into the energy and forces of bi«aoleeular
species (Becker) to the observation of morphological
changes induced by light (Olson) .

Methods employed? Technical advances play an important
part in the efforts of this section » Contributions have
bean made in several fields - linearization of spectra,,
oxygen recording for metabolic rates^ scatter detemi=
nation of refractive properties,, These original develop-
ments make available new kinds of information in fields
i&iere it is most needed,,

Nuclear magnetic and electronic resonance provide
new approaches and greater insight into the mechanisms
under study s both in vitro and in vivo.

Major findingss See other projects reported in the
Section on Photobioiogy0

Significance of program to the Institutes This field of
basic inquiry into the biophysical mechanism of energy
exchange has very little direct bearing on immediate
health probl®as0 Nevertheless, a Esaningful appraisal
of the importance of radiation to national health and
welfare requires that this kind of mechanism be understood,,

Proposed course of projects a) Attack on basis problems
of hydrogen bonding will be strengthened with additional
personnel in order to use effectively both spect rose© pic
and aagnetie resonance approaches0 b) More aefcive work
in the ultraviolet s both in the action on cells and the
paralleling photochemistry will be possible with more
emphasis on the chemical aspects of the problem.,

al No, NIAMD-101
Page 1

Proposed coots© of project (Coab<>d)s

It is hop@d that provision can b® ssado for the us©
of single cell tissue cultur© for thes© studies- It is
especially important to compare the action of ultra~
violet >dth that of higher energy radiation.

Part B teeluded X@s /jT7 m £J

>~101

Page

Part Bo Honors,, A*sards3 aid Pub3J.eatd.oas

Publications other than abstracts from this projects

1* ■ Matter^ Carl Fo To, F„ So Bracks tt, and Bjron J„ Ols

"D®fc ©miration of number and siz© of particlss by
<3i©etrical gating? Blood cedls," ,»„ App0 Physiol 0 M,
56-70 (1957)--. Reported as bs&ng in press in 1956 rapert„

Zo Bradntt, Fo SB„ J, Ho Daniel and R, 6. cxickard,

"Recording o^gesi concentration and rat© of exchang©/8
R©v» of Sei, Inst, 28^, 182-18$ (1957). Reported as b®ing
in press in 1956 report,

3o Braetosttj, Frodsrids 3o9 "Linearizing infrared spectra/3
Jo Opt, Soco of Am05 ££, 636=638 (1957)o

Tts> additional pap@rs sh©i&sn on project ieTh© action of
radiant ensrgy on structure and ch®mosynth©sis in Living
ceils™ war® joint studies t&th this project 0

Honors and A'wards relating to this projects

Masahor ad hoc coaiaitte© on diagnostic instrusamtation
of the African Cancer Society^

jldual Project Re
Calendar Year 15

Sexlal Ho, HIMD--102
1, Physical Biology
2o Photobiology
3* Bethesda

Part An

Project Title; The action of radiant mergy on structure

and diemc synthesis in living cells.

Principal Investigators Rodney A, Olson

Other Investigators; Charles L, Grsenblatt and
Estelia Ko Ei^el

Cooperating Units; Hon©

Man Tears (calendar year 195?)
Total; 2=1/2
Professionals 1-1/2
Others 1

Project Descriptions

Objectives; To study the effects of radiant energy on
cellular metabolism arsd Chen© synthesis. To correlate
the priKsry light process^ the ensuing ehamical
changes^ and the morphology of photoreceptors at
different levels of organisation.

Methods eaployeds Ths us© of the unicellular organisms
ChloreHa and Eujgjeraa have introduced problmas of
physiological variation under routine culture eon-

- ditionsc At pressit xtfe are developing techniques ©f
constant culture to produce homog«aeous populations.

In previous studies w® have used the rate measure
ing ossygesi electrode (see Brackett), Unfortunately
certain metabolic poisons iispose limitations ^iich require
isolation of the reaction system f mm the deteetor ,
Studies are under ^y toward utilising t9ion exclnading"
mssibr sne s 0

Serial No, NIAMD-1Q2
Page 2

Methods employed (Cont'd):

Techniques of critical absorption and fluoref

microscopy have be® applied to study pigment organi-
zation id thin tha chloroplast. The combination of
line sources and narrow band filters allows convenient
study of the image due to pigment absorption.

We have been studying pigment changes in
Euglena in an attempt to correlate the in vivo
spectral changes with those of th© extracted
pigments.

Major findings? The oxygen transients and steady
state photosynthesis are inseparable fay metabolic
poisons thus far used0 These studies will ne@sssi»
tate the application of newer methods as noted.

In studying culture to culture variation of
Chlorellag chloroplast lamellae were observed
similar to the '"osmophilie11 structure found in
electron microscopy. Application of pigment
absorption micros copy has led to the measurement
of the optical density of individual chloroplast s0
Correlation of this data with th© known molar ex-
tinction of the pigments allows estimates of the
number of pigment monolayers , It may be possible
to gain insight into the effect of orientation on
the absorption of pigments as coapared to the random
orientation of these molecules in solution.

We have successfully broken the Chlorella cell
and isolated the extruded chloroplast for further
study. The microscopic appearance of the isolated
€nd disintegrating chloroplast correlates well with
the in vivo structure.

Another aspect of our \&vk concerns th® action of

light 9 darkness and. metabolic poisons on pigment
synthesis and disappearsneep ©specially in Euglena
where the chloroplast is extremely sensitive to these
alterations,, This wsrk has involved the study of
pigment chemistry as well as the underlying meta-
bolic relationships.

102

Significance to the program of the Institute; 'TJha
same basic structure is found in the important
molecules involved in energy transfer processes in
all living cells, with the chlorophylls and the
heme pigments these similarities are especially
noteworthy. The chemical identity of the molecules
as well as their spatial, orientation must nlay a
role in these universal mechanisms 0

Proposed course of projects In th© rear future^ with
better optical equipment,, it may be oossibl® to
observe the action of inhibitors directly an the
chloroplasto Furthermore the use of enzymatic
alteration may permit a « Mo chemical dissection"
of this structure. We hope to correlate inhibitor
action and induced structural change with the degree
of localised fluorescence within the chloroplast,

Stud3.es are planned in following microscopically
the growth and loss of the chloroplast and relating
this to the pigmait changes of the organelle. The'
critical control of the wavelength of incident light
will allow us to assay its role in the dependent
and semi<=depende&t mschaaisas of photemorphogenesis0

Part B included les Jff jfo £J

Serial Ho- KEAKD-102

Part B-,

Publications other than abstracts from this projects

1„ Braeketts P, So, Ro Ao Olson and R, G« Cri«fcard, "Tran-
sients in Og solution by Chlorejla during light and
darkness • !<, Phenomena and method. s,M Researeh in
Photosynthesis , Interssdence Publishers,, InCo9
K©w York , New York (1957) , PP» 412-418.

20 Olson,, R„ A»# Fo So Braekett and R„ Go Grickard, "Tran-
sients in O2 solution by Qhlorella during light and
darkness,, II. Influence of Og concentration and
respiration,,'8 Research in Photo sjynthe si sa Inter science
Publishers g inc., New York, New York (1957), PP* ifl9°429

phs=:
Individual Project Report
Calendar Xear 195?

Serial No,, HIAMD»103
In Physical Biology
2o Photobiolegy
30 Bethesda

JasLi.

Project Titles Molecular structure and organization in
biologically important organisms,,

Principal Investigators Elliot Charney

Other Investigators; Hon®

Cooperating Units? Hon©

Man Years (calendar year 1957) s
Totals 1
Professionals 1
Others 0

Project Descriptions

Objectives; The objectives of this research are to
explore the relation of msleeular organisation to
biologic activity 3 ©specially to the transfer of
electro magnetic energy in pho&o synthesizing structures
and to ©32p lor© some spectroscopic techniques useful
in pursuing this objective.

Methods employed; A nunber of investigations have been
undertaken, On© of these Involves mea earing the
spectral dependence of scattering from single ©ell
algae 6 Another is the investigation ©f the structure
of molecular complexes hj solution spectroscopy and
polarised absorptioa spectroscopy in the solM state o

In addition to Interfering tdth absorptioa
measurements used in studying the kinetics aod quantum
yield in these algae^, the scattering phenomenon itself
may provide information concerning the esdstence and
structure of particulates in the ehloroplast of the
living cello a few measurements by other investigators

-103

Pag© 2

Methods employed (CoRfc"d)s

have indicated th© possibility that scattering is
strongly enhanced near absorption bands (anomalous
dispersion) leading to th© possibility that th©
positions and intensities of in viro absorption
are not corrected by the expedient of using a
monotonie correction for background scattering 0
Scattering measurements are being made and compared
t&th absorption measurements made by conventional^,
or variations ©f conventional techniques,,

Major findings: A device fbr collecting and measuring
scattering from a micro-sample (o014 era3) has b©«n
designed^ constructed and used to make some preliminary
measurement s0 The device is based on an ingenious
suggestion by Dr0 F„ So Brackett that a dask-field
microscope condenser ■would act as a scatter collector
if used in reverse „

The investigation of th© structure of molecular
eoEgplexes was started with th® aid of a summer student ^
Mr» Warner Fit©9 with the extraction of chlorophyll^
lipoprotein complex from higher plants {clover and
spinach) „ These complexes were obtained recently in
Japan by Yasutane Chiba as micro-crystals,. It is hoped
to grow crystals of this material large enough to
make diehroisaa and birefringence studies aimed at eluel=
dating their structures It is recognized that these
crystals are to some extent artifacts of the method of
preparation^, but because the chroisophore is still eom=
plexed to the protein there is reason to hop© that they
may give more illuminating evidence em molecular
organisation in the ehloroplasto

Complementing these researches^, th© nature of the
effect of molecular organisation in th® ehloroplast
on absorption and siergy transfer is being examined
from a phenomenological or semi-theoretical point
of viewo

Lai No c, KIAJ5D-103
Pag© 3

Significance of the prograsa to th© institute; It fes long
been recognized that sub-micros copie (molecular) organi-
zation is the basis for such of the structure of iiidjtjg
organisms,, Th® role organised ifiolesular structures play
in biologic activity in general and in energy transfer
in particular is only partly elucidated „ Using th© probe
of electromagnetic radiation with biologically active
ehromophores such as porphyrin pigments or mors siaple
analoguoss w® hope to make further advances in a funda-
mental understanding of these phenomena.,

Proposed co tsps® of projects Th® msasureaenfcs of scattering
from algal calls containing dlff orarfc concentrations
of pigments and in ra&dia of different refractive indices...
and th© analysis of th® results obtained therefrom is
expected to csmplet® th® first phase of this project.

The chloropfayll-iipopsretein crystal spectroscopy
tdll be pursued and probably broadaied to eneorepass
problems of general and mars fundamental signif leasee
from th® point of view of Molecular structures and inter-
molecular foreesj, namely questions concerned with the
origin of intaisity changes and spectral shifts in geing
from solutions to solids csr smi-solid organised states.
Studies tdth relatively sisple ehromephores are envisaged,,

Part B included Yes £J No jgj

PHS-
Individual Project Report
Calendar Year 195?

Serial Noo HlAMD-lol;

1, Physical Biology

2, Phot ©biology

3o B©th©sda

Part A,

Project Titles Investigations of the action of radiant era

on biologically important compounds n

Principal Investigator; Norman E„ Sharpies©

Other Investigator sg Dolores A« Gregory s until transfer to
National Science Foundations 9/3/57 o

Cooperating Units: Hone

Man Years (calendar year 1957)?
Totals 1;>67
Professional: 106?
Others 0

Project Descriptions

Objectives: The objectives of this project ar© the
establishment of various biologically important int©r=
raadiates in photobiology and to study their kinetics
and other physical ehemlcal properties 0

Methods eaaployeds Ultraviolet or visible radiation is

the means employed to effect any alterations in the materials
under investigation s and spsctroseopy in the ultravlel
visible and infrared regions is the yaajor method used
evaluate any changes occurring e

Major findings: Infrared spectroscopy as a tool in
biological and medical research suffers a severe
limitation in that many compounds of biological interest
such as amino acids^ proteins and carbohydrate© are
insoluble in the standard infrared solvent s« Recently
th© us© ©f an infrared transparent pellet, usually
potassium bromide^, has been proposed as a suspending
medium for such compounds,, The composition of such

Serial Mo., MI AMD- 10^
Pag® 2

Major findings (G©nfc»d):

pellets can be controlled for quantitative det©rmi=
nations^, bit other factors which influence the true
representation of the spectra must also be considered*
Tw of these factors -which have bean investigated in
this laboratory are the sizes of the particles of material
used and the refractive index difference between the
material under investigation and the matrix material,,
It has beesi found that refractive index differences ess
distortion in absorption bands in the infrared end the
degree of such distortion can be used to construct
anomalous dispersion curves in this region.. Particle size
differences have been showi to affect the molar extinction
coefficients (the fundamental intensity factor) by
increasing the value with decreasing size* The values
of such coefficients obtained as functions of particle
size lie between those of dilute solutions and those
©f a single crystal 0

An additional factor which could affect the spectra
of solid materials is orientation of the crystals in
the pellet o It is conceivable that the high pressures
necessary to form the pellets could cause the materials
to flow in such a manner that the same face would tend
to be presented to the light bessa in the spectrometer.-.
This possibility is currently under investigation;
using X~ray technique So So far it has been noted
that compressed discs of potassLum bromide are rather
opaque to X-rays of 1<,5405 A« The diffraction patterns
obtained from such discs show marked back reflection
of X-rays with normal short spacing lines (cao 1°A)
superimposed on the reflection blackening „ Logger
spacing lines are either abaaat cr very broad and
diffusa

Tho ©xt®tt of compound formation between calciferol
and propionamides representing a simple protein^ is
under investigation0 The results so far are incon~
elusive,, Infrared spectroscopy has shoisi no apparc&t
evidence of compound formation and thermal studies
seem to indicate primarily solid solution formtiono

Pago 3

Significance to the research of th® institute! Fund&<=
Kienfcal investigations into the structures and rea<
ties of biologically teportant molecules are basic
to the understanding of their actions In the cell or
on the organisnio Studies on the effectiveness of th©
new techniques of investigation are a necessary
preliminary to such wsrk*

Proposed course of projects A continuation of the
physical chemistry of the steroids of th© ^itaisin
series^ utilising infrared and ultran&olet speetros=
copy for establishing structures of Tarloues inter-
mediates and related compound s0

Part B included Yes

Serial Ho. HIAMD-lQk

Part Bs

Publications oth€<r than abstracts from this project;

Sharpie S3, L SM and Jc So Murala^, «Th«5 infrared spoetra
of son© hstsropoly acid 8alt©5» Anal, Ch(«» J2S
1619-1622 (195?) o

FHS~KXK
Individual Proved

Calersd&r Year 195?

Serial KOo NIAMD-2C5

lo Physical Biol©®?-
2o Photobiolcgy
3 « Bethesda

Project Title? Molecular structure determined by spectroscopic
methods

Principal lavestSgator? Edwin D„ Becker

Other Investigators? None

Cooperating Units? None

Man Years (calendar year 1957) t
Total? 1
Professional? 1
Other? 0

Project Description?

Objectives? (1) An understanding of the forces within and
between molecules, especially those of biological importance,.,
(2) Development ef spectroscopics sssthcds fog? studying
molecular structure and analysing for materials of biological
interests

Methods ©replayed? Abscrptioa of infrared radiation provides
valuable information on the composition and structure of
molecules* Sines the infrared, spectrum is highly s«aisi~
tiv® to the formation of hydrogen tondss %m have utilised
infrared techniques for studying such interactions a

The technique of nuclear magnetic resonance also
provides a sensitive tool for studying male@ular structure
and molecular interactions,, During the past year we have
purchased equipment for conducting such ajqierlmeats and
have begun to colore its potential applications*.

Major findings? Much of our effort has been directed toward
the determination of equilibrium constants and heats of
formation for hydrogen bo wis in several alcohols, We have
be®a able to extend and improve the correlations reported
previously between these thermodynamic ard spectroscopi@

Serl-
Page

Major findings (Cont>d):

qaan.titd.QS found for hydrogsa bonded systems,, is a
result of this investigation we have concluded that
hydrogen bonded ale© hoi disers exist in a cyclic form,
and that the tw 0-4looo0 hydrogen bonds of the diner
are necessarily non»linear0 Hydrogen bonis in several
other systems are kxmm to be linear, sad it ted been
commonly assuissd deviously that all intermleeul&F
hydrogen bonds are linear.,

Eaplaratory '«©rk with the nuclear mgnetic resort
apparatus has indicated that this technique will be
useful in attacking a vide range of problems in laolesu-
lar structure,, Our MB, studies of hydrogen bonding in
ethanol have pro -sided stroasg stupor t for the existence
of non°lin®ar hydrogen bonds in alcohol diners „

Significance to research of the Institute: An understand"
ing of the properties of the hydrogen bond my fa® of

considerable significance to many areas of biocheMcal
and biophysical study o For e2Staples the structure of
proteins is thought to be largely determined hy the
extrsit to which it csn be stabilized by hydrogen bonis.
Sob® of the chemical properties of proteins (their
sensitivity to changes in temperature and chemical
environment) parallel the behavior of siiapl© hydrogen
bonded systesas, such as the ones that haare been studied «

The development of spectroscopic techniques for
studying molecular structure 9 especially those involving
nuclear magnetic resonance^ is of potential interest
to raany NIAMD scientist s« Such techniques sasy ultimately
find application in the analysis of steroids and other
conplex molecules of biological inter est o

Proposed course of project: W© plan to continue these
inwstigatiens along several lines: (1) studies *dll
b© Bade of hydrogen banding in more ©oseplex systsaso
These studies will utilise both infrared aai nuclear
magnetic resonance techniques and -«dil be directed
toward the establishment of better and sore complete
systessaties of hydrogen bonding,. (2) Further
exploratory work tdll be carried out with HMR »thods«
(3) A comprehensive bibliography of nuclear and
electron resonance literature is being eoraplied on
punched cards as an aid to research in this field by

Serial No. HlMD-105
Page 3

Px© posed course of project (Cont^d):

investigators in this end other laboratories , (4) Modi-
fications of equipment will be made to facilitate the
planned experiments,,

Part B included Tea £kj Jfo £J

Serial No,. NIAMD~105

Page

Publications other than abstracts from this projects

1. Lid del „ Umers "Sore® simple hydrogen bonding systeais
studiod by infrared absorptions Asm, N» To Aead-
Ssi* 6g, 70-85 (1957),

2, Liddolj, Umerj, ard Edwin B<, Becker 5 ^Infrared Spectro-
scopic studies of hydrogen bonding in saethanolp
ethanol arid t~bufcanol„M Speetrochin&ea Acta (In press)

3« Becker s Edvin D,„ Uxner UddaL and James N, Shoolerys
"Nuclear magnetic resonance s-ttsM.es of hydrogen
bonding in ethanol9" J, Molecular Spectroscopy
(in press) o

4^ Becker^, Edwin D„9 '"Infrared studies of hydrogen bonding
in methanol,, ethanol and t-butanels» Pr©e0 Inter-
national Syaposium on Hydrogen Bonding,, Ljubljana*
lugosla-adap July 29~Augusst 2S 195? (in press) 0

-hxh

Individual Project Report
Calendar Year 1957

Serial Ho, HIAMD-1Q6
1.

2. Mathematical Research
J. Bethesda, Maryland

Part A.

Project Title: Mathematics of kinetics and reaction -transport
systems.

Principal Investigator: John Z. He&ron

Man Years:

Total: 1

Professional: 1/2

Other: 1/2

Project Sescription:

This description is best understood against the background
of these facts: The Office of Mathematical Research has only
recently been established. Some time has thus been consumed in
recruiting efforts, etc. In line -with the prescribed role and
general philosophy of the group there has been consultation
with investigators from the various Institutes, ranging from
advice as to mode of attack to research done with or in behalf
of the investigator. Most often such research will fall
naturally under the project title here, but occasionally it
will be tangential or even separate. It would be untenable,
except in the case of really major items, to attempt to report
such work under separate projects.

Objectives: The main objective is to conduct a systematic
study of the mathematical problems of reaction systems and open
systems in which reaction and one or more types of transport
(diffusion-permeability, convection, active transport, etc.)
occur.

lajor findings: The major results consist of a general
formulation of the steady-state rate of complex reaction
schemes (arbitrary number of steps involving an arbitrary number
of cof actors and substrates). The general solution can be
exhibited in a form such that by mere inspection of the proposed
reaction scheme the rate equation can be written down. Further
the effect of any type of inhibitor entering at any step in the
sequence can be taken into account by simple modification of
specified terms in the equation. These results are to appear
in the 2nd Edition of The Enzymes.

Serial No. MIAMD^1Q6.
Pas© 5

For the special case of one substrate these results have
"been extended (in connection with work on eholinesterase by
Dr. B- Witkop, LO, and Dr. S. Friess, Naval Medical Beseareh
Institute) to superposition of the effect of an inhibitor
upon inhibition hy excess substrate.

An extensive memorandum on a certain type of integral
equation has been prepared for Br, M. Pollisar. Dr. Pollisar
is working with the Biometrics- Branch, NCI, on the mathematical
theory of cancer induction. The integral equation connects the
actual incidence of cancers, the observed or reported incidence
and the distribution of detection times.

Significance: The steady-state formulation is sufficiently
general that the classical cases of Michaelis and Menten and
strictly competitive or non-competitive inhibition are deduced
as special cases. It is in fact shown that the typical Line-
weaver-Burk plots in these classical cases result from a curious
mathematical coincidence and that these typical results do not
usually follow in more general cases. On the other hand, in
complex cases a strictly non- competitive inhibitor may give
typically competitive kinetics. These results therefore furnish
solutions not heretofore available, cast new light on those
previously available, and force •&■ complete reinterpretation of
an entire area of enzyme kinetics.

Proposed Course; In the future, preliminary results will
be followed up along the lines of temperature dependence of the
-generalized steady-atate rate, the possibility of deducing
- ■ ■ ■ inhibition by temperature (thermal denaturation) from the
generalised inhibitor results, and devising practical methods
for deciding in which steps of a complex sequence cofactors and
inhibitors enter.

Preliminary results on the kinetics of intact cells
indicate certain problems of a purely function-theoretic
nature which must be settled before practically applicable
results can be obtained.

The course of the integral equation investigation depends
upon success in application of what has thus far been done.
However there is considerable interest in the general mathe-
matical problem since the same formal equation is relevant to
the determination of the mean life of the f armed elements of
the blood.

Part B included / Yes/

ial No. NXAMD=.106

Part B: Honors, Awards, said Publieations

Publication;

John Z. Heaxonj S. A. Bsrohard, S. L. Fries®, J. Botts,
and M. F. -Morales. Enzyme Kinetics , Chapter in The Enayaes,
2nd Ed., edited by Lardy, Boyer and Ifyrback. Kew Xork,
Acadeinie Fress. In press.

PHS-NIH

Individual Project Report

Calendar Year 1957

Serial Ho. MXAMD~1Q7

1. OABR

2. Mathematical Research
5. Bethesda

Part A.

Project Title: Biometrics for HIAMD
Principal Investigator: Dr. R. L. Stearman
Man Years (calendar year 1957):

Total:

1

Professional:

1/2

Other:

1/2

Project Description:

Objectives: Solution of Diametric problems arising in
HIAMD and studies on novel and improved fitting procedures.

Major findings: The main research efforts have been
directed towards devising and testing a serviceable, yet
statistically valid, method for fitting linear combinations
of exponentials. This method exploits the fact that the
normal, equations are linear in certain parameters. Elimin-
ation of these parameters yields a surface in the remaining
ones and a systematic, iterative method has been devised
for searching for the minimum of this surface. The method
is such that for the ^-parameter ease hand calculations by
computational personel is feasible.

Significance: This problem arises in some problems
of chemical kinetics, all problems of tracer kinetics and
many problems in target theory (hit theory of irradiation
damage, etc.). It is important in a certain class of
problems not only to have decent estimates of the parameters
(pool size, rate constants, etc.) but to also have valid
estimates of error to serve as a rational basis for reject-
ing or accepting the hypothesis giving rise to the mathe-
matical model.

Proposed course: Some aspects of this problem are
under continued investigation by Hr. J. Cornfield,
Biometrics Branch, Division of Research Services. The
Burroughs Company is interested in exploring the method
for programing on small (Eleetrodata E 101) computors.
They have agreed to work on this aspect in liaison with
Dr. J. Z. Hearon.

Part B included Ho.

Analysis of NIH Program Activities
January-December ,j 19$7

National Institute of Arthritis and Metabolic Diseases,
Laboratory Research.

INTRODUCTORY COMMENTS'

The continued research activities of the six laboratories and
twenty=one sections comprising the basic science area of N0I0A„K0D„ are
summarized for the calendar year 19f?7 0 Although there has been some
growth of N0I,A0MoDo in personnel and in budget, this growth process is
decelerating as the laboratory space assigned to this Institute approaches
saturation,, In certain defined areas, notably in physical biology and
bioraathematics, some growth has occurred and is expected to continue,, The
gei'm~free animal venture has been initiated and some future expansion is
anticipated in this area alsoc

The late Justice Brandeis fathered the view that for a given type
of project there was an optimal size, that expansion beyond this size
resulted in impaired efficiency and reduction of returns,, N„I0Ho is very
large for an institution devoted to medical research, and in some regards
would appear to have approached or exceeded optimal size, The very
bigness of N„IGH0 carries with it certain encumbrances quite evident to
its scientists,, It does provide, however, certain benefits which may not
be available in smaller institutions,, Prime among these is the opportunity
for any scientist to secure counsel and collaboration from experts in
virtually every field of basic or clinical science 0

Many scientists in N0I0A0M0D0 have discovered this opportunity and
have learned to make use of it„ By taking advantage of the diversity of
skills represented on the campus, both within our Institute and in our
sister Institutes, scientists have initiated experimental studies, in
collaborations, which would have been outside the scope of any individual
investigator 0 New hybrid sciences have thus evolved and answe.rs to
questions have been secured which would otherwise have been unattainable 0

It is our belief that such hybridisation of sciences is a good thing
It should be noted that the three major domains of expected growth, physical,
biology, biomathematics and germ<=free mammalian life, all represent investi-
gative areas in which two or more traditional disciplines are merged 0 The
pure breeding of a scientific discipline may be expected, with the exception
of an infrequent mutational event, to yield more of the parental strain. It
is by hybridisation, by the marriage of a plurality of disciplines, that
tne new and exciting variants of science are most likely to be produced„ A
number of examples of this evolutionary process will become apparent to the
reader of the ensuing pages.

-fcaln Studies,. During the past y»ars selenium was identified a«
an ©a, dmponetnt of Fact*.- a prevents dietary livar neer
s fad diets ©oatalniog large amounts of Torula yeast s The act)
of different selenium compounds varies,, abactor 3 isolated from natural
ces being 3 to 0 times as active as any other compound tested. Cert
inorganic selenium compounds are active at dietary levels as low as 0„QS
Vitamin K also prevent© dietary liver necrosis but at a level that is
M&mately 1000 times greater than Factor 3„ Mitocnondria Isupplemei/'
or ATr) Isolated from the livers of rats fed the ToruU yeast d
supplemented with vitamin E maintain their rata of succinate oxidation to a

extent than those from rata on the same diet supplemented with
Factor }r. This and other evidence suggest that vitamin E and Factor 3 may
act independently of each other 3

The fact that dietary liver necrosis is produced by a simultaneous
deficiency of vitamin E and Faster 3 raisea the question as to whether
sttmt functions of the vitamin can be replaced by Factor 3 or sselea&um,,
In cbieks, a deficiency of vitamin E produces either exudative diathesis
(a subcutaneous serum exudate) or eacephalomalacia^ depending upon the
of diet used0 The reason for the difference became apparent when

selenium as a replacement for vitamin, E in the cniek was studied 0
The addition of small amounts of selenium (0,1 ppm) to the diet prote
Lve diathesis but has no effect on eneephaiomalaal*
..ogs suggest that the selenium content of the older diets used for
studies in the chick determined the type of deficiency sec

Ctaly a small fraction of the dietary intake of vitamins can b*
?»he fate of the remainder is practically unknown., During the past few
work was started to determine the sites of destruction and to el
"vegr&datlve pathways,, fleeent work has shown that vitamin k is n
•■ oyed when It is incubated with blood,. The reaction appears to
and the enayme appears to be specifically associated

ties on the relation b etween a fol:
mouse and the protection afforded thereby against lymphoeyti
are growing in interest,, Recent work indicates that even though ths
mouse doss not succumb to the virus luf

the virus.. For soma reason,, folic said must be pres<
to manifest its virulence. 'Mies raised on a histl"
■v fcha same response to the virus as the fo3
to say why both folic acid and
lere is a possibility I

■ ■■

- 3 -

Other work on folic acid relates to the "native" forms of this
vitamin ass they occur in liver. Two of the s,pre°folicIJ acid compounds
exist in liver 0 They differ in their solubilities Q Preliminary evidence
suggests that the wpre~follcn acid compounds are more active than folic
acid (chick assay) , If a similar situation exists in man, the npre=folicn
compounds may b® of clinical value in treating patients who have been
subjected to therapy with folic acid antagonists. The earlier observation
that a deficiency of folic acid is associated with ths excretion of formimino~
glutamic acid has been used in a field study among Alaskan Eskimos where
moderate anemia is endemic Preliminary evidence indicates that a signifi-
cant percentage of these people excrete f ormiminoglutamic acid which has
been associated with a deficiency of folic acid. Therapeutic trials are
underway to see what effect folic acid has on the anemia 0

It has recently been shown that soaking corn in a lime solution
increases the availability of niacin0 The steep liquor must be fed with
the corn since some of the niacin is leached out during soaking,, fchen corn
so treated Is uised in rations fed to dogs, chicks, and rats, they show no
signs of a niacin deficiency whereas the animals on the untreated corn d©
develop a typical deficiency,, It has been recognized that although many
people in Latin America consume large amounts of com, they have no pellagra.
Corn used in that part of the world is soaked in a lime solution. The above
work offers an explanation for the absence of pellagra among the people who
subsist largely on tortillas.

The laboratory which provides microbiological analytical s@rvi.ce for
other investigators has, among other projects, collaborated with the Cancer
Institute on a study of induced pantothenic acid deficiency in human subjects.
Clinical work suggested that inoculation of cancer patients with certain
viruses produced almost complete lysis of tumors if the patients were unabl©
to produce antibodies to the virus. In order to prevent the formation of
antibodies, patients were kept on a pantothenic acid=free diet since animal
studies showed that this vitamin was required for normal antibody production.
Although this work is not completed, it does indicate that a pantothenate
deficiency as manifested by very low urinary levels had no influence on
antibody production in the human in response to a variety of antigens,

Amino Acids and Proteins, The use of an amino acid mixture as a source
of protein for chicks" showed that good growth was secured only when small
amounts of a purified protein were also present. This observation suggests
that the intact protein contains an unrecognized factor required by the chick.
It has been shown that in the guinea pig the requirement for tryptophan by
the lens is much higher than that for growth. Normal rates of body weight
gain were secured with 0,12$ of tryptophan in the dtet but 0o2$ was necessary
to prevent cataracts,

Requirement of Guinea Pig for Zinc, During the past few years an
increasing incidence of hyperkeratosis la scaly, dry skin) has been reported
among swine raised on commercial feeds. Work at a number of laboratories

showed that the increased levels of calcium in the diet produced a
conditioned deficiency of zinc0 In our laboratory, an uncomplicated
deficiency of zinc in guinea pigs resulted in eczema of the face and
occasionally of other parts of the body0 The guinea pig appears to have
an unusually high requirement for zinc (2 mg./lOO gs diet) which may be
related to the high requirement for such minerals as potassium and magnesium,,

Studies with Germ-Free Animals 0 The growth-stimulating and vitamin-
sparing effects of such substances as antibiotic and large amounts of
vitamin C were attributed to the action of tiiese compounds on the gastro-
intestinal tract of the animal 0 The compounds could produce their effects
either by altering the flora ©f the gastrointestinal tract or by changing
the physiology of the host animal 0 The use of germ-free animals is the
only method of securing unequivocal proof to the exact mode of action,, In
order to use diets composed of purified substances such as casein, sucrose,
fats, minerals, and vitamins, a special procedure had to be developed for
sterilizing the ingredients and for mixing them in the germ-free tanks „ The
solutiqn of problems associated with preparati n of diet and collection
of blood samples has permitted the initiation of nutrition work using
germ-free rats0 These studies show that whereas the penicillin-supplemented
diet (at a level of £ rago %) prevents the development of a pantothenic acid
deficiency in conventional rats, it is ineffective in germ-free rats. The
same is true for high levels of ascorbic acid {$% of diet),, Studies are
under way designed to clarify the role of intestinal flora in the production
of folic acid„ Sulfonamides have in the past been fed to conventional rats,
and have been reported to aid in the development of signs of folic acid
deficiency „ Such studies are now being repeated in germ-free animals „

Experimental Obesity^ Rats made obese by the ad libitum consumption
of a high fat diet show histologic changes in the thyroid. The I1-* uptake
by the thyroid glands of thiouracil-treated rats was greater for the laan
than for the obese rats„ Although the exact significance of this observa-
tion is not understood, it does provide additional evidence for physiological
as well as histological changes in the thyroid of the obese rats„ Blood
lipid levels in the obese rats were slightly higher than those in the controls,
A method has been developed for the homogenisation of the entire rat carcass
which should prove valuable for many types of studies „ The skin has been
the stumbling block to all previous attempts to grind the entire animal „
Autoclaving the rat for a half hour in a sealed conlainsr softens the skin so
that considerable disintegration is secured in a large blendor0 After passage
of the material ' through a colloid mill a smooth emulsion results from which
representative aliquots can be withdrawn for analysis „ Duplicate analyses- for
moisture can be done with a variation of less than 1>»„ This technique will be
used to establish the constancy of the water to protein ratio and to extend
the carcass analyses to many other components.

Nutritional Effects of Sterilization with Ethylene Oxide „ The studies
of the effect of ethylene oxide on diets have been continued. This compound
might be used in food processing despite the fact that it produces
nutritional changes in the food even though the compound is totally
eliminated after treatment „ Collaborative work with Virginia Polytechnic
Institute shows that th© primary compound formed from niacinamide as a
result of ethylene oxide treatment is N»<»ethoxyniacinamide chloride 0 The
latter compound has none of the biological activity of the vitarain0 When
the reaction between niacinamide and ethylene oxide dissolved in water is
carried out at room temperature, th© pH rises 0 If the pH is permitted to
rise during the reaction, a yellow compound is formed which appears to be
an amine formed after rupture of the pyridine ringc

ENDuCftlNQLOGI

Studies on Experimental Diabetes 0 In th© totally depancraatised rat
it has been shewn that disturbances in the lipid metabolism ensue very
rapidly upon the alteration of carbohydrate metabolism,, Ketosis develops
ia such rats simultaneously with the hyperglycemia,, Two hours after
pancreatectomy th© rat has a high blood ketone and lipid level 0 These
abnormalities are corrected at a rate which is similar to th© reduction
in blood glucose when insulin is given0 The lipid disturbance i3 not
completely dependent on insulin since the intravenous injection of fructose
partially corrects th© ketonuria but has no effect on the high Hood glucose
level 0 Since ketogenesis occurs primarily in th© liver, this tissue was
further studied 0 It was observed that if adequate amounts of fatty acids
(acetate, butyrate, and octanoate) were added to liver slices, the production
of ketone bodies was the same whether the livers came from diabetic or
nondiabetic rats9 An extension of the ketosis study relates to the transfer
of ketone bodies across th© placenta „ VJhen pregnant rats were depancreatized,
the blood ketone bodies in the mothers and fetuses were markedly elevated
17 hours after the operation,, Lipemia, fatty livers and fatty kidneys
occurred only in the mothers e Additional evidence that ketosis is not
always linked to disturbed carbohydrate metabolism came from the studies
with fasted, pregnant rats where ketosis occurred in the presence of
adequate amounts of insulin and normal blood glucose levels. The studies
on the possible relation between growth hormone and insulin were extended
to the hypophysectomized^pancreatectomiaed rat0 In this animal it was
also apparent that no more insulin was required for the increase in body
weight produced by growth hormone than was required for the control of
diabetes o

Further studies on the glucose tolerance factor (GTF) have indicated
that it is a waters-soluble, low~molecular weight compound which., when absent
from the diet of rats, results in a slower than normal reduction in the
blood sugar following an intravenous glucose injection,, Vjhen 0o0f> to
0„1 mg0/LOO g„ of body weight of the best GTF concentrate is injected into

rats2 the rat© of decrease in blood sugar is restored to normal „ The
effect appears to be mediated through the liver 0 In addition to the
glucose tolerance factor there i8 a substance in liver which appears to
increase glucose uptake „ It can be distinguished from GTF by its activity
in eviscerated rats where GTF is inactive 0 The liver factor does not
appear to occur in other tissues j it is water soluble^, relatively stable
to boilings and is not a proteine Other work indicates that the decrease
in blood glucose following its intravenous infection is influenced by a
number of factors „ In the absence of the pituitary ^ the amount of food
consumed is one of these factors „ tvhen the intravenous glucose tolerance
test is carried ott on hypophysectomized ratss the rate of disappearance
of excess sugar from the blood is lower than in the normal 0 The rat© can
be restored to normal by tube feeding „ During the intravenous glucose
tolerance test periods of faster-than-normal removal rates in hypophysectornised
rats have been observed,. This suggests that the decreased removal rates in
hypophyseetomized rats fed ad libitum are due to an increased rate of
addition of glucose to the blood 0 These studies emphasize the difficulties
associated with the interpretation of glucose tolerance data9

By means of the insulin assay developed in the laboratory it was found
that the level of insulin in the blood of normal adults is approximately
£0 micro=units per ml0 of plasma 0 The hypophysectomized^ alloxan diabetic
rat; as well as the mouse ^ has been adapted to the insulin assay „

The Hole of the Hypothalamus in Metabolic iieaetions0 Accumulating
evidence suggests that the activity of various endocrine glands is under
neural control 0 When the dog's brain is transected in the midbrain^, an
interruption is established in the pathway which norsaally activates the
release of TSH and of ACTH in response to stress 0 Mow that it has been
established that transection of the midbrain destroys the neural pathways
involved in the control of endocrine activity ^ efforts are being made to
pinpoint the areas involved 0 Stereotaxic lesions placed in the midbrain of
cats has shown a tenfold increase in adrenal steroid excretion in some
cases; while in others there was no change „ Anatomical studies are underway
to delineate the specific areas of the midbrain associated with the inhibition
of the steroid excretion 0 A difference in the creatinine excretion of dogs
with different neural lesions has been observed. The dogs with midbrain
transection shos» an immediate decrease in creatinine excretion to 66$ of
normal^ while the dogs with transected spinal cords maintain their creatinine
excretion at the control level for two weeks when a gradual reduction to
66$ of normal sets inc Isotopic studies are under; ay to elucidate the
mechanisms involved in these phenomena.

Excretion of Corticosteroids 0 A micro method has been developed for
the determination of a number of steroid fractions in urine „ by this means ^
urine samples from a variety of endocrinopathies are being analyzed. In
the dog the evidence suggests that the chief corticosteroid is tetrahydro-
Cortisol (pregnane=3a.lipi7a.21=tetrol'=20°>one):, The excretion of this

7 -

compound increases sixfold for several days after a severe stress such as
surgery,, The corticosteroid excretion profile in the dog differs from
that isi the human, especially in the absence of the 17=ketosteroids which
are present in fairly large amounts in human urine „

Isolation of Anterior Pituitary Hormone sa A simple method has been
developed for extracting TSS (thyroid stimulating hormone) from pituitary
glands, A series of solvents are percolated through a column containing
the glands 0 by this means a $0% yield can be secured of a fraction having
a potency of 1 USP unit/mg0 The method permits the isolation of the other
hormones from the same batch of pituitaries, thus effecting a considerable
economy 0 The application of this method to human blood indicated the
presence of 1|0 USP rajllinnits of TSH per 100 ml, of plasma „

Preparations have been secured in which the TSH concentration was
1000 times that in the original beef pituitaries0 by this means prepara°
tions with 20 USP units/mg0 have been prepared 0 The luteinizing hormone
which is a frequent contaminant of TSH preparations has been separated
from TSH and concentrated 0 Contrary to previous reports in the literature,
the luteinizing hormone appears to be a basic protein or peptide 0

Application of the above procedure to mouse pituitary tumors has re=>
suited in a preparation that has 8~10 USP units/rag 0 The TSH in the mouse
pituitary, although similar to beef TSH in many respects, differs from it
in its partition coefficient during chromatography 0

PATHOLOGY AND HISTUCHMISTKI

Experiment Pathology 0 The investigation of human chronic rheumatic
conditions has been considerably hampered in the past by the lack of a
reproducible similar condition in small laboratory mammals 0 Hence the
discovery of a spontaneous osteoarthritis in rats and mice and the elucica-
tion of its causation assumes a considerable importance. Obesity-producing
high fat diets have resulted in production of osteoarthritis in rats, but
not consistently in mice0 In the latter the arthritis is shown to depend
on a genetic factor. This genetic factor did not correlate with that
inducing thyroid dysfunction,,

Infectious diseases „ A strain of Streptobacillus moniliformis isolated
from rats in oculated with material from joints of a patient at Georgetown
Hospital quite regularly produces an acute osteoarthritis in rats, and the^
organism is recoverable from the lesions after a negative phase beginning
with its disappearance from the blood at 2=3 days. These animals give some
of the serologic reactions found in human rheumatoid arthritis 0 when it was
observed that intermittent exposure to very low barometric pressures,
equivalent to those at 18,000-25,000 feet altitude, produced heart valve

lesions in rats, and that inoculation with green streptococci would produce
vegetative endocarditis imposed on the previously damaged valve, the way
was opened for the study of a regularly inducible bacterial endocarditis 0
Bacterial endocarditis in the dog with multiple infarcts and glomeruli
nephritis has been induced with Staphylococcus aureus „ Streptococcus aureus
and StrejAococcus mitis after surgical creation of aortic insufficiency
and is not prevented by the use of a Hufnagel valve, thus indicating the
relation of infectivity to valve damage rather than to abnormal pressure
variation of aortic regurgitation,, As in rats, a similar lesion is produced
in dogs when infected during discontinuous exposures to very low barometric
pressures „ It is indicated that on infarction alkaline phosphatase is
liberated into the blood stream,,

When the Laboratory of Pathology and Histochemistry first arose as a
section in the Division of Pathology and Bacteriology of the Hygienic
Laboratory, its principal investigative function was to supply morphologic
criteria for the reproduction of induced infectious diseases, which forms
the third postulate of Koch in identification of etiologic agents for
infectious diseases. This function has been continued in cooperation with tho
Laboratories of Infectious and Tropical Diseases of the present NIAID, with
the various Laboratories of the Division of Biologies Standards, and with certain
investigators in the National Cancer Institute who are concerned with the
role of certain infectious agents in the causation of a number of malignant
(cancerous) diseases,, Infections so studied have included: (l) (with LID,
NIAID) Lymphocytic choriomeningitis during amethopterin therapy! (2) (with
LLP, DBS) itabbit myxomatosis and the effect of chloroform, ether and heat
upon itj (3) (with LID, NIAID) Fungus infection in connection with the
therapeutic and toxic doses of a new chemotherapeutic agent j (U) Polio-
myelitis, as part of the control (DBS, LVP) of the manufacture of the
Salk vaccine o Spinal, cords and, when necessary, parts of the brains of
some U-£000 monkeys have been studied pathologically for absence of lesions c
The pathology and pathogenesis of polyomyelitis strains in South African
vervet monkeys is being studied with view to replace the usually employed
Indian rhesus monkeys 0 This is intended to overcome the objections by
certain groups in India to the exportation on monkeys for experimental
laboratory use0 In the search for an even more effective vaccine than the
Salk preparation, an assay of the effects of an attenuated living strain
of virus is being made0 Since it is thought that polyomyelitis may at
times be transmitted as a food infection, a study is being made for the
presence of lesions in intestinal tracts after gastrointestinal infection
of monkeys „ (5) Fibroma virus. Pathology in suckling and adult rabbits
indicates a far greater pathogenicity in the former (with PA, NCI).,
(6) S0 Ac virus0 This agent, though producing clinical neurologic symptoms,
has failed to induce definite central nervous lesions in chicks and hamsters
(with LID, NIAID). (7) A recently isolated filterable agent from the
Ehrlich ascites tumor . This material has produced intense splenomegaly of
mice, often leading to fatal splenic rupture (with LID, NIAID) „ (8) Studies
(with LID, NIAID) of the pathology and virology of the Russian type IV polio

virus 0 The identity of this virus with Goxsackie A7 virus has been
established,, This repeats the American experience „ However, her© the
identification as a new virus v&s made, without first reporting it as
polio IV 0 (9) A new gastroenteritis virus „ This agent produces both
visceral and neural lesions (with LIL, NIAIL))„

The Public Health Service has long had a peculiar and particular
interest in the investigation and control of leprosy, and has consistently
maintained some laboratory investigation of this disease, both in the
leprosaria and at NIHC At present a study is in progress on the effect of
vitamin IS deficiency on the pathology and pathogenesis of rat leprosy „
Attempts are also being made to transplant lepromata to brain tissue, and
to cultivate Ma leprae murium in Ehrlich ascites tumors 0 In the deficiency
experiments the development of amyloidosis is being studied0

Germ~Free Lifea The use of germ- free animals has read© possible the
investigation of the effects of deficiencies, intoxications, parasitisms
and single pure line organism infections without the complication of the
spontaneous infectious processes which are so common in small laboratory
animalsn Specifically folic acid deficiency depresses bone marrow at least
as much as in control animals (with LBN, NIAEi))0 Infestations with germ-
free helminthic parasites are being studied for their pathogenic effect „
Hound worms have been found to complete their cycle in normally insuscept-
ible hosts (with LTD, NIAID) 0

Toxicology,, Toxicologic studies (with LCr, NHI) have shown acute
fatty changes in heart muscle after large doses of noradrenaline and
adrenaline, which correlates with arrhythmias 0 The role of vascular changes
in the genesis of the spermine nephrosis is being explored (with LPT, NIAMD)„

Hematology. Hematology studies have been devoted to the isolation and
purification of hemopoietin and the study of its effects on the turnover of
red and white cells, utilizing P^2 and Cr?l labels of erythrocytes and
thymidine H^ labels of lymphocytes „ It is probable that the latter circulate
only for a day or two though they may persist in spleen for long periods0
The existence of a humoral factor other than tissue hypoxia, which stimulates
production of erythrocytes, appears to be indicated. Isolation and identifi=
cation of this substance might prove to be of considerable therapeutic
importance, as well as contributing materially to our understanding of the
mechanisms regulating production of new red corpuscles & The occurrence of
more than one hemoglobin in man was first demonstrated by the discovery of
a distinct fetal hemoglobin,, Then the association of a distinct hemoglobin
(S) with the hereditary sickling trait and the observation that V.est African
Negro children with this trait were relatively insusceptible to malignant
malaria lent positive clinical significance to the multiplicity of hemo=
globins, Just now another as yet unnamed variety has been reported as an
apparent concomitant of the hereditable thalassemia (Colley's anemia) factor,,

10

Heiaoglobin studies hy moving boundaries electrophoresis have separated
intermediate compounds of ferrihemoglobln and its cyanide derivative 0
These observations give final experimental proof to the ancient specula-
tions, based upon interpretations of oxygen dissociation curves, that the
four ircn=porphyrin residues of hemoglobin react separately 0 Hemoglobins
of types A, S and C have been observed to undergo reversible degradation
to molecules of one half the initial molecular weight,, l.hen this proceeds
in mixtures of hemoglobins, no hybrid molecules are formed0

Renal Studies „ A highly specialized distribution of esterase activity
in the rat kidney has lead to exploration of the mechanism of urine concen-
tration, probably by countercurrent exchange, in the renal papilla „ Evans
blue=labeled proteins and isotopically tagged erythrocytes and albumin have
demonstrated shifts in medullary blood composition and erythrocyte popular
tionD Fluorescence and autoradiographic techniques are being used to
supplement these studies 0 The study of the precise distribution of this and
a number of other enzyme systems in the cells of the tubules and stroma of
the kidney is gradually leading to a better understanding of the mechanisms
at work in this organ in the production of urine and the reabsorptlon of water
from it0

Histochemical Reactions 0 Other histochemical studies include those of
polysaccharides with special reference to renal and bronchiolar epithelial
glycogens, chondroitin sulfuric acid and amyloid 0 Methylation and alkali
demethylation of carboxyl groups, methanolysis of sulfate groups and
resulxation are being employed for the more precise identification of these
substances in tissue,, Histochemical methods applied to these substances
contribute materially to the recognition of chemically distinct materials
lying in close proximity to each other , but nevertheless separated and
probably functionally as well as morphologically independent of each other0
Thus a closer appreciation of the true biological significance can be
attained than by biochemical ©>•■ strictly morphological methods alone0

The existence of a phenolic reducing substance in certain usually
scattered cells of the gastro~intestinal mucosa has been known for 2$ years
and the cells themselves have been recognized as a distinct type since l8700
Because of the prominent property possessed by some of them of forming colored
complexes with chromates, they have been designated as enterochromaffin cells
and the phenolic granules are called enterochromaffin 0 The identification
of the enterochromaffins with the pharmacologically interesting and
clinically important internal secretion serotonin (5~hydroxytryptamine) had
never appeared adequate, and it was in an attempt to complete this identifi-
cation that new indole reactions were evolved. This identification has
failed, under circumstances wnere the failure appears significant „ In
some species two chemically distinct substances appear to exist in the
stomach and intestine, as demonstrated by differences in the azo coupling
reaction,, Neither of these substances is demonstrably indolic in nature0

11

Pigments 0 Studies of th© melanin and lipofuscin figments have con-
tinued 0 Improved reactions for identification of indoles, especially
tryptophan, tyrosine and high concentrations of carboxyl have been evolved,
A final o~quinhydrone nature of cutaneous melanin has been established by
closer definition of the silver reduction reactions and by application of
graded oxidations and reductions to tissues „ It is concluded that a
chemically distinct neuromelanin, skin melanin and yellow hair pigment
exist 0 It is hoped to conclude the characterization of the pathologic
pigments within the ensuing year0 The functional significance of th© neuro=>
melanins and lipofuscin pigments remains unknown but the existence of
histochemically and morphologically distinct types in various organs and
even in the nerve cells of various nuclei and areas of the central nervous
system supports the view that these are functional substances and not mere
evidence of "aging" and general senescence of cells, as has been advocated
by many0 The cutaneous and eye melanins, in addition to the obvious
function of excluding light, may well serve other purposes,,

Services 0 besides the diversified experimental studies just recounted,
the .laboratory has maintained since 1920 a small "mail order" diagnostic
service in human pathology0 This presently includes materiel from perhaps
200 autopsies and 2J>00 surgical operations per year and is derived chiefly
from smaller hospitals of the Indian Service and the b apartment of Justice,,
This service is operated jointly by most medical members of the staff and
serves the essential purpose of keeping them alert pathologic diagnosticians,,
This is highly necessary to prevent the possible development of the attitude
which sometimes appears among experimental pathologists that the lesions
we observe are necessarily the result of what was intentionally dors; to th©
animal 0 The existence of this service has further enabled us to collect
series of human cases exemplifying particularly the infectious diseases 0
Tularemia, psittacosis, ttocky Mountain spotted fever are conspicuous examples0
Currently this m terial is enabling us to make a study of the prevalences
of specific organ tumors among American Indians and to contrast these with
the distribution prevalences among the white population0

CHEMISTRY

Carbohydrates 0 Studies are continuing on th© chemistry of D~ribos©
and of 2=deoxy=»]>=ribose0 New methods for th© synthesis of 2«dQoxy-D-ribo-=-
furanosides and those phosphates of 2~deoxy°D-=ribose which have bean
demonstrated to be metabolic intermediates ar© being developed0 The
synthesis of 2=deoxy»D'=ribose was markedly improved, the physical constants
of the highly purified sugar redetermined and a large quantity of the
sugar furnished to the Laboratory of Biochemistry and Metabolism, where the
details of its fermentation by Lactobacillus plantar urn and degradation by
2=deoxyribose aldolase have been studied0 A new and general synthesis leading

12

to D°arabinofuranosides was elaborated from the anomerie tribenzoyl=D~
axabinofuranosyl bromides, Aside from the synthetic possibilities thus
opened, these tw> substances are of theoretical interest as the first
pair of furanosyl halides to be made available „ The occurrence in nature
of nucleosides containing D<=xylofuranose residues prompted a study of new
synthetic approaches „ It was found that a properly substituted D=xylose
dimethyl aeetal (readily obtained from D<=xylose) may be utilized for the
preparation of these naturallyoccurring furanosides, Other studies show
promise of providing novel syntheses of 2=ketosss, diketosugars as well as
monoraeric nonreducing arihydrosugars0 A considerable amount of J^deoxy^D"
ribo-hexose was prepared for studies on tolerance tests in diabetics and
for clinical investigations in general „ There is evidence that this novel
deoxyglucose is transported and metabolized by hamster gut„ The synthesis
of 3"deoxy°D~xylO"hexoBe and 3*°deoxy~D°lyxo°hex08e , and their anhydrides,
are in hand0 The metabolically important amorphous sedoheptulose has been
characterized by its hexaacetate, the first crystalline derivative from
which the heptulose can be regenerated under gentle conditions „ Considerable
theoretical significance attaches to the configuration and conformation of
anhydrides of hexoses, heptoses and heptulosese New members of this class
have been prepared and their equilibria studied0

Amino Acids „ Amines, Peptides and Murines „ The finding that natural
hydroxyamino acids, such as hydroxy^L^proline and o^hydro^^L^lysine have
an inhibitory effect on the growth oT plant cells grown in tissue cultures,
has been extended to the growth of animal cells in chick embryos 0 A
systematic search for special inhibitors of H>=hydroxytryptophan decarboxylase
and serotonin oxidase has yielded a number of potentially useful inhibitors,
which has started a program on the correlation of the MAO=dnhibitory effects
of drugs with their psychopharmacological actions 0

The synthesis of the novel catechol metabolites, 3=-methoxytyramlne,
normetanephrine and metanephrine has aided their identification, pharma=
cological evaluation and recognition of their metabolic significance „ The
conversion of dopamine to noradrenaline, one of the crucial steps in the
biosynthesis of catechol hormones, has been worked out in vitro and serves
now as a guide for the study of the in vivo synthesis utilizing dopamine
labeled in various positions 0 The enzymatic cleavage of catechol by
pyrocatechase to yield cis,cis~muconic acid has been studied with regard
to the role of an especially labile intermediate hydroperoxide „ On a
dimeric level such a hydroperoxide was synthesized and shown to searrange
to a (tetra)carboxylie acid in an analogous fashion,, A very reactive labile
intermediate in the bacterial metabolism of xanthine to formiminoglycine
has been synthesized and furnished information en the enzymatic require^
ments for its cleavage to f ormiminoglycine „ The analogous intermediate
between histamine and formiminoaspartic acid, though equally labile, has
also been obtained in solution0 Interest in the synthesis of that part of

- 13 -

insulin which is subject to species variation has led to improvements in
the construction of sulfur<=containing peptides with one or more cysteine
groups 0 The study of the bonded or non=bonded interaction of peptide groups
with ester carbonyls in medium size rings is being continued,, Models for
stable free radicals resembling intermediates in the metabolism of tyrosine
and the biosynthesis of thyroxine have been synthesized 0 Hew selective methods
for the oxidation of the indole part of tryptophan as such5 in peptides and
proteins have been applied to lysergic acid derivatives^, gramicidin^,
lysozyme and other tryptophan~containing natural products. The interesting
enzyme<=inhibitory properties of polytyrosine prompted the preparation of
polytryptophans with hydroxyl groups in the 2= and ^positions 0 A new type
of ester labilisations dependent on the over- all shape of the molecule^
was detected in certain polyacetates of tertiary and quaternary aminoinositolsc
It was possible to utilize the new phenomenon to construct molecules in
which the spontaneous hydrolysis of ester groups matched the enzymatic
hydrolysis by cholinesterase 0

Steroids 0 The anthrasteroid rearrangement^, discovered by NIAMD
scientists, has been considered as a possible pathway for the metabolic
conversion of steroids to carcinogens (e0g0, methylbenzanthracen0)o Con«=
tinvxing investigations have disclosed the subtle but important influence
of certain positioned double bonds on the course of this rearrangement 0
The synthesis of 5s7i)93llU'=°anthrapregnatetraen-20-one mad© available the
anthrasteroidal analog of progesterone for biological evaluation by the
endocrinology section of NGI0 The possible utilization of solanum plants
as commercial steroidal sources was demonstrated in principle by a novel
one-step deamination process which converted the steroidal alkaloids
tomatidine and solasodine to a mixture of steroidal sapogeninsD The latter
could conceivably serve as intermediates in the synthesis of hormones of
endocrinological importance. Pregnancy urine has be&a carefully examined
in an attempt to deterirdne the effect of complications such as toxemia^,
diabetes and rheumatoid arthritis on corticoid excretion, While definite
effects have been noteds more data are required before the excretory patterns
can be defined „ The known sparing solubility of biologically active steroids
prompted an attempt to prepare the more soluble 21-glucosiduronate deriva-
tives of these substances. The first glucosiduronate to be prepared was
derived from 11 -deoxycorticosterone „ A collaborative study (with the
Laboratory of Tropical Diseases^ NIAID) on the steroidal content of a
number of parasites^ protozoa and bacteria has demonstrated the presence
of cholesterol in Trypanosoma cruzi„ Considerable progress can be reported
in regard to the elucidation of the stevioside aglycone structure (referred
to in the 1<#6 report) 0 Clarification of the stevicl-isosteviol isomerism"
has made it possible to relate it to the allogibberic acid=gibberic acid re-
arrangement 0

Analgesic Drugs „ dince the separation of analgesic action and
addiction (as well as side-effect) liability in substances having the same
or greater potency than morphine has,, as yet, not been attained, the

elaboration of such a drug has been the continuing objective of numerous
investigations,, Morphine, in optimal dosess will adequately control
only 80 per cent of severe clinical painp therefore, the need for a drug
affecting areas not reached by morphine has still to be met0 At the organic
chemical level the approach to these problems has been through alteration
of the natural alkaloid architecture and by the synthesis of novel structural
systems „ Pharmacological activities which originally centered about
screening and assaying new arsalgesic agents in animals as well as at the
clinical level have now been expanded to include investigations of the
metabolic and enzymatic degradations of narcotic drugsa as well as studies
on the development of tolerance to drugs as measured by various behavioral
responses „ Collaborative studies (with Smithy Kline and French) of 2° -hydroxy*
2|,53,9'='trimeth<yl<=6s7-benaomorphan have demonstrated for this substance a
conditioned response-blocking action (in mice and rats) superior to chloro-
promazine0 Replacement of methyl on the nitrogen by phenethyl (in this
substance) enhanced the analgesic activity to ten times that of morphine %
while alteration of the position of N-closure from carbon<=10 to carbon=l
in 3-hydroxy~N~raethylmorphinan decreased activity £0 fold.

In the light of reports that the N=methyl group undergoes oxidative
degradation in vivo,, the evaluation of replacing this group in morphine
by N~formyl or carboethoxy was undertaken „ The potentialities of trans=>
forming the medically useless opium alkaloid thebaine into pharmacologically
beneficial drugs are by no means exhausted and research c ontinues in this
area0 Information^, in regard to the chemotherapeutic spectrum of selected
substances prepared in connection with this section" s activities,, is being
obtained in cooperation with industrial laboratories,, Thus strong anti°
bacterial activity was shown by several phenanthryl amino alcohol s9 and a
few acridine amino alcohols are to be screened as antiviral agents e Clinical
analgesic studies of analogs of nalorphine have demonstrated that
N°propylnormorphine is hazardous in that it precipitates disturbing
abstinence phenomena in individuals somewhat dependent on their regular
narcotic „ Another analogy N<=>methallylnormorphinea is presently under study 0
Enzymatic studies with liver preparations of rats receiving various
morphine-nalorphine mixtures have shown the existence of a parallelism
between the depression of enzyme (N=dealkylation) activity and the depression
of analgesic response 0 further enzyme studies on the effect of st©reo-=
isomerism of these drugs, as well as on the purported potentiating effect
of quinine on morphine will be investigated. A behavioral response (in rats),
namely $ swimming a fixed distance^ has been newly employed as a measure
of drug action and as a tool in studies on tolerance to drugs0 It is hopeds
thereby, to gain information bearing upon the placebo reaction as well as the
hypo=responder in man„ .An important continuing function of the section
is its advisory capacity to other governmental agencies,,, as well as to the
United wations and World Health Organization on problems related to narcotics
and addiction,,

• 15 -

Analytical and Other xtesearch Services 0 The resources of the micro-
analytical laboratory haves as usual^ been fully utilized by the scientific
staff of all the Institutes on the caucus „ Upwards of 8^,000 elemental and
functional group analyses ware performed,, In addition^ infrared and
ultraviolet spectra in excess of 3^000 were taken in support of the various
investigations in the Laboratory 5 and mention should be made of the fabri-
cation of a novel micro illuminator ("infrared microscope") which made
possible satisfactory spectra with as little as 1$ micrograms of substance 0
A synchronous recorder was constructed which permits the transfer of spectra
to a punch card system simultaneous with the taking of the large scale
spectrum,, This promises to be of great value for the documentation and
classification of spectra on a large scale 0 The Editorial Committee during
the past year processed and approved for publication U50 manuscripts , As
in the past tactful and thoughtful criticism^, where required^, served to
improve manuscripts prior to public ation,

BIOCHEMISTRY ML' METABOLISM

Carbohydrate Metabolism (a) Pentoses „ The central role of D-jsylulose
5>~phosphate in carbohydrate metabolism has been established^ which makes
discovery and purification of an animal D~xylulokinase of significance „
This completes the delineation of a metabolic pathway involving the steps?

/. a.

D=glucuronic acid — -^ L~gulonic acid =—=£ 3<B,keto~L=gulonic acid

=»L^ L-xylulose ==£==» xylitol =4=|, D~xylulose =^ D-xylulose-5-phosphate

I*
pentose cycle

We thus have a basis for understanding the metabolism of L-atylulose^ an
intermedj.ate accumulating in the urine of pentosuria patients 0 An enzyma
system has been purified 15>»fold which catalyses formation of L=sylulos©
from L=gulonic acid0 There are two steps (Numbers 2^3^ above) successfully
separated,, The 3<=,keto acid is closely related to ascorbic acid and could be
an immediate precursor. Incidentally s since the pentose cycle leads to
glucose-^phosphate which can go to D=>glucuronic acid^ discovery of th© kinase
for D-xylulose completes the last link of a new cyclic pathway for glucose
oxidation0 Isotopic studies with l^C-^ labeled D=glucuronolactone (CHO group)
and 6-^C1^ labeled L^gulonolactone (CH2OH group) , are consistent with the
pathway outlined above 0 The isotope is recovered in carbons 1 and 3 of
glucose from liver glycogen when rats are given these compounds 0 A series of
studies on pentose utilization by l0 plantar urn have illuminated further the.
enzymatic pathways for the metabolism of <?=carbon sugars 0 L-ribulokinas©
has been purified s and an enzyme discovered which converts L=ribulose
phosphate to D=xylulose phosphate. The latter is split to acetyl phosphate
and triose phosphate by another newly discovered enzyme, phosphoketolase,
which has thiamine pyrophosphate as a c of actor. These reactions ^ in conjunction

- 16 -

with the one catalysed by Lauipen's arabinose iscmsrass, give us the
following pathways

ATP
L~arabinos© ^p* L^ribulos© .=^> L~ribulos© phosphate ?=*

D-xylulose phosphate «=^ acetyl phosphate + D<=glyceraldehyd© 3-phosphat©

More recently, the metabolism of 2~d©02jy™D~ribose by L0 plantarum has b@en
studied „ A kinase converts this sugar to deosyribose-fUphosphate 0 This
in turn, is split to acetaldehyde and triose phosphate by an adaptive enzyme
"DR^aldolase", similar to an enzyme found in jC„ coli by Hacker „ The adaptiv©
nature of this enzyme when deoxyribose is the^substrate suggests that its
function is to catabolize deossyribose, not to synthesize it0 In another study
a soluble enzyme was discovered in K0 coli which converts ribose 3>=phosphat©
to 2°keta~3-deo2yglucoheptonic acid! The phosphorylated form of this compound
is a precursor of shikimic acid which in turn forms aromatic ring compounds
in nature e

(b) Hexoses „ The enzyme uridine dlphosphoglucose^U-eplmerase
(u^^pimsraseT^^Gal-U^epimsrase which catalyzes the conversion of nueleotide-
bound galactose to nucleotide°bound glucose has been subjected to further
investigation. The n&mmalian enzyme has been purified to a high extent and
the requirements for diphosphopyridine nucleotide (DPW) are even more striking
in the purified fractions 0 The role of DPN is still unknown 0 DPN and
reduced DPN labeled in the para position with tritium have been prepared 0
Although DPN is necessary for the reaction, no tritium was found either in
UDPGai or UDPG0 The question of the mode of action of DPN in this reaction
is, therefore, still open0

Studies have been continued on the bacterial mutants (E0 coli) which
lack ability to grow on galactose (galactose negative mutants) „ Eight
genetically different mutants were subjected to ensymological analysis 0
Three were found to be defective in the first step enzyme of galactose
metabolism, io@oj> galactokinase0 As many as four mutants were found to
be defective in the second step enzyme of galactose metabolism, i0e0,
Gal=\L~P uridyl transf erase 0 This is the same enzyme which was found through
work at tnis Institute to be deficient in congenital galactosemia 0 because
of the serious effects of galactose in this disease, it was thought of
interest to stucy the effect of extra addition of galactose to the various
bacterial strains „ It turned out that growth of the wild type (galactose
positive) and the strains defective in galactokinas© were not suppressed by
extra addition of galactose 0 The growth of the strains which were blocked
in Gal»l=P urldyltransferase was seriously suppressed by the addition of ^
galactose to the medium,, Moreover, under the same conditions, only the latter
strain accumulated galactose l=phosphat® in the cells <, It seems clear that

- 1? -

galactose 1-phosphat© rather than free galactose should b© suspected as
the agent responsible for the toxic symptoms which appear upon addition of
galactose to living organisms which have a hereditary block in galactose
metabolism,. If Gal-l-P does not accumulate, no distrubances seem to occur 0
The toaac effect of galactose 1 -phosphate was further studied on the exizyms
level and it was found that it is an inhibitor of the enzyme phosphoglucomu-
tase0 Tiiis inhibition is due to a formation of galactose diphosphate which
may be the immediate inhibitor „

UDFG pyrophosphorylase, the enzyme which catalyzes the biosynthesis
of UDPG is found not only in animal tissue and in microorganisms , but also
in plant tissue0 It has now been highly purified (about 800-fold) from
mung bean seedlings by a successful application of the Sober-Peterson
column 0 The role of UDItial in synthesis of mucopolysaccharides, including
the bipod group substances, is under investigation. Preliminary experiments
with C^=labeled UDPGal suggest that this nucleotide functions as a
galactosyl donor in mucopolysaccharide synthesis in the microsome fractions
from guinea pig gastric mucosa 0 It has been possible to produce antibodies
which act as antienzymes to mammalian Ul)PGal=U°epimerase by immunication with
the latter0 This antiensyme may be a valuable tool for the study of
galactose metabolism in vivo and in vitro „

Work on the human disease congenital galactosemia was continued. The
following new facts were added: (1) The defect in Gal-l-P uridyl transferase
is not only confined to red cells, but also to the liver j (2) It is possible
to detect the enzyme defect in blood from the umbilical cord, thus establish-
ing that the enzyme defect is congenital} (3) There are cases which show a
marked defect but not necessarily a complete lack of the enzyme 0 This was
revealed by using C^-labeled galactose both ±n vivo and in vitro 0 The
residual low activity might, however, be due to the development of an
accessory pathway0 A program including studies of UDP and UDPGal metabolism
in families which carry aberrant genes is under way0 i''or this purpose more
sensitive and accurate methods are being developed 0 Soros of these methods
are also planned for use in a study of the metabolism of the above mentioned
compounds before and after birth in tissues of mammalian species 0 The
methods already available from work in this Institute turned out to be
applicable as a diagnostic assay for the disease congenital galactosemia 0

In order to understand the biological role of UDP«glucose and UDP-
galactose during development, and the pattern of biochemical differentiation,
work on induction of enzymes in microorganisms such as yeast may furnish
fresh ideas 0 Studies on the adaptation pattern of enzymes responsible for"
galactose metabolism in yeast has furnished the following informationc Studies
on a yeast mutant, defective in galactose metabolism and identified as
galactokinasele3s, gave particularly interesting results 0 Free galactose
initiated a striking induction of the enzymes, Gal»l»P uridyltransferase and

- 18 -

UDPGal°lj~epimera;8©0 This is at variance with the classical hypothesis of
sequential differentiation 0 Because of the hereditary block in this yeast
strain which makes it unable to synthesize galactose l=pho8phat8^ an
induction of the two succeeding enzymes should be impossible according to
the hypothesis of sequential adaptation,, Induction of an enzymatic mechanism
for transport and concentration of galactose in the yeast cell has also been
obtained; this is the first permease described for free h®xos©0 Investi-
gations of a glucos© permease mechanism are bsing carried out by a member
of this laboratory who is spending a year at the Pasteur Institute 0 In
connection with this study ^ various glucose analogues are being investigated,,
Energy~uncoupllng agents inhibit the system,,

Using variously labeled substrate it has been demonstrated that
Aerobaeter aerogenes can convert D=-glucose into L=fueose without rupture
of the hexos® carDon skeleton „ This suggests that epimarizations about
carbon atoms 2S 3 and $ and reduction at C~6 occur in the over-all conversion,,

^G^ Cyclitols „ Several investigators in this laboratory are investi-
gating the TmetaDoTisra of myo=> inositol and other cyclitols in living organisms „
These compounds are widely distributed in nature^ but little is known of
their functional importance „ The first rapids sensitive and specific
enzymatic assay for iryo=inositol has been developed in order to investigate
its metabolism,, The assay enzyme is inositol dehydrogenase a Scyllitol a an
isomer of myo-inositol occurring in fishes^ was prepared in quantity for
metabolic work and a new complex^, scyllitol diborates was isolated,, This
complex is of unusual interest because from ordinary stereochemical considera-
tions one would not expect scyllitol to be capable of binding two borate
molecules „ Biosynthesis of inositol from radioactive glucose has been
demonstrated in slices from rat seminal vesical and prostate 0

(d) Glycolytic frnzymes,, Glycogen and Insulin,, In an attempt to obtain
more information about the manner in which ensymes function the mechanism
of action of four enzymes of the glycolytic cycle has been investigated*
Fhosphoglucose isomerase (PGI) and phosphomannose isomerase (IKI) convert
fructose 6~phosphate (F<=6-P) to glucose 6-F and mannose 6~Pa respectively,,
Both enzjir.ss activate C-bound hydrogen on C-1 of F=6-F as a proton^, but the
H which is activated by PGI is not the one which is activated by PKI0 Phos-
photriose isomerase likewise activates only one of the 2 C-bound hydrogens
on the primary carbinol of dihydroxyacetone phosphate (DHAP)„ Aldolase can
activate one of these hydrogen atoms in the absence of glyceraldehyde
phosphate^ but the hydrogen which is labiliaed is not the one which is
activated by the triose isomerase „ Moreover^ identification of the H-atoms
of DHAP which are involved in these two actions can now be made in an
absolute sense, A DHAP°aldolase complex has been detected spectrophotometri-
cally„

It has b&en found that incorporation of glucose in vivo proceeds
more rapidly into the larger molecules of glycogen of muscKHand into the
smaller molecules of glycogen in liver „ In an attempt to understand these
dif ferences^ studies have been carried out in vitro with liver phosphorylase
or muscle phosphorylase and liver and muscle glycogens of varying average
molecular size0 The results indicate that each enzyme has a preference
for polysaccharide molecules of a specific sise0 Muscle phospnorylase
reacts preferentially v.ith larger glycogen molecules than does liver
£ hosphorylas® 0

In order to evaluate more rigorously than heretofore the role of
insulinase in mammalian physiology the intact liver and hind limbs of rats
have been perfused with oxygenated whole rat blood containing added traces
of insulin labeled with radio=aodine „ Experiments to date have shown that
the intact liver causes rapid and extensive degradation of insulin whereas
the hind limbs are far less effective in this regai-d0 Kidney perfusion
studies are in progress e

Murine Metabolism0 The interesting story of the enzymatic steps in
xanthine utilization by certain anaerobic bacterial cells was presented in
last year8 s annual report „ i?'ormiminoglycine reacts with tetrahydrofolic
acid to form 0°foriaiminotetrahydrofolic acid0 The latter s in turn,, goes
to 5j, 10=methenyltetrahydrofolic acid and N=10=formyltetrahydrofolic acid„
N^lO^formyltetrahydrofolic acid^ in another enzymatic step^ reacts
reversibly with ADP and inorganic phosphate to give ATPa tetrahydrof olic
acid and formic acid0 The enzyme catalyzing this 3a st reaction has now been
crystallized 0 It is useful ©s a higiily sensitive and specific assay tool
for formic acid in biological sources 0 1-ith this assay 5 it has been shewn
that an increase in formic acid excretion occurs relatively early in the
course of folic acid deficiency in rats0

Nucleic Acid Ketabolism0 The role of polynucleotide phosphorylase
in tiNA metabolism has been held somewhat in doubt because the enzyme acted
in such random fashion to polymerise any nucleoside diphosphate 0 Klsos unlike
Romberg's DNA system^ there was no primer requirement and a poor affinity
for substrate # so that O00lj M ADP was needed to saturate the enzyme 0 It
has now been found that highly purified preparations of the enzyme^, made
available by Ochoaa require presence of a primer for activity0 Polymers or
small polynucleotides serve as primers, jf'urther^, with primer a the affinity
of enzyme becomes greatly increased (lower K.m) so that a rapid rate of
polymerization is observed with low concentrations of ADPfl UDPS etcos such
as exist in tissues 0 The primer molecule must contain a minimum of two
nucleoside residues. Thus 58=Al!iP is inactive but the dinucleotide pApA
(p » phosphate^ A » adenosine) is a primer 0 The reverse reaction^
phosphor illy sis s occurs only until the polynucleotide chain is reduced to the
dinucleotide,, which is therefor® a "limit polynucleotide" comparable to limit

- 20 -

polysaccharides in starch enzymologys

(primer)

pApA •:• n(ADP) — =$ pApApA — £ pApApApA =A Poly A

P0Ii (liridt polynucleotide)

Poly A -^ — -> pApApApA ===4 pApApA =4 pApA

Oligonucleotide primers show no specificity,, so polymers of the structure g
pApApUpUpUpUpUpUpU (U ■ uridine), can be made, Such molecules, with specific
labeling at the "front end" will be very useful for study of the mechanism
of action of phosphorylase and nucleases, kith polymers as primers a marked
specificity is manifesto Thus Poly A primes only AD? polymerization, and
this specificity may have biological significance 0 Study of the specificity
of nucleases and diesterases is being continued,,

Recently Begun Microbiological Projects „ Investigation of th© biosynthesj
of theTMasoie ring of thiamine is underway, using thiamineless yeast mutants,
Torylopais utilie9 and various S-labeled precursor compounds 0 Another investi-
gation is concerned with the general mechanisms involved in the metabolism
of amino, hydroxy, carboxyl and sulfhydryl groups, using enrichment culture
technique to isolate bacteria which utilize various simple organic compounds 0

PHAKMACOLOGY

Burn Shock and Bacterial Infection,, The successful treatment of a
sisable population of severely burned patients in Lima, Peru by th© oral
administration of saline solutions was reported last year0 Whereas this
therapeutic regimen protected patients very well against shock, it was noted
that a significant late mortality was associated with Pseudomonaa septicemia.,
Laboratory animals proved quite resistant to systemic infection ty. this
organism unless subjected to prior burn or treatment with cortisone, or
unless the organisms were suspended in mucin0 Since no entirely satisfactory
antibiotic against Pseudomonas was available, human gamma globulin was tested
and was found to protect mice not only against this organism but likewise
against E0 coli, Staphylococcus a etc„ The possible usefulness of this
discovery in human clinical material is currently under test in Peru„ The
project on clinical evaluation of therapy in shock for the past two years Jias
compared large volumes of saline in one group with large volumes of saline
plus plasma in the other group,. With around ?0 eases in each group saline
plus plasma has a slight but not statistically significant lower mortality
from shock than saline alone,. It has been previously shown that large voluntas
of saline are equally as effective as plasma plus glucose solutions,.

- 21 -

Amino and Amino Acid Metabolism, The demonstration of the ubiquitous
preseneeofpolyamineB 'lputeescines spermidine, spermine) in living matter,
and the pronounced pharmacological activity of the latter two, has led to
a continuation of work on the physiological significance of these compounds
and their relation to disease 0 In cooperation with other workers, the
enzymatic degradation has been shown, and in the past year the biosynthesis
has been elucidated,, kith purified baoterial enzymes and carbon1^ labeled
compounds the following scheme was founds (I) methionine * ATP » "active
methionine" (II) decarboxylation of "active methionine (III) condensation
of the propylaniino group with putrescine to form spermidine „ This is a
novel reaction for methionine, and in the systems studied constitutes an
important pathway of methionine metabolism,.

The enzymatic degradation of histidine has been shown to proceed through
(1) urocanic acid to (2) formiminoglutamic acidQ The further metabolism of
formiminoglutamic acid has been elucidated as a three step reaction with tetra=>
hydrofolic acid, and the enzymes involved have been purified from liver „ The
formimino TFH compounds have been characterized,, This important vitamin
function has been demonstrated by the excretion of compound (2) in experi<=
mental and clinical folic acid deficiency,, Based on the above, an enzymatic
assay for this compound has been perfected and has been applied clinically
as a guide to antifolic therapy in leukemia and related diseases 0 It has
been further shsaai. that large amounts of formic acid derived from tryptophan
and other sources are excreted in folic deficiency, Picolinic acid has been
shown to be a product of tryptophan metabolism, and the enzyme involved has
been purified from liver 0 The level of this enzyme was found greatly increased
in diabetic animals,, and this level has been shown to b© under hormonal
control, Insulin or adrenalectomy reduces the diabetic levels to normal, while
cortisone elevates them again „ A study of sulfur metabolism in yeast has
revealed several new compounds and new enzymatic reactions s (a) the methyl
thiol ester of 3-phosphoglycerie acid is formed enzymatically from methyl
mercaptan, phosphoglyceric acid and ATP, and this compound is dephosphorylaied
by a specific phosphatase (b) 3~phosphogiycerie acid synthesis from glyceric
acid and ATP (c) S-methyl cysteine from methyl mercaptan and serine0 It is
believed that these findings have biochemical implications of importance 0

tfydroxyiation Mechanisms 0 Using oxygen°l8 it was shown that certain
enzymes activate atmospheric oxygen and incorporate it into organic compounds
(enzymatic oxygen fixation) „ Three new enzymes that were concerned with
oxidation of aromatic compounds were isolated and shown to be "oxygenases"
(pyrocatechase, imidazolacetic oxygenase, kynurenine hydroxylase) 0 Four known
enzymes have been similarly characterized? tryptophan oxygenase, 3 hydroxy-
anthranilic oxygenase, aromatic hydroxylase and lactic oxidative decarboxylase,..

Drug Action,, The effect of drugs which affect nerve conduction, e0g0
cocaine, upon transfer of electrolytes between nerve and surrounding medium
has been studied. It was shown that the effect of such drugs is attributable
to changes in membrane permeability 0

- 22

PHYSICAL blOLOGI

The Laboratory of Physical Biology has the broad interest of relating
various physical aspects of biological processes and states: at times by
analysis of the role of structure and function and again by investigating
the energetics of interaction between the environment and living substance
or its constituents 0 A further interest lies in the relation of structure
to the conversion of chemical to mechanical energy and a fourth category of
interest is an extension of these basic studies to more applied aspects such
as ionic and molecular exchange between the biological entity and its
environment^ falling under the category of physiology0

Spejgjxosoopic Studies a Basic approaches to the knowledge of the highly
complex structure of the components of living matter are being made by various
physical means which in some cases do not alter material under study and
in others produce effects which can be assessed quantitatively „ Thus rela-
tively simple substances such as alcohol have been studied for their infrared
absorption spectra under various conditions of temperature and concentration 0
These conditions permit the demonstration of the fact that the forces causing
aggregations of molecules., l0e0 hydrogen bonding, are not linear in some
substances^, contrary to earlier implications^ and that both temperature and
concentration determine the type of bonding exhibited c In another instance
the structure of molecular complexes and its effect on energy absorption and
transfer is underlay using a chlorophyll»llpo-protein which is available as
a crystalline substance derived from the photosynthetic structures in plant
cells. Ultraviolet light absorption and light scattering modes by such
structures are being studied to find the characteristic detail which is
related to specific absorptive activity,, Problems of absorption spectroscopy
in this field have been made soluble by the determination of the relation of
refractive indices and particle size to anomalous dispersion => an error factor
in measuring specific absorption of various wavelengths of radiant energy0

Photochemistry and Photo-biology,, In connection with these studies the
transient phenomena of photosynthesis which are felt to be a common denomin
in all energy ©xehang© systems in living tissue are being analyzed by specific
cally designed oxygen sensing equipment and by visualization of the structures
known to contain, reactive substances e In other studies it has been shown
that definable structural and molecular changes are effected in substances
absorbing radiation when it is presented in resonant conditions „ The study
of calciferol=propionamide reaction under irradiation has been carried on
with the hope of determining factors favoring compound formation but both ^
infrared spectroscopy and thermal studies show thus far only solid solution0

While the major effect in our research on the effects of radiant energy
on the living substance is on resonant energy and concerned with synthesis
or modification and activation, a growing need for knowledge of the destructive

23

action modes of high energy radiation has been felt, The initiation
of studies is underway,. Studies of destructive radiation have been
cari'ied on for some years in connection with cosmic ray phenomena at
very high altitude and cooperative -work with the military has shown some
correlation between cosmic ray paths and changes in brain tissue „

Transport Phenomena,, In a physical-chemical approach th© behavior of
charged^moTeeular substances passing through highly selectively charged
membranes has been shown to permit ion accumulation which may give th®
basis for the mechanism of ion accumulation in living eellsa The inter-
action of similarly charged ions has teen demonstrated under c onditions
of electrical transport across dense and highly selective membranes 0

Proteins „ further studies in structural aspects of chemical behavior
in bioXogical reactions have been concerned with the sites of activity
on such important substances as the clotting protein, fibrinogen,
whose seemingly minor interaction with thrombin results in fibrin
networks 0 It has been shown that arginine pairs are related to the
selective action of splitting off peptides which permit polymerization
or network forma ti on 0 Diisopropyl phosphofluorldate-thrombin in which
radioactive phosphorus has been incorporated is inactive in clotting
but has been shown to b® rapidly acted upon in the lungs after injec<=
tion into animals in such a manner that the radioactivity is sequestered
there differentially to other tissues 0 It ha® long been known that
the lungs play a very important part in the control of the clotting
mechanism and these observations are expected to yield clues to the
basis for this function,, Other studies of functional changes in biological
structures are being carried out with contractile proteins which, like
fibrinogen in bloody undergo changes in configuration due to relatively
minor changes in structure 0 In the case of muscle, part of the energy-
freed is dissipated as mechanical work0 Studies on the energetics of
muscle fibers indicate that release of energy from adenosine triphosphate
by interaction with muscle is far greater than that actually converted
into work by the muscle fiber on stimulation by this substance „ Studies
of the variations and similarities of contractile proteins among the
various phyla of animals show that, beyond variations in amino acid
content and distribution, th© major differences are due to the
aggregation of subunits in some forms (Tropomyosin B) and their separate
existence in others (Tropomyosin A) „ Various means for the characteris-
ation of the complex structure of these proteins have been applied and
the discovery of carboxy=peptidase~B, a new enzyme, permits the
demonstration of carboxyl end groups on polypeptide chains „ It is
suggested that this enzyme is responsible for the release of the
important dietary amino acid lysine to the body after tryptic digestion
of proteins Basic studies of very simple proteins are also underway,
demonstrating that such a protein as toe protamine, salmine, although by
physical methods a homogeneous protein entity, is actually heterogeneous

21;

in a chemical sense even after special preparation to avoid multiple
origin of the biological product 0 Theoretical considerations derived
from studies on binding reactions of various ion3 to proteins., such as
those from muscle and serum, have been gathered into a general theory
accounting for the binding reactions and charges of proteins in general
and current assessments of this theory have corroborated the scheme 0
Further studies on metallo^protein reactions have shown that anions
affect the binding of zinc to serum albumin^ specifically that thio<=
cyanate as compared to nitrate 5 increases the binding by a factor of $Qn
This implies that the structure of serum albumin is characterized by a
clustering of imidozole groups 0

Extensions of such basic concepts to disease states have been
made by studies of proteins and peptides by specially sensitive chromato-
graphic means from tissues and excreta,, Thus hydro xyproline,, chemically
related to collagen j, and ganima=guanidinobutyric acida a close analogue
of gamma~amino=>butyric acid found in nerve tissues have been assayed
under various conditions 0 Immunocheaistry is similarly being used both
as a laboratory tool and a diagnostic means 0 In rheumatoid arthritis
studies the sensitized sheep cell agglutination (SSCa) factor has been
quantitatively estimated by its hemolytic activity inhibition of sensi<=
tized Newcastle disease virus „ By this method for selection of high
titers^ it was shown that such sera contained two activities associated
with macroglobulins of sedimentation coefficients of 19 or more as pro=
posed by Kunkel and associates,, A simple relationship of activity of
sera for predicting the distribution of sensitised virus between erythri
cytes and SSCA factor has been developed0

Electron Microscopy and X°-ray Diffraction 0 Studies of the mechanism
of virus elaboration have been carried forward by visualization by electron
microscopy of infective particles of vaccinia and it has been shown that
there is a high dependence of resolution and integrity of morphology
on suitable fixation and embedding 0 The involvement of mitochondrial
structures in virus formation is implied in the most detailed visualiza-
tion Efforts to extend the resolution of electron microscopy have
yielded favorably to demonstrations of molecular separations in organic
crystals at the level of 9 Angstroms e i'urther studies intended to
elucidate the experimental basis for such fine detail are now in progress
by means of X-=ray diffraction measurements 0 un the other hand differentia-
ting and molecularly identifying means of visualization of fine structures
are also being carried on by development of soft X°ray microscopy,, Iietail
equal to the best optical magnification have boen attained and can be
expected to improve. Identification of localized accumulations of sub<=
stances exhibiting different degrees of absorption and fluorescence by
X=ray are readily carried out by using various hardness of X~rays0

25

General^ Physiology 0 While effects of environment on a molecular scale
have been studied in various sections of the laboratory other studies in
physiology have developed effects on organ systems and functional behavior n
Thus the effects of hypoxia due to hi^h altitude have been studied in various
phyla of animals up through mammals „ Current studies show that beyond
the expected polycythemia there ar© detrimental reactions increasing the
susceptibility to bacterial endocarditis and also altering the pattern
of the distribution of fatty deposits in the body,, particularly within the
circulating system. Other studies in progress have shown th® adverse
effect of hypoxia on the reproductive tissues and demonstrate that th®
most drastic effects are on various stages of differentiated cells and
the least effect is on undifferentiated tissue <, other studies on altitude
exposure effects have been carried out to analyse the hemodynamic changes
by means of simulated blood volume increases0 Substances which by injec^
tion increase the plasma volume (Dextran and Polyvinylpyrrolidone) cause
changes in capillary permeability and vascular tone varying in magnitude
among different spe des of animals are being studied to compare the effects
on organ functions such as the work of the kidney0

Hespiratory studies in various phyla of animals have been used to
analyse the factors which control access of oxygen to the respiring
tissue and loss of carbon dioxide from it0 A theory of combined dif=
fusion and flow through the smallest respiratory passages has been elabor-
ated and shown to account for the special behavior of respiration in
insects and to relate to some effects in mammal s0 The use of insects to
demonstrate different metabolic pathways for energy supplies to muscle
tissues has shown that the disaccharide trehalose is a counterpart of the
mammalian glycogen and that a specific enzyme controls the in tercon version
of glucose and this product „ The effects of various substances on this
mechanism are expected to shed light on the insulin problem in mammals,. An
extension of ventilatory studies to mammals has been continued in the work
of breathing against various resistances in the external airway and a method
to estimate internal resistance has been suggested by this procedure 0
Measures of the work of breathing indicate that it may form the basis for
a pulmonary function test*

In the course of much of this work in this area of physical biology
it has been apparent that the necessity for predictive methods of great
complexity were developing and required the assistance of staff trained in
mathematical analysis and synthesis of multiple factors 0 luring the course
of the year mathematical research into biological problems was initiated
and has contributed to various problems of ensyme reactions and to the
predictive formulations of complex structures which require electronic com-
puters0 Mathematical consultation has been given to projects in N„0oIOi) N0h0Ic
and NcI0fc0H0 The problems have included considerations of mathematical theory
of tumor induction^, spherical lung models and variations of sedimentation
constants with concentration „ Problems of steady°state kinetics of complex
systems and the general inhibition thereof have been topics of special
mathematical inquiry „

26

CONCLUDING JffiHAKgg

Inextricably associated with the research activities which have bean
reviewed are the various training and educational activities at NoIoAoKoD0
In the American culture, the highest quality of research has generally
been associated with a conscious training function,, There are various
reasons why this should be so0 The presence of young investigators in
training serves to keep th© intellectual milieu of the laboratory young..
Few inature scientists manage to preserve the energy, enthusiasm and dedi-
cation which they exhibited during their training and as a consequence a
very large portion of the most original research is published in doctoral
dissertations „ The very act of teaching, in addition to benefitting the
student, may also benefit the teacher 0

Incidental training of younger scientists has always been a part of
N0IoHo activity 0 All th® clinical associates and the various types of
fellows working in our laboratories and patient areas derive, we hope,
education in a real sense from the more exp©rienced investigators with
whom they are thrown in contact, as well as from each other,, That this
training, though not specifically progransned, is effective is indicated
by the many and flattering outside offers of employment which our junior
scientists receive 0

As of July, 1?£7 , an additional deliberately planned training program
was initiated at N0I0II0 Five of the seven Institutes, MoIoAoMoD0, N0H0I0,
W0IoA0IoDo, w0I0H0H0 and N0I0N0D„BC, are currently participating in this,
the Research Associate Program,, Young clinicians with about two years of
postdoctoral hospital training are brought to these laboratories for a
two°year period, during which time their major responsibility is to secure
training in laboratory research. In a modification of the usual xh0D0
curriculum, each associate is assigned to a preceptor, under whose immediate
guidance he conducts research in a problem carefully selected for its
pedagogic merit e For one or two hours each day he attends lectures on
various topics in basic sciences given jointly by members of the staffs of
all the Institutes,, In addition to teaching staff, N0I0A0M0D, has contribute
positions to this program. Two exceptional young men are currently in
training and two more will be added in i!'0Xa !5°0

Early indications are that this program is very successful 0 Each
position available was sought by 12=»l5 applicants all excellently qualified
and the popularity of the program is growing,, The lecture series, which „
is open to the scientific public, is attended by many scientists who are
not enrolled in the program. The entire project, which is considered highly
e^erimental, is under continual scrutiny and will doubtless undergo many
changes 0 It is hoped by this device to assist in alleviating the ever-preser.

shortage of highly trained clinical investigators^, a shortage in part
attributable to the existence of ova- intra- and extramural programs 0 As
is the case in other research institutes,,, two products are generated?
research^, in the form of published accomplishments^, and men capable of
continuing to do research,, We believe that both of these products ar©
important, that they complement each other., and that the Research Associate
Program is a healthy assumption by K0I0H0 of a part of its educational
responsibility 0

NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES

Annual Project Report

Calendar Tear 195?

Summary Sheet

Arthritis and Rheumatism Branch

Estimated Obligations for FI 3ff$8

Total: $919g939
Direetx $282i,O0O
Reimbursements* §69$$l93&

Projects number 108© thru H8o included 0

MIS-SIB

Individual Project Report

Calendar Year 195?/

Serial Hoo M&$D~ inn^q
la Clinical Investigations

2o Arthritis and Rheumatism
3o Bethesda

F&rt A.-

Project Titles The Bentonit® Floeculation Test in Rheumatoid Arthritis

Principal Investigators Br0 Joseph JQ Buniis

Other Investigators? DrQ Kurt Bloeh

Cooperating Units* MrD John Bosicevich (NIA.ID)

Dr„ Jules Freund (K3AID)
Dto Robert R* Williams (ARB) 98

Man Years (calendar year 1957)8 Patient Days (ealendar year 195?) s
Tetal* 2-1/6 -j,-

Professionals 1
Others 1=1/6

Project Descriptions

Objectives s To evaluate the Bentonit© Floeculation Test (BFT)
as a d£agnostic~a£d in rheumatoid arthritis o

^^gJJLJJggjjg^S JfaJ^-Mg^jggl This t®st originated in the
laboratories @f 3jimunol@gy Section af NIAID and the Arthritis and
Rheuaatlem Branch of HIAJffl in May„ 195? „ Thus far it has been
well reeeivsd in other laboratories in this country and abroado
The test is based on the flocculatien of gamma globulin=e@ated
bentonlte particles by the ^ rheumatoid faster (s)w circulating in
sera 'of patients with rheumatoid arthritis <> The BFT has the
advantage of simplicity,, speed and ease of performance and interpreted
tion as compared to the Sheep Cell AgglutinatiGsi and latex fixation
testa In this test absorption of heterophile agglutinins and
preparation of euglobulin fraction are unnecessary <>

To determine the reliability of BFT as compared to the
euglobulin SCA test;, sera from 85 'patients were subjected to both
testso A very high correlation (9h%) between the results of the
BFT and SCA was found., A third serological test, latex fixation v
test 9 is currently being done on these serac

3h 72 patients with verified rheuaatold arthritis the BFT
was positive Sn 85$<> In 163 ©ontrol patients it was negative In
9U,

Part B included lea £J H© JTJ

Serial too B3IAMD~ 1Q8«C
Bage 2

It was found that storage of sera at 2(fQs $\s and
minus 20®C did not adversely affect the titer of BFTQ Further-
laorej it appeared that storage initially enhanced the BFT titer |
occasionally false negative or doubtful tests became positive
on storage o

Attests were road© to increase the sensitivity of the BIT fey
employing the euglobulin fraction of patients sera0 Euglobulia
fractions prepared by ©eld distilled water dilution ©r cold wat@r~
carbon dioxide pro©ipitatioas increased the sensitivity ©f the
teste, but also increased the tendency t© false positive reactions*
Several sera giving negative BFT with whole sera gave positive
tests with euglobulin fraction „

"False positive" reactions were ©btained in leukemia and
raacreglobulinemia (Kunkel has recently sh©wn that ^rheumatoid
factor3 is a macr ©globulin of approximately MW lj>0009000)D
Although ultracentrifugation studies on the leukeaaie sera have not
been done so far, it is probable that jaaeroglobuliiis are present
in these sera and are responsible for the positive test results^

Attempts to use two commercial garana globulin preparations
©f bovine and percira® ©rigin in place of human gamma globulin^
have been unsuccessful so far0

Significance to HIAMD Researchs The BFTp crSgto&ted at HIHP
is a valuable laboratory aid in the diagnosis of rheumatoid arthritis
and has important advantages over the other serological tests
currently in use0

Proposed Course @f Projects (©) It is proposed to study the
effect on th® BFT of other members of the serum isaeroglobulin group
including the macroglobulin of syphilitic sera, ©f biological false
positive sera and that occurring as a ©@ld agglutinin,,
(b) Further attempts will b® made to increase th© sensitivity
of the testo

(e) Immunologic studies of rheumatoid factor and rabbit antlsera
employing th® Ouchterlcxy gel technique are planned,-,

PKS«NIH
Individual Project Report
Calendar Year 19$1

Serial No0 NlAMD~iQ9<=c

lo Clinical Investigations
2, Arthritis and Rheumatism
3c Bethesda

Part A.

Project Title? Glinieal Trial of Mew Anti^Iaflaassatory Agents

Principal Investigators Drc Joseph JQ Bunim

Other Investigators? Drc Roger LP BXaekj, Br0 Ernest R0 Steon0

Drc Kurt Bloch, Bra John Bavidp and
Dr0 Ralph Ee Peterson

Cooperating Units? There were not aay cooperating units

elsewhere in the Public Health Service

Man Years {calendar year 1957) a Patient Bays (calendar year 195? 5 *
Totals krl/6

Professional! 3-1/3 S-HJ^f

Other i 5/6

Project Description?

ChjectjVQ&j The new anti-inflammatory compounds have been
evaluated with several objectives in viewc.

&q To measure relative antirheumatic potency-

bn To study metabolic and hormonal effect g0

c.-, To detect j, characterize s and assess toxic effects o

do To relate chemical structure to biologic activity o

Methods Employed g Patient Materials The evaluation ©f drugs has
been conducted almost exclusively on an in-patient population,,
Detailed studies of the hormonal,, metabolic g and clinical effects
of the various agents have been performed0 The methods employed

have been described in previous reportSo

Part B included Yes MJ *® £3

Serial Noe M^ffiglQ9=C __
Rig© 2

The following confounds have been studiedg

A„ The synthetic 112 oxygenated steroids
Prednisone
Prednisolone

Triamcinolone (l6ae21 desosy=>5>a fluoro prednisolea©)
21 desoxy~9& fluoro prednisolone
21 desexy<=9<a921 defluoro hydrocortisone
21 desoxy 21 fluoro prednisolone
21 dQsoxy='9aj)21 difluoro prednisolone
16a methyl prednisone (MK~1G9)

B, Phenylbutazone derivatives
G~33

Q«2?202

0o Ghlorequin

Ma.1@r Findings 8 Prednisone and prednisolone have now been
evaluated in a group of £9 patients p each receiving the minimum
maintenance dose of the drugj, only two requiring more than 1$ sagsns
dai3y0 On such a regimen ? @f these Hi are enjoying full
functional capacity 8 2 others only minimally handicappedo Only
1 of the remaining 5 ipassaina confined to a bed^hair ©xisten©©,-,
Side effects have been a problem in 26 of the 59 patients and
include 2k peptic uleers* 9 fractures^ diabetes in 29 severe
infections in 20 and mental changes in 7«

Triamcinolone was evaluated in a total of h rheumatoid patients
Metabolic studies revealed sodium diuresis and slight potassium
retention on a 20 mgm daily dose0 This drug was effective as an
antirheumatic agent with about the same potency , mgm for mgm as
prednisone,, Side effects included severe sweating in 3 patients
and epigastric pain (no ulceration) in onec

A group of h synthetic steroids^, characterised by loss of
the oxygen atom at 21 position and fluorinatsd at various sites
(refer to list itemised under k)s were found to have potent
pituitary suppressive effects at doses of E>0 to 1!?0 mgms daily 0
The alteration at the 21 position apparently overcame the mineralo^
e<§a?ticoid effeots of fluorinated compounds studied previ@ra@ly0
Only 2 of these "21--desoxyw compounds produced sodium retention
and this only to a mild degree 0 Three of these compounds had slight
antirheumatic properties at the dosage employedc.

Metabolic studies of S=6a msthyl prednisone in daily doges of
10 mg revealed no sodium retention^, n© nitrogen or potassium lossn
a potent pituitary suppression^ and antirheumatic properties equivalent
to that of prednisone Con weight basis) in 9 patients o One patient

Serial No. NIaMD~iQ3sQ_
Rage 3

developed a gastric ulcer on this abdication and another showed
slight edema0 No other serious side effects were demonstrated,.

In clinical trials with phenylbutazone derivatives (G=>33 asad
G-272G2) it was found that 2 rheumatoid arthritis patients taking
G=272G2 showed slight decrease in pain and swelling „ Four others
©n this drug were not improved and no <§ne taking G-33 improvedo
The erythrocyte sedimentation rate failed to decrease; ;in any
patieato On© patient taking G=2?202 developed skin eruption and
another decreasing white bl©@d eount0

Chloroquin has been administ<sre€s in a 2^0 tsgm daily dose
t© h patients,, Qae discontinued the drug after 1 month because
©f abdominal pains which disappeared shortly afterwards One other
patientB although showing no toxicity, discontinued therapy after
2 months o No patient has experienced significant relief while
under this form of therapy,.

Serial Mo„ NIAJ®:
Page k

Part Bfj Honors , Awards , and Publications

Publications other than abstracts from this projects

le Bunim, Jo J,, and Black* Hr LC; Connective Tissue (Collagen)
Diseases, AnaJey^jfJfed^ 8s389, 195?o

2a Black;, RG Lng Yielding^ Ka L0ff and Bunim0 J» J0 Observations
on New Antirheumatic Steroids and Critical Evaluation of
Prednis@ne Therapy in Rheumatoid Arthritis 0 JpOhronoDiSo n
5s?5l„ 1957 o

3* Spicknallp C„ G0, Bozicevich, Jos Black, RQ Lot and
Terry B L8 L„ The Complement Fiction Test in the
Diagnosis of AraabiasiSc Gastroenterology » 36sll31j> 195? •

1»0 Rodnan, G0 P„8 Sehreingrj, G0 E0J and Blasks Ra L0
Rsnal invols?em8»t in Progressive Systemic Sclerosis
(Generalised Scleroderma) „ AnoJ0Med«, 23%WxS8 1957 0

5o Bunim, Jo Jo Clinical Uses and Hazards of the Adrenal
Hormones and Their Analogues in the Management ©f
Rheumatic Diseaseso Bull,, of NvY„Aead,;Qf Medt,n 338k6lfi 195? o

60 Bunime JD Jo, Editor => Proceedings - Second National
Conference on Research and Education in the Rheumatic
Diseases o McGregor and Werner s InCoS Washington, D„ Cos 195? -.

To Bunimj, Jc J0, Editor = Introductory Statement to Symposium
on Rheumatoid Arthritis „ JnChron0Diaj. 5:609s 1957 „

80 Bunim, Jo Jc, and Shulraan, L0 Disorders of the Joints,
Chapter in 3rd Edc of Principles of Internal Medicine^
Tc R0 Harrison, Editor The Blakiston Company, Ineog
New York (in press )<,

9o Bosieevichj, J., Bunirae Jo Jo, Freundg Jop and Ward , S0 B„
Bentonite Floeeulation Test for Rheumatoid Arthritis o
Proe.SoefExDtl.Biol0&Medo (in press )„

100 Ebaughp F„ G.5 Jr0, Peterson, Rc E„, Bunim, J0 JD, and

Rodnan, G„ Pc The &tsEsslf\ ®£ Rheumatoid Arthritis P (in press),-:

Honors and Awards?

Dr0 Jo Jo Bunim was elected First Vice-President of the
American Rheumatism Association for the year 1957 to 1958
and President for 1958 to 1959o

PSS<rJIH

Individual Project Report
Calendar Tear I^S?

Serial I©0 ESIAICU 11Q~G
io Clinical Investigations
2Q Arthritis and Rheuisatlsjn
3o Bethesda

Project Title? Metabolism of Adrenal Steroids in Man and Anisal

Principal Investigators DrQ Ralph Eo Peterson

Other Investigators? Charles Pi®rces Janet Sehuck,

Margaret Bollierj, George iotoss
Drso Bernard iiiEjaa,, William Zorfcaei^
Roger L0 Blacky and Joseph Jc. Bunisi

Cooperating Units? Dr0 Bcsaald Sa Fredriekson (SHI)

Dr0 Jamas 0o Davis (MI)
Dr0 William Wc Miner (HCI)
Dr„ Rudi Sehs&d (NIAMD)

Man Years (calendar year 195?) i Patient Bays (calendar year 1957) i
Totals 6 ,0_,0

Professionals 3 1252

Others 3

Project Descriptions

Objectives s Disposition^, sset&bolic fates and rate of synthesis
of adrenal steroids in masi0

.MthodgJ^DJfflr"^; Maajor Findings s

Development of Methods for Isolation and Assay of Steroidss
A great deal of tia» and effort has been devoted this past year

to the develepassnt of a news very sensitive and specific method for
the assay of steroids 0 The Kathode include us© of tritium and radio
carbon labeled acetates of the aeetyl&ble steroids o A considerable
number of new paper ehrosaatogr&phie methods have been designed for
the isolation and purification of th© steroids „ In addition,, mm
column chromatographic methods have been developed for th© Isolation
and purification of th© steroids in biological sis-tares 0 Th©
development of these methods has sssd© it possible to extend ®ur
studies on the Esstabolism of th® steroids in both man aed aniajsis
to areas previously not readily accessible to study

Part B included Xes JFJ M@ £jj/

Serial l%o0 HI&MD. raw* _

Pag® 2

Metabolism of Aldosterones. As th© result of the deva!
of a very sensitive asssy for aldosterone,, and the preparation of high
activity tritium labeled aldosterone (101 pe/sxg) it is mm feasible
to study the physiological disposition and metabolic fats of aldo-
sterone in aan0 These studies are nos? in progress,, Furthermore ,
through the use of these same tools it say be feasible t© usasure th©
rate of secretion of aldosteron® by th® adrenal cortices „ and an
attempt at this is now being Hade„ This involves an entirely &®w
technique for measuring the miseibl© pool and turnover rat© of a
coBpound jn viyo0 In collaboration with DrQ Jamas Davis studies are
being carried out9 in d©gss on th® factors controlling the secretion
©f aldosterone by the adrenals , These studies entail the us® ©f
dogs with hyperaldosterenemia resulting from constriction ©f the
thoraeie vena eava$ and measurements of adrenal vein plasma &M©=
steron® with th© new isotope derivative method0 These studies to
date seem to indicate that th® liver ,> through either a hUKoral ©r
neurogenic stimuli* plays an important role in the regulation of
aldosterone secretin The extracellular fluid volume does not
appear to be a prime regular ©f aMosteroaa secretiono

Metabolism of. Corticosteronet, Turnover rate studies in
noraal subjects have demonstrated that the eorticosteron® miscibl®
pool is 200 to 300 pgs and that the pool is turned over about
10 tisses per day0 AGTH administration produces a proportionally
greater increase in th© rat® of turnover ©f eortieosteron© than of
hydrocortisone o In liver disease there appears to be some moderate
decrease in the rate of turnover of corticostsroa®0 It has been
possible to use high activity tritium labeled ©orticostef one for
the turnover rat© studieso However s through th® cooperation of
Dr0 Roebert StearmWj, ©n experimental saodel was developed for
ssaasuriag th® turnover rat® with much larger than trace quantities
<s£ ©arbon<=lU labeled e©rtic©steron@0 This procedure gm® results
that compared favorably with th® tritium 2s©thod wing th® conven-
tional trace quantities of labeled e@rticost©rone0 Bata collected
to th® present have established that the tritium labeled steroids
are metabolized in the same Banner as the earb©n=>lU labeled steroids0

Salicylate Aetion and Adrenocortical Functioni The short
term administration of therapeutic doses (3 to 6 gms) of salicylates
ha® not been found to alter the rat® of secretion of hydrocortisone
by th® adrenals 0 Following salicylate administration the urine
steroid excretion (corticosteroid and 17<4tetost«roids)« plasms,
steroid Isvels (hydrocortisone and corticosteroids and rate of
turnover of hydrocortisone were found to be unehanged0 Furthermore,}
no ©hang® was observed in the metabolism of infused hydrocortisone
@r c©rtieesteroas0 Preliminary data indicate that long term
administration of larger quantities (6 to 7 gas) ©f salicylate nay
actually prepuce sob® slight depression of adrenocortical function*,
Alsos the salicylates produce their amti=>toflaasjat©ry effects in
the absence of asy adrenal cortical secretion ©f hydrocortisone 0

Serial Hoa KIAMD^lift.c
Pag© 3

S«!esL^JBgtefig»Be-qn . Corticosteroid Motebpligmi. Administration
©f estrogen© (ethynyl estradiol 0„5 sag/Say) causes'. a too to three
fold increase in the plasma levels of hydrocortisone and eortiso=
st©ron©0 This is associated with a gradual fall in the urine
corticosteroid excretion, and a delay in the rats of metabolism of
infused hydrocortisone and eorticosterene,, In addition, a
significant decrease in the rat© of synthesis of hydrocortisone and
csrticosteron® occurs as measured by turnover rate studies 0 The
■wry high plasma hydrocortisone levels may tee maintained for sany
weeks with estrogen administration? however, these subjects do not
show any signs of Cushings syndroms 0 Thus, estrogens appear to
interf®r®2 in seas® ssanner, with the biological action of hydr©=
©ertison® in the- tissues,, Also, the anti*4nflammat©ry activity of
therapeutic doses of hydrocortisone appears to be interfered with

estrogen is administered to subjects with rheumatoid arthritis 0

Effect of Agphenone on Adrenal Steroid Secretions In collabora-
tion with Drc William Tullner studies have been carried out on the
effect @f ampfoenon© on the secretion of various adrenal @@rtical
steroids in different species of animaiSo These studies have
involved measurements of adrenal vein plasma steroids by the isotope
derivative method,. The studies to date show that amphenon® say
produce different effects in different aniatals, and that the
synthesis of the 17-hydrosy corticosteroids seems to b© more readily
inhibited by amphenon© than the 17-desQsysteroids such as
aldosterone, eortieosterone, and d©sQxyeertieost@ron©0

Effect of Gholestenone onjldrenal Steroid Secretions In
collaboration with DrQ Donald fredriekson studies have been
initiated on the influence of cholestenone on the synthesis of adrenal
steroids in vlvan In th® rat fed cholestenone a asarked depression
of adrenal function results with an associated adrenal hypertrophy 0
The adrenal cholesterol;, a precursor of adrenal steroids, is
replaced by dihydr ©cholesterol, a satabolit© of cholestenone 0 The
adrenal vein plasma of th© cholestenone fed rats shows a marked
depression in the levels of eortioosterea®, aldosterone, and
hydrocortisone, . as asasured by th© isotope derivative 'method,, It
has -net been possible to demonstrate any alteration in adrenal
function in patients fed ©holes tenon® <,

Steroid Glucuronjde Formations Studies have been performed
in collaboration with DrQ Rudi Schmid on a patient with congenital
non<=>herao3ytie, nonobstructive jaundice. This patient, in addition
to the defect in bilirubin glueuronid® formation, showed an iapai&sd
eepecity t© conjugate certain steroids with glucuronic aeidD
Attempts have been made t® study steroid glu©ur@nide formation
©mymati<sal2y jn vitro but without su©@ess0 Although it has been
possible to form giucuronides of various csrapotaads eneyssstieally, in
<®ur laboratory it has not been possible to form th® steroid
glucuronidase This suggests that steroid glucuroaide formation
may involve different ©msymeg or eo^eagymeso

Serial lo„ MIAMD- HQ»c _

■ :

Syjafchegja of Steroids and Steroid Derivative.^ Attempts have

3 of th® analogs said <

been sad© to synthesis® various of th® analogs sad derivatives of
th® steroids for use in studios ©f their meit&bellsno Attssapts were
n&d® to synthesis© 2G=>hydr©sy derivatives of hydrocortisone but
to date tSsss© have been unsuccessful A water soluble derivative
©f d®0sye©rtie©st©s,on© was prepared for clinical us©„ Various
derivatives of steroid acetates kv© bean prepared for possible
use in th© isotope derivative isethodo Work is now in progress
©n the synthesis of the 21=3B3thyl ether of hydrocortisone using
an improved raathodef synthesis developed for 21<=*G8theKff3rogest@r©n®0
Studies are now in progress on the synthesis of cortioold
21=.glye©sides0 The synthesis of blocked l^&alides of 2=de©syglue@e©g
2<=de®»3rallose and 2j,6^dideo2yallos© are being investigated as
possible intermediates in the glyeosidation ©f the steroids0 A new
rout® to 21-=glueuronid®s and glye©sideg has been investigatedo
This has involved tritylation of the 21=p@sltion and then treating
with a blocked l~hal© sugar in th© presence of silver perchlorat©0

Prgpojed Course of ^o^^L

lo The possible physiological significance of th© protein
bound- and free hydr©c@rtisone in th© plassa warrants additional
study©

20 Additional data will be collected on the nstabolism and

physiological disposition of aldosterone in rcaae especially studies
©f aldosterone turnover rate0

3o Further w@rk will be den® on the aeehanism of the
secretion ©f aldosterone using the intact dog0

ilk Th© nature of the aetabolic defect responsible for
suppression ©f adrenal steroid synthesis in the rat fed

cholestenons needs further clarification,,

So Further work should be don© of perfecting and extending
the usefulness of the isotope derivative aaethod for assay of '
steroidso This will include the synthesis of additional analogs
and derivatives @f th© aeetates0

60 Further work on the synthesis ©f 21-»thyl others ©f the
eee'tieoids should be carried onto Also, -fee synthesis of th®
21=glyeoaides of various corticoids will be pursued as these
water soluble derivatives say have interesting pharmacological
aetivity0

Serial I@0 MIflHD~no°C

Pag® 5

Fart B„ 8 Honors, Awards,, and Publications

Publications ether than abstracts from this projects

lo Peterson,, RD Ec, Karrerp A02 and G?zarras S„ Le Evaluation of

the Silber<=Port®r Procedure for th© Determination of Plasma
%drceertl3©ne0 ^SilaHHfea 29!lW», 1907 e

20 Peterson,, R„ E0 The Identification of Corticosteroid in Emms.
Plassaa and Its Assay by Isotope Dilution* J.Biol.Chea. ,
225s25, 19S7o

3o Petersen,, R. Eop Herts,, H„, and Lvfo@B HQ A0 Suppression ©f
Biosynthesis of Adrenal Cortical Steroids in Man by
m0 aroc.Soc.ftro«r;JloL,«fedU. 94sla21s 1957o

Ijo. Petersen, R0 EOB Fierce^ 0o EOJI Wyagaardea, J0 B<,B Bunisa, j0 J»g
and Brodie, B<> Ba Th® Physiological Disposition ■ and M©tab©lie
Fata of Cortisone in Haa0 J.Clla, Sweet.. 36:1301, 1957 o

So Petersen, R0 £«, Pierce, C." E0, &ad Slioan, B0 Isolation- of
^Progneas^UB^lTSpSCagSl^TstroI^^on® from Urine of Mano
Areh.Blochea.aBiophye^ 70»6% 2957o

6C Peterson, R„ E„ %p©pltnitarisms Clinisal^Pathologieal
Conference at the National Institutes ©f Healtho Aann ®f
InbJledo. li6sHo06s 1957 o

To Peterson, Re E0 Plasisa Cortieoaterons and Hydroeortisongi
Levels in Man. JoCltoeEndoaafetabo. 17 s 1X50, 19?? ,

80 Peterson, Rc E., and Schmid, RQ A Clinical Syndrom© Associated
with a Defect in Steroid 8Xucurcaide Fonaati©n0 JaClin0Endo.,&
letabo, 17sDeeember, 195?0

9o Pet®rs©ns Rc E0 The Influence of th© Thyroid en Adrenal Cortical
Function* J,Clia„Invegt0 , (to press )0

10o Peterson, R0 Eos Black, R0 L0, and Bunias, J„ J0 Salicylate© and
Adrenocortical Function in Man0 Arthritis&RheuiBatiam (impress) 0

Honors and Asrardss

DrD Ralph E0 Peterson isas elscted to aubsrsltip in the
American Scciety for Clinical Insrestigatioaso

Calendar fear 19^7

Serial So.,_NIAi-33~ X11~C
lo Olinio&l Investigations
20 Arthritis and Rheumatism
3o Bsthesda

Part A„

Project Titles Study of Familial Generalized Osteoarthritis

Frincipal Investigators Dr0 Roger La Black

Other Investigators s None in NIAMD

Cooperating Units; Dr0 Carl Hitkop (NIDR)

Man Tears (calendar year 195?) s Patient Days (calendar year 195?):
Totals 1/3 Entirely an out=patient study

Professionals 1/|
Others

Project Descriptions

Objectives 8 Since very little is known about the cause,-:
predisposing factors or even the course of osteoarthritis s a
detailed study of a large family having a high prevalence of
osteoarthritis was begun in order to learn something of the

genetic factors involved 0

Methods Employed and Patient Materials A family of &%
individuals is being evaluated for the occurrence of generalised
osteoarthritis o This family is a fairly stable group j, resides in
a small geographic area, and exhibits some degree of in°breeding„
Previous information revealed a peculiar incidence of "back
trouble** and a preliminary survey showed severe generalised
osteoarthritis with obesity and short stature in a proportion of
individuals o Factors of gene tics „ obesity „ and the occurrence
of generalized osteoarthritis are being evaluated,?

Ma;jor Findings 8 Although no patients under this project
have been admitted to the NIH, t® date $2 individuals have been
examined^ and pertinent laboratory work obtained,.

These patients may be divided into age groups as follows?

A) Under 20 = 2h = (11 males and 13 females)

B) Between 20 and 39 = 16 <=■ (5 males and 11 females)

C) Between I4O and 79 - 12 => ( h males and 8 females)

Part B included Yes / 7

Serial H©„ NIAMD ■=> m-C
page 2

None in Group A were found to have osteoarthritis D It is
of interest that 6 of the 16 in Group B had symptomatic
osteoarthritis of the spin®s bat no peripheral joint involvement,,
All 12 patients in Group C had symptomatic osteoarthritis of
the spin®s and 10 of ths 12 had involvement of psripheral joints
as w®llo

Significance to IIAMD Research &ad Proposed Course of Projects
Since osteoarthritis is sueh a common disease, it is important
that the NIAMD have an interest in soma of the facets of this
problem,,

It is anticipated more members of this family will be
investigated and the pattern of occurrence of generalised osteo°
arthritis batter identified,. Detailed evaluation of endocrine
and metabolic functions may be desirable s involving in-patient
studies at a later date* It is hoped that a better understanding
of osteoarthritis may be achieved^

PUBLISH

Individual Project Report

Calendar Year 195?

Serial No,, NIAMD I3_p<u;
Clinical Investigations
Arthritis & Rheumatism

Bethesda

Part A

PROJECT TITLES Studies on Alcaptonuria In Wan

PRINCIPAL INVESTIGATOR : Bert N, La Duff J. F. SeegmUler

OTHFR INVESTIGATORS s ¥6 Zannoni, Le Laster, J. MeGuire, Fe Love

C00PFRATIM3 UNITS* None

MAN IFARS (Calendar lear 1957): PATXFNT DAIS (Calendar

Totals >2/3 let? 1957):

Professionals l»2/3 -a^

Others 2 . " OJ

■ PROJFCT DFSCRIPTION:

Our aims in studying patients with aleaptonuria have been
several: (1) to determine the exact nature of the metabolic de»
feet in this condition! (2) to study the hereditary pattern of
this disease^ ands if possible^ to develop a test which will detect
the heterozygous state in relatives of alcaptonurics carrying the
trait? (3) to study the formation and deposition of the pigment
derived from horeogentlsie acid and how it produces the pathological
changes in the connective tissuess particularly the joints? (h) to
study the cause of the arthritis nearly always associated with this
condition^ and (5) to attempt various means of treatment of this
Metabolic disease *

Nature of Defect to A quantitative assay of each of the enzymes
involved"" is tyrosine degradation was made in liver horaogeaates
(liver biopsy samples) of alcaptonuric and non»aleaptonurie patients t
No significant differences were foussS in the activities of any of
the ensyirses except for HGi oxidase. This activity was high in the
mn^aleaptonurie liver and undetectable in the alcaptonuric liver 0
Further experiments indicated that this lack of activity was not
due to the presence of an inhibitor nor the lack of a cofactor© It
is concluded that the defect in aleaptonuria is a failure to syn-
thesis© liver- EGA oxidase*,

The liver enzyme which catalysas the formation of homogentisic

acid (HSA) from p^hydro^pheiTylpyruvic acM (HIPP) has been purified
over 300 fold from dog liver and some of Its characteristics have

Serial Nb. jgAED..13g5C,
Page 2

been studied e The need foira Vitamin C can be completely replaced
by 2s6~&ichl©:rophenolindophenol dye (296=>DGPP) in the purified
systera and the determination of the role of these agents in this
oxidation are wider investigation,, A need for catalase was dem**
onstrated in the purified system aM its action appears to be "bo
protect -Hie ensyme from hydrogen peroxide arising from the oxidation
of PHPP rather than to take part as a peroxidase,, Further studies
on the native of the .purified' ensyme in regard to metal components
and essential^ulfhydryl groups,, are under investigation. Kris en=»
syme system in human tissue was foujgi" to "'be- similar" to' that in
animal tissue, including the effect of activators and iiahibiters,,

Part B included s YES

Serial Ko9 MIAMD ug-G
Pag® 3

Fart B? Itoaorsp Awards s and Pablieatiens

1«, La T)us B» NB, Zanr«oni9 V., Lastera L8J> and Reegniiller, J„ fi„
The nature of the defect in tyrosine metabolism in aleapton-
uria„ Jour, of Biol, Chem, In press 0

2e Btmira, J., McGuire, J.„ KHbish, T. F„ Laeter., LeJ La Bus
B„I„ Seegraillers J. E„ Clinical Staff Conference on
Alcaptonuria,, Ann. of Int. I'fed0 In press«

..dual Pro
Calendar Tear 195?

Serial NoQ JK3AMDJQ.>>C
Clinical Investlgat i - i
Arthritis and Rhe
Bethesda

Part A

PROJECT TITLEs Studies of the Metabolism of Uric Acid Riboside

PRINCIPAL INVESTIGATE s Leonard Laster

OTHER INVESTIGATOR: Alberta Blair

COOPERATING UNITS;

Man Years (Calendar year 195?): PATIEW QAXSs (Calendar fear X95?)s
Totals 1-3/3
Professionals 1/3

PROJECT DESCRIPTIONS

In order to determine whether uric acid riboside,, which occurs in
human red blood cells s is involved in uric acid metabolism in the
humans the metabolism of this substance was studiedo

We found an ensyme that catalyses the reactions

(1) Uric acid riboside 3BSSBS3& fflgBteSfr Uric acid * .j.

An analogous ensyme 9 purine nucleoside phosph©rylases has been shown
to catalyze the reversible reaction:

(2) Purine riboside * Inorganic pho3phat©<s5=3i Purine * Ribose°l~

We have not yet been able to prove that "X" in reaction (1) is rihoss-1-
phosphate^ nor have we been ;able to detect the biosynthesis of uric
acid riboside when ensyme s uric acidr, and ribose~l=phosphste are
incubated under the same conditions required for reaction (l)0 It is -
possible that the enzymes for (l) and (2) are not the same arid ti
the reaction mechanisms are also different,,

We have purified uric acid ribosidase from several sources and
have investigated its properties., Because our substrate was isolated
(making use of a chromatographic procedure we developed) from beef
red blood cellss we first studied the enzjms in beef liver0 Although
our best preparations s ten^fold purifications s... still contained nucleo=
side phosphorylase activity j & highly purified preparation' of nuclei

Sggialjjp^ ;^;^2:SM>n

side phosphorylas© from beef liver contained no uric acid ribosie
activity „ Moreover ^ although we found the ®qw.ilibri.«ffi for reaction
(l) t© favor synthesis very strongly s we could not demonstrate sya~
thesis with rih©s©~l=>pfa©sphate0 ^his evidence gives us reason to
believe a new enzyme is involved o

The opportunity t© obtain fresh human tissue arose when a spiea=
©etoeijv was perfozned 'on a patient with congenital hemolytic jaundite©
in our institute o The enayme was detected in extracts ©f acetone
powder preparations of this tissue and it was purified five~foldo
Splenic tissue from other patients (with hyperspleaisira) sis® contai
urie acid ribosidase activity 0 The properties of the ©myme differed
in masy respects from those of nucleoside phosphorylase of human red
blood ©ells and the enssyroes appear to be dissimilar o Other ribosides 3
©specially those of eytosine and uracils inhibit urie acid riboside
and this finding my have implications regarding the function of pyri-
saidi&gs in control of urate metabolism

Seeking more active sources of ensyme we surveyed other organs and
other animalso The following distribution was found in the rats

Organ

A@tivi%

Heart

k*

Kidney

!■♦

Intestinal mucosa

3*

Liver

2*

Huscle

1*

Lung

0

Testis

0

We ar® now-purifying the ensyme from rat ..and dog intestinal mucosa
and have a preparation pure snoiigh for studying" the reaction mechanism ..
but highly unstable o We are attempting to. stabilise this preparation
for future usec The high levels of activity of this ensya© in kidney
and intestine raise in our minds, the question ©f whether it is involved
in the transport of uric acid across mesiDraneso ^e hope to investigate
this possibility in the f utureQ

W© plan t© study the possible conversion of urie aeid riboside to
the ribetid© by phosphorylations or the conversion of uric acid to
the ribotid© by reaction with phosphor ibosylpyrophosphateo Thereafter
we plan t© study these enzymes in noraal humans and in those with
abaomalities of purine metabolism

Part B included! No

his <= n:

:idml Projsc

Year 19$7

Serial Noc SIMP llt^C
Clinical Investigations
Arthritis & Rheumatism
Befhesda

Part I

PBDJECT TITLES Metabolic and Therapeutic Studies of Gouty
Arthritis and Hyperuricesiiao

PRINCIPAL INVESTIGATORS s Jo E. Seegmillerj, Leonard Laster*

OTHER INVESTIGATORS t K0 Lenone Yielding, Joseph McGuire,
Lois Liddls5 Ethel Low*

COOPERATING UNITS?

MAN YEARS (Calendar Year 1957) t PATIENT DAIS (Calendar

Totals 2-1/3 Year 19$'?) :

Professionals 1 *>,»(>

Other* IM/3 ^

P30JECT DESCRIPTIONS

For the past two and one half years , we have studied the seriM
urate levels and urinary uric acid excretion of 38 patients with
various stages of gout while receiving a constant dieto W© have
found that until severe renal impairment appears^, the average value
for urinary uric acid eaxsretioa by gouty patients is not eigni£i<=
eantly different from nine normal controls studied,, Patients with
tophaceous gout and sever© renal disease excreted about half ta©
normal amount of uric acldo

We found that the new uricosuric agents G«283l59 & sulf©3d.de
analog of phenylbutazone, improved the treatment of three o^t of
five patients with tophaceous gout sad sever® renal isapairaisait who
have not previously responded satis factorily to probenecid ©r
so dim salicylate administratione

A systematic ©valuation of the effectiveness of intravenously
administered colchicine for treatment of acute gout in 1*0 oases
has hmti concluded,, Undesirable side effects observed ware a
febrile response in on© patient which, may have been attributable
to this BJ©dications a frequently observed local irritation at the
site of injection,,, an occasional transient drop in polymorph® <=,
nuclear leukocytes and a temporary elevation in BSP retention which
occurred in a few of the patients treated « Howaver9 no lasting or
serious 'toxic effects were encountered in our entire series*. In
the majority of patients intravenous colchicine .produced relief of

Serial Mo0 MIMDJJJ^

the acute gputy attack sj©re promptly and with fewer gastrointestinal
sjRaptoms than ©rally administered colchicine <>

An unusual patient with hypouriGssda (sensE urate labels as
low as Oolj iag$) was studied wife renal clearances to 2bbf& whether
©eeretian of urie acid by r enal tubules can b© d emonstrated ia th©
human.. No clear-cut e-eidene® of tubular s@cr®tioni®8 founde

Part B included i Ho

H3-NIH

Individual Project Report

Calendar Year ±9$J

Serial Noo NIAMD Hg-C
Clinical Investigations
Arthritis 4 Rheumatism

Bethesda

Part A

PROJECT TITLE: Studies on the Mechanism of Action of Steroid
Hormones „

PRINCIPAL INVESTIGATORS Gordon Tomkins

OTHER INVESTIGATORS ; K„ Lemone Yielding* L„ Eo Le©

COOPERATING UNITS s

MAN YEARS (Calendar Year 19$7 ) s PAIXJiKT DAIS (Caltmtiar
Totals 1 lea? 195?) i None

Professionals 1
Others

PROJECT DESCRIPTIONS

In connection with a study of the biosynthesis of hyaluronic
acid (see 195>6 annual report) <, it was found that several adrenal
corticoids may inhibit the entry of glucose into the synovial tissue
cells o In order to investigate this phenomenon more closely s liver
tissue was also studied and5 instead of the expected inhibition,,
a marked stimulation of the conversion of gluCose^C^- to C^K)2
resulted from the inclusion of ^1 hydrocortisone (prednisolone)
in a reaction mixture with either liver slices or homogenates0 To
our knowledge this represents the first stimulatory activity of a
corticosteroid in a cell=>free tissue preparatibho Further studies
indicated that the stimulation was not as a result of increasing
the rate of the hexokinase reaction^ nor was it at the level of
the Krebs cycle c The mechanics of the effect is as yet unknown ^
but -it is under investigation o

Similarly s an inhibition of pyruvate oxidation catalyzed by
liver mitochondria has been observed when the incubation is carried
out in the presence of adrenal corticoids o This could serve as a
biochemical explanation for the elevated blood pyruvate level in
patients treated with steroids or with Gushing 8s disease,. The
mechanism of this effect is likewise being studied o Several features
of these experiments should be noted 0

-Serial K60 KZfiMD 115
Page 2

1) The steroids display the same structural specificity in vitro i
as in Jivo., i0e0j> the hydrocortisone derivatives are active while
the cortisone derivatives are note

2) These effects are produced by th© addition of steroid to a
cell-free extract in vitro and not as the result of pre^treatment <£
the animals « For these reasons^, the present investigations seem
especially promising,,

Part P included s Yes

Serial Mbo MIAHD il£-C

PggfrJB

PUBUGATIOKSa

Helding, Ko L»9 Tomklns* Go H.s Bunisij, J, J03 Synthesis of
Hyaluronic Acid foy Human Synovial Tissue Slices 0 Science «, 12£.
1300 only, 1957,

FHS-HIH

Individual Project Report
Calendar Tear 1957

Serial Ho0 NIAM) 116~C
Clinical Investigations
Arthritis & Rheumatism

Part A

PROJECT TITLE? Studies on the Enzymatic Metabolism of Steroids

PRINCIPAL INVESTIGATORS Gordon Tomkins* M.D.

OTHER I WESTI GATORS s Joseph McGuLre, A. Donald Merritt, Jean Curranf

Patricia Michael., Robert So Adelstein

COOPERATING UNITS :

MAN YEARS (Calendar Tear 1957) : PATIENT DAIS (Calendar

Totals 3»2/3 Year 1957) s

Professionals 1-1/3 "om
Others 2-1/3

PROJECT DESCRIPTIONS

Ao Steroid Hydroxylation

The enzymatic mechanism of steroid ll^=hydrosylation
have continued -with the following advances s

1) A new heat°stabile coenzyme necessary for the hydroxy^
lation reaction has been found in liver s placenta, adrenal
and testis o Preliminary studies suggest that it is not &
compound previously implicated in biochemical reactions and
its isolation and characterisation are in progress at this
writingo

2) Evidence has accumulated that 3 separate enzymes are
required for hydroxylatlon and each of these are in the
process of purification and individual study0 Preliminary
data indicate that one of them may be obtained in crystalline
form from adrenal mitochondria0

Serial NOs MAMD 116-C
Page 2 — — —

3) Modifiers of physiological and pharmacological significance
have been studied and it has been found that ions are inhib-
itory in the hydroxylation reaction „ Since aldosterone , the
salt retaining steroid^ is n^hydroxylated,, the implications
of this finding for the homeostatic regulation of intra-
cellular ionic strength is apparent o

The adrenolytic drug^, amphenone^ and a host of other aro~

matic compounds have been found to inhibit the hydroxylation

It has also been found that digLtonins in a "whole series
of surface active agents tested^ can stimulate the reaction^
but the same was not true for the cardiac~active glycosides 0

k) Studies have been started, on the mechanism of the hydroxy-
lation reaction in fungi /but these are still in a very
primitive state 0 Several experiments suggest that it is
possible to demonstrate the hydroxylation reaction on cell=>
free mold extracts and that the enzymes may be substrate*
inducible o

Bo Steroid Reduction

Further work on the reduction of ring A of the steroid
nucleus has suggested that the reaction mays in facfs be
catalyzed by a flavin-conbaining enzyme^ In contrast to our
earlier findings^ it has now been found possible to substi-
tute dithionite and safranine=T for TPNH as the enzymatically
active reducing agento

Furthermore^, it has been found that liver homogenates pre=
pared from thyroloxic rats are capable of catalyzing mush faster
steroid reduction than comparable preparations from normal
animals «, The possibility that this difference is the result of
a relative TP3SH excess in the thyroloxic animals has been ruled
outo The increase in activity has been traced to the microsomal
fraction and a study of the properties of the "thyroxine ^induced"
enzyme(s) is a matter of current Investigation*.

Co Studies on a Cyclic Alcohol Dehydrogenase

The purification and properties of a pyridine nucleotide-
linked enzyme from mammalian liver which catalyzes the reversible
oxidation of cyclic $s 6^ and T^oarbon alcohols to their corres-
ponding ketones are being presently investigated,, The test
system is the cyclohexanol»cyclohexanone couple s but the struc-
tural relationship between these compounds is prompting this
invest! gation o

Part B included - Yes

Serial Mb0 MIAMD 316=
Page

Part B

PUBLICATIONS s

Tomkinsj, Go Mo, Enzymatic Reduction of <^t*-3=Setosteroids5) Jc of
Biolo Chem0p 225j, 13~2lia 1957«

Tomkins9 Go, Michael^ Po Jo, and Currans Jo Fo, Studios on the
Mature of Steroid ll-^Hydrosgrlationo Biochiaxo Biophys0 Acta^ 23,
655-^56. (19$1) o

Tomkins, G„, and Michaels, POJ Inhibition of Adrenal ll~j^Hydroxy=
lation by Ions0 Nature, 180, 337 ©*»lys

SilH
Individual Project

Calendar Year 195?

Serial Ho<» NIAMD 117«>C
Clinical Investigations
Arthritis & Rheumatism

Bethesda

Part A

PROJECT TITLE? Studies on the Biosynthesis of Thyroid Stimulating
Hormones (TSH)

PRINCIPAL INVESTIGATOR: Peter Go Condliffe* Gordon Tomkins

OTHER INVESTIGATORS: Robert Bates

COOPERATING UNITS s

MAN XEARS (Calendar Year 1957) s PATIENT DAYS (Calendar
Total: 1 I©ar 195?): None

Professional: 1
Other :

PROJECT DESCRIPTION:

The chromatographic purification of thyroid stimulating hormone!
(TSH) on ion exchange columns developed by Condliffe and Pates
suggested that the biosynthesis of this specific protein hormone
could be studied and TOuld be of particular interest since its
synthesis is under hormonal control (i0eo9 inhibited by thyroxine) „

Incubation of TSK-=producing mouse pituitary tumors with C^
glutamic acid followed by chromatographic isolation of the hormone
suggest that there is forme d, in vitro ^ a radioactive protein com=>
pound with the biological activity of TSPo

These studies will be extended to cell-free extracts in an
attempt to study their mechanismo

Part B included: No

PHS = BIH

Individual Project Report

Calendar Year 195?

Serial N©P NJAMD 1I8«C
Clinical Investigations'
Arthritis & Rheumatism
Bethesda

Part A

PROJECT TITLE j Intermediary Purine Metabolism in Gout

PRINCIPAL INVESTIGATOR? J„ Bc Seegmiller

OTHER IWESTIGATOISs Leonard Laster9 Lois Liddle, Ethel Lovep
Irwin Sehuman

COOPERATING UNITS':

MAN YEARS (Calendar Year 1957): PATIENT DAYS (Calendar

Totals ' 3^2/3 - Year 1957) :

Professional? 1/3 J.81
Others 1.2/3

PROJECT DESCRIPTIONS

The metabolic origin and disposition of uric acid in goaty
patients is being investigated toy administering isotopieally
labeled precursors of uric aeidg or labeled urie ©eid itself to
normal subjects and patients with gout and hyperuricemia without
gout and determining the extent and pattern of incorporation of
isotope into urinary uric acidc Although the majority of patients
with non^tophaeeous gout so far studied have shown a pattern and
extent of incorporation of glycine N^S into urinary uric acid
similar to that of the normal g all of the gout patients studied here
by Dro Wyngaarden were found to show an excessive incorporation of
glycin@=l=C^ into urie aeidQ This seeming discrepancy has been
investigated hy administering doubly labeled glycine (l^C11* M*5) to
one patient who had been studied previously hj both methods o Prs«
lirainmry results indicate that both isotopes of -the glycine Molecule
were diluted to the same escfcest upon being incorporated into uric
aeido The disparity in results obtained by the two procedures ean°
not be essplained by a preferential incorporation of carboxylcarbon
over amino nitrogen of the glycine molecule o The explanation for
these divergent results may lie to the large dose of glycine #?
(7 gsa) requipd for such studies compared to the trace amount of
glyeine^l^C1^ (008 Kg) requirado Gout patients «ho had previously
been found to have a normal pattern of glycine N15 incorporation
have been studied with glyeine^l-C1^ but results of these studies
are not yet available 0

Serial Moo NIMSD 218-q

Page 2

Studies using the purine precursor li==aiHino=>5==isaida20l© carbox-
amide^ij^C1^ jjaTC been completed during the past year and all of $
gouty subtests studied incorporated larger ©Mounts of this compound
into uric aeid than did norssal sublets even those patients who
had shown a pattern of glycine Nl5 incorporation identical to that
of the norssal subject® 0 All 5 gouty subjects showed an accentuation
of the rapid pathway for incorporation of this compound into uric
aeido The failure of this compound to suppress de koto synthesis
of uric acid from glycine ?$5 to the low levels previously found
in the normalj supports the view that some gouty patients have a
defect in the regulatory mechanism for endogenous purine biosynthesis <

In an effort to determine whether or not excessive production of
uric acid is characteristic only of gouty subjects 9 we have inves-
tigated the pattern and extent of incorporation of glyeine^l-C-^
into urinary uric acid in two patients with urate renal lithiasiss
but without stigmata of clinical goutQ The one patient whose studies
are now concluded showed excessive incorporation of isotope into
urinary uric acido

Part B included? Yes

Serial Ho0 IjIAMjLiJJIgCL

Pag® 3

PHS - NIH

Individual Project Jfeport

Calendar Tear 1957

Part B% Honors 8 Awards, and Publications;

10 Seegaiiller, J„E0, Stetten, DeW„, Buni% J.Jo, Sokoloff, Lo,
Laster,"1 L0, Wyngaarden, J0B0, Clinical and Metabolic Aspects
of Gouto Amo Jo of Medicine, 22, 807~82it, !9$7Q

2o Se@gmiller8'Jo E«, and Laster s hOS) Th© Metabolic Origin of Uric
Acido Progress in Arthritis, edited by Dr®^ John Talbott and
and- Maxwell LockLe, published by Grrae & Stratton, Ince, Men
lorko In press o

3. Stetten, DeW., Talbott, J. H„ SeegriLller, J. Ee, tfyngaasden, Je B0

and 'Laster/ L. The Pathogenesis of Gout. Jfetabolism, VI, 88»9XS '
1957,

NATKJNAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES

Annual Project Report

Calendar Tear

Summary Sheet

Clinical Endocrinology Branch

Estimated Obligations for FY B>58

Totalt Ib3b,000
Direct t $199^000
Reimbursements* $235^000

Projects number 139c thru 127c includedo

Individual Project Report
Calendar Year 195?

Serial llo, E1MD ng.,n

lo Clinical Investigations

2. Clinical Endocrinology Branch

3o Bethesda

Part_Aa

Project Title: Studies ©f Labelled Protein Matabolisa

Principal Investigator: Charles G0 Lew&llaBs, M0 D„

Other Investigators : J0 E0 Rall8 H0 DB9 Ifeses Ber©an8 PhoD0 sad
Betty Sfersdan

Cooperating Units: Slosn-Kettering Institute for Cancer
Research,, New Yoarka H. Ya

Man Years (calendar year 195?): Patient Days (calendar yeas 195?):
Total: 2 2/3

Professional: 11/3 Koae

Other: 1 1/3

Project Description:

Methods - The ssethods of cold ethane! fractionation said sons
starch coluasa ©leetropheresis have been applied to rat seres l!3l „
* Isabel ling of the isolated serum protein fraction has been carried out
by a ssodific&tion of the jet iodi&ation saethodo A ssaatthod for deterain-
iag total retained label by whole body counting of the rat has fees®
applied to tracer data.,

Results - It has been possible bj starch eotasa electrophoresis
on a preparative Scale to prepare rat albumin, g globulin,, sad j glofeulia
in a state of alectrophosetie hose©g<gaeity0 Best overall separation of
eosiponants was obtained with pH 8„S barbital buffer in comparison to
e wide variety of other buffer® tried* Addition of a asssbar of anions
including this eyanat®9 iodide,, borata8 and caprylate did sot facilitate
the separation ©f albuaino

Data previously obtained ixsm labelled albussia studies in ssan
required for kinetic analysis a aaiatem of three exchanging coaspartsentSs
The general three eoapartront solution of tracer data has been e&tandgd
to include a ssappiag procedure by which is is possible to eosapute the
limits of the rase constants for all physically pemissibl© aodals
when the e&pertaaatal data is insufficient to permit a unique solution..

Pert B included Yea is. I m» /_/

isgisl ma mump it^e
Page » 2

Faster® Stediets => Teetaieal difficulties bgwe feaea eas©tia£ered
in ©bfcaiaia® s-eprodueifela degrees of iodisagioa using She jet aetfaodc
Oaca Ehis is overcomes, is is proposed So study £h@ effeefea of a
variety ©£ iiorsaoags in the rat ©a £&e s&ega&oliasa of £fee plasms
profceia fractioaated aad labelled, ui£h particular eaapt&asis ©a
slowly aasehaagiag feody profceia e©sapa£teea£So

Serial Hoo fM£BU3g£L
Pag© 3

taxt B; Hcmo3ras JwaardSj, easd P&ifelicatiosas
Publications oth©? thea abstracts fs-oa this project:

Lippinc©tt8 So Wo 8 Lewallea,, C0 G„ aad SheilabasgeSs Co Jo

Pathology of Radioisotopic J&latioo of the Thyroid ia the Doge
MA Archives of Pathology 5 63? 540-556? 1957 .

PHS-N1H
Individual Project Report
Calendar Year "1957

Serial No0 EliMD 120»S

lo Clinical Investigations

2o Clinical Endocrinology Breach

3c Bethesda

Part A.

Project Title: Studies on Iodine Metabolism fey jybnomal Thyroid
Tit

Principal Investigator; Jacob Robbing, M. D0

Other Investigators: J. E„ Rail, M. Do8 Daniel D„ Federman8 M0 Do and

Dorothy Jermany

Cooperating Units; NCI « oq£

Man Years (calendar year 1957 ); Patient Days (calendar year 1957)?
Total : 3

Professional: I 1/3 1713

Other: 11/3

Project Description:

The possibility exists that certain abnormal thyroid glands
sight differ qualitatively from the nonsal in the manner by which
iodine is metabolized. Knowledge of such differences would vary
likely lead to a better understanding of the normal ssechaniams of
thyroid hormone synthesis » In an attempt to investigate this problem
studies have been undertaken on abnormal thyroid tissue r®aov©d
from human subjects at operation and on thyroid iaaoplassss la &&%&?■

In the human studiess &b© thyroid tissue was sliced and in~
cubated with a radioactive iodine solution,, These surviving slices
continue to orgaoify iodine for scus® hour 3 „ After varying ti&e
intervalss the incubations were stopped and ghe products of organic
iodine 3 synthesis were asssBinedo The analyses may be divided into
two maia catagorlas: Those dealing with the nature of the protein
substances into which iodine was incorporated e and those dealing
with the iodinated compounds released fro® these proteins by
hydrolysis.

Twelve patients have thus far been studied, Of thase8 g had
colloid adenesaa8 2 had fetal adenosa®, 1 had H®shimato8s thyroiditis,
2 had thyroid carcinoma and i had Graves" disease . In sows patient s8
normal thyroid tissue was available for comparison. The protein
studies have indicated that, in ail cases except one with thyroid
stardoma, the snajor iodinafcigd component srasesbiad n©»sl thysroglobsslis:

P«rt B included Yes faTI Ko £jl

Serial Ho.. MIAMD 22Q=&
tag® <= 2

fey the criteria of solubility and eleetrophoretic mobility* In -at ■
least. 3 cases (on© with carcinoma, one with fetal adenoma, and one
with colloid adenoma), an abnormal protein component was present «
These preliminary findings indicate that thyrogiobulin need not be
the only iodinated protein in thyroid tissue, as was heretofore
believed,,

Analysis of the hydrolytic products of these thyroid glands

revealed the presence of a masher of components which were different
from the well known iodinated md.no acids and which are, at present,
unknowno In one instance where it was possible to compare normal
and abnormal tissue (colloid adenoma) in the same individual, un
abnormal iodinated substance, present in considerable amount, was
found only in the abnormal tissue*

In the animal experiments, investigation has been started
on a group of transplantable thyroid carcinomas in rats developed by
DrD So Wollman of the National Cancer Institute (project ao„ NCX-906).
One of these tumors, studied in considerable detail, has been of
great interest „ This tumor was able to concentrate inorganic
iodine in large amounts, but lacked the ability to convert this to
organic form,, As a result, it was possible to evaluate controlling
factors in the Iodide ^trapping" mechanism which cannot be studied
in aorsaal thyroid tissue., It was found that this iodide "'trap'* did
not respond to fer©®teesKi: with thyroid-sttaulatlng hormone of pituitary
origin, or to triiodothyronine 0 The tumor was found to contain very
little thyrogiobulin {i0e„, in uniodinated form, as determined by
ultracentrifuge analyses). Addition. ": of unlabelled rat thyrogiobulin
did not result in iodlnation of this material „

Serial Ho. NXAM) Ign^G
Page 3

Part B; Honors, Awards, and Publications

Publications otter than abstracts from this project:

Bobbins,, Jo and Rail, J. E„ The Interaction of Thyroid Hormones
and Protein in Biological Fluids. Recant Progress in Hormone
Research; 13; New York8 No Y., Academic Press, Inc., 1957.

Bobbins, J., Petermann, M. L.9 and Rail, J. E. Thyroxine-binding
by Serum and Urine Proteins in Nephrosis. Qualitative Aspects.
J. Clin. Investig.; 36; 1333-1342; 1957.

Berman, M., Rail, J. E. and Heslin, J. Some Physical Considerations
Governing the Choice of Internally Administered Radioisotopes for
Therapy. Physics in Med. & Biology; 1; 243-257; 1957.

Alpers, J. Bo and Rail, J. E. The Effect of Thyrotropin on
Thyroglobulin Hydrolysis. Endocrinology; 61; 111-112; 1957.

Ralls J' E. and Barman, M„ Correspondence - Choice of Radioisotopes
for Therapy. Physics in Medicine & Biology; 2; 71; 1957.

Rail. J, E., Radiation and the Medical Profession, AMA Archives of
Internal Medicine; 100; 347-352; 1957.

PHS-NIH

Indivi&aal Project Report

Calendar Year 195?

Serial JSo0 BMM) 121-*C

lo Clinical Investigations

2a Clinical Endocrinology Branch

3o Bathesd®
Pars A-

Project Title; Mechaaissa of the Biosynthesis of Thyrossine

Principal Investigator j Hans ChansLanns Ph0Do

Other Investigators: Ter*s© Matsnsura0 Ph<,Do

Cooperating Units;

Man Years (calendar year 1957): Patient Days (calendar year 1957):
Total: 15/6

Professional: 11/2 Biene

Other: 1/3

Project Description:

Most of the investigations have dealt directly or indirectly
with the still obscwre mgehanissa of the biosynthesis of thyroxine
In this synthesis two saoieeales of 395-dii©dotyrosia© condense to
form one jaoiecsal© of thyroxine and she alanine side chain of one
of the diiodotyrosine molecules is lost.

Hoaens^aic incubation exp^immtst la order to stady the
course of the reaction in vitro H-chlaroaeetyl-395-diiodotyr©sina
-JHgffl incubated 3?°C| pH 7„5 and 11.9). "The fate of the smarting
material was studied by Beans of paper chromatography and of high
voltage electrophoresis 0 Spots obtained ware identified (or
tentatively identified) as starting saaEeri&l, M-ealor©ae©£yl~3»
iodo»Kyrosine and N-chloreaeetylthyro&io@o A nonpheaolic spot was
thought to be or derived from the "lost alanine side chains
However8 it was neither K~chloroaeetyl serin® nor chloroacetylalaainee
nor chloroacatyidshydroalanine*

The investigation of the iucabation products of N~ehloro-
aeetyl-385-diiodotyrosine presents difficulties becaase mssas-OHS
reaction products are forrod within less than one hoar's in-
cubation. Therefore deEaaiso-39 5-diiodotyrosiae sad hoss&logeous

Part B included Yes /__/ No / x /

Serial Ho, NSiSMD 121=0
Page - 2

acids (formic, acetic » props? Ionic acid side chains) were
iacuba&ed (37°Cj pH 7*5) is another set of axperissgntso la the
ease of She acetic and proprioaic acids crystalline substance •
which appear to be the corresponding thyroxine analogues (thyro-
acetic and thyroproprionic acid) precipitated from the incubation
mixture within less than one day and ware obtained in yields of
6-77. after a few days* incubatioa. The' filtrate contained starting
materials, partially deiodisaated starting material (moneido^), one
or two unknown phenolic acids (believed to be hydroxylated ia the
side chain) sad polymerized ffiat@rial0 The fate of the "lost side
chain"9 is under investigation . In contrast to the acetic and propionic
acid aaalogue8 the formic acid analogue (p^hydro&y^S-diiodofeeazoic
acid) reacted only slowly «, After about 2 weeks' incubation a small
amount of crystals formed which were gtot thyroforaic acld0 Therefore P
it seems that a - C&2 " or »CH group adjacent to the phenyl ring
is required for the formation of thyroxine analoguas0 Butyric8
GMaethyl-propioniCj, and acrylic acid analogues of 395»diiadotyrosina
are now being prepared so that the influence of the side chain on
the coupling reaction can be studied* N-scetyl-ajS-dibroaotyrosine
did not undergo any detectable change within two weeks* incubation.

Oxidation experiments - Oxidation experiments were carried
out with analogues of 395-diiod©tyrosine ia order to sea whether the
formation of a free radical could be demonstrated (a possible
first step in the coupling reaction)., Potassium ferricyaaide8 lead
dioxide 8 and silver oxide were used as oxidant sa When H=chloro®cetyl-
3,5-diiodotyrosine was treated with alkaline ferricyanideB iodine
was liberated and polymerization took place. When its aethylester
was treated with lead dioxide again iodine was liberated and a
variable mount of polymerisation took place depending upon the
conditions of the reaction*. Although polymerization may be free
radieal reactions, no stable free radical could be demonstrated
through the appearance of a typical free radical color. As stable
free radicals were obtained from 29488*tri-s~butyl phenol (il*t$+ )

and similasusterically hindered phenols, 2,g»diiodo-4-t°butyl^
phenol ( gggffihLjL ) was treated with f errieyaaide and with lead
dioxide. ^F^ la both cases a dark blue free radical color
appeared whtel*8 however 8 was rapidly displaced by the color of
liberated iodine > Again much polymerization took place. Small
amounts of two crystalline substances,, oae yellow (quiaoao) and
-j one colorless were isolated from the reaction mixture. The
oxidation of 2sg-dibromo-4ȣ-butylphanoi lead only to polymerisation,,
From the various oxidation reactions that were carried out it can be
concluded that asy free radical formed ia the course of the bio™
synthesis of thyroxins rasust be very unstable because otherwise it
would lead to extensive deiodiaation. Extensive polyissrisatioa is

Sarial No. TslimD 123-qC

Page • 3

prevented in vivo through a yet un&spla&aed sseehsnisss (possibly
the reaction of the 3j,5~di£odotyrQsiaa within a protein chain )0

Tissue slice aKperissents - to attempt was Bade to determine the
fate of the lost side chaixr by analysing the hydro ly sate from s
thyrogiobulin into which labeled tyrosine had been incorporated.
Thyroid slices were incubated for 5 hours in the presence of
Dj,L-tyrosine~2-C14, Thyrogiobulin was then isolated from the
slices p An aliquot of the thyrogiobulin was then hydrolysed and
the hydro ly sate was chroaafcographed on paper « Radiegaitography
revealed only the presence of radioactive tyrosine „ Neither
radioactive thyroxine nor other radioactive reacton products
could b© detected,. Studies are under way using higher specific
activity tyrosine.

iteother experiment was started in order to study the
eatent of the incorporation of C*^ - labeled tyrosine not only
into thyrogiobulin but also into a sasaber of other thyroid protei®
fractions. At the assise time the influence of (a) propyl thiouracil
sad (b) iodide ion on these incorporations is being studied.

PHS-NIH
Individual Project Report

Calendar Year 195?

Serial No, NXMD 122-C

1= Clinical Investigations

2o Clinical Endocrinology Branch

3o Bethasda

Part

Project Title: SSslecular Characterisation of Proteins

Principal Investigator : Harold Edelhoca8 Ph„D0

Other Investigators: Roland Lippoldt

Cooperating Units:

Man Years (calendar year 1957): Patient Days (calendar year 1957 ):
Total: 11/6

Professional: 7/6 (l 1/8) ®
Other :

Project Description:

Our principal concern baa been related to those properties
of proteins responsible for their stability*, Kbrk is continuing
on the characterisation of the native and denatured state of pepsin*,
Since very interesting phenomena have been observed with various
proteins in concentrated solutions of LiBr we are currently
evaluating its effect on both forms of pepsin by means of viscosity
and optical rotation „

Claims have been made in the current literature that
differential ultraviolet spectra can serve as a means of identifying
tyrosyl hydrogen bonds in proteins „ To critically evaluate this
hypothasi69 and since pepsin also shows this type of differential
spectra,, the spectrum of tyrosine has been investigated in the following
solvents: HC1 (Od M)e H20°Mc (0°iOS^)8 H20-Dioxaaa@ (0°8GK)» WBr
(«l), «ffid LiBr (6 M)°HAe (lOfc^H^O, Ml these solvents sfeow very
similar differential spectral effects and east considerable doubt en
the claims that have" been- vm&e<> It is thus apparent that a change in
the environment of tyrosine groups in proteins (as probably occurs on
denaturation) will- result in spectral shifts. If the rupture ef a
tyrosyl hydrogen bond does produce a spectral chsage9 it seems likely
that it will be ©bsc'ured to a considerable degree by
solvent effects.

Pare B included Yes / / He /x_/

Page. - 2

A prelisainary study ©g thyroglotmlin has been perforated on
frastioned sheep thyroid extracts «. Sadisaaatatioa and electrophoretic
(pH - raobility) data have beaa obtained. Our data ax© in agreeiaent • '
with those obtained in other laboratories.

finally, ia collaboration with Br0 Jo C0 Rabiaowits (HI^d)
She issoleeular properties of crystalline fonaylase essyae8 isolated
fey Dr0 Rahinowits, has beaa studied by mums of
electrophoresis and diffusion,,

PRS--NIH

Individual Project Report
Calends Year 1957

Serial No„ NSAHD 12>C

lo Clinical Investigations "

2o Clinical Endocrinology Branch

30 Bethesda

SSL

Project Title; The Effect of Testosterone in Thyroidal Activity

Principal Investigators Daniel D0 Fedemaan, M„ D<,

Other Investigators: Jo E„ Rail, M. Doa Jacob Rofebinsp Mo Do and
Jane Heslin

Cooperating Units:

Msm Years (calendar year 1957); Patient Days (calendar ye&r 195?):
Total: 2

Professional: I 1/3 2l|lt

Other? 2/3

Project Description:

It has been noted that methyl testosterone (Ml) causes a fall
in carta precipi table iodine (PBI) in children and certain subjects
with cancer ° The mechanism of this effect was examined in four
individuals with normal gonadal and thyroid function (3 males, 1
female )<, A fall in PBI was confiraad0 a marked decrease in the
thyroxine binding alpha globulin of serum (TBP) and an increased
fractional rate of thyroxine degradation were observed under treat =
■ant with m. An increased level of '"'free" thyroxine in serum and
a slightly increased turnover rate of thyroxine were also observed*
These abnormalities returned towards normal after treatment o There
were inconsistent variations in thyroidal iodide and renal iodide
clearance and in -the BMR0 A rise in serosa cholesterol during treat-
ment and a fall afterwards was seeno

These data are interpreted as indicating that the primary
effect of MI is to depress the level of the thyroxine binding
protein of serum and changes in the other parameters of thyroid
activity are secondary fee this0

Part B included Yes / / Ho /* I

PHS-M1H
Individual Project Report

Calendar Year 1957

Serial Ho. M1WD I2h°C

1* Clinical Investigations

2. Clinical Endocrinology Bsaach

3. Bathesda

Bffit_A-

Project Titles Effect of Diabetes Mellitus on Vitamin
Metabolism

Principal Investigators James B„ Field, M. D.

Other Investigator: Minnie Woodson

Cooperating Units;

Man Years (calendar year 1957): Patient Days (e&endar year 195?.) s
Totals 1 2/3 235

Professional : 1
Others 2/3

Project Description:

Excretion patterns of sozne of the B vitamins were studied in
normal subjects and in diabetics with and without degenerative comp**
Heat ions. Each patient was given a constant standardised diet for
two weeks before the administration of the first test dose to minimise
the effects of previous diet- The second test dose was given one. week
later when insulin had been withdrawn from the diabetics and the norms!
controls had been given a restricted caloric and fluid diet. There
were no significant differences in the excretion pattesso in the three
groups before the first test dose. The diabetics with degenerative
complications excreted less of the first test dose of niacin than did
the other two groups. During the period of insulin deficiencyB the
diabetics without complications excreted more of the thiamine test dose
than those with complications and more of the pantothenic acid than
the other two groups. The excretion of riboflavin and niacin was
greater after the second test dose than after the first in all gronpe.
This was probably a manifestation of negative nitrogen balance and
weight loss. It w@s not possible to determine any definite relation?
ship between the development of the degenerative complications of
diabetes and the patterns of vitamin excretion.

Part B included Ye« ^x_/ Ho l__i

Serial Ms, mm-22k&
Page 2

Past- B; Honors 9 Awards 9 and Publications

Publications other than abstracts fress this project;

Fields J0 Bo and Federsjans D» D„8 Effect of C&rbutaaide in the
Diabetic Associated with Acromegaly* Diabetes $ 6? JQ*>72-e 195? »

Field9 Jo B* and Federman9 D» D» Oral Preparations of Antidiabetic

:s, Mad0 Ann, of District of Colus&ia; 26j 297-3005 1957 •

Fieid9 Jo Bo9 Tiefcze, F0 and Ststt@n0 Do0Jro Further Char act erisatlom
of an Insulin Antagonist in the Sena of Patients in Diabetic Acidosis .
Jo Clin0 Investoj 3S8 1588~1593j 1957 o

Shohl8 J0 and Field9 J0 B0 Insulin Binding la Vitro by Leukocytes
from Konaal and Diabetic Subjects, J0 Lab0 & Clin* Bed,; In Press,

Fieldg J, Bo Studies on the Circulating Insulin Inhibitor Found in
Scans Diabetic Patients Exhibiting Chronic Insulin Resistance 0 Jo
CliOo Investoj In Press*

Field8 J& Bo9 FederiBan8 Do D.9 McDaniei8 S. and Bakensaa, Ha
Urinary Excretion Patterns of Sosae B-Vitasias in Diabetics with
and without Degenerative Complications „ Diabetes j In Press*.

FHS-HIH
Individual Project Repose

Calendar Year I95?

Serial No, ESXAMD 12S-C

lo Clinical Investigatioas

20 Clinical Endocrinology Branch

3= Bethasda

MIA

Project Titles Pentose Sfetabolisa in Normal and Diabetic
Man

Principal Investigator ; Stanton SegalB Mo D„

Ofcher Investigators; Joseph Foley

Cooperating Units:

Man Years (calendar year 195?); Patient Days (calends year 1957 k
Total:- 2

Professional: 7/6 (1 1/6) U*9

Other: 5/6

Project Description:

The nfetabolisa of D-Ribose in ©an has been studied by Mans

of intravenous infusion of varying sssounts of the sugar and deteralaa----
fcioa of blood levels of ribose9 glucose, pyruvate and phosphate „
Urinary excretion has also been determined,, Ribose disappears froa
blood rapidly and when over three grass are given the system for
handling the sugar appears to be saturated. Of particular interest
is the pronounced lowering of blood glucose levels by as such as
5C%, Blood pyruvate and phosphate are altered only slightly
Ribose has been found to be insul in-responsive B in thag IV insulin
acutely lowers blood ribose level®,, this alteration being independent
of she blood glucose response to insulin *

Studies with Ribose ~1-C14 have been carried out in both taor»&l

and diabetic subjects. The normal subject metabolizes ribose rapidly
and to a considerable extent, to CO2. The diabetic subject appears to
have a block in C**02 production from ribose „ This block apparently
is due to the fact that the ribose is converted to glucose and the
diabetic is unable to oxidise the glucose ssoraally. Chemical de-
gradation of the C*4"glucose isolated from the arias- of" a diabetic
given ribose-l°C14 showed a pattern of labeling of the nolecule which,
would result froa operation of the pentose phosphate pathway,, The
cause of the ribose induced hypoglycemia has been investigated and it

Pare B included Yes £/ WsTJ

Serial No, HIAMD 12£°C

Page - 2

has been found that a possible explanation may be that these is en
inhibition of liver glyconolysls due to the inhibition of the enzyme
phosphoglucomutase by ribose»5~phosphates © metabolic product of
ribose metabolism..

14
The second problem has been a study of the metabolism of C

labeled pentoses D=xylose, D-arabinose, L-arsbinose and D-lyxose

in man. All except L-arabinose gives rise to radioactive carbon

dioxide in expired air„ Much of the radioactivity is excreted in the

urine in 24 hours, but a large proportion of activity is due to

metabolic products of the sugar and not to excretion of the

unaltered pentose „ The nature of the urinary metabolites remains

unidentified,, A somewhat unexpected finding in several insulin

responsive sugars (D-xylose for example) was the absence of change

in volume of distribution of the sugar from tolbutamide administration

The occurence of hypoglycemia without a change in level of xylose

after tolbutamide suggests that the hypoglycemia is not secondary to

insulin release »

Third, the biosynthesis and metabolism of L-Fucose has been
under consideration, both in bacteria and man.. Isolation of Fucose.
from the capsular polysaccharide of aerobacter aerogenes grown on C
glucose has been carried out and the molecule degraded chemically to
ascertain the patters labeling „ It has been found that the labeling
pattern is consistant with the view that the Fsacose is derived from
glucose without disruption of the carbon chain,, This same result
appears when fucose is isolated from the milk of. a human female given
C glucose. Experiments have also shown that C fucose is rapidly
and extensively metabolised when given IV to man,, The rabbit also
utilises this sugar a Studies are at present in progress regarding
fucose metabolism in tissue slices and tissue homogenates „

Serial No, HMMD 19<~C
Page 3

Part B; Honors, Awards,, sad Publications

Publications other than abstracts from this project:

Segalj So 8 Foley,, J«, and Wyngaardeng JoB= Hypoglycemic Effect
of D-Ribose in Man0 Proc0 Soc0 Exper= Biol, & fed, ; 9_5; 551-555?
1957.

Segal<> SoD Wyngaarden, J«B« and Foley « Jo The Effect of Insulin
on Blood Levels of Infused Pentoses in Man0 J» Clin* Invest., ;
36; 1383=1394; 1957.

SegalB So 9 Fr®?ley8 T»Fo and Foley, Jc A Comparison of the Effects
of Tolbutasnlde and Insulin on Infused Pentoses A Study in ten.
Diabetes; 6; 422-425; 1957 .

Wyngaarden0 J„ B„, Segai9 So and Foley8 J. Physiological Disposition
and Metabolic Fate of Infused Pentoses In Man, Je Clin0 Invest «;
36; 1395=1407; 1957 .

Segalj So and Topper8 Yo To. On The Biosynthesis of L=fucose by

Aarobacter aerogeneso Biochiaiica at Biophysics ACTA; 25; 419-420;
1957 o

PHS-N'IH

Individual Project Reports

Calendar Year 1957

Sesial Noc HIAMD . 126-C

lo Clinical Investigations

2. Clinical Endocrinology Braach

3o Bethesda

Par£_A„

Project Title: The Effect of Thyroxine on Isolated Dehydrogenases

Prinicpal Investigator: Jan Wolff „ M. D„9 PhBD0

Other Investigators: None

Cooperating Units:

Man Years (calendar year 1957): Patient Days (calendar year 195?}:
Total: 5/6 „

Professianal: 1/2
Other: 1/3

Project Description:

A study on the effect of thyroxine on isolated dehydrogenases
has been completed (Bioeheanica & Biophysica ACTA, November 1957 ).
Because of the fact that isany dehydrogenases appear to contain sine
and because thyroxine forms sine completes,, the nature of the thyronine
dehydrogenase interaction was investigated. Thyroxine inhibits purified
or crystalline sialic, glutasaic8 lactic,, triosephosphata, yeast alcohol
and yeast glucose*6=phosphate dehydrogenases » The apparent Kj values
are 1° 10° ^ wish malic dehydrogenase , a-lO^M with glutamic dehydrogenase 8
and 1~5°1Q°5M with the other dehydrogenases „ Triiodothyronine 9 eriiodp-
thyroacetic acid„ and other thyroactiv® congeners of thyroxin® are also
dehydrogenase inhibitors,, Various halogenated phenols (e0go pentabross=
phenol „ apparent Kj = 5° 10°%) and x&athena dyes (eogo Rose bengal;,
apparent Kj = 1°1Q- 5m) inhibit glutamic dehydrogenase „ Other
horaonesB and a variety of uncoupling agents are not inhibitory <>
Dehydrogenase inhibition by thyroxine appears to be intermediate be-
tween competitive and' noncoiapetitive in type vs. both substrates and
the pyridine nucleotides „ The inhibition is reversible, Attempts to
demonstrate a thyroxin® interaction with specific functional groups of
the ansysaes were unsuccessful „ Although all dehydrogenases found here
to be inhibited by thyroxine are reported to contain sinc8 no direct
interaction of thyroxine with this astsl eould be obtained, Carfeoxypeptidase
isB however8 not inhibited,, Other sssaias of studying the interactios
of thyroxine with bound or partially eo®plexed sine are now under
investigation..

Part B included Yes 7x7 No /~

Serial Sfeo MAMD 126-C

Pago

Part B; Honors,, Awards,, and Publications

Publications other than abstracts from this project;

Goldberg. Rs C08 Wolff 9 J0 sad Greep0 R„ 00 Studies on
the Nature of the Thyroid Pituitary Interrelationship,
Endocrinology | 6Qj 38-52; 195? ,

Wolff, J, and Goldberg9 R6 C« Disorders of Iodine IfetabolissSo
Biochemical Disorders in Huraan Disease, RD H„ So Thompson and
E„ Jo Kings Editors^ 289°351j Acadeaic Press,, New York9 195? »

Wolff9 Jq and Wolff 8 E0 C, The Effect of Thyroxine on Tsolated
Dehydrogenases 0 Biochemica et Biophysics ACTA|
265 387-3965 1957c

PHS~NIH

iBdivkdual Project Raporft

Calendar Year 1957

Serial No, NIAMD l£?^C .

I. Clinical Investigations

2= Clinical Endocrinology Brand

3, Bethesda

Part Ao

Project Titles The Effect of Sodium Salicylate on Thyroid
Function

Principal Investigator: Jan Wolff „ M„ D,s Ph,Do

Other Investigators; Capt, F, K. Austin;, MCS USA
John Goldsberry

Cooperating Units: Walter Reed Aray Hospital

Man Years (calendar year 1957): Patient Days (calendar year 195?};
Totals 11/2

Professionals 1/2 Bone at NIH

Other : 1

Project Descriptions

Thyroid hormone e 2„4~dinitrophenolj, and sodium salicylate
can all raise the metabolic rate and act to uncouple oxidative
phosphorylation,, Both thyroid hormone and dinitrophenol may
suppress thyroid function, presumably via an inhibition of the
pituitary gland. The similarity of salicylate action suggested the
possibility of studying interference in thyrotropic action in man
by an agent other than thyroid hormone , Euthyroid male patients
with rheumatoid arthritis were treated with approximately 8 gm> of
sodium salicylate per day to attain a serum salicylate level of
30~40 mg, percent. Under these conditions the BMR is raised 30% and
is correlated with the blood level of salicylates,, Serum cholesterol
was not significantly depressed except in on® myxedematous subject t
There is a 30-407. fall in the serum protein bound iodine associated
with an accelerated rate of disappearance of exogenous l!3l labeled
thyroxine from the circulation. Paradoxical ly0 thyroidal l!31 uptaks
and clearance is depressed 30-407.0 There is a marked depression in
the "release" of accumulated 1^31 from thyroid glands of salicylate
treated patients or rats* Sodium salicylate leads to an incomplete
suppression of goiter formation in the rat treated with the goitrogen8
propylthiouracil o However, patients respond normally to exogenous
thyrotropin with an increase in the "release6' rateg and elevation

Bart B Included Yes

Seri&i No<, HX^MD 127~C

Pag© - 2

of the PBI and I uptake „ Studies In rats and in vitro have

shown that salicylate has no signif leant direct affect on the thyroid

gland's ability to concentrate iodide or to convert it to organic

Various analogues of salicylic acidp some of which are
supposed to possess antirheumatic activity, hut which do not un-
couple oxidative phosphorylation, also block release of I^3j
from the thyroid,, It is concluded that sodium salicylate, like
2j,4-dinitrophenol and thyroid hormone inhibits thyroid function
indirectly by an effect on the pituitary gland = thyrotropic
mechanism. The mechanism of this effect appears not to be due
to adrenal activatio»8 antirheumatic properties 9 uncoupling of
oxidative phosphorylation ©£ fever production., The study is
being terminated with an investigation of the distribution of
carbosyl labeled salicylate in the rat„

NATIONAL INSTITUTE OF ARTRKETIS AND METABOLIC DISEASES
Annual Project Rupert
C&lemta Y»ar W$7
Suimaary Shwet

Estliaatod Obligations for ¥1 p$8

Totals $£37*000
Direst.! $18U»000
B»i8fcun»inenfcst $3?3»000

Projects number 128© thsra 136© ineltadtado

Individual .ft?© jest Report
Calendar Year 1$S?

Serial No« NIAMD-128-C ^^

lo OlinicalTSve^MglS^nir
20 Metabolic Diseases
3o Bethesda

Part Ae

Project Titles Study of the Iirananology of Blood Call Deficiencies,-,

Principal Investigators Pr* N0 Raphael Shiftman

Other Investigators? Bra* Thomas Cc Bithall and Joel H0 Feigon

Cooperating Unites Dr„ Terrell L. ffillj, Department of Chemistry,

University of Oregon „

Man Tears (calendar year 1957) « -Patient Days (calendar year 195?) s
Total 2 . 1

Professionals 2/3 32$

Other? . 1/3.

Project Descriptions

pbjectircss To 'study the nature of ©®rtaia -iOTEmologie reactions nh
affecvspscifie blood cells and lead to their disappearance from this
circulation,,- Of special Interest are the biochemical reactions which
'result in formation of complexes between c@ll&9 antibodies^ and drug
hsptenesi and" the physiologic processes" which result in seauestration
of sella with attached antibodies*.

Methods Employed? Techniques of quantitative immunoehes&stry

including protein fractionation and characterisation., micro<=ana3ys®s
for nitrogen and alkaloid drugs§ precise measurements of complement
fisation^ and quantitative measurement© of cellular agglutination and
lysis o Jfethods of provoking antibody, responses in man and animals
and methods of separating specific cell types from whole blood are
also us©d0

Msffor Findings 2 It has been found that the antibody responsible
for drugp^pura^in this case quiriidlne purpura) reacts with platelets
in the preaens© of the drug to form ant±body=drug=>platelet eompleses.?
the composition of which can be ©hanged by varying the relative eon-
eentr&tion of each of the reactions in the test system0 The attach"
snsnt of an antibody Bsoleeul© on a platelet surface can be ®ff acted
by 1, 2 or 3 quinidine molecules depending on the concentration ©f
qulnidinso Antibody molecules attached to platelets with one or three
quinidine molecules do not cans© complement fixation or platelet &ggluti~
nation^ but antibodies attached with two qainidine s»lecules provoke

Part B included Tes £J No g/

Serial Ho» SI|MD» 128-C

Page 2 «=— --c-;;— -

both of these reactions <» VJhen complement is fixed toy antibody=dmg»
platelet complexes^, it ia not fixed at the sit® of attachment of
individual antibodies but is fixed between antibod3.©s on the platelet
surface© Quinidine appears to function as a typical haptens in its
combination with antibody, and the protein portion of the antigen
does not appear -bo b© of platelet origin 9 Platelets appear to
enter into the reaction non-speeifieally by virtue of having sites
on their surface appropriate for adsorption of certain haptene«»antlbo&y
complexes©

!Sy comparing conditions necessary for various in vitro reactions
with conditions present in vivo when thrombocytopenia developed during
controlled intravenous adMnx^ration of quinidine to patients with
the antibody of quinidine purpura., factors of significance in the
development of throirib@eyt©penia could be deduee&w— Jrfcontrast to
previous suppositions it appears that the various-factions of the
antibody which are measurable in vitro have nothing to do with the
development of thrombocytopenia in v£vo«, Attachment of minimal
amounts of antibody to plat ele tsHTnlflvb appears simply to increase
their susceptibility to normal mechanisms of sequestration., and
reactions such as complement fixation^ platelet agglutination and
direct lysis of platelets are not involvedo

Significance to N2BMD Keseaz-afos It is of special significance
in the f i eid~©f ""Km&^Io^Wa^WiesQ studies provide a clear under-
standing of the pathogenesis of drug purpura^ a sounder basis for
interpreting the nature of various idiopathic purpuras^ and a' rational
for experiiientsl approaches t© the study of diseases ©f sensitivity
resulting in deficiencies of other types of blood cells ? The basie
studies have provided for the first time a detailed model for sn
isssnuasreaction involving &ntib©diesa calls and hsptene^ aid a specific
mechaniesa for complement fixation by an antigen-antibody complex© These
findings have numerous implications in various diseases of suspected
sensitivity involving cellular damage (a o go* idiopathic throisbocytopenie
purpur®5 eollagen diseases 9 nephritis) and indicate the types of
immunologic studies which msgr lead to detection of aatibody^eell
complexes in such diseases Q

Course of Studyg Further studies will b® directed toward
the nature of the protein component of the antigen which
provokes antibody response in drug purpuras Idiopathic thrombocytopenic
purpura and certain instances of agran&teeytosis will be studied in an
attempt t© determine whether similar immunolsgie mechanisms are
responsible for these diseases©

Jbdividual Project

Calendar Tear 1S$1

Serial No« -glfiMD- 129-Qj—
lo (&inicalT3ivestigatIons
2C Metabolic Diseases
39 Bethesda

Part, Ac

Project Title; Study of Blood Coagulation arid Diseases of
Hsasorrhage -and Thrombosis©

Principal Investigators Dra N9 Raphael Shuliaaa

Other Xawestigatora s Xteso Thomas G0 Biifaall and Joel H0 Foigpn

Cooperating Units? lone

Mas Years (calendar yeas? 19$7)s Patient Days (calendar year 1951) s

•Totals 1

Professionals 2/3 2?5

'-Other? 1/3

-Project Descriptions

Objectives? 10 Study of the affect of proteolytic ensyaes
and proteolytic enssya© inhibitors on the conversion of prothrombin
to thrombin M vitro md in viro©

20 Study ©f fibrinogen metabolism*,

3o Determination of abnormalities in blood e@agulation factors
in unusual bleeding disorders 0

Methods B^lqredg lc. Techniques of protein purification and
characterlssHon7iS.nding the fractionation methods of Norfchrup
and Kun.lt z and of Coh©np electrophoresis and ultrmcentrlfugationo

20 Precise quantitative techniques for measuring proteolytic
and 8ster<=splitting enzymes o

3o Various specialized experiaeatal pharmacologic procedures
in man and animals o

ko HI of the usual tests for evaluating blood coagulation „
abnormalities as well as specialised experimental procedures for
quantitative determinations of coagulation factors 0

Part B included Tes £J $® jfj

Serial Hoo NlfiMD^ 129~G
Page 2

Ma^or Findtogss lo Praliarlnaiy results indicate that proteolytic
ensyme HffiffiTSorsexert their anticoagulant action by combining with
a product intermediate between prothrombin and thrombin0 'This inter-
mediate product which has hitherto not been described in the biological
conversion of prothrombin to thrombin was identified by its reaction
with proteolytic ensyme inhibitors in a coagulation system consisting
of highly purified reactantse Crystalline trypsin was found to act
as a coagulant by exerting a two-fold effect both in vitro and
ill ^J°j> directly converting prothrombin to thrombin and in lower
caheei&rations* enhancing the activity of thromboplastic material
by neutralizing the anticoagulant effect of proteolytic ensyme
inhibitors o

20 Study of patients with hypofibrinogenemiag which is frequently
the end result of a hyper eo&graable state* has indicated that excessive
find prolonged intravascular coagulation may result in refractory
state of the liver to further stimuli for fibrinogen production o
It was found that fibrinoSysis., which has generally been considered
to be the cause of hypofibrinogenemias was not a significant factor,,

3© ^he clinical study of patisnts with severe hemorrhagic
manifestations yet no abnormality in the usual clinical coagulation
tests has pointed out the limitations of currently available clinical
coagulation tests in predicting the severity of hemorrhagic tendencies o
By using sensitive experimental techniques for measuring the generation
of blood thromboplastic activity s it was found that these patients
had a coagulation defect which ??es@^)led most closely a mild form
©f classical hemophilia! in contrast to the usual cases of mild
hemophilia*, however* their hemorrhagic manifestations were not
corrected by administration of massive doses of antihemophilic globulin e
These observations suggest the possibility of differentiating a new
disease entity within the syndroms of hemophilias

Significance to WB.H) Research. 8 1« The types of in vitro and
in vivo coagulation studies being done represent a fundamental
approach to the understanding of the nature of diseases of hemorrhage
and throAosis <, Tfaes© diseases comprise a major segment of teratologic
disorders^ which have been a categorical interest of NIAM)0

20 Farther progress in diagnosis and treatment of a number of
hemorrhagic diseases depends on understanding the nature of the
metabolism of various coagulation factors o Such studies are appropriate
to this institute and have direct bearing on the understanding of
protein metabolism in general o

3o Identification of factors of significance in clinical hem©stasis
and evaluation of diagnostic tests and of effectiveness of therapy
depend primarily upon the clinical study of patients with bleeding
disorders*, These studies provide information concerning the basic
nature of the clotting process^, and in addition affordp through con-
sultative gervlce§ highly specialised diagnostic procedures useful
to the support of other research in the ear® of various obscure
hemorrhagic conditions occurring throughout the Clinical C@nter0

Serial loc mKD- jgSM;

Pas© 3

Proposed Course of - ;'Pjrojegtg lo It is planned to define more
preeiseTLyH^~nsEure of the intermediate product between prothrombin
and thrombin formed during the conversion of. prothrombin in the
presence of proteolytic ensyms inhibitors* and to study further the
effects of plasma proteolytic ensymes and their inhibitors on blood
coagulation in vivo and in vitTOe

2a An attempt rf.ll be made to duplicate the type of hypo-
fibrinogenemia seen in patients by producing a hypereoagaable state
in dogs using intravenous thromboplastin* These studies will be
directed at determining the pathogenesis of hypofibrinogenegaia and
at the same time the physiologic factors which effect the over-all
metabolism of f Ibrinogens o

3o Cabined clinical and laboratory studies of patients raitfa
obscure hemorrhagic diseases will be continued in order to define
more precisely boisb of the obscure abnormalities which fall within
the syndrome of hsmophiliaj and in order to develop mora sensitive
tests for predicting hemorrhagic diatheses

fisdlvl&tsal Project Report
Calendar Tear 195?

Serial No0 SIAMD^O^G
lo Clinical Investigations
2c Metabolic Diseases
3o Bethesda

Part 1«

Project Title? Studies in Bone Metabsllsa

Principal Jmrestigators Dr0 Go Donald Whedon

Other fiwestigators* Dra Hobert P« Heaney and Dre Lso Lutwak

Cooperating Unites none

Man Tears (calendar year 195?) s Patient Bavs ^calendar year 1957) s
Totals 6<=>2/3

Professionals l|- 1562

Others k^S/6

Project Descriptions

^3_egtlreag 10 T© investigate the factors affecting aineral
storage and loss in deraineralising bon© diseases^, with particular
attention to the relativ© influeneeaof adrenal cortical ^feeroids^
gonadal steroids and dietary levels of minerals o

2© To investigate the rates of mineral deposition and ansoua&s
of bone undergoing active exchange with body fluids^ in various bon©
disorders o

fe^od^^^lojjd.s lo Metabolic balance studies under rigid <&ietary
eontroTlLn^patients with various deaineraliaing bone diseases^, noting
the effects on nitrogen^ calcius and phosphorus balances of adrenal
cortical steroids^, of gonadal steroids and of various dietary levels of
calcium and phosphorus 0

2o Determination of pool siss©s turnover rate and deposition rat©
of calcium in patients with various bone disorders., using teaser doses
of radioactive ealciumo

Part B included Tes gj Ho £J

Serial Noo l^^l^C^
Page 2

Major Findings s lo Osteoporosis has long been regarded as a
disorder of protein tissue metabolism^, involving inadequate formation
of osteoid matrix^ without impairment of mineral metabolism 0 ^or
this reason^ in traditional therapy dietary minerals have been
scorned and gonadal steroids advocated,, yet beneficial results with
the latter mode of treatment are not often seen0 A minority opinion
has suggested that matrix formation and mineral deposition occur in
close sequence and that adequate mineral supply must be available
for bone formation and repair of des&neralisationo In the 1956
report we indicated that we had obtained data supporting the latter
hypothesis from metabolic balance studies in a±x patients with
osteoporosis^ in that progressive elevations in dietary calcium
intake levels resulted in consistent shifts from negative calcium
balances to positive 0 In addition^, gonadal steroids had not eon=
sistently favored calcium storage in uncomplicated osteoporosis and
h?d had no beneficial mineral effect in two patients with osteoporosis
and active rheumatoid arthritis requiring adrenal e§s?tie&L steroid
for control of their joint disease 0

During the past year# patients with active rheumatoid arthritis
on prednisolone were placed in positive calcium balance by high
calcium intake^ a protective effect not accomplished by -gonadal steroids
in previous studies! in an additional patient with uncomplicated post-
menopausal osteoporosis , improvement was demonstrated in calcium balance
on increased dietary calcium intake^ a very high level being requiredo

The metabolic effects of a new synthetic saaboiie steroid^ 17=
19°nor testosterone were studied in k female pati®itsc This steroid
has been introduced because of a suggested dissociation in animals
between androgenic and anabolic effects 0 In two patients who had
post=men®pausal osteoporosis© complete metabolic balances showed
significant calcium st@rag3 asaounfeing to 5>0 to 100 mgmc per day on
100 mgmo orally of the steroid dailyo In the second of these patie.-
an equivalent influence on calcium storage was achieved by raising
the calcium intake by 1©2 grams daily to a level of \<S grams o Xn a
third patient with osteoporosis but also with active rheumatoid
arthritis requiring 15 isgifflo daily of prednisones a eoa^arative study
of the effects of the new steroid and methyl testosterone revealed a
closely similar degree of decline in urinary calcium on administration
of the two anabolic steroidso In a child with osteogenesis imperfecta*,
the new steroid at a dosage level of only 20 mgm0 daily teought about
significant calcium storage e Only one of the three adults showed
maseuliniz&tion on the new drugj, the dosage level givaa being such
that had methyl testosterone been usedp this serious side effect would
uniformly have resulted o

Serial Ho* 1HAMPk3L30-C

;e 3

20 At the time of the 1956 report of caldujn-Ii5 studies a
preliminary ® count was given of calcium pool results and anticipation
expressed that the data could be deployed to calculate rates of new
bone deposition© The hope has been realized* resulting in important
information on the dynamic nature of bone * To date isotope analyses
have been completed on 12 patients with various disorders of bone
metabolism* 5 of whom had osteoporosis a

Once considered to be altogether inert, the adult skeleton
has recently been shown to be subject to continual turnover* just as
are otter body tissues » The more important type of turnover has been
thought to be physical exchange processes between the solid phase of
bone substance and the soluble phase of the body fluids with no net
local change in structures The present studies* however* demonstrate
that the predominant bone turnover mechanism* in the adult as well as
in the growing skeleton^ is actual physical destruction of structure!
bone units and their replacement by formation of new bone units©
^termination of true miscible calcium pools revealed them* in a normal
individual* in hyperparathyroidism and in hypoparathyroidisms to be
approximately the same sise as the extraosseous calcium* indicating
that the bulk of the bone calcium in the adult skeleton does not
appreeiably-pffitieipate in surface' physical exchange processes with the
body fluids o •

The magnitude of the bone formation rates obtained in the
investigations is indicated by the figure of 600 milligrams of
calcium going into bone each day for an adult of normal weight and
without bone diseases In Paget5 s disease* in which bone turnover
is prodigious* nearly 9 grains of calcium daily were found to be going
into new bone construction o A striking finding in the work was that
is osteoporosis* the thinning bone condition occurring frequently in
the aged and in post^menopaus&i females * bone formation rates were
found to be normal? this consistent observation in five patients
with osteoporosis seems to require that bone physiologists review
and recast their present ideas of the pathogenesis of this disorder*
until now considered to be due to diminished bone formation 0

3© . In calcium methodology* real progress has been made in our
laboratory' in that a) a technique"- has' been" perfected for utilisation of
flame ' photometry for accurate and rapid measurement of calcium in
urine* and b) a method has been devised for counting ealclu»-2|£ samples
in a scintillation counter^ resulting in a %mmad@m advantage over
the previous method of gas-flow counting for processing samples con-
taining this radio=»isotope0

Serial Io0 NIAMfe» lio^c ^_^

Page k

Significance to NIAI^D Research; Bemineralisation of the
skeleton is a common accompaniment of many incapacitating chronic
diseases at all ages, including severe rheumatoid arthritis o Al-
though gonadal steroid honaones administered parenterally are
reported as favoring mineral storage, . universal agreement on this'
•point doe® not exists nor is there adequate information as to the
effects of oral gonadal steroids which ©re more commonly prescribed
in practice, for the treatment of osteoporosis 0 Since a high inc±d«me@
of the latter bone disorder has been particularly noted in patients
with, active rheumatoid arthritis under management with adrenal
cortical steroids? and the latter compounds are being more and more
widely prcseriJbedl, it is becoming increasingly important to obtain
information which will aid in understanding more clearly the relative
influences ,©£ adrenal cortical and gonadal steroids and of dietary
sainerals upon mineral metabollssno

The development of a means of determining the rate of ealdiBH
deposition in actual new bone formation^ as distinct from salciusa
entry into bone by surface exchange processes and from net calcium
storage ^measurements from balance studies^ gives every promise of
providing a means of obtaining truly basic information on the
mechanisms of bone formation and demineralization is various diseases?
of bon©o The finding of an appar®atly normal rate of calcium deposition
in osteoporosis has already led to as extensive reconsideration of the
accepted concept of the pathogenesis of this disorder*,

Prgposed Course of Projects Continuation is planned of both
E^tabo^cTaalance and 'isotopie "studies* which can be earried ©n
siimsltaneausly in the same patient^ in an effort to determine
the effects and mod© of action In calcium metabolism of adrenal
cortical and gonadal steroids and of the mineral level of the
di®ta and also to obtain understanding of the processes of bone
formation and regorp&ion in various bone disorders©

Serial No* NIAffl) - 13Q-G

PARTJB*_ Honors s> Awards^ and Publications
Publications other than abstracts from this projects

lo Thonjpsdn, D„ D, and Hiatt, H<, Ho Renal Reabsorption of Phosphate
in Normal Human Subjects and in Patients with Parathyroid
Disease,, J, Clin* Invest,, 36s 550-556, April 1957®

2e Hiafct, Ho H„ and Thompson*.. D0 D„ The Effects of Parathyroid

Extract oh Renal Function in Mano J9 Clin, Investo 36* 557-565®
April 1957b

3e Thompson^ DQ Dc and Hiatt, H6 H0 Effects of Phosphate Loading

and Depletion on the Renal Excretion and Reabsorption of Inorganic
Phosphateo J„ C0Lino Invest* 36* 566-5?2, April 1957o

ko Hiatt, H0 Ho and Thompson,- De T)a Some Effects of Intravenously
Administered Calcium on Ihorganic Phosphate MetaboHsiSo Jo dinc
litest o 36s 573»5S09 April 1957*

5<» Whedon, Go D© Steroid Hormones in Osteoporosis o Hormones and the
Aging Process j, Proceedings of a Conference held it Arden House,
Harriaan, lew York, fey 30-31, 1955, edited by E0 To Engle aid
Go Pineus, Academic Press, &ic0, New York, No To, March, 1956©

60 Star, Eo, Lasslo, Bo, Shock, No We and Whedon, Go De Panel
Discussion on Osteoporosiso Jo Am0 Geriato Soec, 5s 363»382j.s.
April 1957 o

?0 Whedon* Go Do and Shorr, Ee Metabolic Studies in Paralytic Acute
Anterior Polioa^elitigo lo Alterations in Nitrogen and Creatine
Metabolisms Jo Clin0 Investor 3&t 9bX~96$s June, Part II, 195?^

( SupplSHSSEEb } o

80 Whedon, Gc Do and Shorr, E» Metabolic Studies:' in SteOyti© Acute
Anterior PolioBQrelitis . ' II* Alterations in Calcium and Phosphorus

Metabolism o Jo Glin0 Investor -36s 966-981, J,une, Part II, 195? a
(Supplement )o

90 Whedons. Go D0 and Shorr, Eo Metabolic Studies in Paralytic Acute
Anterior Poliomyelitis 0 nio Metabolic and Circulatory Effects
of the Slowly Oscillating Bedo Jo Clin0 Investor 36s 982=994,
June;, Part II, 195? (Supplement )o

10o Whedon-, Go Dc and Shorr, Eo Metabolic Studies in Paralytic Acuta
Anterior Poliomyelitis o IVo Effects of Testosterone Propioaate
and Estradiol Behasate on Calcium, Phosphorus, Nitrogen, Creatine
and KLeetrelyt© Metabolism., Jo CHk0 Barest ■„ 3§s 995-1033, Jots,
Part II, 19?? (Supplement )o

Serial Nbo SIflMD-130-C

Ho Heaneyj) R. P„ and I%edon5 Gc T)e Measurement of the
Miseible Calcium Pool and the Rate of Bone Fomatiois
in Man» J0 01in« Investor In presso

Honors and Awards relating to this project? .

lona

; »hih

-reject Re ,
Calendar'' Year 195?

Serial No, NIAMD»1 jj=b c-

lo ClinicalThvestigatians'
20 Metabolic Diseases
3o Bethesda

Part Ao

Project Titles Metabolic Effects of Adrenal Cortical Steroids

Principal Investigators Br0 G3 Donald Whedon

Other Investigators t Dr0 Robert P„ Hsaney^, Dr., Leo Lutwak

and Mr0 Preston omit fa

Cooperating Units? Thi8 project complements (and is cooperative

with) "Trial of New Anti«>Rhsumatic Brug@s0K

Mara Ieare\ (calendar year\l9!>7)* Patient Days (calendar year 19f>7)s
Total « \2/3

Professionals 1/6 • 159

Others l/2

Project Descriptions

Objectives s To evaluate the metabolic effects of various sot
syathSHc sdrenal^eortieal steroids with respect to sodiu% potassium
and nitrogen excretion and in selected instances with respect to
ealeium and phosphorus balance o 'Effective anti«inf2&mT,iatory action
does not qualify a new steroid, f oe -aide clinical trial in rheumatoid
arthritis unless certain metabolic ®id©=>©ffests can b© show to be
minor ©r absent 0 'She particularly tp3esirable effects most often
encountered ssr® sodium and water retention,, and potassium aid nitrogen

lOSSo

Methods Bmpleyeds Under rigid dietary control short-term
2©taboTic studies (e£e weeks) are made of the effect of new synthetic
adrenal steroids on the urinary esteretioa of nitrogen^ sodiuia and
potassium and on the blood levels of the latter two elements© VJhen
short-term studies suggest acceptability of the compound with respect
to the metabolism of these ©laments^, more lengthy studies are carried
out in selected patients for the long-term effects of the steroids
©n the complete metabolic balance of these elements and of calcium
and phosphorus o

Part B included les £J N© JjJ

Serial Io0 MIMD^ 131^0
Page 2

Major Findings; Five new antirheumatic compounds were evaluated
for th^dlTmetabolie effects^ each compound in a patient with active
rhsuisatoid arthritis 0 Results .were as follows?

lo A fourth member of the Zl-desoxy series of adrenal cortical
.steroids., delta-lg 21«desosy« 9=«alpha 21 di-fluoro hydrocortisone (SQD)
was given for six days at 100 mgm0 daily^ then for six additional days
at 150 mgm« This steroid^ which had less active anti^inflajamatory properties
than certain others in the series^, brought about a mild retentiea of
sodium and nitrogen with no change in potassium excretion <>

2e Belta^l^ 9=»alpha flttorOg lo^alpha hydroxy' hydrocortisone (known
variously as lEks Triamcinolone and Aristoeort) was given for 12 days at
a dosage of 30 mgm0 daily 0 A distinct sodium (Heretic effect* less marked
after the first six dayss a modest potassium retention and a definite
nitrogen loss was noted with this steroid which has sufficient -anti^rheus-
■.activity to have led to fa2rly broad clinical us©0

3o A new derivative of phenylbutazone kno%m as G=33 was given for
only 6 days & a dosage of 800 mgaio daily o A slight loss of nitrogen and
potassium occurred and no significant ©Iteration in sodium excretion o

lie BgQ.ta~i§ 6=BS8thyl hydrocortisone in a dosage of 10 ragmD daily
was given for 18 days© No significant change ira nitrogen or potassium

■ excretion resulted,. The sodium data indicated a slight loss of this
element but analyses are being checked »

So Metabolite 1 of phenylbutazone (0=27202 ), 600 mgmo daily^ was
.given for two periods^ the first 20 daya aid the second 10 daysg with
.comparable effects with each administration,:, There was a marked but
transient retention of sodium during Va® first few days on the drug
accompanied by body weight gaina As sodium ssassflntion returned toward

■ control levels^ body weight declined slightlyi when the drug wan dis=
continuedj, a diuresis of sodium occurred accompanied by ©harp weight
loaso Nitro-gen and potassium were retained during administration of the
compound j less markedly but more. persistently than sediumo

Significance to H1AMD "Research s This study is cooperative with 'Trial
of NewJurtEffieiimatie Drugs/' NIAMDS and is important primarily in indicating
whether effective antirheumatic steroids my be safely given t© patients
over considerable periods of time with respect t© metabolic effeetso Of
additional importance is the fact that determination of the metabolic action
of steroids under study may yield information rfiich will give useful leads
t© ehesnists engaged in the synthesis of various cortisone-like ster©ids0

Prenosssd .Course of Project* This project will be continued during the
coming year along present lines, with particular stress on electe©2yte effects

of new steroids and attention to mineral effects in the more promising com-
pounds shown to lack significant undesirable action on eleetr@3yteso
Additional study is necessary to clarify the action of prednisone and pred°
aisolon® on calcium usstsfcolifflio

Calendar lear I:

Serial Noo >g£Sh~^r<Lra
1© Clinieal^nvestigations
20 Metabolic Diseases
3« Bethesda

Part A«,

Project Titles Studies in Anemia

Principal Investigators Dre Rudi Schiaid

Other Investigators? Bre Ted Cleisens*, Jr©

Cooperating Unit as none

Man Tears (calendar year 1957) : Patient Days (calendar year 199! )"
Totals U/12

Professionals 2/3 588

Others lA

Project Descriptions

Ob jeetives s lo Development of a method for quantttaiing and
more accurately localising the source of blood loss freaa the gastro™

intestinal traeto 20 Characterisation of a new type of congenital
and familial hemolytic anemia c

Methods EgBplgyeds lo Quantification of gastrointestinal blood
loss was studxedTbymeans of oral administration of radioactive
ehromate tagged red blood cells to normal ' sub J ects and ^asuresient
of amounts of radioactivity in the stools 0 In patients with knoun
blood loss their own red blood cells were tagged with radioactive
chroaate, put back into their bloodstream and the radisaetivity of the
stools measured o Study was made of the usefulness in bleeding sours©
localisation of the passage of a Cantor tube down the intestinal tracts
followed by periodic aspiration and testing for presence of blood and
radioactivity as the tuiae was passed dom the tafcestinse

2 0 In addition to standard teaatologieal testsg supravital
staining and red cell lifeXspan technique s^, various chemical procedures^*
including eolism chromatography^ absorption spectra analysis and .
elemental analysis!, were used to characterise the specific dark brown
urinary pigment of familial heas&lyfcie aneada0

Part B included Xe@ JfJ Mo £J

Serial Nb0 NM^Isi?.?^

Page 2

Major Findings t 10 The development of a valuable clinical
diagnostic tooTTor detection and quantification of gastrointestinal
blood loss was described in the 19$6 Annual Reports The life»saving
"value of this procedure has been confirmed in three additional patients
whose sites of bleeding escaped discovery by repeated routine methods
in other hospitals

2„ Characterisation of the clinical features and red cell morphology
and life span of a previously unknown and rare form of congenital
hemolytic anemia with red cell inclusion bodies was described in the
195'6 Annual Reports An unusual aspect of the disease which was found
was the excretion in the urine of a dark brown pigment^ never before
isolated from urine and preliminarily, identified as a mesobilifuchsin^
presumably resulting from a defective mode of breakdown of the .anemic
calls o This year* separation of the pigment into fractions and
analysis by various methods have indicated that the pigment is a py.v
probably a di^pyrrolo Insight into a possible different mechanism
of formation of the pigment was afforded by the occurrence in one
patient of a smave hyporegenerative crisis with fall in hemoglobin
3 8 8 grass % this cessation of blood formation im accompanied bj a
disappearance of jaundice and of the dark pigment from the urinee the
latter reappearing promptly with the first indication of return of
red cell regeneration 0 This observation suggested that the , pyrrol
pigment say foe derived from abnormal anabolic processes of hemoglobin
metabolism rather than from foreakdowi of hemoglobin as previously
assumed 0

Significance to HMD Research? 10 Although initiated by
hematologisis in pursuit of more precise diagnosis of blood loss anemia
and of better detection of sources of blood losss the procedure deve]
is pertinent to NIAIffl11® plans to initiate gastroenterological research
intramurally and support substantial extramural research in this
specialty,, Assumption that the gastrointestinal tract is the most
frequent location of insidious and undetected blood loss was materially
confirmed by this woric Although the Cantor tube and serial aspire:'-:
had been suggested by earlier workers,, the NIAMD investigators have-
conclusively shown the method °s importance by quantitative demonstrai
with isotopes of the unsuspectedly large amounts of blood which may
be lost and have dramatized the method's value by detection of blood
logs sites enabling life-saving surgical procedures to be undertaken
in several desperately ill patients o

Serial Mo9 Ml^hlS?^
Page 3

20 The categorical interest of NIAMD in disorders of the
blood has been furthered by the discovery by NIAMD hamatoXogists
of a previously unknown form of congenital hemolytic anemiac In
a fashion characteristic of both the strong biochemical bent of
this institute and of the modern trend in hematological research*
the NIAKD investigators have sanded out chemical characterisation
of the newly discovered rare urinary pigment as part of study of
the abnormalities of hemoglobin metabolism presumed responsible
for this type of sraemiao

Proposed Gouree of Projects Project discontinued in June.,
IP^Ts" when" Drs « "SchmiT^d^emens left NIH to take staff positions
at Harvard Medical School and Oklahoma School of Medicine j, respectivel;

Serial H6* Mi^B^JMdL

PARTJSs Honors*, Awards-, and Publications

Publications other than abstracts from this projects

18 Ebaughj, F0 Sos JrQ and Olesensg, T.s Jr© Quantitation and
Sourc© Loe&Ltsstion of Blood Loss from the Gastrointestinal
Tracts Am© J0 Medo» In Press©

Honors and Awards relating to this projects

Ifon©

Individual Project. Report
Calendar Tear 1957

Serial N©0 jCTAMDgj^jgg

1© ClinicalTSwest^ations
20 Metabolic Diseases
3e Betfaesda

Part A0

Project Titles Metabolism of Bile Pigments

Principal Investigators Dr0 Ru&i Schraid

Other Investigators: Dr0 Ted Clemens, Jr<, and Miss Lydia Hammaker

Cooperating Units: lc Dr9 Julius Axelrod, Section on ^harasacologyj,

Laboratory of Clinical dciencesS Basic
Reseai-chj, National Institute of Mental
Health o
20 Mr„ Samuel Pbiley, Animal Production Section,
Laboratory Aids Branchy Division of
Research Services* NIR%

Man Tears (calendar year 1957): Patient Days (Calendar year 1957) s
Totals ■ 2/3 166

Professionals '' 1/3
Other : 1/3

Project Description:

Ob j ectives s To study the bio*>chenilcal processes involved in
excretion of bilirubin in bile9 with special emphasis on possible '
enzymatic defects in the conjugation of bilirubin with glueuronide e

Methods Basployed: Jn vitro conjugation of bilirubin, with UDPGA
and liver microsomes (by mooXfieation of Strosiinger method) s diasoti-
sations extraction and analysis by paper chromatography! breeding of
rats with a congenital defect in glueuronide format ion| study ©f
patients with congenital non-hemolytic nonobstructive jaundice for
glueuronide conjugation of bilirubin and of adrenal cortical steroids*,

Major Findings : lo A liver ensyme system catalyzing the biosynthesis
of billruBIn ' glueuronide was demonstrated in vitro g the conjugation was
obtainsd by the incubation of crystalline bilirubin with uridine diphos-
phate glucuronic acid and normal guinea pig liver microsomes 0

2a In collaboration with T)r<> Axelrod and Mre Poileyg a e©l©^r ©f
mutant strain of rats was bred which have congenital nonhemolytic
jaundice o The liver microsomes of these rats were found to be exbremely
deficient (reduction by 10 to 20»fold from normal) in the enzyme system
necessary for bilirubin conjugation «, Formation of UDPGA from PDPG
was found to ba within normal limits., but the defect in glueuronide
conjugation included, in addition to bilirubin,? ortho-aminophenol5
anthraailic acid and menthols

Serial Ko0 jffl^g>-°133gC__
Pag© 2

3a Clinical studies of three ehildrea and one adult with this
type of jaadlee repealed that the bilirubin in the serum was of the
unconjugated type and that other char act eristics of bilirubin
metabolism were virtually identical with those of the jaundiced
rates including marked depression of glucuronide conjugation with
mentholg, salicylate* etc In collaboration with !&■„ Ralph Peterson.,
infusion of cortison©°related steroids revealed narked delay in
their removal from the blood in comparison with normal subjects
and with patients with liver disease and that appearance of the
glucuronides of these steroids in the plasma and urine was markedly
delayed and in reduced concentrationso

he Studies8 in both rats and patients having the conjugation
defect^ of the rate of removal from the plasma of infused bilirubin
glucuronide in comparison with bromsulphthalein removal strongly
indicated that conjugation of bilirubin with glucuronic acid is essential
for excretion of the ^Ljp&ob in the M2©o

- Slgnif icanee to HIMD Research g These studies of bilirubin metabolis
represent a derailed characterisation of a biochemical disease by
coordinated investigations utilising in vitro msysologleal taehn3,qu@sfl
in viv® and "in vitro studies of animals and clinical research procedures
in patient so AHbTgir degree of intra and inter^institute collaboration
was also ""domonstratedo These studies represent sa addition t© a growing
list of so-called "molecular diseases.," characterised by & hereditary
defect resulting in the impairment @r absence of an en^rm© which
catalyzes an essential biochemical processo One of the earliest
reports of an ensymatic defect in one of these diseases^ galactosemia^
from NI&MD three years ago0

Proposed Course of Projects Project completed o Dr0 Schsaid left
HIH forHarvara"ledicaI SchoSaTin June,, 1957 •

Serial !fo0 NLgp ^Bj^^

PART B; Honors9 Awards., and Publications

Publications other than abstracts from this project:

lo Schmida R», Hamruaker, Le and Axelrod* JG The En^paatie
Formation of Bilirubia Glucuronide0 Archive BiocheBto
Biophyso 70 t 285»2889 July 1957®

20 Schmidt Rs Some Aspects of Bile Pigment Metabolisms
Clin* Chem„ 3s 39h$ 1957 •

3* Watson* C. J8J, Varco, R» L« and Schmidg R© An Unusual
Case of Acute Porphyria with Volvulus and Gangrene of the
Cecum* Abu J„ Med« 22: 98G-985* June 1957 •

k* Schmidt R«> Neuera Gesichtspunkte auf dem Gebieta das

Galleafarbstoffw©ehs«lsp Jislvstiea Mediea Actag 2kt 273=281^ 1957 <

$0 Az»lrod9 J„j Schmid., R© and HammakePj, L© Bioctemical
lesion in Congenital Non«@bstpucti'^e^ lfon«heE©lytie
JauaSie®e Mature^ 180« 2h260 Deceiver 219 1957c

6o Schmidg Rs The Identification of Direct-reacting Bilirubin
as Bilirubin Gluouronid@<, Jo Biolo Gheme &* Press©

individual Project Re]
Calendar Year 195?

Serial No* NIflMD- X#HL^
lo Clinical"aEiii^HgaSSis
20 Metabolic Diseases
3o Betheada

Part Ae

Project Titles Study of the Formal and .Abnormal Physiology of the
Formed Elements of the Blood*

Principal Investigators Dr0 Frederick dtohlman,, JrQ

Other Investigators? sons

Cooperating Units t DrB De Bergenstal^ mi$ Endocrinology Branch,

is collaborating on a study of the relation."
ship of the pituitary gland to red cell
production and erythropoietins formation,,

Man Years (calendar year 195?) s Patient Days (calendar year 1957 )s
Totals 2/3
Professional t 1/3 2kk

Obhers 1/3

Pr© j ect Inscription t

Cfe^ eetives g Study of factors contributing to the production and
destruction of formed elements of the blood in normal and disease
states o

Methods Employed? Aside from routine determination of formed
elements in the peripheral blood., methods consist of s^asursmest of
red cell survival with Qr51 and differential agglutination.? of red
cell production with F©59 uptake^, and of platelet function with a.
variety of standard coagulation tests « 3h addition*, the ability of
the saarrow of patients with refractory type anemias to respond normally
to standard stimuli (phlebotomy^ hypsrtransfusion and steroids) is
being studied*, Assays for erythropoietins from suitable patients be?:
and after various forms of treatment are being conducted in conjunction
with our basic research project*,

Major Findings? 10 Some instances of refractory anemia are due
to derangement of the humoral regulatory mechanism and respond normally
to such stimuli as bleeding and hypsrtransfusion 0 Assays for erythropoie-
tine in human anemias have demonstrated that certain aplastic anemias
are not the consequence of depressed erythropoietine production but
due to the failure of the bone Harrow to respond to the stimulus >> much
like the situation seen following high doses of radiation inhere eryfchre=
poietine production can be stimulated but damage to the erythroid pre-
cursors does not permit a response 0

Part B included Yes £J Sfe (x[

(See Beport of Laboratory of Pathology and Histochemistry
for publication ©n erythropoietic <>)

Serial Moe NIftMD-13U-C|M
Page 2

2Q EJypophyseetomy does not produce an anemia provided
adequate substitution therapy in the form of cortisone and
thyroid is provided »

Significance to NIAMD Research? inesnia is a common complication
of arthritis and" eertain isietabolic" diseases and nay be refractory to
treatment o A better understanding of the regulation of erythropoiesis
is of basic interest and should eventually result in improved therapy©
Establishment of the role of the pituitary in the regulation of red
cell production is essential since a pituitary erythropoietic hormone
has been postulated based on observations in animals » It is important
from the metabolic standpoint to determine in man whether the pituitary
plays a primary role or is involved secondarily 0

Proposed Course of Projects Continued study of the relationship

of eryShropoietine to refractory" anemias 9 polycythemia^, and hemolytic
disorders and of the relationship of ©2^-gen demand and of the pituitary
gland to the release of erythropoietine0

Individual Project Report
Calendar Year 195?

Serial No« HIAMD- l^«,c

lo Cliiiicalc=E^sSgatiorffl
2Q Metabolic Diseases
30 Bethesda

Part ft«

Project Titles Total TCnargy Metabolisms Studies in Health and Disease
Principal Investigators Br3 G0 Donald Whedon

Other Investigators? Ronald H„ Thompson^ Ernest S0 Bober^ Jre and

Richard Moor©

Cooperating Units s none

Man Tears (calendar year 1957 )s Patient Days (calendar year 195?) s
Totals , -,3-3 A

Professional j2f' : 0

Other 2 ij

Project Descriptions

Objectives ; 10 To establish a technique of total energy
bal mcewiIcE™can b© applied to various clinical problems and to
fiandassental physiological problems of energy ssatabolissa not now
understood©

20 To study the influence on total energy eonsuisption and
balance of various factors^, including clisate and the endocrine
horaones©

3o T© investigate toe characteristics of energy balance and
their influence on the nutritional state of patients in pertisent
disease conditions^ sueh as obesity and cancer,,

Methods Employed s Indirect hraaan cal®riiEatry by means of complete
continuous expired air analysis in the Metabolic Chamber,,, metabolic
balance determinations^ caloric analysis of dietary intake* and exereta0

Part B included yes £J no gf

Serial SFoc, NTWg^l3SgC__
Page 2

Major Findings s 10 Yarious pilot and preliminary but considerably

detailed observations have been carried out in order to determine
with greater definition the feasibility and precision of the metabolic
study system^ these have included continuous observations during sleep
throughout the sight and over 2k hour periods o These observations
have demonstrated f easlbili^r of the system for long term studies^,
but have indicated eertain deficiencies with respect to the degree
of precision expected to be neededo Various revisions of the analysis
apparatus,? therefor e9 have been and are being made*, including^, a) con<=
version of analysers to operation at atmospheric pressure s under rigid
temperature control and on the most stable form of amplification.,
b) alteration of sampling airlines so that oxygen and carbon dioxide
analysers will have nearly identical response times (greatly simplifying
both data integration and R0Qo calculation) and so that analyzers may
be frequently referenced to standard gas samples during the course
of experiments© Provision has also been made for continuous recording
of heart rate and respiratory rate6

2e Collaborative investigation with Glinical End@crin©l@gy Branch.,
NIAMD^ has j«st been initiated of the effects of thyroid hormonal
analogues on activity metabolism *fhsn a patient with severe syxedOTa
was raised to normal levels of basal metabolism on triiodothyronine
acetate., oxygen consumption during standard exercise consistently was
not increased over that found on exercise in the myxedema stat©e At
euthyroid levels of thyroxines howaver9 the patieat showed the expected
greater utilization of oxygen on standard exercise (or decreased
metabolic work efficiency) <> These differing preliminary findings with
respect to the uncoupling of oxidative phosphorylation by thyroid homone
predicted from in vitro experiments of others await confirmation hj more
precisely ©ontrolled stndieso

Significance to HIAHD Research g Previous ■ studies of total energy
B^tabolism were concerned wiSi the influence of various diets on normal
metabolism and were carried out with chambers in which oxygen eonsump=
tion could not be measured directly 0 Other total energy data have been
based ©n measurements of food consumption or on brief measurements of
activity aataboliss extrapolated to tw©nty°four hours o The HIMD
Metabolic Chamber presents an unique facility for the exact and complete
measurement of all important expired and excreted products in the
stiady of energy metabollsa over long periods of time0 3tudy with this
techniqu® has not been made previously of the effects on total energy
of various physical and endocrine factors or of disease stateSo Ah
example of a disease in which energy metabolism may be altered;, to be
approached with the Metabolic Chamber^ is ©besity or overweights

Serial lo0 gIAHD<» 135~c
Page 3

fcserous other NIAMD laboratories are engaged in studies of the
©nergy changes of biochemical processes at the cellular levelo Metaboli
Chamber studies nill report ' energy changes for the entire human
organism,, Although the latter represent oily the net result of
a multiple number of metabolic processes^ it Is hoped that by novel
techniques and by various devices of standardization and control
some coordination may be achieved between these methods operating
at opposite extres©s of metabolism and pertinent basie information
obtained concerning certain key metabolic processes or groups of
processes o

Proposed Course of Project? Investigations will initially be

condueSeooT certain selectecTrelatively circumscribed problems of
work efficiency and activity metabolism^ studies of the effects of
thyroidal saaalogues on activity metabolism will be continued on a small
scale o In conjunction with bhes© studies and in Reparation for long
tera energy metabolism studies^, techniques for determination of body
composition will be s@t up» Work will also be undertake® in normal
subjects to establish the technique of energy balance over periods of
several daysf .later this technique will be applied in studies of
diseases Additional developmental work will be carried out toward
construction of the direct gradient calorimeter within the existing
sjhambe?o

^ddu&l Reject Report
Calendar Year ±957

Serial ?fe>o NlteD-

lo Clinical fwestigatl

2e HetaboH.c Diseases
3 c, Bethesda

Part As

Project Titles The Effect of Sulfonylurea Derivatives ©a Intermediary
Metabolism in Man

Principal Investigator s Dr0 Thomas F0 Frawley

Other Investigators s none

Cooperating Units? Br* Stanton S©@8>2g Clinical Endocrinology

Branchj, THAI®

Man Tears (calendar year 1957)? Patient Days (calendar year 195?)?
Totals 1
Professional? l/2 191

Others 3/2

Project Descriptions

Objectives? T© compare the changes in carbohydrate metabolism

accompanying tEe hyp©glyeen&c<==>indueing effects of the sulfonylurea
derivatives with those observed following insulin as a possible means
of determining the locus or loci of action of the sulfonylurea
derivatives*,

-Methods Employed s In clinical gtudies^, the short^tarm effects
of the sulfonylurea derivat^e^orinasej, have been investigated using
intraveacro administration of sodium orirmse0 Glucose loads hav®
been given both before and after the administration of orinas@o Blood
levels of glucose^ phosphate^, potassium* pyjfwie acid and orinase have
been determined,, In the same subjects the studies have been repeated
casing insulin and^ except for blood ©rinase*, making the same determii
In vitro studies using rabbit liver slices have been made for the
comparative effects of ©rinsse and insulin on liver glycogen^ oxygen
eonsufflption and medial concentrations of pyruvate and laetateo

M or Findings s 1© A new technique has been introduced for the
study of mechanisms of action of the orally active drugs now
being used in management of diabetes „ The procedure involves rapid
intravenous infusion of tolbutamide (Orinase) in a fashion similar to
that used for studies of the action of insulins producing an hypoglycemia
identical in onset and degree with that produced with intravenous
insulin^ and making possible a variety of studies giving direct comparison
of the action of insulin and of tolbutamide e>

Part B included Yes JJ I© £J

Serial loo MIAHD<*> 136~C
Page I

2n After the initial comparable fall of blood sugar Taken
insulin or tolbutamide are infused intravenously a msaber of clear-cut
differences oeeumsd which, indicate that tolbutamide does sot work
merely by enhancing the action of endogenous insulins a) return of
blood sugar to normal levels is considerably delayed following
tolbutamide^ b) when glucose is administered following a hypoglycemic
agent (in tolbutamids^glucoae tolerance tests versus insulin-glucose
tolerance tests),? with tolbutamide blood sugar does not later over*
shoot normal levels as it does with insulin^ c) following tolbutamide^.
blood pyruvate falls whereas M&ih insulin it rises*, and d) conversion
of infused fructose to glucose is reduced by tolbutamide 0 These
findings suggest that tolbutamide exerts its hypoglycemic action by
interference with release of glucose from the liver o

% 3h vitro studies supported '^m above indicated action by
showing i3B.F£H~on of liver glycogenosis by tolbutamide c In vitro
studies also showed that disappearance of added pyruvate from media is
enhanced by tolbutamide©

lie Comparison of results in a considerable member of oral
glucose- insulin tolerance tests and oral gLucose*t©lbutamid® tests
showed conclusively that the new agent does not alter or impair
absorption of glucose from the intestinal tract o

5o A ©talking difference in peripheral action between insulin
and tolbutamide was shown in collaborative steadies with Drc Sggal
©n disappearance curves of infused psatoises© Insulin facilitated
the disappearance rata of D-sylose and L-arabinose but fcolftatsssid®
was found to have no such effect^ strongly suggesting that sulfonylureas
do not act primarily by enhancing the peripheral action of endogenous
insulin0

Significance to WlkW Research % These studies are along the lines
of the Strong categorical interest of NIAMD in research in diabetes
and carbohydrate ssetabolisao 3a addition to the obviousu immediate and
practical implications of the work in contributing some of the first
definitive material on the mod© of action of a new hypoglycemic agent
already in clinical use in the management of certain diabetic patients^
the research contributes valuable basic informatics! both from patients
and from tissues which hare increased bur understanding of the altered
carbohydrate metabolism in diabetes and of the mechanisa of action of
insulino

Pag© 3

Mors speeiflea32ys the work clearly shows certain significant
differences in the processes of glucose utilisation icnas^ the in=»
flusnce of insulin and ©rims© so that it is possible to conclude
that these agents are not interehamgesblegttafc experience with insulin
cannot, be used to anticipate the full effects of orinase and that &
considerable period of eHssiogX utilization of orinase and much
additional information on its effects must be obtained before it can
be decided whether or not this compound, will be a -taa3y useful
addition to methods of n*asag©Hie2st of diabetes patients 0

Proposed Course of Project a Project discontinued with dspartur®
of iW Frawley for Albany Se32cal College (Associate Professor" in
Medicine )o

Serial Ho0 «gjMD~i36-p

PAftT-Bt Honors, Awards^ and Publications

Publications othar than abstracts from this projects

1* Frawley, T9 Fop Sagal, S«, Camus," M. M0 and Foley, Je A Concept
of 1^ie Mechanism of Sulfe>Bylure&~>indueed Hypoglycemia Eased on
Studies of Glucose and Pentose Disposition in Km<> Annals of the
Ks I,-' Acad, of Med09 71* 8l-96# July 195? »

2© Frawleya T„ F„ Th® IhtraTOnous Use of Sodium Tolbutsmid© in Acute
Studies of Carbohydrate Metabolism. Th® J0 Cline Endocrine & M®tabos
17$ n2k°°n.279 Septet er 19S7 .'

Honors and Awards relating to this project 5

Mono

.ARTHRITIS AND RHEUMATISM BRANCH

I

and conjunc I i ■■ ■ ■
in a bow J, J to 2 percerai have n©n-§pec

is m d is seen most f requenfcly in iniHi
The polyarthf itis may be acute- subacute, chronic of :
siany ®f the clinical features and radio]
those seen in rheumatoid i
is mistaken for rheumatoid arl

d 1 1 ®ee« not unlike
of one may yield a ' o the e £ the other- Dienes h:

orted observations which led to the cons
PPLO organism as a possible etiological agent I
drosise but Dienes concluded thai a definite causal rela
could not be e ; ed

Of twelve :,
us during tl jar at Walter Reed Army He-
Naval Medical. Center, two were select sion to

-ical tenter ox intensive baciteriologic aac
One patient recovered spontaneous! a few days and

cipated studies co I be douse In the
prel ■> . findings have \ quite i«

; on of ■?
By spec Hal cull I in two-

other collaborating laboratories recov<
from this patients" blood, synovial fluid,

and conjunctival secretios done to

determine whether or not the patient teas developed specific
antibodies lo this Microorganism- Similar cultural and iiamtt=
nological studies will be done in other cases of Heller s
syndrome &nd. in cases of rheumatoid arthritis asscJ traumatic
arthritis-

.
» i d a ? t h 8

id Use latex fixation I bentoni

which have ar.

ve electric charges „ are era{
ead of * , eryilu ar polj

The bent on He I on test C8FT
sevei ■

t as?.d read liable- The tc iple and
does not require f rsctionation of th
'■ion by sheep erythrocyte?* or the addition
rafc": in 72 case.® c ied rheumatoid art

she BFT was positive in S5%- In 163 controls, fa
taction occurred in less than 2% and these cow
be explained in each case- sheep cell agglu-

tination tests and BFT were done in 65 cases -1" rheumatoid
m©R nd the s ; agreed in 94%- Reports

received from work i other laboratories in this count
who have adopted the BFT confirm our finding:*.

During the past few attempts have been made
MiAMD and ot Late and chemically chs

terize the ■ oid"9 factor responsible for sheep c
agglutinate etion. To attain these ©bjec has

been deemed desirable to develop a precise method ior measuring
the activity of : heen achieved by NIAMD

scientists daring the past year The method utilizes a hemo-
lytic agent., the Newcastle Disease Virus sensitised with a
quantity of rabbit antibody. The degree of inhibition of
hemoU i 'esulting from the addition of euglobulin from
rheumatoid serum is measured- It has been established tSi
inhibition of hemolysis is a function of the quantity of
sheep cell agglutinating factor and that results can be di
Heated with an error of only *5% By methods previo;
employed, the error ranged from =100% to i'200%.. Frsct
by eel lu.8 rase i&n exchange column chromatography has yielded
pre;. 25s which have 800 £o 2000 times the specific
activity fas measured by the new method) of the serum from
which the fractions were isolated- Correlation of acfciV'
wish ul fcracentrif uge patterns indicates that the highest
specific activity is present in fractions with the greatest
concentration of approximately S 19 material -

Synthetic anti-inflammatory compounds have been studied
with several objectives in view: Ca5 to measure relative anti^
rheumatic potency, (h) to study metabolic ^nd hormonal effects
(ci to detect, characterise, and assess toxic effects* and d'd)
to relate chemical structure to biologic activity- The com-
pounds were of three types.- CO 11,17 oxygenated sterol.

phenylbutazone derivatives and C35 ehloroquin. The new
synthetic steroids included prednisone, prednisolone.

■

prednisolone, and 16a~roethyl predispose Cat!
and prednisolone have now been evaluated in a group of 59
patients with rheumatoid arthritis, each receiving minimum;
maintenance dosage and only two requiring more than 15 rag
daily-. Of these 59, 14 patients are being followed regis-,
in a long-term study. Seven of these 14 have attained fu
functional! capacity, 2 others are only minimally handicapped-,
4 are moderately handicapped, and one is confined to a bed"
chair existence. Side effects have been a problem in 26 of
the 59 patient* arsd include peptic ulcers in 14 patien
frg«: si5 9, diabetes in 2, severe infections in 2. and
some mental changes in 7, Triamcinolone was evaluated fin 4
rfeeuma&oid patients- Metabolic studies revealed sodium
diuresis and slight potassium retention on a daily dose of
20 mg- This drug was effective as an antirheumatic agent
ar»d had about the same potency h&gin for ragraJ as prednisone.
Side effects included severe sweating in 3 patients and
epigastric pain irao ulceration) in oner A group of 4 syn-
thetic steroids., characterised by loss of the oxygen atom at
carbon 21 and fluorinated at various sites was found to h
potent pituitary suppressive effects at doses of 50 to 150
rayms daily. The loss of oxygen atom at the 21 position a
parensly overcame the mineralocorticoid effects of the 21 ■
oxygenated fluorinated compounds studied p Only

2 ©f these ''il-desoxy'" compounds produced sodium retention
and this to but a mild degree. Three of these compounds had
slight but unimpressive antirheumatic properties at the

Pie employed. It was concluded that compounds in this
group would be of little value in the treatment of rheuma-
toid' a*= Metabolic studies of 16a-- methyl prednisone
in dail y doses of 19 rag- revealed no sodium retention, no
nitrogen or potassium loss, potent pituitary suppression
and antirheumatic properties equivalent to that of predni-
sone Con weight basis'? in 9 patients. One patient developed
a gastric ulcer and another slight edema No other serious
side effects were demonstrated. In clinical trials with
phenylbutazone derivatives (G-33 and G-27202) it wa$ found
that all of 6 patients experienced moderate objective and
subjective improvement on G-27202- in contrast, G-33 ex=
hibited no significant antirheumatic effect-, The erythro-
cyte sedimentation rate did not fall in any patient- One
patient taking G=272©2 developed a skin eruption and two
others severe Seucopenia- Chloroquin has been administered
in 250 mgra daily doses to 4 patients- One discontinued the
drug after I month because of abdominal pains which disap-
peared shortly afterward- No patient has experienced sig-
nificant relief while receiving this drug

e of pa- th salic

ceivsn.j Ceroid therapy have* been noted since adrenocort.- .
steroid? and corticotropin have beers used in the treatment of
rheumatic disease®.. It has been suggested that salic.}
act through stimulation of the pituitary adrenal system.
Presen* methods for study of adrenal function in man have
made It possible to determine the relation of the effects
salicylate to adrenocortical function. No increase in p]
hydrocortisone,, corticosfterone , urinary corticosteroids, or
17-ketosteroids was noted, following the administration of
single large doses «.3„6 gin- 1) or during periods of continue
<2.7 to 8 gm, per day for 3 to 50 days'! administration of
salicylates to' normal subjects and patients with rheumatoid
arthritif Salicylate therapy also failed to alter the rate
of metabolism of intravenously administered hydrocortisone a
cortieosterone In (addition, studies of turnover rate of
hydrocortisone were conducted in two normal subjects -and in
one patient with rheumatoid arthritis; in none did salicji
therapy increase the turnover rate, Oise subject with rheums
arthritic clinically responsive to 3,6 gu, aspirin daily was
given £$ 9a~f iuorohydrocortisone in quantities '12,0 rag per
day) sufficient to suppress hydrocortisone production,, >
inadequate to exert an antirheumatic effect, Good clirai*
response to 3-6 gm, of aspirin was observed even though
adrenocortical function was suppressed, These studies suggc
that antirheumatic effects of salicylates are not mediated
by increased adrenocortical activity..

Administration of estrogens tethynyl estradiol 0.-5 rag,' day?

caused a two to three-fold increase in the plasma levels of
hydrocortisone and corticosteroner This was associated with
a gradual fall in the urine corticosteroid excretion, and a
delay in the rate of metabolism, of infused hydrocortisone
nnA corticosterone.^ In addition, a significant decrease in
the rale of synthesis of hydrocortisone and corticosterone
occurs as measured by turnover rate studies. The very high
plasma hydrocortisone levels may be maintained for many weeks
with estrogen administration,1 however these subjects do not
show any signs of bushings syndrome- Thus, estrogens appear
t© interfere, in some manner, with the biological action of
hydrocortisone in the tissues- The anti---inflaramatory activr.
of hydrocortisone also seems to be antagonised or inhibited
when estrogen is administered to subjects with rheumatoid
arthritis

ALKAPTONURIA AND OCHRONOSIS

A quantitative assay of each of the enzymes involved
in tyrosine degradation was made iss liver horaogenate:; sliver

!

S ack of

■' an inh ,: --

defect in a e to

z e ! I v e r BG A o x i d a •<■ e - T k e © t

\ i © 1 g t h e $ y ss t n e & a § © f HG A ox a d a & e a s
defec iiwes of tonurics who carry

fcrajjr relatives should appear

but might show 21 lower tolerance %l
noinaal if given a large amount of horaogentific acid tc
1 nee tests using phenylalanine or hoti
made is a few re^ of alca ic pat-
1 and have so far shown no a on ©if ma 11 plasma HGA 3. e^i
her tests are planned,-, and. direct en ay ma tic assay of
live] specimens will fee ear.? lied out when they become
anil a b 1 e ,

The liver enzyme which catalyses the forma? son of
homogeni cid (HGA) from p~hydr©xypnenylpyrivic acid
tPHPI been purified over 300 fold from &&q liver and

airacfceri have been studied- The weed
for V ,i; oat; be completely replaced by 2,6-dfictolo
phenolindophenol dye C 2 , 6«Di. PP 5 in the purified system and
t fee determination ©f the role of these agents 1st ■■ ■.! =
da t ion are under investigation* A need for ej was
demonstrated in the purified system and its srs

to be to protect the enzyme from hydrogen peroxide arising
frora the oxidation ©f PHPP rsther thass to take part as a
peroxidase- Further studies on the ©i ied

ensyme ins regard to metal components &n& essenf Ifhy-
dryl groups are under investigation. This 1 stem
& 8a faiSisn tissue was, found to be similar to that in animal
including the effect of activators and Inhibitors

GO 01

Studies on the nature of the metabolic defect lira go;
continue and In the past yes- ielded results of unusual
interest It teas been found that 4~ami»©«5'=iraidfflzolecas>li»«
raide~C CAIGJ is incorporated into uric acid by all gouty
patients to a greater extent than normal ,, e.ve» in those
patients who had shown a normal glycine W'-"3 fliscorpoffai
It has been concluded that increased de, novo biosynthesis of
uric scfd is at least one of the raetabolTb "'defects in gout-
As reported previously,, the simultaneous administration of
Alt. and glycine to normal subjects results in suppression of
glycine incorporation into uric acid,. Although some suppi
$ion of glycine incorporation occurred in all gouty paties
the magnitude of suppression was not as great as in norma]

:

who - a normal pattest of glycine^1 ° incorporat

showed a pattern of. excessive incorporation when
glyci^e-J-C^4 was used Study of .this discrepancy was pui
by administering glycine labeled with both isotopes- Pre

r,s show &he two isotope® appear la isric acid
in the proportion as in the administered glycine., For this
reason the discrepancy in results cannot be explained by
preferential incorporation of carboxyi-carbon over aminos.''
gen„ but is probably due to the difference fin the. dose of
glycine required for the studies with glycine-N11*5 ?7 grams)
compared with the trace quantities required for the glyci
Ci4 studies CO. 8 milligrams). It is important to settle
point, because in previous experiments it was found that i
gouty patients studied showed urate overproduction when
glyci»e-US4 was administered. Several patients with urate
renal Hthiasis but without stigmata of clinical gout are
being investigated in order to learn whether the inet'abo;
defect characteristic of gouty patients can also be detected
in these subjects- Two such patients have been studied by
the administration of glycine-C14,. The results ^available
fchus far in only one case) Indicate increased incorporation
of glycine into urinary uric acid,

For the past two and one half years „ we have studied
the serum urate levels and urinary uric acid excretion of
patient? in various stages of gout receiving a constant
diet, It was foscrad that until severe renal impairment ap=
pears, the average value for urinary uric acid excretion
was not significantly different from normal! controls- Pat-
ients with tophaceous gout and severe renal disease.- ex-
creted about half the amount of uric acid found in norma'
A new uricosuric agent, G~2831o, a sulfoxide analog of
phenylbutazone,, was found to induce uricosuria in three out
of five patients with tophaceous gout and severe renal im-
pairment who have not previously responded satisfactorily
lo administration of older uricosuric agents, probenecid
or sodium salicylate,

A systematic evaluation of the effectiveness of inti
venously administered colchicine for treatment of acute gout -
in 40 cases has been concluded- Undesirable side effects
observed were a febrile response in one patient which may
have been attributable to this medication, a frequently ob=
served local irritation at the site of injection,, an occas-
ional transient drop in polymorphonuclear leukocytes and a
temporary elevation in BSP retention which occurred an a few
of the patients treated. However,, no lasting or seri®us
toxic effect® were encountered in our entire series. In the
majority of patients, intravenous colchicine produced relief

jck

aether uric acid riboside
©coiiijfi in human erythrocytes in invoked in uric acid met
li/m an man,, the metabolism of this substance wa ? studied-
ly laboratory ass enzyme was fouwd which catalyses the
■winy reaction:

So acid riboside ^JMXS.PALS^PlSlfiiM^^ Oric i

An analogous enzyme, purine nucleoside phosphorylane , tea-
shown by others to catalyse the reversible reaction'

12) Purine riboside * Inorganic phosphate--*-^. Purine * R

We have not yet been able to prove that "X" in reaction
ribo:v,5=-l-pfeo'sphate . nor have we been able to detect the biosj
thetif: of isaric acid riboside when enzyme- uric acid and
bose-U-phosphate are incubated under the same conditions
required for reaction i I ) It is possible that %be enzymes
for <fl'1 and £2) are not the same and that the reaction mech-
anisms are also different. We have investigated the prop
of purified uric acid ribosides© obtained from the following
sources' beef liver, human spleen and from heart,, kidney and
intestinal mucosa of other animals. The high levels of activity
of this enzyme in kidney and intestine raise the question of
whether it \$ involved in the transport of uric acid across
membranes

GENERALIZED OSTEOARTHRITIS

Since v>.ry little is known about the cause, predisposing
factors or ever; the course of osteoarthritis,, a detailed study
of a large family of 400 having a high prevalence of osteo=
arthritis was beiun in order to learn something of the genetic
factors involved. This family is a fairly stable group,, re-
sides in a small veographic' area, and exhibits some degree of
in-breeding. Previous information revealed a high incidence
of "back trouble"1 <nd a preliminary survey showed severe gen-
eralized osteoarthritis with obesity and short stature in a
proportion of individuals- Although n© patients under this
project have been aonitted as in-patients to the Clinical
tenter, to date 52 individuals have been examined,, and per-
tinent laboratory wok-k done,, The patients have been subdi-
vided into 3 age group-;,, (A J Under 2© years of age,, 24
patients (11 males and 13 females!) ? (B) between 20 and 39.,
16 patients (5 males an4 11 females),: and it) between 40 and
79, 12 patients C4 males and 8 females)- None in Group A
was found to have osteoarthritis. It is of interest that 6
of the 16 in Group B had osteoarthritis of the spine- but no

of p

t ne-
on osteoartfou
osteoarth- b genet i

so susceptible , obesity con o the occurred-

• disorder.

THE ENZYMATIC METABOLISM OF STEROIDS

The enzymatic mechanism of steroid 11 pi elation
have continued with the following advance?- sew hea
stabile co-enzyme necessary for the hydroxy! at lorn react'
has been found in liver: placenta,, adrenal and testii
liminary studies suggest that it is not a compound previou-
implicated An biochemical reactions and it's isolation and
characterization are in progress at this, writing,. (2} l
dence has accumulated that 3 separate enzymes are required
for hydroxylation and each of these is in the process of
purification and individual study. Preliminary data indie.,
that one of them way be obtained in crystalline form from
adrenal mitochondria- C3) Modifiers of physiological and
pharmacological significance have been studied and it ha§
been found that ions are inhibitory in the hydroxylation
reaction- Since aldosterone,, the salt retaining steroid..
1 l"hydroxylated, the implication of this finding for the ho-
raeostatic regulation of intracellular ionic strength is
apparent, The adrenolytic drug, araphenone , and a host of
other aromatic compounds have been found to inhibit the
hydroxylation reaction.. It has also been found that dig; -
tonin., in a whole series of surface active agents tested,
can stimulate the reaction but the same wa<? wot true for the
cardiac~active glycosides- (4) Studies have been started e
the mechanism of the hydroxylation reaction .in fungi foe
are still in a primitive state, Several experiments sugge
that it is possible to demonstrate the hydroxylation reacti<
on cell=free mold extracts and that the enzymes may be sub-
strate-inducible,

B Ite^ojiKeJujaMl

Further work on the reduction of ring A of the steroid
nucleus has suggested that the reaction may, in fact,, be c .
alyzed by a f iavin=containing enzyme.- Is* contrast to c
earlier findings, it has now been found possible to sufostitu&
; dithronile and safranine-T for TPMH as the enayraatically
active reducing agent-.

Further©: >'e , it has been found that liver horaogenates
prepared from thyrotoxic rat are capable of catalyzing much
; faster steroid reduction than comparable preparations from

ion

in the
The increase in sk has been i
to the 'microsomal fraction and a study of the properties of
the ""thyroxine-induced"' enzyi.: is a matter of current
investigation..

^ !JlJ^JJidiej=cjLn_j^

The purification and properties of a pyridine nucleoli^
linked enzyme from mammalian liver which catalyzes the reversible
oxidation of cyclic 5-,, 6-, and D-carbon alcohols to their cc
responding ketones are being presently investigated. The t<-
systein is the cyclohexanol-cyclohexanone couple, but the struc-
tural relationship between these compounds is prompting this
investigation „

STUDIES ON THE idECHANISil OF ACTION OF STEROID HORMONES

In connection with a study of the biosynthesis of hys
uronic acid J' see 1956 report), it has been found that several
adrenal corticoids may inhibit the entry of glucose into the
synovial tissue cells- In order to investigate this phenome-
non more closely,, liver tissue was also studied and, instead
of the expected inhibition, a marked stimulation of the con-
version of glucose-C14 to C140-2 resulted from the inclusion of
A^-hydrocort isone 1 prednisolone.) in a reaction mixture with
either liwer slices or homogenates. To our knowledge this
represents the first stimulatory activity of a corticosteroid
in a cell-free tissue preparation. Further studies indicated
that the stimulation was not as a result of increasing the
rate of the hexokircase reaction nor was it at the level of
the Krebs cycle,, The mechanism of the effect is as yet un-
known but is under investigation.

Similarly,, an inhibition of pyruvate oxidation catalyzed
by liver metochondria has been observed when the incubation is
carried out in the presence of adrenal corticoids. This could
serve as a biochemical explanation for the elevated blood
pyruvate level in patients treated with steroids or' with
bushing's disease. The mechanism of this effect is likewise
being studied. Several features of these experiments should
be noted, 11 The steroids display the same structural spec-
ificity in vitro as in vivo„ i,e,., the hydrocortisone derive-^
lives are active while the cortisone derivatives are not. 2)
These effects are produced by the addition of steroid to a
cell-free extract in vitro ■= not as the result of pre-treatment
of the animals. For these reasons, the present investigations
seem especially promising.

!

u r i d p hate glucuronic acid i 00 P 6<A

cep? -en demonstrated- The apparent

eatalyzed by the same liver microsomal enssyrae which forms
'"ether*" and ""ester"' glucuronides and may represent an im-
portant new metaboiic pathway for amino»coinpound metabolism.
It Is of interest that the basic dimer of hyalurSc acid,
N~acetyl hyaluronic acid, was apparently not synthesized
this enzyme,

STUDIES ON THIS BIOSYNTHESIS OF THYROID STIMULATING HORMONE

The chromatographic purification of thyroiditis

hormone CTSHD ion exchange column developed by Uondliffe and
Bates suggested that the boisynthesis of this specific protein
hormone could be studied and would be of particular interest
since its synthesis is under hormonal control Ci-e, inhibited
by thyroxine.*. Incubation of TSH--°producing mouse-pituitary
tumor® with o*4 ylutanin acid followed by chromatographic
isolation of the hormone suggest that there is formed, jhi
vjt.ro, , a radioactive protein compound with the biological
activity of TSB, These studies will be extended to cell-free
extract:?- in an attempt to study their mechanism

A NliW PHYSICAL METHOD FOR THE
DETERMINATION OF STEROIDS IN BIOLOGICAL EXTRACTS

During the past year NIAMD scic i developed in

this laboratory a newc very sensitive and specific method
for the assay of steroids- This is a quantitative assay pro-
cedure using a modification of the isotope derivative dilution
technique previously applied to amino acids. The extract com-
taining the steroids is first acetyiated quantitatively with
tritium-labeled acetic anhydride. An authentic sample of the
steroid .to be assayed is reacted with acetic anhydride-carboxyl-
cJ4 and a measured amount added to the extract as an indicator.
The mixture is chromatographed repeatedly until freed from other
tritium-Sabered materials- The tritium and carbon*4 content of
the puire -steroid is assayed by simultaneous counting in a Ti
Carb liquid scintillation spectrometer, From the determination
of the amount of indicator steroid lost during the purificat
and yield of tritium radioactivity plus a knowledge of the
specific activity Ccpsn trit iuia/micromole^ of the steroid to be
determined, it is possible to calculate the amount of steroid
present in the original extract- The procedure has extremely
high sensitivity (will determine 0,01 /xg of steroid)),,, and has
the inherent specificity and accuracy characteristic of isotope
techniques.

chemical metho
del.' ram and gub-ralcrograra quasi

b biologic?.1 selective assay of

dividual steroids in biological extracts usually requ-
extei purif icatlon , and this cannot as a rule be accom-
plished without significant and unpredictable losses-, The
development of this method has; made it possible to extend
our studies on the metabolism of the steroids in both man
and animal , to areas previously not readily accessible to
study,

M^il.boJJ^m_®XJ^^£M^rjiLne_ The isotope derivative assay, in
cop-binaiion with the use of very high activity tritiuiu-isbeled
aldosterone, have been used to study the physiological dispo-
sition and metabolic fate of aldosterone in man- The labeled
aldosterone was prepared by exposing aldosterone monoacetate
to tritium gag and this material then purified extensively I
paper chromatography to a constant specific activity,, 1,1
microcuHe* /microgram- Preliminary data indicate that, aid
terone bolized XS, vjvo. at a rate about twice that of
hydrocortisone. The metabolites of aldosterone appear in the
urine very rapidly and more than 80X> have been excreted within
the first 24 hours- Aldosterone appears in the urine as free
aldosterone and as a conjugate bydrolyzed with acid- It was
not possible to demonstrate the presence of ®n$ aldosterone
conjugated with glucuronic acidt. Attempts are now being made
to determine the raiscible pool and rate of turnover of aide
terone using these same general techniques;- With the isotope
derivative assay, it has been possible to measure .the total
free and acid«hydrolyzed conjugated aldosterone in the urine,
and the free aldosterone in the plasma- With this method
normal subjects have been found to excrete between 5 and 20
micrograms of aldosterone per day-. The blood plasma contains
about 01 micrograms of aldosterone per 100 ml,-

JUL^L^^SJkiM^SLLJdAaJLkMJ^SJ&S." In collaboration with
ientists studies are being carried out, in dogs, ok the
factors controlling the secretion of aldosterone by the
adrenals- These studies entail the use of dogs with hype
aldosteronemia resulting from constriction of the thoracic
vena cava- and measurements of adrenal vein plasma aldosterone
with the new isotope derivative method.. These studies to date
seem to indicate that the liver,, through either a humoral or
neurogenic stimuli,, plays an important role in the regulation ^
of aldosterone secretion- The extracellular fluid volume
does not appear to be a prime regulator of aldosterone secretion
ikffeja^£.J&>atfe£JlSJ^^ In collab-

oration with He I scientists, studies have been carried out on
the effect of amphenone on the secretion of various adrenal
cortical steroids in different species of animals. These
studies have involved measurements of adrenal vein plasma
steroids by the isotope derivative method. The studies to
date show that auphenone may produce different effects in

J2£j£iUiUj

NHI sscih

... dies

■
one a marked
sioM ed ad re

hypertrophy. The adrenaS. cho ol -. a preci-

replaced hy dihydrocho
choice lenone . The adrenal v< jsraa of the cho]
fed liow§ a marked depression in the levels of c

i e rone,-, and hydrocortisone, as mean
isotope derivative method «ot been p ■-..■

my a* tera t loss i
chole^tenome?,
■

iUbjl

pool Q to U00 /i.g , and t:

10 tiroes per day.. ACTH admj

a J K y ■ in the

than of e<, Xn liver diseas

some de mover a

high a<: d labeled c

an exp
mental model wais developed for measuring the
with much larg« of carbon-14 lab*.

corticosterone. This procedure

favorably with the tritium m e conventional

trace o abeled cort rone, '

to the pro. seal have established eEed

steroid,? are metabolised
labeled

CLIN HAL tiNMiKINOLOGX ....BHAjjffg

The Clinical ■ lindocrinalogy Branch of MIAMI)

major ef.i
carbohydrate me
me 1 3

i>a rboh vdra te .motabo
carbohydrate me

on the effect of diabe Issra, have he

completed Thiss w olved a ssitsdy of the metaboJ:
several b--v&tarains in normal subjects and In indiv
diabetes? meilitus who we

those without degen I icationg •

diabetes were lest
controlled with ins
been withdrawn- On a standard

I

degenerg than did th<

two , During a period of insulin deficiency, d
without complications excreted wore thiamin and panthothenic
acid than did the other two group®. There wag no clear c
relationship between the degenerative compl ications of disbet.e'
and the pattern of vitamin excretion..

been

up to
(more

for h

large

level

pho®p

The m

pears

as du

phate

with

the s

I n d i v

along

norma

giluco

shows

Wo
cent

J a

rap
andl
r do

of
hate
ecfaa

not
e to

a
ribo
ugar
idua

thi
Uy
*e i

a 1

rk on met
inued- I
rams of U
idly than
i n g t h i §
^es of U«»
pyruvate

and a p r

nisra for

ta be du

inhibiti

known int

se !-c!14

..-letab
Is wit
s pathway
met a bo Hz
s o 1 a t e d £
abeling p

a bo H sun in
t has been
~ r % bo s e c a
D»g2 ucose
sugar appe
ribose pro
in blood a
onounced d
production
e to I n $ u 1
on of pfeos
erraediate
have shown
olized to
i a betes me
uraabl
ed via gits
rom isrlne
attern con

ma n o f a

found tha
n be roe tab
K Above
ars to be
duce incon
nd a very
epression

of hypogl
in and is
phogl ucoiau
in ribose

that in
c09 to an
Hitus ha
y due to t
cose. Deg
of a diabe
sistent wi

v a

I h

I h

riety of pent©',
in normal individu

ized very rapidly
is level the sj
turated- Somewhat
ant changes in the
fight faH in serum

blood glucose lev
eraia from D~rlbo$e
ntatively identifi
se by ribose<-5,pbo
tabo'lism- Studies

normal individual
lent greater than
a block somewhere

fact that rifoose
dation of labeled
c given ribose-1-t

this notion,

50%

The metabolism of the tr*1 labeled pentoses- U-xyl ose
D^arabinose 0 L-arabinose and D-lyxose have benn studied- All
except L-arabinose are metabolized to carbon dioxide. in
addition. as yet unidentified metabolites of these sugar®
have been found in urine.

tuiaai
leea

iminary
i to a
i the v
rupt ion
i e me t a
so been
*hat pe
i me tab
iiiolo^y
si ucida
id towa
' the t
led on
< norma
s and t
i a 1 an
ind par

trk h
;tat i
? tha

' the
I i Sill
sted,

I? to i
Stud
an of
s obt
°©id
> syn
ind g

I 6 ;;?i 1 a

iopro

illy

as been

ng woman

t fucose

carbon

of label
In gen
represen
n ma n .
iei on t
the exa
aining s
hormone,
thesis o
oitrous
ntable r
tein , wh
characte

done with
A Isbel
is derive
chain has
ed xucose

thes
% an impor

he thyroid
ct mechani

owe insigh

To this
f iodoprot
human thyr
at thyroid
i c h i -
rized from

carbon labeled
ing pattern con-
d from glucose
been found- Rapi<
in normal individ--
e investigations
tant pathway for

have been direct
sin of synthe
t into the inechan
end studies have
ein& Jn, yi.vo and
oids and in norma
tumors- In the
thyroglobul in . h
the standpoint o

ed

f

certain j £« addi;

I dirt e have bee a demons!
dinated compounds are at present; under Jn the
piantable rat thyroid tumors, very gene

VvoUman from the National cancer Institute, two separate
tumors? have been intensively studied,. The first tumor con-
centrates iodide as such, but is incapable of forming any
organic iodinated compound®. Although this tumor contain®
negligible quantities of thyrogi ffibulin0 the absence of this
protein has- been shown to be probably secondary to absence
of the enzyme responsible for the formation of the active
iodinating species- Studies with thyroid stimulating hor-
mone and triiodothyronine have shown that at least in this
tumor the iodide concentrating mechanism is unaffected by
variations in the level of pituitary thyrotropic hormone
This agree* with studies reported last year in a case of
goitrous cretinism in man The other thyroid tumor ttudijed
has been found to synthesize two abnormal iodinated proteins
and one species of iodinated material associated with mito»
chondria These appear to be alternate pathways for iodine
metabolism. Their significance is not, at this time en-
tirely clear.

in v itr o, studies have been directed towards elucids1
of the mechanism of condensation of iodotyrosine to form iodo-
%ro»"Js?eS: it has been shown that the acetic and propionic
acid derivative of tyrosine will condense to form iodothyro

acids in. y_i_tro . The formic acid derivative of tryosine does
not undergo such condensation. The compound 2 ,6~diiodo»4-

tertiary-butylphenol has been synthesized and shown, under
certain conditions,, to be capable of forming a free radical
stable for 30 seconds or so- This finding lends support to
the idea that a free radical mechanism may be involved in

the condensation of tyrosine.

Work has continued on the transport of thyroid hormone
in serum and various biological fluids, It has been shown
that the administration of methyl testosterone to the human
causes a marked depression of the level of thyroxine binding
protein in serum. An increase in the level of free thyroxine
in serum occurs concomitantly „ An increase in the rsste of
metabolism of labeled thyroxine and fall in the level of
serum thyroxine which have been noted,, are presumed to be
secondary to the change in level of free thyroxine, This
study serves to emphasize the importance of thyroxine bind-
ing protein in thyroid physiology and is compatible with the
theory previously advanced that the level of free thyroxine
is a major factor In determining the rate of degradation and
physiologic effects of thyroxine, Lxtensive studies in co-
operation with Walter Reed Array Hospital,, have been done on
the action of salicylate in human subjects. A fall in the
serum thyroxine level., and £®tl in thyroid iodide clearance
appear to be due to primary depression of output by the

ate i ie concentration o -janification- .An

rate of degradation of thyroxine uraed to be
an increase in metabolic rate. Studies with carbon labeled
salicylate in the rat hai*ec. however,, failed to demonstrate a
marked concentration in the pituitary,

ivork has continued on the effect of a variety of thyro=
active materials on isolated enzymes systems- The sine con-
taining dehydrogenases which are inhibited were noted in last
year"s report, inhibition by thyroxine appears to be inter-
mediate between competitive and noncompetitive in type ver=
sus both substrate and pyridine nucleotide- No reaction
between zinc and thyroxine was demonstrated. Carboxypeptidase
is not inhibited by thyroxine- The in. y&^o implication of
these enzyme-hormone reactions is not clear although such
study seem® a necessary part of the quest to determine the
manner in which hormones affect the organism.

METABOLIC DISEASES BJ&MCH

BTNAKIC NATURE OF BONE

The adult skeleton until recently was considered to be an inert frai
work for support of the body which would demonstrate appreciable liability
only under conditions of trauma,. Lately isotople studies have gugg&stsd'a
continual turnover of mineral in bone? but mainly by physical exchange pro-
cesses between the solid phase of bone and the soluble phase of body fluids..
A new technique for handling radioactive calcium data, however!, devised in oui
laboratories j, has demonstrated that the predominant bone turnover mechanism j,
in both the adult and growing skeleton s is actual physical destruction of
structural bone units and their replacement by new bone formation «, Normal
adult bone formation lias been found to result- in the deposition of approximately
600 milligrams of calcium daily., and in Paget Bs disease, where bone turnover
prodigious s 9 grams of calcium are deposited in bone each day0 The conclusion
that surface exchange processes do not contribute greatly to calcium turnover
is derived from the observation in these studies that true miscible calcium
pools are generally less than 1$ of the total body calcium and approximately
equivalent in slse to the extras-skeletal calciums

This new technique is expected to contribute vitally important basic
information regarding the pathogenesis of certain bone diseases and on the
mode of action of various hormones upon bone deposition and resorption,, The
initial striking finding with respect to disease pathogenesis has been that
five patients with osteoporosis* a disease hitherto regarded as due to dimin'
bone formation.,,, have had normal rates of calcium deposition in new bone formation
This finding requires recasting of our concepts of osteoporosis and has suggested
a new theory of the role of minerals in bone formation,. It is anticipated that
data bearing significantly on this theory will be obtained in studies during the
coming year0

MINERAL METABOLISM BALANCE STUDIES

Possible protective action on skeleton of high mineral intakes Data
has been obtained in^ietaboDjc~Dalance studies over tfie" past" year further
supporting the minority notion that abundant mineral intake is essential for
mineral storage in desalner&lising conditionso Two of these studies were con*
ducted in patients with active rheumatoid arthritis requiring adrenal corti,
steroid for control of joint symptoms „ Previously in a limited number of stu
in our branch^ gonadal anabolic steroids had been ineffective in favoring eal
storage in this clinical situation^ but positive calcium balances have now been
obtained when the calcium intake was raised to levels of 1600 mg&o daily or
higher o Reservation must be made that the areas in -ahieh net calcium gains
occurred are unknown^ it is ejected that lengthy follew-up studies on rheumatoid
arthritic patients on high-caldum diets will be needed to determine whether the
skeletal area of greatest vulnerability on adrenal steroid therapy^ the spine p
will be protected by this regimem©

.^ataboli
chemists are also attempting to produce testosterone-like sterol:
have diminished androgenic or virilizing effects in proportion to anabe'..
activi; ly o A steroid of the latter variety $ 17-ethyl 19»nor testosterone
has bj>en tested for its clinical and nitrogen»retaining effects in several
hospitals., The relatively unique rcetabollc balance facilities afforded by
Clinical Center,, however fl have been utilized to measure carefully the pol
ealci'an retaining effects of the new steroid0 Consistently effective saineral
storage activity was demonstrated in three varieties of demineralizing dis

Metabolic affects of new cortlsone^like^ steroids s Because of the importa
of d( >termihin g* wh^her~eff e ctiv e antirheumatic steroids may be given to patien
without concern for untoward asstabolic effects* in. selected patients five new
compounds ware tested for their influence on sodium9 potassium and nitrogen
SBBtabollsau This study was collaborative with and supplementary to observation
of the antirheumatic activity of the drugs in numerous patients hy the
Art .'iritis and Rheumatism Branch o In g@naral« compounds with significant
antiinflammatory activity ware accompanied by appreciable metabolic changes
which would need to be 'taken into consideration during therapeutic use0

ACTION OP ORAL DIABETIC DRUG (ORIHASE)

Basic studies of the mechanism of action of the sulfonylureas^) oral
e.vugs now used In the control of adult mild diabetic patients^ have been
enhanced by the introduction of a new technique developed in an MIAMD clinical
:i nvestigations laboratory 0 The procedure involves rapid intravenous infusion
of tolbutamide (Cscinase) into normal subjects and patients in a fashion a'.
;o that used for studies of the action of insulin., producing a hypoglycemia
Identical in onset and degree with that produced with intravenous insulin^ and
:.aking possible a variety of observations giving direct comparison of the actio:
of insulin and of tolbutamide,, This safely tolerated technique has already
brought out certain clear-cut differences in action between these two hyps-=>
ijlycamic agents indicating that tolbutamide does not work sorely by enhancing
the action of endogenous insulino The data demonstrate absence of a peripheral
action of tolbutamide on sugar metabolism such as occurs with insulin and
suggest Inhibition of release of glucose from the liver as one of its principal
mechanisms: of lowering blood sugar0

! PEJK'JST M3PAB0LISHs NSW "H0L2OJLa?t" DISEASE DEFINED BIOCHEMICALLY

a well«planned combination of laboratory in vitro % animal and
clinical studies* another in the growing list of "Molecular" diseases has
been characterised as due to a deficiency in an enzyme needed for an
important biochemical process o Investigators first demonstrated in vitro
a liver enzyme system catalysing the biosynthesis of bilirubin glucuronide^
the "direct bilirubin" form in which bile pigments are excreted into the
intestinal tracto Next, a colony of a mutant strain of rats with congenital
nonhemolytic Jaundice was breds and it was determined that the abnormality
causing their icterus was a deficiency in the liver enzyme system necessary
for glucuronide conjugation of bilirubln0 Then clinical studies of three
children and one adult with a similar type of jaundice were carried out which
revealed that their disease was also due to a marked depression in glucuronide
conjugation of bilirubin0 This metabolic abnormality was a general one which
resulted in delays and decreases in the excretion of glucuronide conjugated
drugsj such as msmthol and salicylates^ and cortisone^related steroidso These
in vivo studies substantiated the hypothesis that conjugation of bilirubin
with glucuronide Is sssonfcia! for excretion of the pigment in the blle0

HEMATOLOGY STUDIES % PATHOGENESIS OF DRUG PURPURA

Although it has been known for many years that antibodies^ formed as
a result of sensitivity to certain drugsj, act in some way to produce throiufoo^
cytoptsnic purpura;, the nature of the processes involved in platelet disappeara?:
has not been understoodo By studying quantitative aspects of reactions which
take place between platelets^ antibody and drug (quinidine) in vitro5 the
specific inraino^chemieal reactions leading to formation of pSateTelPdnig=°
antibody complexes and to fixation of complement hy the complexes were
demonstrated o Attachment of an antibody molecule on a platelet surface can
be effected by one, two or three quinidine molecules depending on the con-
centration of quinidineo Antibody molecules must be attached to platelets
with exactly t*ro quinidine molecules to cause complement fixation or platelet
agglutination^ and complement is fixed spatially between antibodies on the
platelet surface 0 __„„„_„_____,_

(^T^SStiSo^ZI) ( 'antibody "^

^^quinidine
^ aoleeulss

platsiet surface

Thens in patients with the antibody of quinidine purpura8 quantitative studies
of the effects of intravenous quinidine identified the nature of the in vivo
reactions and physiologic processes resulting in development of tiirombocytopeniao
Attachment in vivo of minimal amounts of antibody to platelets appears simply to
increase theTr^iuaTeaptibllity to normal mechanisms of sequestration* and rep-
such as comnlemonfc fixation* platelet agglutination and direct lysis of plat
are not involved „ This work has special significance in hematology in that it-
clarifies considerably the pathogenesis of drug purpura and has direct bearing
on obscure purpuric diseases of suspected sensitivity* The basic aspaeta of
the work have mors general implications in the field of cellular ir?wsnology0

L SflTDI :

In previous work some instan ces of refractory anemia appeal
due to derangement of the humoral regulatory mechanism for blood format"
and such patients have been found to respond normality to such influences
as b3.eeding and hypartransfusiono In current studissj, assays for erythro->
poietine in human anemias have demonstrated that certain aplastic anemias
are not the consequence of depressed eyyiihropoietine production but are
to failure of the bone marrow to respond to the stiimiXuso The situation i3
similar to that seen following high doses of radiation cohere erythropoiei
production can be stimulated but damage to erythroid precursors does
permit a response© Investigation of anterior pituitary factors has revealed
that hypophyeectoK&r does not produce an anemia provided adequate substitution
therapy with cortisone and thyroid are giveno

NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES

Annual Project Report

Calendar Tear 19$1

Summary Sheet

Review and Approval of Grants

Estimated Obligations for FY 3g58

Totals $271^00©
Directt $153»000
Reimbursements* $121c,OQ0

NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES
Annual Report - Extramural Programs

January 1 - December 31 , 1957

The grants-in-aid programs for research and training
in 1957 again reflected the concept of support for both basic
and applied activities in the areas for which this Institute
has been assigned responsibilities. To the traditional
efforts in arthritis and diabetes have been added a rapidly
developing program in gastroenterology and a small but
promising program in physical biology. In both old and new
areas of interest, awards made during this year financed
research on both clinical and fundamental aspects resulting
in a well balanced attack on the disease problems involved.,
ranging from investigations of etiology and prevalence to
treatment and rehabilitation. Concomitantly, the training
programs supported will develop increased numbers of more
thoroughly trained investigators and clinicians in the
immediate future. Budget increases in both training and
research permitted orderly expansion of existing efforts while
simultaneously allowing the initial support of other worthwhile
activities .

It is anticipated that these programs, especially those
in gastroenterology and physical biology, will be allowed to
expand at a reasonable rate . It is also expected that the
research programs in these new areas /will, as is occurring in
arthritis and diabetes research^ influence closer cooperation
between the clinical investigator and his counterpart in the
basic disciplines.

Research Grant Activities

The accomplishments in arthritis research may be high-
lighted by noting developments in the immunochemistry of the
rheumatoid factor which give great promise of a better under-
standing of the etiology of certain arthridites as well as
the probable development of a more accurate diagnostic test,
particularly important in the early detection of this crippling
affliction. For example, the work of Doctors E. C. Franklin
and H. G. Kunkel in New York on the isolation of the high
molecular weight proteins responsible for the serological
reactions in rheumatoid arthritis has shown that the active
factors represent further examples of 19s gamma globulins
with biological activities and that they exist in serum
primarily complexed to a second type of protein . Similarily,

= 2 -

advances in the elucidation of the chemical, physical,
and pathological nature of connective tissue give rise
to the expectation that more efficacious treatment and
rehabilitation of patients may soon result. In this
connection Doctor Bernard S. Gould, Cambridge , Massachusetts ,
studying the role of ascorbic acid in collagen biosynthesis #
has postulated that more than one pathway of collagen bio-
synthesis may exist,

The possible mechanisms of insulin action continue to
claim the attentions of many competent investigators who
have provided valuable insight into the role of this hormone
in the metabolism of glucose and in the understanding of the
causation of diabetes. Evidence continues to accumulate, such
as that from the work of Doctors Levine and Goldstein in
Chicago and Doctor Park in Hashville,, which Indicates that the
primary function of insulin concerns the facilitation of glucose
transport across cell membranes. Other investi gators , such
as Doctor Robert Williams and his colleagues in Seattle,
Washington continue to investigate the mode of action of the
sulfonylurea compounds both as laboratory tools and possible
therapeutic agents. Doctor Williams has demostrated that the
probable primary action of these drugs involves the stimulation
of insulin secretion by the pancreas but that there may well be
an additional effect resulting from inhibition of glucose release
from the liver. Also of interest in this area of the Institute's
responsibilities is work on the fundamental relationships of the
various endocrine organs from which may be derived information
on the causation of diabetes. Hhe studies of Doctors MeCann and
Iszo at the Xtoiversity of Rochester have shown that there is a
definite increase in the excretion of 17-keto steriods in
diabetics after the administration of oral cortisone.

Although a coordinated program for the support of research
in gastroenterology was initially undertaken during this past
year, this Institute in the past has supported projects in the
general areas of metabolism and nutrition^, many of which would
now be included in a general program of gastroenterological
research. An example of such investigations is the work underway
at Columbus, Ohio under the direction of Doctor Robert Zollinger.
In their studies on the pathophysiology of acute and chronic
pancreatitis 9 these researchers have been able to reduce the
mortality in patients suffering from acute pancreatitis by the
administration of large quantities of albumin. Of particular
interest also was the initiation during this past year of support
for research in ulcerative colitis and regional enteritis , both
of which have been described as neglected areas by experts in
this field.

3 -

Ho. Amount

March 1957 Council 276 $ 3,10U,l49

June 1957 Council 8©0 10,1^2,513

November 1957 Council 327 4, 385^738

lpJ03 $17,632,^0

Approvals^/

Mo.

227
7^3
250
,220

Amount

$ 2,1*21,702

8,97857790/
2,689,291^/

Of those research grant applications recommended for
approval,, 1077 have been paid or earmarked for payment by
the National Institute of Arthritis and Metabolic Diseases
in the amount of $12,437,U66. These grants are to 189
different institutions, in kO states, the District of Columbia;,
Puerto Rico, Hawaii, and 5 foreign countries. Ihey support
both clinical and fundamental research related to arthritis,
rheumatism, and various metabolic diseases including diabetes,
thyroid disease, liver disease, nutritional diseases, and
kidney diseases, also research in physical biology. As of
December 1, 1957* this Institute -was supporting 911 active
research grants in the amount of $10,900,1*08.

1/ Excluding all multiple-assignment
applications and withdrawals.

[other-prefix first]

2/ Due to lack of funds, the originally recommended amount
was reduced from $2,960,77^ to $2,689,291.

- k -

TRAINING GRANT ACTIVITIES

Of particular significance to the training program
supported by this Institute was the first award in physical
biology. A grant to the Massachusetts Institute of
Technology provides funds for staff, student stipends,
equipment and supplies . This year also witnessed an increase
of fourteen institutions in the number to which an NIAMD
training grant had been awarded. Active grants by category
in 1957 were as follows: Arthritis , 35; Diabetes , k°J;
Gastroenterology , 12 j Hematology, 11; and General Metabolism
and Endocrinology, 6.

Bequests Approvals

flb« Amount Mo. Amount

210 $3,299,311 20a $2,604,095

Of those recommended, 195 have been paid or earmarked
for payment by the National Institute of Arthritis and
Metabolic Diseases in the amount of $2^,52^,561. These training
grant© are to 71 different institutions, in 3**- states, the
District of Columbia and Puerto Rico, and support Hk indirect
trainees .

Direct Traineeships

Requests Approvals

No. Amount Bo. Amount

80 $373,768 6k $272,063

Of the direct traineeship applications recommended for
approval, ^9 have been paid or earmarked for payment by the
National Institute of Arthritis and Metabolic Diseases in the
amount of $203,819. Tnese direct traineeships are to k$
different institutions, in 17 states, the District of Columbia^,
and England.

NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES

Annual Projeet Report

Calendar Year 2$$1

Summary Sh@et

Administration

Estimated Obligations for FY !$$&

Total* $89,000
Directs $89,000

Reimbursements* ©

Calendar Year 1 $1

Serial Ko0 NXAMB 133
Of fie© of th© Director
Bethesda

Part Ao

Project Title? Administration

Principal Investigator: Dr0 Floyd So Daftg Director

Dr0 Go Donald Whedon* Assistant Director
Mr© Wo Go Baylis* Executive Officer

Project Descriptions

Admini str atlon: The activities of the National Institute of
Arthritis and Metabolic Diseases include two
major areas: (1) Intramural Programs (Laboratory and Clinical Research) j
and (2) Extramural Programs (Fellowships* Research and Training Grants)©
The Office of the Director is responsible for planning and directing
the overall administration of the Institute* in conducting* fostering
and coordinating investigation of the cause* prevention* diagnosis* and
treatment of arthritis* rheumatism* and metabolic diseases! for maira=
taining effective operating relationships with other Institutes* with
other units of the Public Health Service^ with the Department of Healthy
Education and Welfare* other Governmental agencies* and public and
private agencies carrying on related funotionso The Office of the
Director also participates in determining policies governing the
National Institutes of Heal the

During 2$$1» the Director, with the cooperation and advice of his
Staff* sponsored several cooperative conferences in collaboration with
officials of the American Rheumatism Association and the Arthritis and
Rheumatism Foundationo Chief among these was the Fourth Interim
Scientific Session of the American Rheumatism Association which was
held at Bethesda* Maryland o Also* the National Institute of Arthritis
and Metabolic Diseases Board of Scientific Counselors was organized during
1$$1$ and held its first meeting in Bethesda on November 15 and l6o
Current interests and recent accomplishments of NIAMD's intramural
research projects were described by the six Laboratory and three Clinical
Branch Chiefs of the Institute o

The Office of the Director sponsored one of the two successful
NIH Candidates nominated by the Department of Health* Education and
Welfare for toe Interdepartmental Management Intern Program which is
conducted by the Civil Service CommLssioao Our candidate fully met all
admission requirements which included a written test* an oral interview
and a qualifications review* and was accepted by the Commissiono The
2$5l Fall program was especially designed to serve the management needs
of Federal agencies engaged in the actual prosecution of research or
development in any branch of science or engineering by providing special
training for employees who display potential in the Administrative field °

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