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Clarence F. Qgcker

A THLSIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in partial fulfillment of the reeuirements
for the degree of

RASTLn OF SCILRCE

Department of Physiology and PharmacolOgy

THESIS

11.0 £21270. 'LEDG-LEEIT

The author wishes to express his sincere appreciation to
Dr. Joseph Meites for his kind assistance during the course
of this work and in the preparation of the manuscript. Thanks
are also due to Dr. Lester F. Wolterink for his valuable and
kind suggestions in the use of the 0060 labeled-vitamin.Blg.
The author wishes further to acknoaledge the constructive
criticisms offered by other members of the Department of
PhysiolOgy and Pharmacology and especially to thank Dr. B. V.
Alfredson for the use of the facilities of the department.

t‘té‘i‘ji‘nf‘f;

.‘~‘g ‘ . P.“

TAJLE OF COthHTS

IIM‘TEfIClfit-JVCTIOPJOOOCOOOOOCOOOO0....O'COOOOOOOOOOOOOOOO
hQVIEN OF BIOLOGICAL mFFECTS OF hfiOfiTH hUthLZ....
hffects on Body Growth and rotein Metabolism
Effects on Carbohydrate hetabolism...........
Effects on the Skeletal System...............

VIT‘UAJ'll-lq B12 HDVV‘ILLI'I00.000.000.00.oooooooooooooooooo

Effects of Vitamin 812 on Erythropoesis......

Vitamin 513 and body Growth....................
Interrelations between Vitamin 513 and Endocrine Secretions.

Efl’th-ILI‘JTAL

Experiment I:

melation Between Anterior Pituitary Growth
hormone and Vitamin 512 in the Growth-Plateaued Female hat

ProcedmneOOOOOOOOOOOOOOOOOOOO0......OOOOOOOOOCOOOOOOO.

hesuj-tsoooo3.00.00.00.00.000.00.000.00oooooooooo-oone.

Experiment II:

helation Between Anterior Pituitary Growth

hormone and Vitamin 512 in the Immature hale hat.........

Proced'ure.0.0.0...0....OOOOOOOOIOOODOIOOOOOOO0...00...

fiesul-‘LSOOOOOOOIOOO00.00.000.000.OIOOOOUOOOOOOOOOOOOOOO

Experiment III:

Pituitary Extract in the Ioung hypophysectomized hat...

Proce'dU—I‘eoooooooOOOOOtoooooooooooooooooo
heellltso0.0.000...00000000000000.0000...

DIaCUSSIOIQOODOIOOOOOOCIOCOOOOOOOOOIOOO.00....O....
Sljl'thrhiiY........,,.................................

BISLIOLErQ";lJi-LIOOOO0.00..ooooooooooooooooooo000000000

‘.
_)

I“ T"
.LI’I

,,,~_~‘—.-r
”labikoooooooo0.000000000.000.000.000...0000.000

nelation Between Vitamin B12 and Anterior

4'1]

P) fl ,5
u all:

\OCT‘I: l3"

13

13
15
16

[\J
C7 “

gum
Q (3

LIsr or TiaLis

TABLE PndE
I. Lffect of Vitamin B12 and/or Growth hormone on Body
1eirdt and bood Intake in the Growth— Plateaued Female
fiat.0......00......00.000.00.000.00IOOOOOOOIOOOOOIIOOOOO 20

II. Effect of Vitamin.513 and/or Growth hormone on the Or1an
iVK'it; :llL/S Of UY‘LfJUll-f‘ldbe 8.119 «d Fclllale fiatsooooooooooooooooo 2?

III. Effects of Vitamin B12 and/or Growth Hormone on Body
eight and Food Inta“e of Young male hats............... 33

IV, Effect tof Vitamin 513 and/or Growth hormone on the Organ

b‘ie iézl‘ts Of Your-lgllhale ILatSOOOOOOOOCOOOOOIOOOOOOOOOOOOOO. 34

V. hffect of Vitamin 313 an d/or GrOWth Hormone on the Ce60
L% beled Vitamin blg Content of Tissues from Ioung.Male

hats.................................................... 35

VI. Effect of Vitamin 812 and/or GrUWth Hormone on the Percent
Moisture of Tissues from Younthale mats.,,,,.....,.,,.. 36

VII. Effect of Vitamin 512 and/or Growth Hormone on the Percent
Ash of Tissues from loung Rats.......................... 37

VIII. Effect of Vitamin B12 and/or Anterior Pituitary Extract on
Average Bodylieight and Average Food Intake of Young
H3rop;\ily’SeCtomized Rats . . . . . O . . . O . . C C . C . . . . C O . . . C O C O O O . . . Lil

LIS” 0F EIGUnLS
FIGURE PAGE

1. Effect of Vit1nin 512 and/or growth hormone on the body
VJeigilt Of IIIatU.re iii/{Thalia ratsoooooooooooooooooooooOOIOOOO

|'\3
\f,

2. Effect of vitamin.B12 and/or growth hormone on the bva
weigirit of il—Ilmature nlale rats . O O O O O C . . . O C o C . O . . . . . . O . . C O C .

be
m

3. Effect of vitamin 512, aureomycin and anterior pituitary
extract alone or in combination on the growth of
liflDOplxbl'sectonxizeci rats 1:0

h. Self absorption curve for Coeo labeled-vitamin 512......... iii

u.-

in'lLLCI pt 3'? It. 1.

INThOUUCTICN

A number of factors other than inadeQuate dietary intake can cause
malnutrition, including conditions that interfere with absorption or
utilization of nutrients, or those that increase their requirement,
destruction or excretion. "These 'conditioning factors' as Jolliffe
terms them have one point in common: they increase body reuuirements
for specific nutrients or precipitate nutritional deficiencies on diets
that would otherwise be adequate were such factors not operative."
(Ershoff, l9h5). A general group of factors (stress factors) which in-
crease body requirements for essential nutrients include physical exer-
tion, fever, drugs, toxins, abnormal environmental conditions, pregnancy
and lactation.

In recent years considerable information has accumulated concerning
the vitamin requirements of animals subjected to stress brought about by
marked changes in hormone levels within the body. It is a matter of some
importance whether marked changes in the level of any particular hormone
cause a concomitant increase or decrease in the need for various dietary
nutrients. Such an effect of a particular hormone might conceivably be
of aid in (l) helping to elucidate a possible interrelationship between
the hormone and the dietary nutrient in normal metabolic processes,

(2) differentiating between the ill effects caused directly by excessive
amounts or deficiencies of certain hormones and those due to an accompanv-

ing increase or decrease in squirement of dietary nutrients and

(3) establisning the level of hormone secretion at which supplementation
of a particular nutrient is required.

The most extensive investigations concerning the relation between
endocrine secretions and dietary needs have been concerned with the
thyroid. hyperthyroidism has been shown to increase the need for calories,
certain vitamins such as A, thiamin, riboflavin, pyridoxine, niacin, folic
acid, ascorbic acid and minerals. hypothyroidism presumably decreases
the need for these nutrients. Hyperthyroidism has also been shown recently
to increase the need for vitamin 812. It has further been demonstrated
that marked variations in estrogen or cortisone levels in the body can
also increase the requirement for vitamin Bl2 and possibly other dietary
growth factors in rats. Supplementation of the diets with vitamin B12
and antibiotics partially or completely prevented manifestation of dietary
deficiencies induced by hormonal changes.

'he present experiment was undertaken to determine whether or not
the reauirement for vitamin 812 could be altered under the stimulus to
growth induced by administration of anterior pituitary growth hormone.
It has already been shown that growth hormone increases the requirements
for vitamin A (hrshoff and Deuel,l9t5; Margitay-Becht and Wellner, 1937)
and pantothenic acid in rats (Lotspeich, 1950). It was postulated that if
vitamin 512 were required for optimal growth, then the additional stimulus
to growth incurred by the anninistration of growth hormone would result in
an increased need for this vitamin. Such an increased need, it was believed,
would be indicated by the precipitation of the symptoms of a vitamin 513

deficiency, the most obvious of which would be retardation of growth rate.

The actual data obtainec in the experiments reported here indicate that
growth hormone may not increase the need for vitamin.812, and may even

reduce requirements for this vitamin under certain experimental conditions.

‘ a 11 ' "1 '7' ~' x-r 'v. . 1.1-. 1.’f'1iT\(" 11 (fly! "rn‘j 1.7- 43' ’(‘1‘3‘ '
ILQJIJJ Ul‘ DlCLOLLLUJ..L mfflblo C'I.‘ ulLJJili iiUlu'iuhu

thIflJ OF BIOLOGICAL EFFUCTS OF GnUJTH QOthhn

I. Effects on Bodv Grtwth and Protein.hetabolism.

 

As early as 1912 Cushing reported that removal of the anterior
pituitary in growing animals stepped growth and precipitated a loss in
body weight. Later, numerous reports indicated that replacement therapy,
either by injection of the extract (ovens and Long, 1922; Fracnkel—Conrat
at 31., l9hO; Marx, Simpson, Reinhardt and fivans, l9h2) or by daily im—
plantation of Whole pituitary (Smith, 1923, 1927, 1930) resulted in a re-
sumption of growth.

The administration of anterior pituitary extract or implantation
into normal animals was shown to result in gigantism in young animals
(Putnam at 31., 1928; Smith, 1923, 1927, 1930). Similar treatment of adult
animals gave esults which were species dependent. In dogs but not in rats
acromegaly occurred. however, in both species a marked visceromegaly
occurred (Putnam at 31., 1928).

One early report (dryan and Gaiser, 1932) indicated that the ultimate
body size of rats following anterior pituitary "growth hormone" injection
was not larger than could be obtained by proper dietary means. The changes
in body composition of animals after treatment with pituitary extracts
causing accelerated growth have been studied by several workers (Downs,
19303‘wadehn, 1932; Bierring and Nielsen, 1932; Lee and Schafer, 193A).

Lee and Schafer (193h, 1935) made the rather important finding that on a

given quantity of the same food, animals treated with anterior pituitary

\JL

extract grew faster in weight than untreated con rols. Their data indi-
cater that (1) treated animals on an.§3 lihitum diet consumed 10 to 15
percent more food than the non-treated controls, and (2) the treated
animals gained more weight than he untreated, even if pair-fed.

The interpretation oy these authors that the anterior pituitary growth
hormone stimulated true growth was substantiated by Brues' work in 193d.
his data showed a more rapid liver regeneration after partial hepatectomy
in rats inyected with anterior pituitary than occurred in untreated control
animals. The liver of the injected rats contained more water, asn and
protein than did the liver of the controls. It was also found that all
Visceral organs grow more rapidly following hormone injectiors than did
the body as a whole.

In contrast to the results obtained by the above mentioned workers,
Kleiber and Cole reported (1939) that they did not find any significant
difference in the ash, fat or protein content of injected and control rats.
Since true growth is generally interpreted as accumulation of protein, it
was reasonable to expect that an important function of growth hormone
would be the retention of nitrogen, which in turn increased the protein
content of the body tissues.

Early reports (Teel and Watkins, 1929; daebler, 1933; harrison and
Long, 19LO) have indicated that growth promoting pituitary extracts caused
a reduction in both the blood non—protein nitrogen and urinary nitrogen.
Later work (narx, Nagy, at 21., 19h2; Fraenkel-Conrat at 31., 19L2) with
partially purified growth hormone have confirmed these results. In 1939

douchin succeeded in reducing the alkali soluble protein components of the

liver following the indection of anterior pituitary extract ar c suggestec
the existence of a prote in metaoolizing hormone distinct from the lacto-

qenic, thvrotropic, carbohydrate metabolizing

U

, fat metabolizing and
gonadotropic hormones.

The administration of anterior pituitary extract to dogs was reported
to have a Sparing effect on protein, i.e., here was a decrease in protein
catabolism (nirsk and So «ish,193t). It was also demonstrated that if
the extract were administered to dogs subsequently eviscerated, an in—
crease in the rate of non-protein nitro5en accumulation resulted in these
dogs as compared to the untreated eviscerated dogs. This was interpreted
as indicating that the extract increased the rat e of protein metabolism
in muscles, but that the effect was not apparent in the presence of the
aodcminal visce ra The sa.me workers reported that in the absence of the
pancreas, the anterior pituitary increased rather than depressed the rate
of nitrogen metabolism. It was postulate d that a dual action of such
extracts on protein resulted in (1) direct stimulation of protein ca tab olism
in the muscles and (2) an indirect stimulation of protein anabolism,

through stimulation of the pancre

n3
C)
(+-

Gae Ller and nobinson (19’2) reported that anterior pituitary extr
could induce nitrogen retc -ntion in a dog lacking thyroid, pancreas and
both adrenals. An earlier report by Gaeoler and Bartlett (1939) indicated
that the administration of anterior pituitary extract to normal dogs caused
an increase in urine volume, wate intake, body weight and oxggen consump—

tion A cscraase in phosphate and nitrogen excretion in the urine was also

In a review Soskin (l9hl) concluded that the anterior pituitary
extract accelerates protein catabolism and gluconeogonesis from protein
by the liver, but that in the presence of the pancreas the effects of the
anterior pituitary are masled by the opposing action of insulin.

Harrison and Long (l9hO) demonstrated that anterior pituitary ex-
tract reduced urine nitrogen excretion, non—protein nitrogen of the blood

and blood sugar, although hetonuria was increased. l addition, these

workers made the important finding that the above effects persisted even

Li and Evans (19h?) in a review stated that although their early
experiments were performed with crude or partially purified preparations,
it appeared likely that the growth promoting action of the pituitary was
accompanied by either an increase of protein anaboliSn, or a decrease of
protein catapolism, or both. The work of Fraenkol—Conrat at al., (19t3)
demonstrated a decrease in liver arginase content in rats stimulated with
a purified growth hormone preparation and S'emed to indicate that protein
cataoolism had indeed been inhibited. Gacbler at al. (19h?) studied the
effect of growth hormone upon several enZyme systems, including liver and
kidney D-amino acid oxidase and muscle succinic acid dehydrogencse, Their
data indicated that, with respect to these particular enzyme systems, the
concentration of the enzyme in the respective tissue was constant. They
concluded that this indicated synthesis in direct proportion to tissue

grtwth .

' I

Ihe effects of growth hormone upon body growth in general and nitrogen
retention with accompanying protein synthesis in particular have been

summarized by Li (lth) as follows:

(a) retention of urinary nitrogen

(o) lowering of blood amino acid concentration

(c) elevation of the protein content and a decrease in the fat
content in the carcass and thymus

(d) elevation of the blood alkaline phOSphatase anu inorganic
ph'"phate levels

(3) elevation of the ribonucleic acid content of the liver

(f) enhancement of amino acid uptake into protein in the skeletal
muscle.

Through what mechanisms growth hormone performs these protein anabolic

functions is as yet unknown.

ll. Effects on Carbohydrate hetacolism.

 

the mechanisms by which growth hormone stimulates carbohydrate meta-
bolism are‘asye unknown; that such stimulation does occur can scarcely
be doubted. The work of hirsky and Swadish (l93o) has already been cited
as indicating a possible relationship between growth hormone and pancreatic
activity.

Bennett and Li (late) demonstrated that in rats made diabetic by
alloxan and maintained on a constant food intake, a significant nitrogen
retention and gain in weight were obtained upon injection of pure growth
hormone. hora evidence that growth hormone and the diabetogenic factor
in anterior pituitary extracts may be the same has been provided by
noussay and Leloir (1935).

In a very recent report by haben andliestermeyer (1952) evidence

is offered that the diabetogenic factor of the pituitary is not identical

with growth hormone. Thea thors assert that :rowth hormone prepared

“I

from a glacial acetic acid extract was egual in growth promoting e ficc ts

to other purified extracts, but did not produce diabetes. Russell (l93o,

91/)

) after havinr done a considerable amount of work on the effects of

L
growth hormone on carbohydrate metabolism, concluded that growth hormone
administered to rats is concerned not only with the preservation of body

‘ l

vcarbohyorates during fastinr but also with the diaposition of this sub-

U
1‘ 3
‘34

stancr vhen *eL. In a later report, Illingworth and hussell (1951) re-

t

ported that a single intraperitoneal injection of growth hormone in
adult fasted rats caused an increased accumulation of glyco 5en in the

g strocnem ius, heart and diaphragm muscles.

111. Effects on the Skeletal System.

 

The influence of the pituitary on the growth of the skeleton has
been known for some time (Dott and Fraser, 1923; handelsman and Gordon,
1930) but the sp ific effects of growth hormone on the epiphyseal
cartilage of hypophysectomized animals have been described more recently
(Freud _e__t _a_l_., 1939; Ray gt _a_l_., 19w), Kibrick _e_t 5a., (191a) concluded
that these effects were as follows: "nypOphysectomy rapidly initiates a
loss in the dimensions of the epip hvsee 1 plate, despite the fact that
growth of the cartilage and bone may continue for a short time in the
young animals after removal of U18 pituitary. This loss in thickness

reflects the ir i cial disturbance of the equilibrium that normally exists

r‘d

yetween chondrOgenesis and osteogenesis. Administration of pituitary

growth ho1mone rapidl» restores the dimensions of the c artilat-:e plate by

10

stimulating, first, chondrogenesis aim then osteogenesis until an equi-
librium is reestablisned."

Becks et 31., (1953) have shown that administration of growth hormone

Q1

ZS

IN! a

to hypo Mhysact m rats, even after postOperative intervals of a year
or longer, was able to reawa ken chond1o:enic and oats eo5enic processes

in the epipnbseal cartila5e of the tibia to an extent comparable to that

seen in normal, Joana, growing rats.

In early studies concerning the relation bet en the growth promoting
ef ots of anterior pituita.ry and the thyroid, Evans, Simpson arid Penoharz

(1939) concludtd that the growth effect obtained with anterior pituitary
extracts were not dependent upon the thyroid but that they were 5re eater if
the thyroid were resent harx Simpson and Eva ns (19h2) reported that

3
purified therprotein did not itself possess a growth stimulating action,
although it did exert a true synergism with growth promoting pituitary
extracts in hypophysectomized rats. Such synergism is not mediated by the
thyroid. since it occurs even in the absence of the thyroid gland.

More recently, a great deal of information has been gained concerning
the possible mechanisms involved in the action of growth hormone and of
thvroxin on bone growth and differentiation. Most of the work in this
are a has been done with rats thyro—parathyroidectomized at birth. Several
reports (Salmon, l93ba, l93‘t 3 ow and Simpson, l9h5) indicate that such
animals exhibit a marked retardation in growth and maturation. Later,
several workers (Scow and harx, 19L 5; Becks . eta ,19h8) showed that the
administration of growth hormone caused an increase in rate of 5rowtn,

both in body weight and length, without an increase in the rate of

ll

maturation. Apparently the growth hormone is essential for growth, i.e.,
increased dimensions, but is not able to stimulate the process of dif—
ferentiation or maturation in the thyro—parathyroidectomized animal. The
thyroid was found to be necessary for the latter process.

It was suggested by Scow et al. (l9h9) that the mechanism by which
thyroxin therapy corrects the rctardeo growth in thyro—parathyroidectomized
animals was to restore in part at least the production of growth hormone
by the pituitary. The non—endocrine viscera of such injected animals were
maintained in proportion to body weight with the exception of brain and
eyeball. The endocrine and reproductive viscera developed only in the
rats receiving thyroxine.

Further evidence by honeff 53 El- (1959) of the histological changes
in the pituitary of thyro-parathyroidectomized animals tended to support
the theory that thyroxin therapy caused the release of endogenous growth
hormone. It was found that thyroxin administration alone was responsible
for restoration of the acidOphiles, the putative source of growth hormone.
Histological evidence for the changes in individual bones also indicated
that thyroidectomy markedly retards skeletal growth and differentiation,
and that growth hormone stimulates growth without differentiation, (decks
et al., 1950). Thyroxin alone was demonstrated to stimulate growth and

an
.L

differentiation. There was no augmentation of either of these e1 acts when
both hormones were administered together.
These relationsnips between the effect of growth hormone and of

thyroxin have been summarized by Asling at al. (1951) as follows: "It

appears, that, in rats, growth hormone is a potent stimulus to skeletal

12

growth, but has little effect on maturation, while thyroxin directly
advances maturation but stimulates growth only slightly. By administering
these hormones concurrently in preperly balanced dosages, a normal balance
between growth and differentiation may be established. The necessity of
both hormones for this balance is best shown by studying their actions in
thyroidectomized-hypOphysectomized rats."

Growth hormone has also been shown to be independent of the tnwmus
gland, although thymus hypertrOphydoes occur in the intact animal upon
the injection of growth hormone (neinhardt, harx and Evans, l9hl). This
may simply be due to an increase in protein anabolian causing an increase
in thymus protein. One other action attributed to growth hormone may be
mentioned, and that is the so-called "renotropic effect". It was reported
by Selye and Jensen (l9ho) that crude extracts of the anterior pituitary
caused pronounced enlargement of the kidneys, and that this renotropic
effect was greatly enhanced by the simultaneous administration of thyroxine.
The increase in size of the kidneys was much greater than could be expected
from the growth hormone content of the extract, although other anterior
pituitary preparations failed to exhibit any significant renotropic
potency. hochakian (19h?) reported that androgens markedly increase the
size of the kidney, while purified growth hormone does so only slightly.
It seems possible that the crude extract used by Selye and Jensen (lth)
contained sufficient gonadotrophic hormone to cause stimulation of androgen

production, which in turn elicited the increase in kidney size.

l3

Vila-.11“; 512 nlfiVInl'.’

In l9ho Smith, hickes et al. (l9hba) announced the isolation of the
anti-anemia factor in the semi—pure state and gave it the name vitamin 812
or erythrotin. Vitamin 812 was found by Shorb (l9h8) to be identical

with one of the two unidentified growth factors required by Lactobacillus

 

lactis Burner, and both of these unidentified factors were present in re—
fined liver extracts.

The subsequent work on the actions of vitamin 812 may be most con-
veniently divided into several categories. One main line of study has
been the effects of vitamin Big in the treatment of pernicious anemia.

A second has been concerned with the effects of vitamin 512 on body growth
and its specific role in metabolic processes, and a third has dealt with

the relation between vitamin 812 and various endocrine secretions.

I. Effects of Vitamin 313 on fithhFQQQGSiS.

The work dealing with the effects of vitamin 512 in the treatment
of pernicious anemia is primarily of a clinical nature, but many of the
findings have contributed to the elucidation of its functions in various
metabolic processes.

Crystalline vitamin B12 prepared from liver extracts gave a positive
response when injected intramuscularly into patients with.Addisonian
pernicious anemia in doses as small as 3 pg (West, l9hb), although the

/ I
actual amount required to produce a maximum reticulocytOSis response was

e to 10 pg (hall and Campbell, l9hb; Ungley l9hta, l9h8b). An average
maintenance dose was 10 ug every two weeks (Ungley, l9h9). Vitamin 312
. was also shown to be effective in nutritional macrocytic anemia and
tropical Spree (Patel l9LE; Spies, Suarez et al., l9h9). Vitamin B12
.prepared from E. griseus cultures (DunlOp andlvilson, l9h9, Miller and

Moorhouse, l9h9) and vitamin blgb prepared from S. aureofaciens cultures

 

(Lichtman et_al., 19h?) were apparently as effective in pernicious anemia
as vitamin 813 prepared from liver extracts.

In a later report (hausmann, l9h9) it was indicated that vitamin 312
existed in certain substances in the form of conjugates, inactive in
pernicious anemia until they had been digested with hOgS' stomach mucosa
or with pancreatic enzyme extract. It was further suggested that these
conjuga,es might be identical with Castle's "extrinsic factor" (Castle
1929; Castle 33 al., 1930; andiiatson and Castle, 19h6). Ternberg and
Eakin (19h?) showed that the active principle in gastric juice long desig—

ated as the "intrinsic factor" was in reality a protein which combined
with vitamin 512 (extrinsic factor) to form a complex which was resistant
to the destructive changes wrought upon B12 itself by the digestive
processes. It has been suggested that these interrelationships favored
substitution of the term erythrotin for vitamin 512, apoerjthcin for
intrinsic factor, and erythein for the complex. Beerstecher and Edmonds
(1952) have offered evidence for the existence of an apoerythein precursor
in the gastric mucosa and suggested that it was probably identical with
the intrinsic factor.

The availability of 0060 labeled—vitamin 513 has been of considerable

aid in the study of the distribution of this substance under various

13

experimental conditions. Welch E2.§ln (1932) using radioactive 312 have
shown that the intrinsic factor is involved in the removal of vitamin 312
from the gastrointestinal tract.

Lang and Chow (1952) have studied the effect of age on retention of
Co60 labeled-vitamin b1; and have found that in rats excretion is greater
in young animals than in older ones. The rate of disappearance of C360
labeled-vitamin 513 from various organs of the rat has also been studied
(Harte, Chow, barrows, 1952). Following the administration of a single
dose of Co60 labeled-vitamin 312 more radioactivity was located in the
kidneys than elsewhere after the first day, but this amount tended to

decrease Sharply with time. Le

U)

ser absolute amounts but much higher con-
cent rations of activity were found in the pancreas. The amounts of
activity in the liver remained essentially constant, or even increased
with time. They concluded that the vitamin in live r must be associated
with the protein moiety, since the total amount of radioactivity remained

constant but the concentration rose when the liver glycogen was mobilized

as a result of caloric deprivation.

II. Vita3in 3i)? and Bo “ Grow tL.

 

The effects of vitamin 312 on the growth of animals have be 3n widely

stubied in the past few years. Cary et al. (lQhG) and hartman tl9ht)

G;

described a "factor X", the abs 3nc3 of which resu ed in a declin3 in th
growth rate of rats, a decline that bacmne more marked when the protein

F" 7

content of the diet was incl as ed. ihe fact r appeared to be similar to

the so-called animal protein factor (APF), which increased the hatchability

al

of hens' eggs (hestler ., 193:) and the growth rate of chic1zs main—

lkt

tained on an all vegetaole p “otein ration Hammond, l9hh . It was also
similar to a factor in cow manure that stimulated the growth of chicks
(Rubin and Bird, 1933; Bird gt 31., l94t).

Crystalline vitamin 312 was found to exhibit animal protein factor
activit3 for chicks fed.soy bean meal as the sole source of protein; it
was as effective as the cow ma.1ur 3 factor in stimulating the growth of

331
1,,

chicks (Ctt et al., 19h8; Lillie, Benton and Bird, 19ht; Lillie

l0)
C.+.

19L9). It also increased the growth rate of rats on a factor X—de ple ted

diet, showing that vitamin 312 plays a lurowm3nta1 role in the utilization

of protein (oartman, Dryien and Cary, 19h9).

1

Some evidence has accumulated indicating that vitamin 513 is concerneo

.L.
U

with tran33wt1yylati In the first place, on a diet complz'

v

in the

(U

’T

known vitamins, the growth of chic1:s was improveo bys upplemen ntation with
cholihe or etaine; suhseiuent aidition of a liver paste containing little
choline was even more effective and further supplementation with choline
or betaine then ha; little effect (Gillie and Iorris, 19L9). Crystalline
vitamin.312 has also 3c3n.5101n.to iiwu‘e 3 the orowth rate of chicks or1

a diet low in choline. Ee1i 31 in unv in rats due to a low choline and

(D

methionine intake was minim 25: dhy the addition of vitamin 312 to th

V

diet, and th3 gain in weight was al_so increased, but not when adequat
1 , w r 1
amounts of Choline were fed ( Salmon and Strength,1}h9).

It has been shown Uennett Joralemon and halnern 1932) that rats
3 1* 3

on a "lacile meth3xrl fr 33"1omoc38teine diet containing folic acid are

!J
\3

able to maintain normal livers, and that without B13 sucn anin als cease
to grow and fatty livers result, mats fed diets containing 15p casein
showed signs of an acute choline deficiency in ten days, and the addition
of vitamin 513 and folic acid reduced the liver fat and preven ed kidney

damage \Fischer and Ball, 1952),

Vitamin 313 had a maria ed lipotroiuic eff cct when injected into rats

1::

fed a high fat diet (Drill and ncCormick, 19T9). The adminié 1105 Of

vitamin B12 to rats preceding acute carbon tetrachloride intoxication
prevented liver injm m‘y (Po prer et al 19h9).

Several investigators reported that vitamin 312 played a role in
nitrogen retention and presumably in protein metabolism. It was re ported
that the circulat inn blood of ck*.icls recc ivin,: 50 pg of vitamin 513 per
kilogr": of feed conta'ned less non-protein nitrogen and lesso each of
seven amino acids than did the blood of vitanin Blp-de ficiont chicks.

In addition, the birds fed tLe supplement grew more rapidl; and utilized

ed more effici “ ntlv tzlan did the B 13-deficient controls. The authors

as
(D

1‘.
.L

(L:

concluded that vitamin 813 an ears to function in metabolic sm by enh nci ng
the utilization of circulating :triro acids for builtfl Jng fixed tissu:s

(Charqey »'t al , 1950). Vitamin B13-de'ficie12t ch ' icks were less aole to

Q.

withstand the toxic effects of force fe glycine (Stern et al., l9Ll).
Such an effect would tend to support the tt heory that vitamin B12 er anc es
the utilization of’ axniro acids.

more recent work Chow and Barrows 1950) on rats raised on a corn

soy bean diet indicatec that the growth rate but not the exile cjency of

nitrogen utilization was in was ec by the aiditiln of vitxnin B13. These

V 'L ‘ ~1 »v'1 ‘fi‘lF-/; -- h~ . ' r,‘ 3‘. ,3 ,_ _\ - 7‘ ~ 227- .
adoLors COubluuCu that Vitamin B13 does not enhance tne hiclocic value of
M (j ‘ “ "
fi if I ’f‘ ” +5.2“: ~“ I"? B "“ 'Wv' '\ ' '7 ': n‘f‘v' ' "L ' ")1 5" ~‘ "2 “v. ,. I .
so” Dian proteins, oat uaJ plah an incortart role u CurLQLJthtfi or fat

metabolis.

H:

Lie carcass co position o rats fei on a corn—soy diet has also been

J

investigated (Ling GM‘ Chow, 95;) and ate r salts indicate a low fat,

1 .—. ‘ o r- - rs ru -‘ . q n j . VI ,3 .' r ”A - ‘ ' “I: - 7'1 . .‘.‘ , ~ - r\ -'r
niwn water and nornii glotoin Content, while t;3 SUlLUleJl content of

L2
' J

the elood cells was low, due prim Hril to a lowers Ci “lo"aonicne content.
The administration of vitamin B13 corrected these aonornalities. It was
further Lvanpbl ated that a high L&L~Oh,uf te c.ie ,couplec with the in-
iection of glucose could also cause a decrease in the glu athione content

I)
-

of the rec olcoe cells. The autiors surgostec that the effect of the nigh

rt‘

carbohydrate d :t could be cue to an incr ased utilizat on or requiremen
of vitamin 313. lhe fact that Bl3— _Bflol“ht rats had smaller car2okydrate
reserves, mi that irtravenous injections oi gluccse were poorlv utilizes

as supiort for this theory.

III. lntarrelations Between Vita.in 333 and finencrina Seer-tions.

i - - .- P. 2 ' ‘ ."1
Several resorts nave o;~n conctrnou with tnc reia

,-. _ f‘ -.~ . - ., .‘-.fl—: ,.’ v .‘ r\J- . ~. c‘ ' . n ». h -\ 9‘17“ .’ ‘f; '.. I ~
level Ol Various en eciine sacreticns ant Vitamin o1” metacolism. nats
.... 3 n .' vs . -. n. J—‘. ~-v-vnl* . r‘ o . - L ‘ a ' P ‘ . ' I". " In ”DJ ’ I 1 “ L

fed ratici s containing ohy-Uld-aCth€ Lubfl-lLlS rflfiUqup a labbbr tresort

liver, fish soluolcs anfi tomaooes for "ro2;h t'~:=theil and Lardj, 1749).

The factor was present in anti-pernicious anemia active fractions, out

(l9,C;) fc one

’3
f‘
}_J
\O
f
\ .
V
c)
I3
(\
(D
:1].
(-
L)

whereas Letheil and Lard; (l9i9), firerso
vitamin 513 to be active Brshoff \lQLE) found it to be ineffective.

v 1;

Inconsistent results have seen ctthin,o when attsnpts were ma,e to use

l9

hgierthrroid rats for the assay of vitamin £13. The assay was based on

‘D
l. J

tne assumption that nynertneridism induced in rats by feedinz iodinatcd

J

casein caused a depletion of vitamin £13 body‘stores. however, it has

been Claineo that the test is not specific for vitamin r13.
Liver extracts active in pernicious anemia failed to stimulate the

growth of thyrotoxic chicks, and crude liver exgracts discarded from

anti—pernicious anemia fractions were highly active (Nichol, nooblee,

l 19h9). Vitamin 513 replaced the animal protein factor of inject—

in

.1.
co .,

 

isle liver preparations when tested on tiyrotoxic chicks (hichol,

rdcks nathnngjowth wasxmfl;on—

0

al., l9h9). In normal ,

Dietrich, gt
tained until other supplenents were added (Stokstad et al., 19h9),

Vitamin 513 was also shown to be less effective than 1:20 liver
powder in counteracting the growth depression induced by feeding toxic
levels of icdinated casein to normal male and to connorectomized female
rats (hatts it al., 1951), Little differences in resnonse to these two
supplements were noted in similarly fed normal females and castrated
males.

A rapid depletion of the liver stores of vitamin 313 was reported
to be accomplished by a diet containing sulfasuxidine and iodinateo casein,
but free of cnoline and vitamin 513 (Travers and Cerecedo, 1952 . The
growth of these rats was stimulated by the aidition of vitahin 313 and of
choline. Vitamin £13 was found to be without effect if choline were
present in an adequate amount, and_choline could prevent to a lar e
extent the depletion of the liver stores of vitamin 813.

Heitcs and Shay (lel) have snown that vitamin 313 is sole to over-

come the growth retarding effects of diethylstilbestrol and thyroprttein

when given individually or in combination to young rats. The vitamin in-
creased the food consumption but did not alter the ability of either of
the two hormones to depress the testes weight or the ability of diethyl-
stiloestrol to increase the weight of the seminal vesicles. These
authors also stated that vitamin 512 did not alter the turnover of thyro-
protein in the rat, since the increase in the basal metabolism of rats
given therprotein was unaffected by the administration of vitamin 512.

t has also “ee. shown that desiccated whole liver or yeast can overcome

of

U}

the inhibition of ovarian devel pment in immature rats fed huge dose
alpha-estrauiol (Ershoff and Deuel, l9ho; Ershoff and mohilliams, l9ht).
Meites (1950a) has studied the effects of vitamin 512 on thiouracil
action in rats. he concluced that vitamin 312 can overcome the growth-
retarding aetion of thicuracil in female rats, even though the thiouracil

depressed thyroid activity, as measured by the uptake of radioactive 1131.
he believed that these findings suggested that vitamin 512 does not re—
guire the mediation of the thyroid to exert its anabolic effects on
growth. Libby and Meites (1952) studied the effects of vitamin blg and
penicillin on thiouracil action in chicks and found that these two sub—
stances partially or completely counteracted the growth depression and
reduction of appetite caused by thio acil administration to young chicks.
heither the vitamin nor the antibiotic had any effect on the severe
inhibition of comb growth induced by thiouracil. In a study on the effects
of vitamin B12 on normal thyroid function,lfieites (1950b) found that when
the vitamin was included in the diet of normal or thyroprotein—treated

rats, the growth rates of these animals was increased. He found, however,

21

‘

that there was no significant effect on thyroid weight or uptake of

radioactive 1131

, and concluded that vit min 812 does not alter normal
thyroid activity in rats.

The effects of vitamin 513 on the survival and growth rates of young
rats subjected to thyro—parathyroidectomy has been studied (heites, 1952).
Such animals supplied with 50 or 200 pg vitamin 313 per kilogram of diet
showed an increase in survival and growth rates over similarly treated
animals which did not receive vitamin 812 in the diet.

Wells and Kendall (l9h0) and Ingle (1950) have demonstrated that
large doses of cortisone were able to inhibit both body and hair growth
in rats, and that these effects have been partially attributed to the
reduction in the availability of protein (Engel; hoberman, 1950). Winter,
Silber and Stoerk (1950) reported that large doses of cortisone reduced
normal food intake in rats. In 1931, Heites reported that vitamin $13
and aureomycin were able to counteract the inhibition of body, hair and
thymus growth induced by injecting immature rats with large doses of
cortisone. Vitamin biz was more effective than aureomycin and a combina-
tion of the two was more effective than either given alone. The actions
of the vitamin and the antibiotic in overcoming the effects of the admin-
istered cortisone were accompanied by an increased food consumption and a
greater efficiency in converting food into body weight gains.

While it is generally believed that a deficiency of a particular
hormone decreases the need for various vitamins, there are several eports
which indicate the reverse may be true, that is that low hormonal levels

may actually increase the need for certain nutrients. As already noted

{\D
N

extreme hypothyroidism as well as extreme hypertiyroidism appears to in-
crease the need for vitamin 312- Dumm and malli (l9hb) have reported that
in rats subjected to adrenalectomy, pantothenic acid increased survival
and resistance to various stresses. heites (unpublished observation) has
also found that vitamin 812 and aureomycin increase survival time and body
weight in adrenalectomized rats on a vitamin BIZ-deficient diet. ‘haw

and Greep (l9h9) have demonstrated that in rats subjected to hypophysectomy,
survival and growth rate have been enhanced by purified diets adeguate in
all known nutrients and apparently rich in vitamin 812.

To summarize the actions of vitamin 812, it has been demonstrated
conclusively that the vitamin is identical with the antipernicious anemia
factor and exerts a powerful stimulus to erythrOpoesis. Abundant evidence
is also available that vitamin 813 is a powerful stimulus to growth in
immature animals, and is doubtless identical with the so—called "animal
protein factor". Though the mechanisms by which the vitamin exerts its
influence on body metabolism are as yet not fully known, numerous reports
indicate that it acts favorably on protein synthesis. In addition, several
reports have indicated a possible role in fat metabolism. finally, con-
siderable work carried on by Meites and others seems to indicate that
vitamin 813 can counteract some of the undesirable effects accompanying the

administration of large quantities of hormones.

23

 

 

LXPLhIhEki I. nelation Between Anterior Pituitary Growth hormone and
Vitamin big in the drowth—Plateaued Female hat.
PwOCsDUhs:

Forty female albino rats of the Carworth strain, weighing 230 grams
were placed on the following vitamin BIB-deficient diet (Zuoker and Zuckar,

1950) for three weeks:

Yellow corn meal 35m (by weight)
Ground wheat '55
Linseed oil mea 10¢
Soybean oil meal 20»
Alfalfa leaf meal Cfi
Brewers yeast 3»
Sodium chloride 1%

During this period it was determined that tne animals had reached a "growth
plateau" and were gaining only three grams per week. At the end of this
depletion period the animals were divided into five groups of eight animals
each and were placed in individual cages.

‘1

The experimental diets consisted 01 the basal vitamin B12 deficient

diet supplemented with crystalline vitamin 312“ in amounts ranging from
100 to 200‘pg'per kilogram of diet. Animals receiving growth hormone were

injected subcutaneously each day with 1 m6. anterior pituitary growth

., ‘LX- . . , ... . .
hormone.“ On the 2cth day of the experiment, .010» (by weight)

 

J. - a u ‘ - ~ W o a q «,
‘ Crystalline Vitamin 513 was supplied through the kindness Oi merck
and Company, Inc., hahway, New Jersey.

'4
|\

I
h

A" -

The growth hormone was furnished by the kindness of Dr. 1. h.
Bunoing of Armour Laboratories, Chicago, Illinois. One gram of
this crude pituitary preparation dh-3 has an activity equivalent
to approximately LOp of pituitary growth hormone standard 22 Kn-2
and this material is contaminated to the extent of approximately
7b with thyroid stimulating hormone in terms of the new U. S. P.
Standard.

Protamone” was added to the diets of the animals receiving growth hormone.
Body weight and food intake were measured every two days.

After hO days all animals were sacrificed and the kidneys, thymus,
Splean, adrenals and ovaries removed and weighed to the nearest tenth of
a milligram on a holler-Smith balance.

The following formula was used to calculate the stan ard error of the

mean (8. E.) in this and the other two experiments:

 

v

hoSULTS:

Figure 1 shows the average body weight plotted against time in days.
Table I shows the average body weights at the beginning of the experiment,
at 26 days, and at the termination of the experiment. Table I also shows
the average daily food intake and the food intake per gram gain in body
weight. The animals on the vitamin 512 deficient diet (group 1) gained
as much as those receiving 200 pg of vitamin 812 per kilogram of food
(group 2) and were somewhat more efficient in utilizing their food. hats

receiving growth hormone alone (group 3) or with vitamin 512 in the diet

IQ

(groups h and 5) pained approximately 20 grams more weight and were more

1

efficient in utilizing their food than animals on the vitamin 512 deficient
diet and not receiving growth hormone (group 1).

Table II shows the weights of the tissues per hundred gram body-weight.
The weights of all organs from animals receiving growth hormone and thyro-
protein are larger than those from animals either on the vitamin 512 deficient

diet or on the diet supplemented with vitamin 512.

 

* An iodinated casein product containing thyroxine, manufactured by
CerOphyl Laboratories, hansas City, hissouri.

 

1 - Controlo.
2 - 200 as. vit. Blalkg. diet.

.3 - 1 lg. growth hormone.

h - 100 pg. vit. file/kt. diet + 1 an zrowth hormono/day.
* - 200 pg. vit. Blalkg. dict + 1 :3 growth hormone/day.
‘ 0.016 pct. Protamono added to diet.

 

3.“ and ‘.

270» /
.. \/
C.
a
“
CK
3250+
.-
1:
2
3

25
3 WI n
o /
II

1
240- M‘ 2
I
‘J/ o.
2M)

 

no no 20 25 1» EB .b
DAY8

Figaro 1

 

115'

Effect of vitamin B} and/or growth hormone on the body volght

0 store {no}. net.

26

TLBLE I

taraoi or VIT hit 512 awn/bk ensure anemone ow sen! thGHT
ago rooo Ithhn IN The one TH PLATLALED FAEKLE him

 

 

'r * n,~.- .I ’r‘ ”I?" "- -"
nJclR/pr I‘OUd. intake

 

 

Group Treatment Averace Bod; Vt. (Gm.) (Gm.)
Original At Eh Days final Daily Per Gran dain
in Body Lt.
’ A r' :" + r‘ r n r-’ + .’
l Controls (do _ 3 4&2 : h 457 _o lh.7 22.h

 

2 200 pg. Vit.

Diet

OhO : g zhh :h lh.l 36.1

\u
r

 

3 l he. growth
I - r" r“ h V ’ r "r + P
hormone per are I 3 2:9 I 9 8/0 _h 19.7 l3.0
day

 

h 100 p: Vit.
513 per kg. of

diet + 1 mg 27h f 9 27¢ :7 13.3 13.0
E

[\J
P.)
\C‘

I +
1‘“

rowth hormone
per day

 

5 200 pg Vit.
812 per Kg. of
Diet + 1 mg.
Growth hormone
per day

I +

13.3

£4.
[‘0

\1

E-
l +
1:.—
[\3
\‘1

O \
I+
(“X
H
\fl

C, \

{\J
P.)
\C

 

1:21.171;

Y'\

A

II

t

m‘f‘flC’i‘ UF Vl‘l‘xn"..l.\.' £313 .-..~..=J/on ChiOJJ'l‘H eon-om UN ’i‘hh.‘ Oitfih‘a‘q LiulChTS

CF anVIH-FLATEAbDD F;LALE unis

 

 

zldrwrial
'17.; g/lOO £11.

DO C‘J' l": t .

'1': gym us
i‘;.'::/1‘JU 1171 .
body it .

K i C. ne 3;
1". 72/100 em .

50 7.2.7 ‘1‘"; t .

Spleen

\ ~ - f‘.
hé/lOa gm.

haug‘wt.

. ._—..—._-..-—-———.

 

eh.tl2.h"

gr” '3';
ooof3.o

195.0f3.1*

1f_;‘/lOO £711.
:5 O c 3/ 1": t .

 

63.9il.o

750:2.0

Zlh.9:3.l

 

73.Li7.9

étoi3.o

 

1:,

F} i“ {Z} + I“) '
(2 0/ ""f- ok’

 

[\3
\rl

hhfhn’mhhT II. melati01h3tween Anterior T3itiit rv Growth Hormone an:
Vlu-Iln jia if th3 lo HLUT hale hat.

_ q..——. .. .

 

 

f}: )OqLJylLJJ.

Fifty male albino rats of the Carworth strain and approximately
twenty one days of age were fed for two weeks on the vitamin Blg-deficient
diet “sed in Experiment I. At the end of this depletion period the animals
were divided into six groups of ei ht each and placed on the exn.11h intal
diets consisting of the has. al vitamin BIB—de ficient diet supplemented with
crystalline vitamin bl; in amounts ranging from 20 to LO.Pg per kiiog mm
of diet. fill animals in each group were mar;ed by ear slits so that an

‘ 1

individual record of reignt :3ains and losses could be maintained. Body

weirht and food intake were measured every two days. Drinking wet r was

aW ilable at all ti1w All animals were housed in a tanpcr ture controlled

r1 ~ :1

room maintained at {b 3.

Each of the animals which received growth horrone was inje ect ed sub-

\4

cutaneously each day with 1 Na. anterior pituitary growth hormone." lUlS
was i.1c11:2aseC' to 2 mg 3551‘ day on the 13th dav of the egqieriment and to

h mg per da" on the thh day of the experiment.

 

‘33"

Growth hormone preparation no. C—i-IS was supplied through the

kindness of Dr. dobert h. dates of E. h. Squibb and Sons, New

Brunswick, New Jersey; This preparation causes a 15 gram gain
in body weig11t in 2 weeks in hypophy‘ectomized rats weighirv
about 100 grams when in jec t d suicutaneously at rate of 0.1 mg

per day. It contair 5 loss than 1» Prolactin and Eon, and may
contain small amounts oi thyrotropin and Lh.

A7

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L.ut‘-‘;"};._I,oo Hello/Jim; this mi; Elfibdco I :L‘k: do;lt.:0 at £10 u lot“ JUNO HULLQ,

‘. .. .' r ‘r i _‘.,. '.:. 1 _ ’I” .I _. v ,_ . _ i .__,-1 fl ) _ ‘I_ ' ’ "-",\,’ ... _ ,
’_Lrl§-2,!_;l‘ d L";i;_'t;£‘-L-LL'_:1__L:;;L‘ L Lifting ba».‘.,i-3. n 5.2.1. s. LJSL L‘yolu I; bug; ‘31“i1n...ltt Nag»

.OW‘Z OIL

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‘5‘.
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conductco an; all count

y’- 0 lil . Ll: ' 8.1;.-. “al.; .4; due:- ,; i‘o-._1i, Win-4,11 were Int ndec oeo .Jitli L206
\.-' L

‘4 ' r ‘ a r.--—, c ,4 # r'~ ‘1 r I 0 r1 I . _ 1 r‘ .L J '\ o r‘ ‘ _: ‘ 1 _. V _ I ‘ '
l:L;:1bL;*Vlt-.~hifl #19 TV I‘ : :12in oHLI'J- lilo 1 J-L/ Lilla t1-“ ':2 aux; L111" tl'lfl ",5 ,
LlV'ijI’ 3 tGCt 3-8 , Sills Cfl BULL) VLlAijl L15.- U‘ifl’ili' 5 11121113 Vifiii , 14.1]. ”UlC'SUES WC: PG ’v1131;1‘i~(“)(_

.. " . an .,.4. a p ,-..r‘|"n - .7 m1 .. "”31 ‘ L‘,-"1 up ,_._{ h
to tne nearest tenth oi a “iiii.:an on a Abilur-uhitfl oalanCc.

 

" lie CobuifimelLa—v;tmmmislg was*tdfiineo tmmnixitne kthififl oi

.‘ F‘ - x ' fl # -- --. r s ,1 v v"» -. -- ..< I r. -‘l v— ~‘\ ‘ "‘ z r‘,’ nvn/ .‘ i ‘ 'u' n ” {j ~
1J1” , L , b , Loi b‘ql‘ini‘. arm n :15 (.-I‘i,~_1.1”_.:il.i_,y 1‘2”: gulf-3o ii on heron (i110. uo , 5

- ‘ .. «m . "“:.- . 4-- -:~ L p ”511 . Wit“, ruff 3,--
l...,i-'.. fl, , Lg»: o'u-.' o. “j' . iris 1.13 x_..~.l“i~;.,l haul our: 1b-“..-LQt'd 1-11,; 5-7: -,oi.LlC,=.io_ uni-J.
‘3 ~ ,. L” .-“I ’ " v. 1’» -- '- .13, '-..»'- ;' ,4- _.
(‘4‘ oC'LC '1'.) o ) u.-L o 9 J) O :73 1.11:. KILL-\llrg‘s) :1-ch QUlJ. ULUH 01. V11; -Jhlfl Q12 Iqu‘dkl 21L: ‘1‘. 4

A.\

2d

 

The results of this experiment are shown in Eigure 2 and 'ia amla III
through VII. Iacle III shows the average initial and final oody weights,
the nail; iooo intake ano food i talc per gram gain in body weight. It
can be seen that the animals on the vitamin olg—oeficient diet (group 1)
gainei less weight and were less efficient in the utilization of their
food than any other group of animals. The two groups of animals receivinr
vitamin 313 in the oiet Qgrouos 2 and 3) gained approximately the same

\

anount of weight and were at out equally eificient in the utilization of
their food. hats receiving growth hormone out no vitaznin blg Q :roup L)
appar33ntlxr gaineo as much as the controls eroup 1) ate a little more anu
we re slivhtl more efiicient in iooo utilization than animals on the
vitamin Big-deficient diet Qeroup l). mats receiving growth hormone and

either 20 or L0 pg 01 vitamin $12 per kilorram of diet (groups 5 and o)

ained more weixht and utilized their food more efficiently the n animals

."
Q

receiving the hormone alone Qgroup h) but no more than the animals which
received the vitamin only (groups 2 and 3).

‘raole IV shows the weights oi‘xn1ri~ous orcans eXprc ss;3d as either
milligcams or grams per huno ed gram body weight. Apparently there was no
difference in the weights of the adrenal, thgmus, kidney, liver, spleen or
testes regardi ess oi the diet or whether growth hormone zas auninistereo.

Table V shows the avera:e 0060 labo ed—vitamin 312 content of various
tissues. The urine of animals on the vitm win Big-ocfic ierm oi=t Q_jtrouo l)
containeo less radioac tive vitamin blg than oio that of animals receiving
vitamin 512 in the diet eroups 2 ans 3) and those receiving poth vitamin

Biz and growth hormone Qnroups S and o).

‘rhe olood, kidney, liver and muscle tissues from animals on the
Vitamin olg-deiicient diet (group 1) contained more raoioactivity than
did similar tissues from animals receiving vitamin 519 in the diet (groups

‘

2 ans 3). There was no apparent difference in the spleen content.

V

The amount of rauioactive 312 in the urine from animals on the vitamin

SIB-delicient ciet and receiving growth hormone (group h) was less than
that in any other group. The urine of animals receiving both vitamin 512
and growth hormone (groups 5 and c) contained more rauioactivc 812 than
that from animals on the eeiicicnt diet (group 1) but less than that from
rats receiving vitamin 312 in the oiet but not receiving growth hormone
(groups 2 anti 3) .

The blood, kidnvy, liver, Spleen and muscle tissues from animals
receiving ooth vitamin o12 anu growth ho mone (groups 5 and 5) contained
aoout the same amount of racioactive s12 as the tissues from animals re-
ceiving 612 but not receiving growth hormone (groups 2 and 3).

“aoles VI and VII snow the percent moisture and percent ash reapective-
ly of the various tissues and it will oe noted that the values for all
groups were very similar.

In general, it can be seen that the rats on the deficient diet (group 1)

retains" more raiitactive vitamin 312 in the tissues than the rats which

received either vitamin dlg alone or with growth hormone.

32

 

 

 

 

 

1 - Control.
2 - Controlu. hyper.
3 - Hypox. §»200 pg. vit. DIP/kg' diet
h - Hypox. +-0.00*1 aureo.
* - Hypox. o 0.00‘1 aureo. +1P00 he. vit. file/kg. diet
32° ¢ "' MR5. Mao’J“,
'“‘-l
\‘\
2 290/ \
4!
C
3 260-
h 24 >-
I
2 q
: 220 ~
).
O
0 200'
0
ISO -
160 - 2
P /\
140 j 3
IZO 1M6. 2M6. 4M6. 8M6.
1 J, 1 1 1 1 1 1 1 1
IO 20 50 40 50
DAYS
Figure 3

Effect of nus-1m B 2, Out-canyon: und Interior {unitary extract
alone 0} in cambinntion on the growth of
hyrophylectanizod rutl.

ThSLb III

Lo
\U

Mif‘fi‘rs OF V1.1-.1'11N 1513 j"-1:'L2/(J1L LIIJL3111'1 1101‘}. 0111.1 ON DCDI ”111.111.31.13

IC'.) iCCiJ 1111-11.) Oi"

1.1.1.1 G 1-11.1.1 I"..- 10

 

 

 

 

 

 

 

 

 

 

 

Group Treatment Average Eofiy’ (Gm.) Average Food Intake (Um.)
Original Final Daily Per Gran Gain
in 5055 Rt.

‘ r+: 3L ,7 '

1 Controls bc_u.0 lbliu 10.1 b.o

2 20 p: Vit.

- ,_+,

912 per ha Sti3.5 2l(-) 1D.3 3.2
Diet

3 L0 11g Vlt. -+. . .1. -1 [J
512 per kg. Lu—u.) 2UG_L lj.u 3.)
Diet

h Growth
T.“ (",+ ' .'
normone Cv—3.C 17Df7 11.8 b.00

S 20 pg Vit.
B12 per kg. tti3.0 207it 13.b 3.3
Diet + Growth
Hormone

5 DO pg Vit. +‘
513 per Kg. bCi3.2 211.; 13.5 3.2

Diet + Growth
hormone

 

Standard error of

the mean.

311

[ill-1.1. LE IV

EFFECT OF VITLMIN 512 AJD/Ou GnG;“H EOhmOND ON TEA OhGAN'deGHTS
01"“ IUU 1‘le 11:.ng 11.1313

 

 

Group Adrenal Thymus Kidney Liver Spleen Testes
No. hg/lOO gm hg/lOO gm mg/ioo gm Gm/lOO gm lug/loo gm Gm/lOO gm
Body ‘fft . Body 't- . 30 iii if." t . Bogv 1': t . Body If t . 506;] in t .

 

1 13-bi-5* 13h.015.l* 907.5:37* 1.52:.12* h&8.2ih5‘ 1.5..o&*

 

2 12.3..3 152.1:12.3 €32.2i67 b.1ot.001 285.0ih 1 3:.05

 

3 13.3..8 io9.7:5.5 802.ti38 3.h8i.001 32¢.1t7 1.ui.o;

 

h 15.0.1.3 l33.ot15.h 629.5iho u.is:.ooi 268.1:59 1.3:.006

 

5 iu.9i.9 166.8.8.3 732.6ibl 3.72:.51 h7©.6:ol 1.1:.02

 

6 15:93.5 163.1%)..9 712,7:15 3.6035101 331mm 1.3:.035

 

Standard error of the mean.

35

TABLE V

EFFECT OF VITAHIN 812 AND/Oh GROWTH HOMMONE ON THE 0060 LABELED—VITAMIK
512 comrmr or TIb‘b’b 235 m0}; 1’01) M} mm: was

 

 

 

 

 

 

 

 

Group Urine* Bloodifi Kidney¥¥i Liver** Spleen*% Musclefik
1 59.9 bo.éi5.7# 1.uo:.1c# 273.0i.02# 2hb.7i10.2# 91.1:.o3#
2 o9.o 50.5:é.5 1.20:.15 lou.6:28.3 269.2:20.é 53.7:2.5
3 72.3 SS.8:11.5 1.1:.Ot 117.3i§.& 2S9.5i18.2 o2.6ill.0
h 30.7 5u.b:h.u 1.5:.21 - 1Lh.3t22.3 225.6:19.u 70.0iu.1
S c1.7 50.017.6 1.0:.02 132.7:u.1 1hh.7i16.o éh.2f10.b
6 58.6 éh.3ih.6 1.1:.ou 13o.7i17.3 275.1i22.o 158.9i19.h

 

* Counts per sec. per ml. original volume.
*% Counts per sec. per gm, ashed wt.
*** Counts per sec per mg. ashed wt,

# Standard error of mean.

TELL}; VI

LL) /Olt GitOIJ TH HO 5.150}: L. OH Tm; PLraC 5'1" MOI STU i115
SSUDS FnOL‘I 501] 11G l~..»-LE kaS

HOT 05' VIM-.151 512 ‘
or“ »

1'\
IL
T I

 

 

 

Kidney Liver Spleen husole

 

75.07 69.01 77.51 73.65

 

7b.ol QB.t9 70.63 73.82

 

75.89 72.06 76.9h 77.25

 

K
C
O
C
-\l
O
\O
\J'1
\l
C: .
\]
Ox

7011:.

 

77.69 73.71 79.70 76.b9

 

TABLE VII

EFFECT OF VI’L‘m-glm 512 gang/0.4. (mo-am homer 03-; Tue. mull ASH

“E
OF TISSUQS FMUM IUUKG KATS

 

 

Group Kidney Liver Spleen fluscle

 

l l.h5fi 1.5hb l.bOh 1,37p

 

2 1.56 1.73 1.77 1.32

 

3 1.6L 1.59 1.50 1.37

 

u 1.L9 1.62 1.98 1.37

 

5 l.é5 1.70 1.90 1.3h

 

6 1.31 1.70 1.92 l.bh

 

LXELAL. NT III, Hela ion Be .tween Vit 1min dig and Anterior Pi tuitary

.IVV'L ,‘u " ‘ ', “L 1‘ ‘ f ‘. '. j" \“"A '. r—u "‘ '1 L
fl/LCf‘ao o l.‘. wit: 1.11-1111: [till'ciuli’ 0.2.8 uOH.iZ'3u £13. b .

 

PNGCEDUAE:

Thirt; hypophysectomized and l0 normal immature rats of the Sprague-
Dawley strain were divided into five groups and placed on the following

vitamin.813’ deficient diet:

Cerelose s2 gms.
Alcohol washed casein 25
Corn oil 5

Salt Mixture no. 2 h

Cod liver oil 5
Choline lO0.0 mgm.
Calcium pantothenate 2.8
Niacin 1.0
niooflavin 0.5
Thiamin 0.2
Pyridoxine 0.2

2, Methyl l, h Naphthoquinone0.0h

The animals were housed in spe cial cages in which cotton batting was
placed to pm ovide extra warmth.

The experimen al diets consisted of the basal semi-synthetic diet to
which was added 200 pg of crystalline vitamin 512 and/or 0.0055 aureomycin.
The hypcphysectomized animals were injected subcutaneously daily with 1
whole anterior pituitary extract* ior 23 davs and t‘1e dose was then in—

to 2 mg. per day until the 3lst day of the experiment. At this

(D

or ase
time the dose was further increased to h mg. per day for five days, after
which time it was increa dto o mg. per day until the experiment was

terminated on the h9th day, b01‘1y weight and food consumption were measured

every two days.

 

* whole bovine pituitary extract free 0 ACTH supplied 0; Armour

Laboratories, Jhicago, Illinois.

9

 

f1
1'}

1'1‘ ,. . 1 - . .: , ,. 1-. ,. , -, La-
1113 I‘CSaltS Oi tins experiment 81;; seen lIl #15111

~ 1

(D

3 .nd Table VIII.

Q1

Figure 3 shows the body weight in grams plotted against time in days.

P")

O

(0

It will DC noted that all group Lgpophysectcmized animals grc: at a
greatly retarded rate when compared with the intact contrrls. In addition,
it can be seen that igpophyscctomized animals receiving either vitamin
512, aurecmgcin or a combination of the two not only failed to gain more
than animals on the vitamin BLB-deficient diet, but actually gained
Slightly less.

Table VIII shows the average initial body weight, the average daily
food intake and the average feed intake per gram gain in bcdy weight. It
is annarent that the intact control animals were much more efficient in

converting their food into hodv weirht gains than the hw10 ivsectomized
c . s 1 J

animals.

hypophysectomized animals on the vitamin Dig—deficiert diet were more

efficient in food utilization than were rats 1eceiving either vitamin 513,

aureomgcin, or a combination of the two.

BODY WEUGHT

(GRAMS)

+0

 

 

l - C antral.

    
   
  
  
 
 

 

2
2 - 20 a. fit. a lkg. an:
o - M u‘. vit. Eli/kg. dict 5
u - Growth nor-on} 5
S - ?0 pg. vit. BIZ/kg. dict 0- growth hormone
200 ‘0 - 160 pg. vit. B‘a/kg. diet 4. growth hormone 3
f - Grow“! “among mac/Jul
4
ISO
I60 '
IQO
120
I 00 4 9
2 MG. 4'43.
4 To 12 us 20 24 23
D A Y5
Figaro 2

2 and/or growth hormone on the body weight

EffOCt of vitamin 51
of immature molo rots.

 

F!

r

5' ‘L’ "61011

1

TAALE VIII

Lf“LST U1 VITAAIN E13 AAA/3 a AAILAI‘A II1LI1'11 LK AFC” CH1 AVLJACL
BOUItJ LIGHT AND AV ALA LLCD I1T1I‘ OF IOUAG hIPCIA'1 I uiOmlLLD nAIS

 

 

 

 

 

 

 

up Treatment Average do Vt (Gm,) Avera*e rocd Intak? (Gm.)
Initial Fin.al I6? LRJ be -r Q1 Gain in

L ‘3} wt.

 

.5

Cid

,. r. ," +».I\ . .’“
22)-Lh 310-) 12.0

I13" pox .

to u: "it 12A
1

 

d3113<):<.

'1 +1
0 OCE3 Aureo. 121 u l
+ .". .} .4 .‘A‘A'

\_ A.)
\ .V‘l

I '1'-
kg)

0

C

9.L

 

Nypox.,
200 ug Vit.

ale/Kg Diet 126:3 130:2 ,AS 1A.?
+ O .OOEa
Aureo.

+ :1 Cr .14 .

‘ ‘\\A

' 1 v o 0
1,. __ a.“ .‘ A ..._‘ . , Y... .._. , .V
.- .. ._. — 1......1'._ _ . 1‘ a.) ,1 '1 .m‘; '+*"jjt
L

1'11 sot 1551011

In these three Ctgerianus an attempt was made to study the relation
between vitamin 312 and anterior pituitary growth Lo none. In the firs
exneriment grow tn-ti ateaued f1;3::':ale rats Wore used since it is to st: LC :iro.
animal used for tte assay Cf growth hormone, Unfortur1atC-ly these animals
a;1arently had good CCQJ~storcs of vitfmiL1312 and hence it was impCssiCle
to create a dcfiClCflC” state. There is little doub t tLat the growth

1 - ,4 - r {1}". »' ‘y, ' ‘.. “n4. .‘ n a , -. 1*. . D 1,. 1 ,__ .- Cl ,4. ' ~11? 1
normone 11es e11cctive lu tLe iabb as Can QC sCen 1r0m tnc inchrse in bCuJ

(:1

ght anC orfian weiints of Animals recoiVinj growth Hormone over those of

,1-
‘7’
1D
:1

rats not receiving growth hormone. The “3 ate from tnis exzerimth,

U“

indicate that ma M.r fame l‘, ”roan-pliCCwueo animals stiruulatC d with
growth hormone will continue to grow even in the essence of vitamin 513 in

the diet, presunaolg drawing uan aoeguate bodJ stores of the vitamin for

this continued growth. hegister st al. (19A?) have shown thrt a vitamin

512 —oeficie n'y coulo not rsAuil; oe iniuccu in mature rats.

.-n. a .1.“ - r. 1-1-.; ' ~~r‘,~—.1 . ' 1 ~11:
In tne seConu exper €3nt young male rats weiJ usJC in orCCr to th: A

. . .. f- n u A An ’- r, ,‘_' ',_ o - ' f‘ fi_‘ '0 v I‘ 1“! -7‘1‘(‘:‘,1 .fl . c "
ttsinirr a oeLiCiency C1 Vltmhln ”12 in tn: animels, A1119 15 01 this

" :3

CD

strain and age havo been used consistentlJ in this laC‘ratorJ fCr vit.2min

Clo-1J1iCieLC’studies. ihose animals on a vitaAin B12 —Ce'

{-0-

. a J -L w .
lCl,K.a'I-.1U (I16

Cte, gainCd less weight and were ls ss Hf iC i nt in

J.

H‘I‘EBW all 8. FE} CEI‘QCS‘Q l"

l

‘1

utilizing tLeir feeo than similar animals fed a diet s Cole. nt o with
vitamin 512. The results of this experirent indicate tLat young rats on

. , . —-—~| 'V- -. I . o _ » 1 u r a {1 n ,~ .1 ~ I - ..‘p‘4‘ )71 H: ‘r‘ O ‘ 1f"‘ r "‘ ~‘1
a VitsMin Dlg-ueflClanE Ciot, injecteu Nltn biUWbH Lornone guineu doOuL

£43

the same amount of weight as uninjected animals on a vitamin BIB—deficient
diet. This is rather difficult to interpret, since if the growth hormone
were exerting a real stimulus to growth in these animals, such a stimulus
should have constituted ? stress Which would cause the animals to lose
weight in the absence of an essential growth factor. Such a hypothesis
seems logical when one considois the anaoolic effect of growth hormone on
protein synthesis and the many reports indicating that vitamin hlg is

also involved in protein metabolism.

The possibility that the animals were not completely deficient in
vitamin 313 must not be overlooked, There are several possibilities which
arise. The diet might conceivably contain enough vitamin 512 for such
animals to continue to grow at a reduced rate or the body stores of the

1

itly good to buffer tne extra requirement

5

y

animals may have been sufficia
incurred by the injection of the growth hormone. In adaition it is Quite
possible that the growth hormone was not exerting an effective stimulus
to growth in these immature animals, since they were alreacy secreting
optimal amounts of this hormone. Such an effect has been reported previous—
ly (Evan, l9h2). The latter possibility seems most plausible in the light
of the data showing no effect of growth hormone on the organ weights, per-
cent ash and percent moisture in the tissues of animals as compared to
controls not receiving growth hormone. A general visceromegaly is one
of the effects invariably noted with growth hormone injections.

The fact that rats receiving both growth hormone and vitamin B12 grew

no more than those receiving only vitamin 313 further suggests that the

growth hormone was not effective, althougn it is also possible that the

O J)

animals receiving only vitamin n13 were already urowins at a maximum rate.
In any case, the amounts of growth hormone used in this exyeriment did
not appear to adorava e a deficiency of vitamin 312.

The information concerning the distribution of racioactive vitamin £12
after a single suocutanecus injection of this compound seemed to indicate
that animals maintained on a vitamin Blg-deficient diet retained more of
this substance. Thus it was shown that animals on a vitamin big-deficient
diet excreted less of the inJeo'ed vitamin in the urine and retained more

in the various tissues. The fact that animals receiving no vitamin blg

’3

ctel radioactive

r

(1,)

but getting growth hormone excreted less of the in;
vitamin than those on the deficient diet suggests that the growth hormone
might have caused an increased retention of the vitamin. This suggests
then, that growth hormone ma" actually have a Sparing rather than a deplet-
ing effect on vitamin 512 requirements of the body.

In the third experiment young hyponnysectomized rats were used, and
the growth curve and data on body weight indicate the inability of vitamin
and/or aureomycin to increase the rate of growth of such animals. In
fact, the data indicate that animals on the deficient diet actually gained
more and were more efficient in the utilization of their food than were
rats which were furnished either or both of the supplements. This latter
effect may conceivably be due to the large dose of vitamin 812 used which
may have induced a vitamin imoalanoe aid increased the need for one or
several of the other 5 complex vitamins. In any case, the important ob-

F‘

servation remains that vitamin 512, though a potent growth stimulus, was

n
.-

inefiective in the assonoe of the hypOphysis.

1:-
\JT

It is apparent that further work must he done to elucidate the inter-
relationship oetween growth hormone and vitamin 312. The main difficulty
which has presented itself thus far is the inability to create a vitamin
812 deficiency and to secure an effective stimulus to growth by growth
hormone administration in the same experimental animal. Thus in the ex-
periments described it was possible to obtain a vitamin 512 deficiency in
immature animals but growth hormone was ineffective as far as body growth

is concerned. On the other hand, when mature animals were used the effects

0
}-4

growth hormone were readily apparent but it was impossible to create a
deficiency of vitamin blg. a possible solution to this problem may be
the use of young rats from a colony which has been raised on a vitamin
BIZ-de 'cient diet for several generations.

Further work must also he done to determine whether growth hormone
may not actually decrease rather than increase the requirements for vitamin
312 for body growth. On the whole, the experiments which have been
oescribed suggest that this is entirely plausible. If this latter idea
can be borne out by further experiments, he growth hormone will then have
been shown to be uniquely'different from other hormones,most of which

gravate a deficiency of vitamin 812.

rdi

, ‘

-4 111.

.

5m

DE A LL'l.‘ L11 I

{H

ihe effects of growth hormone on rats receiving diets supplemented or
deficient in vitamin 812 Were studied, All animals were placed on a
vitamin Blg-deficient diet for a preliminary'depletion period of
approximately one month. body and organ weights, food intake, and
efficiency of food utilization were measured. 0060 labeled-vitamin 512
was administered in one experiment to study the effect of growth hormone
1

upon the metabolism of this compound in rats fed diets either adeQuate

or deficient in vitamin B12.

In the first experiment, the effects of vitamin 312 and growth hormone
alone or in combination on the growth of mature female rats of th
Carworth strain were studied. It was shown that animals on the vitamin
BIB-deficient diet gained as much as those receiving 200 pg of vitamin
812 per kilogram of diet. hats receiving growth hormone alone gained
more than the foregoing animals, while the addition of vitamin blg did
not result in any further increment in growth. The organ weights of

all animals receiving growth hormone were about the same regardless of
whether or not vitamin 612 was givenjand were significantly greater than
organs from animals not receiving growth hormone. It was concluded that
in the growth-plateaued female rats, growth hormone is ble to cause
continued growth in the abselce of dietary vitamin B13, due presumably

to the presence of adeguate body stores of this vitamin.

b7

3. In the second experiment immature male rats of the Carworth strain were
used. The results of this stud; showed that young animals on a vitamin
Blz—deficient diet grew at a slower rate, gained less total weight and
were less efficient in the utilization of ingested food than were

limals on a diet supplemented with vitamin 312, It was further
demonstrated that the administration of growth honwone to such rats,
i.e., animals either deficient or supplemented with vitamin 512, failed
to cause an increase in growth rate or weight gained above that of
animals not injected with growth hormone, The results of the work with
radioactive vitamin 512 seemed to indicate a greater retention of this
compound in the tissues from animals on a diet deficient in vitamin 812
than in those on a vitamin.Blg—ade5uate diet. Growth hormone also
appeared to increase retention of radioactive vitamin 812. It was con-
cluded that the growth hormone did not exert an effectiVe stimulus in
these rats in so far as body weight was concerned but that it may have

altered the me aeolism of vitamin 312.

h, In a third experiment young hypophysectomized rats on diets either
deficient or adeguate in vitamin 512 and/or aureomycin were injected
with whole anterior pituitary extract. lhe anterior pituitary extract
induced more weight gain on the deficient diet than in animals given
vitamin 512 alone or with aureonycin. This effect may have been
attributable to he large dose of vitamin 513 used in this experiment
(200 pg per kilogram of diet), since this may have created a vitamin
imbalance. It was demonstrated, however, that vitamin 313 though a power-

ful growth stimulus, was ineffective in the absence of the hypcphysis.

5. It is concluded that: (a) growth hormone administered to growth-
plateaued female rats on a vitamin Blg-deficient diet will cause re-
newed and continued growth, (b) in the immature animal the effects of
growth hormone on the reouirements of vitamindl2 are not apparent
from data on body and organ weights, although the possibility remains
that this hormone does influence the metabolism of vitamin blg as sug-
gested by the distribution of injected C060 labeled-vitamin 312 and
(c) vitamin 512 alone and/or aureomycin supplied to hypophysectomized
rats failed to cause an increase in body weight)showing that the

hypOphgsis is essential for the growth action of these substances.

a L-leC 3h *.;r'2'«; I

F .—
\O

bthICGnhrhY

hsling, C. H., Simpson, h, s. and bvans, h. M. 1951. Effects of pituitary
factors and 01 thyroxin on skeletal morphogenesis in the rat. Trans.
Macy'Conference on hetabolic lnterrelations 3: 125.

Aten, A. H. W., Jr. 1950. Corrections for beta-particle self—absorption.
hueleonics o: o8.

Becks, H., Scow, h. 0., Simpson, M. E., Asling, C. W., Li, C. h. and Svans,
h. M. 195 . heSponse by the rat thyro—parathyroidectomized at birth
to growth hormone and to thyroxin given separately or in combination
III. Skeletal chana s: tibia, metacarpal, and caudal vertebrae.

Anat. Record 107: 299.
Becks, H., Simpson, M. L., Evans, H. M., may, n. D., Li, C. h. and Asling,
C.‘4. l9ho. hesponse to pituitary growth hormone and thyroxin of the

I! v

tibias of hypoohysectomized rats after long postoperative intervals.
nnat. Record 9h: 031.

decks H Simpson, h. 5., Scow, n, 0., Asling, C. W. and ovans, n. A.

: . Skeletal changes in rats thyroidectomized on the day of birth
and the effects of growth hormone in such animals. Anat. necord
100: 561.

Beerstecher, E., Jr. and odmonhs, E. 1952. Evidence for existence of an
apoerythein precursor in gastric mucosa. Federation Proc. 11: 1&5.

bennett, L. L. and Li, C. h. 1yho. The effects of growth hormone and
adrenocorticotrcpic hormone on the urinary glucose and nitrogen excre—
tion of diabetic rats. Endocrinology 39: 03.

Bennett, h. n., Joralemon, J. and halpern, P. 1951. Effect of vitamin
812 on rat growth and fat infiltration of the liver. J. Biol. Chem.
193: 265.

setheil, J. J. and Lardy, H. A. 19h9. Comparative effectiveness of
vitamin 512 whole liver suostance and extracts high in APA activity,
as growth promoting materials for hyperthyroid animals. J. nutrition

37: A95.

oierring, E. and Nielsen, 3. 1932. Composition of tissues of albino rats
treated with alkaline pituitary extracts. Biochem. J. 2c: 1015.

bird, n. n., nubin, M. and Groschke, A. C. 19Le. A chick growth factor
in cow manure VII. Its stability and solubility. J. biol. Chem.
17h: ell.

Brues, A. M., Drurv, U. m. and brass, h. C. l93o. A quantative study

of cell growth in re;;ne rating liver. Arch. Path. 22: 656.

Bryan, A. H. and Gaiser, D. W. 1932. The influence of diet and the
anterior pituitary growth hormone on the growth rate of dolescent
rats. Am. J. Physiol. 993 379.

Cary, C. A., hartman, A. h., Drvdén, L. P. and Likelv, G. D. 19ho. An
unidentified factor essential for rat growth.1otoration Proc.

5: 128.

Castle,'w. B. 1929. Observations on the etiOIOgic relationship of achylia
gastrica to pernicious anemia I. “he effect of the administration
to patients with pernicious anemia of the contents of the norma human
stem ach recovered after the ing estion of beef tuscle. Am. J. med. Sci.
1.1c: (Lag.

Castle, V .a., Townsend, 1%. C. and 1m eath, C. J. 1930. Observations on
the etiologic relationolir of ach; lia gastrica to pernicious anemia
III. The nature of the reaction oetv en normal human g9 stric Juice
and beef muscle lea'Ln" to clinical improvement and increased blood
formation similar to the effect of liver feeding. Am. J. hed. Sci.
1&0: 305.

Charkey, L. h}, Eilgus, 5. 5., Palton, A. H. and Gassner, F. X. 1950.
Vitamin 512 in amino acid metabolism. Proc. Soc. Exp. oiol. heo.
73: 21.

Chow, A. F. ard barrows, L. 1930. mole of 813 on hitrogen retention of
rats fed on soy bean protein diets at oifferent caloric levels. Fed.

Proc. 9: 35b.

Cushing, h. 1912. The pituitary body and its disorders. Lippincott,
Philadelphia {cited by drody, S. l9h5. Bioenergetics and Growth.
P. 19h. Reinhardt Publishing Corporation, New fork City),

Dott, h.7i. and Eraser, J. 1923. Quart. J. Exp. Physiol. 13: 10 (suppl.).
(cited by'Li, C. h. and bvans, h. h l9h7. In Vitamins anu ho m1ones

S: 197, Academic ire oss, how lork CiW ).

_\

Downs,ifl. 0., Jr. 1930. An experimental study of the growth effects of
the anterior lobe of the hyp physis on the teeth and other tissues
and organs. d. ental hosearch 10: L01.

[—1'1
)4.
ch, h. h. 19u9. Liootropic effects of vitarin
Proc. Soc. mxp. oiol. Med. 72 : 3ft.
l9hb.1ne critical requirement I r panto-
. undocrinology' h3: 2o3
1.8111031; 10 75 ‘LL.
11D 812 in rats
103 392.

Drill, V. A. and thorni
P.

11cnalectomizoa rat

Vitamin 552.

yreast on the
its}.

113d _
in blanoular Physiolcqy anu fherapy,

estrao iol.
. ano1icailliam 5 h. o.
and liver on ovaries of immature rats feo mas
bio.

513 concentrate
Dumm, M. E. and halli, h.
thenic acid oy he a
Dunlop, D. h. an 'hilson, e. h. 19h9.
Emerson, o..A. 19h9. urthn promoting aCtiVloJ of Vitam'
receiving thyroid substance Proc. Soc. AXp. biol. hed.
Angel, 1 L. Pituita r-Aurenal Function lho horn-Shafer Co., haltimoro
Md. P. o2.
rshoff, a. n. l9hb. Conditioninr factors in nutritional disease. Physiol.
hev. 2t: 10}.
Ershoff, B. H. 19h9. An antithyrotoXIc factor for the rat not 1o ntioal
with vitamin blp. Proc. Soc. Exp. Bitl. hed. 71: 209.
arshoff D. h. and Deuel_ n J., Jr. 19h5. The effect of 5rcwth hormone
on t1e Vitamin A defiCient rat. endocrinolo5 3b: 2c0.
5151011, 5. H. and Deue l J., Jr. l9hb. The beneficiele effects 0:
body anu agcnatial wei1n t of i1m ature rats fed allia—
r1. J. P113810]. 11.1,). 11:15.
19Ao. “ff1cts of B vitamins roast
1i 1 ve doses of alpha-
6?: 323.

D I 14'
, d, n
. Proc. Soc. Exp.
of fee
‘ the rat. Anat

ErShOfl
"rcwth

estradiol
Evans, h. h 19L2. Chapter II,
Ayerican 1 ical Association, Chicado
and Lor11_:,J . A. 1921.
the by popnysis on the estrous
tiarac teristic effects ulm an 5
intraperitoneal administration
t Record 23: 19.
od'en
none.

M

)r‘\ '1‘,
. Ana
n; laticn 0:

Evans,
estrus

Ewans, h. .
lo 1e of
21: 02.
h . 1'1. ariCL LOIIQ, J . :11- . 19%.; .
and ovulation ln\UCCC by t1
rior hypophyseal ubstance
11..o. and Fe ermniarz, It. 1939.
effects of the pituitary and the thyroid ion
of cietary :rotein to signs

1‘1 .

of fresh an
Evans H.1h., Simpson,
the gimn-rth prom0t11,,
Anoocr inolo 5'v 25: 17".
A. a.na hall, S. D. 1952. n lation
ijlol““CV oLf1 3ts of su1jlom nts of vitamin Llp and
. 5.101. 011C111. 17¢: 1(f

Fischer
of cholinc
fOllC acid. «J

C”)

5/1..

.. .

1.”... ‘ .'—1 a "1 h “3‘ - 1 " q A F’ J T- ’-‘...‘y '.'./‘\-' "r T“ l"~-z"“ "1‘ "‘ ‘1_ T-.
1‘1-1.'1“.1.’_:_L-C1)111‘ 1., d ., L1 - Vlf~1-v.)l-l c: L." n ., alarm)?” l; . 1.. ._-..=1.-1 .aVchzs, L1. 1’...
N, 4 '0'. ‘ -1...- ...,- ,. - 4-1...- -
1742. utfects of Durlllc' r14fll1<'3 {reilraticrs on ~1c HQMrfutvlL
-. .~ , ., L - -.L . J— . .o 1 -. ,— I. 2. a ”v7. obs“:
P1 ”JACQ- "A CAJI S '-4.LL.‘."~.,J 15.. ~21 DLVKJL». . 11m . J . I I U LLUl . -L,)i : l.\.l‘~." .
“ "A ‘w - Y«A.T- 7 ‘ é'm,p. M f—~ U ‘ wisn
DI'afgl’l'ri‘Ql‘Cb Frat, H.) 1 11.11596?) U. .14., 1.2115 .1111, .-. u.) L“: J18, 1-. U. —-/'—+Jo
F‘ '1'. ('rx—'L . r ' 1“ ‘1" l‘\ ‘<u "‘4' -‘ v, «.—», ' \ ~ . .+'-_. '.1<
lrtl 1 j9fllillsu4ojallrJf Lab; : c.1-1 hoz=xiig of th 319;; rior Ijltu3.-Iu .
—.. '1 r“ 5. 5 .0 V n A? 5/ C1
bncoc1UJAhaifiy 21: 1C,.
fi- .7 — ~ A W " ' m" ‘»‘.a' ~ . ~ I7 I. " '~ T-T ’5' .1 0‘ . j fl .-.+ 1*
1‘1A3'3111<51'UUIIT1‘L, 11., 01115.;Swn, 1-. All. 73!- . UV 11;), 11. . 1,1,5). afl’-x. u v.1.
. _ _ .L ,. n ...D 1-: i“-. ..: ‘L .__ ..l. . 1 , 1'. .,,_:,.‘..._N
1POJOLflh but:u01b7 81;:111 P u.L.ic '.1LUJ cir. lx.?rtWr-‘ Lul t14>.11v 1“ 81 .11-b’
d; J. u I} .L‘ (0 ‘
. r. J- . -\ -’~ ‘ _, .-vr- . j I} O 17;)
2101.11 '11on ’f Fats , ~17}. J. 11:50.3(11. 11L 0 g), .

Freud, J., Levie, L. E. Hui Krosr , D. 3. 1939. Cbs=rvaticns on fisosth
. ‘ o o - ”L'n‘
(2}ondrotrgghic) 1crnon‘ a“; loca 1L a ion of its poiit of .tthk.
- r”
J. ur.ccri1o1o l: 3..

v \ A P~~‘ ,I‘ “Po-LN 91").1 ‘ “.."‘ ‘ 1‘" A‘ "r" " _ 5” ‘
Gaebler C. A. 1/3). acne SLproO 1 art -ior pituita rJ t; 1acts on
c

La“- ' .,,. .--
an-i enerL; miguflwllbu J. n“ 5

‘ 1_ rs 2‘ " A '\ - \ «(N '1 (10'; ‘ “ I! C ,l .'_' ‘_o (‘55,. ' 5‘ o _ _. l: a"?
see.l3r, O. H. ano oartlstt, l. a. 19 ,. n11»cts 1. anterioi i..itui’r..1J*
. ‘ " . r . /\ r_v I“ 'W are" . ,— \ -1 4 .’ 1 1‘ n 4
yreparations and lJ‘lne an ri+ 0 en dAoLcBl'n, L318c11L11A, and bcs.l
n- -' -3 ‘ f: ‘ ,— ‘ H... r)
netaooiism. J. Liol. CUM“. 1.9: W9.
‘— — ’ v 1.: ». 'r . 4* —. . r r — n .1
Gmblcl", C . f1. , 1281111218, 9 . C . 8.11.. 1-1111, 1 . 191.9 . The effect 01. o1 cwth
a O ‘ _ fl ‘ 5 A ' ,f - w? 2‘ n .—- .3 w ‘ ' v‘ CU“ To
hormone urcn liv r arc kic1e; D-~-ino acio uni as: anu ugon.nusc1u
. . ‘r‘ t'\ ' “ ‘ a“. 1'\-‘. "t-rx— .1 0/
S’dCCll’llC c1011.“ C'- Cr "u JI‘chLLaSEJ . #111,...0L1 ll’luud .5“: 11.): L. 7 .
m. . ”I ”.4. - r 1" fi‘. , :-.- 4- ,. y 1. : ' - a 1 -
GHLJLJlUT , 0 . H . ELM. u-x.‘ 1.3111391". ’ n , L'L . 17L+2 . ufl -;’.C u ‘-‘ f 3.111} 5734-01“ P111111 t*1 J
" ‘r ‘4 ‘ -/-. . ‘ u an“ I: ‘. " '. rs < ‘3 1' r' I/-q
L.t1act oh nitrooch rets.ticn. uni:mc inol 3d 3o: -27.

Gillis, M. B. and Norris,
on the re hlrbhon fo

*St"
(D(J

rJFJ

V

L
m thv atin
3. a11c‘Camgbe11, D. C. 19L8. Effect of vitamin 312 on the
matopoietic and nervous systems in Addisoni an perniCLous an~3mia.
J. Lab. Clin. Dec. 33: liho.

Hammond, J. C. 19LL. Dried cow Retire and dried rumen content-
partial substitute for alfalfa leaf meal. Poultry Sci. 2
Handelsman M. B. and Gordon, B. F. 1‘30. Growth 312 bcL: chank

r
. : » z-I -~n. -‘. ' :I: '.'-I :‘L 7'- ~rJ' 4' . "\~ f‘ ‘. '.-‘-\ - ... ‘N
thCCtsJ filth e_t-:1or ILtUibarJ cAuracc. J. ELJIRALULUQJ )-

- r _ A ' _ - ‘ ‘ 1 -
Harrison, d. C. an; LuL , C. N. h. 1940. The effects 0
extracts in the fasted rut. Lnflocrinoloor 2C: 971.

Harte, fl. A., Chow, 5. F. and Barrows, L. l9F2. Dis A5pc1rince Cf r:.iio—
activity from various or.3ns of n: rmal adult r1 ts folloning musicsncous
injection of terged vitaminq L13. Fee. 51o . 11: LL;.

nartman, A. M. l9ho. Occurrence in foods of an onloent111c factor
essential for rat growth. bed. Proc. 5: 137.

hartman, A. M., Dryden, L. P. and Cary, C. A. 19L . A role of vitamin
B13 in the no1ma1 mammal. Arch. Biocnem. 23: 105.

hausmann, h. 19h9. Liver extracts, V1tm1nin B19, ana,thym1c1nc. Lancet
2° c2.

l\
\O

Hoberman, h. D. 1950. hnaocrine egulation of anino acid and protein
metabolism during fasting. Tale J. Biol. and hed. 22: 3h1.

houchin, O. B. 1939. The influence of anterior pituitary extracts on
the proteins of the liver. Endocrinology 25: 759.

houssay, E. A. and Leloir, L. F. 1935. Diabetogenic action of the
anterior hypophysis independent of the adrenals. Rev. soc. argentina
bicl. 11: man. (from C. A. 30 110o3).

Illingwcrth, B. A. and hussell, J. A. 19:1. The effects of growth hormone
on glyCOgen in tissues of the rat. Endocrinology he: h2 1.

Inwle, D. J. 1930. The biologic properties of cortisone: A review.
Clin. Endocrinol. 10: 1312.

hibrick, E. A., Becks, h., h:‘“X, L. and Evans, h. m. 19h1. The effect
of different dose levels of growth hormone on t11e tibia of young
hypOphysectomized female rats. Growth 5: E37.

1

Kleioer, h. and Cole, H. h. 1939. Body size and energy metabolism in
growth hormone rats. Am. J. Physiol. 125: 7174.

Kochakian, C. D. l9h9. (Cited 0y shite, A. 19h9, in hecent Progress in
hormone Research h: 1t2, Acaoemic Press, Inc., Lew York City).

Koneff, A. A., Scow a. 0. Simpson M. E., Li, C. h. and Evans, h, h
19h9. hesponse o3 t11e rat ttyro—p aratnyroidec+ow12 eo.at birth to
growth hormone ana to th3roxin g:iven separately er in comoination ,
II. Eistolooical changes 1:1 th3 pituitary. nnat. heccrd 104: hoS.

Lang C. A. and Chow b. F.1912. Retention of V1tamin 512 after aominis-
tration to suojects of oifiMEr nt a,: _es . Bee. Proc. 11: to.

Lee M 0. and.Schafer R, K, 1934. Anterior growth hormone and the

a“-

composition of growth . J. Nutrition 7: 335.

Lee, M. O. and Schafer, h. K.1935. lhe eifr ct oi the ant;rior pitu_} nary
growth hormone on pro otein meta;3clism. . Biol. Chem. 10L:

5L

Li, C. H. l9he. Growth and anterior pituitary. Growth 12: h? (suppl.).

Li, C. H. and Evans, h. M. l9h7. The properties of the growth and
aurenocorticotropic normones. In Vitamins and hormones S: 19?.
Academic Press, Inc., new iork City.

Libby, D. and Meites, J. 1932. Effects of vitamin 312 and penicillin on
thiouracil action in chicks. Proc. Soc. Exp, oiol. Med. 79: 370-372.

Lichtman,fi.,'fiatson, J., Ginsberg, V., Pierce, J. V., Stokstad, E. L. n.
and dukes, T n 19h9. Vitamin 0130: Some properties and its

therapeutic use. Proc. Soc. Exp. 5101. Med. 72: ch3.

Lillie, n. J., Denton, C. A. and Bird, H. R. 19:5. Relation of vitamin
513 to the growth factor present in cow manure. J. Biol. Chem.
17b: lh77:.

Lillie, h. J., Olsen, 5. N. and Bird, H. R. 19h9. mole of vitgmin 312
in reproduction of poultry. Proc. Soc. pr. biol. Med. 72: 59c.

Ling, C. T. and Chow, b. F. 1952. metabolic role of vitamin 513.
Federation Proc. ll: 2&9.

Lotspeich,1fi. D. 1950. melation between pantothenic acid and response
to growth hormone in the acult rat. FBd. PFOC. 733 5#.

Margitay-oecht, 5. anohiallner, E. 1937. Gewichtsanderung mit Wachstums—
hormon behanoolter flatten im Zustande der Avitaminose, Z. Vitamin-
forsohung c: 119.

harx,'W., Nagy, D. 5., Simpson, h. L. and Evans, h. h. 19L2. Effect of

\

purified pituitary preparations on urine nitrogen in the rat. Am. J.
Physiol. 13?: but.

Marx, W., Simpson, H. E. and Evans, H. M. l9b2. Synergism between thyro—
tropic and growth hormones of pituitary. Body weight increase in
hypOphysectomized rat, Proc. Soc. Exp. Biol. Med. L9: 59h.

harx,'w., Simpson, M. E., Reinhardt,'w. O. and Evans, h. E. l9n2. Response
to growth hormone of hypophysectomized rats when restricted to food
intake of controls. Am. J. Physiol. 135: olh.

heites, J. 1950a. Effects of vitamin 512 on thiouracil action in rats.
Proc. Soc. Exp. oiol. Med. 75: 193.

Meites, J. 1950b. Effects of vitamin 512 on normal thyroid function
in rats. Proc. soc. mxp. 5101. Med. 75: 195.

\j‘?
UL

heites, J. 1951. Counteraction of cortisone inhibition of oody, hair
and thymus srowth by vitamin 513 and aureomycin. Proc. Soc. Exp.
B131. tied. 7;: C92.

heites, J. 1952. Chan es in nutritional re quirements accompanying
marked changes in hormone levels. hetaoolism 1: 56.

heites, J. and Shay, J. C. 1951. Effects cf vitamin blg on diethyl—
stilbestrol and thyroprotein actio on in male rats. Proc. Soc. Exp.
Biol. Med. 7o: 19o.

killer, C. M. and hoorhouse, E. h. 19h9. Treatment of megaloblastic

anemia with vitamin_olg. drit. Med. J. 2: 1511.

hirsky, I. A. and Swadish, S. 1935. The influence of the anterior
pituitary gland on protein metabolism. Am. J. Physiol. 123: lhB.

Nestler, R. 8., Byerly, T. 0., Ellis, N. R. and Titus, H.'W. 1936.
A new factor, not vitamin C, necessary for hatchaoility. Poultry
Sci. 15: 67.

Nichol, C. A., Dietrich, L. 3., Cravens, W. W. and Elvehjem, C. A. 19h9.
Activity of vitamin 512 in the growth of chicks. Proc. Soc. Exp.
Biol. Med. 70: DO.

NiChOl: C. A~, “ObblGG, 3. fi., Cravens, M. W. and Elvehjem, C. A. 19h9.
The growth response of chicks to antipernicious anemia preparations.
J. Biol. Chem. 177: e31.

Ott, E. h., Rickes, E. L. and Wood, T. a. l9h8. Activity of crystalline
vitamin 513 for chick growth. J. Biol. Che . 17h: 10h7.

Patel, J. C. 19h8. Crystalline anti-pernicious—anemia factor in treat-
ment of two cases of tropical macrocytic anemia. Brit. Med. J.

2: 93h.

Popper, d. ,Kochéheser, D. and Szanto, P. B 19L9. Protective effect of
vitamin 812 upon hepatic injury p oduced by carbon tetrachloride.
Proc. Soc. Exp. biol. hed. 71: bed.

Putnam, T. J., denedict, E. B. and Teel, H. h. 1928. The preparation of
a sterile, active extract from the anterior lobe of the hypophysis,
with some notes on its effects. J. Physiol. oh: 157.

haben, M. S. and Westermeyer, V. h. 1952. Differentiation of the growth
hormone from the pituitary factor which produces diabetes. Proc.
Soc. Exp. Biol. Med. t0: o3.

nay, a. 0., Evans, n. h. and decks, n. l9hl. EffE Ct 0i tue pituitary
growth hormone on the e-ipil eal disk of the tibia of the rat
Am. J. Path. 17: 509.

hegister,'w. D. he u gwar 3r, W. R. and Elvehjem, C. A. 19h9. an improved

3
assay for a growth factor in liver extracts. J. biol. Chan. 17?: 129.

Reinhardt, E. 0., harx, a. and Evans, h. E. l9hl. Effect of pituitary
growth hormone on the thymectomizod rat. Proc. Soc. Exa. oiol. Med.

he: hll.

Rickes, E. L. ‘rink, h. 0., Koniuszy, F. n.,'Kood, T. n. and Folcers, K.
(3.

,
19h8a. Cryst“lline vitamin 513. Science 107: 39$.

’3

C
“9., . ,.
19hbb. Vitamin 512, a cobalt complex. Seience 10L: 135.

m. L., Brink, h. C Koniusay F. h., flood, T. h. and Folkers, K.

Rubin, h. and gird, E. h. 19M . A chick g rowth actor in cow manure.

I. Its non identityw With ick growth factors previously described.
J. Biol. Chem. 1:3: 3o7. .

Russell, J. A. 1936. The effects of hypOphysectomy and of anterior
pituitary extracts on the disposition of fed carbohydrate in rats.
Am. J. Physiol. 121: 755.

husse11,J.n. 19h2. The anterior pituitary in the carbohydrate metabolism
of the eviscerated rat. Am, J. Physiol. 130; 9;.

Salmon, T. N. 1936a. The effect on the growth rate of tiyro-parathyroi-
dectomy in newborn rats and of the subsequent administration of
thyroid, parathyroid, and anterior hypOphysis. Endocrinology 23: ht:

Salmon, T. N. 1935b. Effect of pituitary growth substance on the develop—
ment of rats thy roicectomized a.t birth. 29: 291.

Schaefer, A. E., Salmon,§J. D. and Strength, D. R. 19h9. Interrelation-
ship of vitaminb12 and choline. I. Effect on hemorrhagic kidney
syndrome in the rat. Pros. Soc. Exp. Biol. hed. 71: 193.

Schweitzer, C. K. and Stein, 5. h. 1950. Measuring solid samples of

low-enerjy oeta emitters. hueleonics : c5.

Scow, h. 0. and harx, ‘i. 19h5. Response to

rats thyroidectcmized on the say of bir

pi ituitary Igrowth hormone of

mi Inat. chord 91: 227.

Scow, n. O. and Simpson, M. E. 19h5. Thyroidectomy in the newborn rat.
Anat. hecord 91: 209.

UT
so

Scow, h. 0., Simpson, h. 2., Asling, C. 2., Li, C. h. ano Lvans, L. h.
19L9. nesponss or the rat tny1o—1arat1Wroicrctou17ro at oirth to
growth hormone anc to thyroxin giVen separately or in combination.

I. General growth and organ chan‘es. Anat. hecoru 101: hhS.

Selye, h. ans Jensen, n. 19hn. "The chemistry of the hormones". Ann.
nev. Biochem. 15: 3L7.

Shaw, J. H. and Creep, n. 0.19h9. Relationships of diet to the curation
of survival, bony we i ;ht and composition 01 h; top1ys'ctomizeu rats
bliuocrir molo ha: 320.

‘hOFD, fl. 3.19AC. Activity of vit: min 512 for the growth of Lacto-
oa.ci11us Lac tis. Science 107: 39s.

 

Smith, E. L. 19he. Purification of anti pernicious anemia factors from
liver. hature 1o1: o3c.

Smith, P. E. 1927. Disaoilities caused by antpn"sectomy and their
repair. The tuberal (hypothalamic) synniome in the rat. J. Am.
hed. Assoc. 68: 15c.

Smith, P. E. 1930. 3yp0o1ys ctomy and a re plaCt mwant therapJ in the rat.

I

Am. J. Anat. HS: 205.

S ith, P. d. anc Smith, I. P. 1923. The reSponse of the hypOphys HOL0.1261
tadpole to the intraperitoneal in; ction oi the ViPiOUS 101 es ano
colloid of the bovine hypophysis. ennat. hecord ";: 150.

Soskin, S. l9h1. MEL9)0110 funo tions of the endocrine glands. Ann. nev.
Physiol. 3: SL3.

82165: T- D-: Suarez, h. 3-: LOPEZ, G. U., m11-11ch, 5., StonE, d. L.,

Toca, n. L., aramuur , T. and hartus, S. 1929. Tentative appraisal
of vitamin 313 as a therapeutic agent. J. Am. Med. Assoc. 39: 521

i 1
c

b. T. H. and chinnis, J. 1951. The effect of
icncy on the metabolism of force fen glycine.
pr ~JS€I1L€Q at the 119th meeting ACS, .April 1-5,

Stern, J. R., Levaiit.
vitamin 113 deii
Abstract ct papa
0-12, 1951 p. 370.

\

3

C
.27,

A

,L

Stokstad,m .L. n., dukes, T. h., Pierce, J. V., Page, A. C. and Mr nklih,
n. L. 19L9 . 1‘1e miltiple nature 01 the ar imal protein ia or.
J. Biol. C1 hem. loo: oh7

0. 1929. Th: effect of extracts containing the
.3.

e ‘ the anterior htpcp-vsis upon t11e blood chemistry
‘SiOl. (-9: \JL‘Z'J .

Ternberq
t Qir r
S .

. L. and zahin, R. E. 19L9. ertLPin and agocrgthcin and
n1 1 '-'
O

icn to the ixtip ernicious anemia principle. J. Am. Chem.

0

7‘ A'fl H r1 .5 fl *‘ , - . H' * - ‘fl n ' u» ° - ‘-,— v A'-
irains, J. u. ahd VUFSCC<Z m, L. A. 17v2. lutLTPciathhSLlpS cu dean
Cuoiine ang vitamin 413 ix ‘ ‘ “ad. Prcc.

ll: bSY.

H
c+
@
f—j
co
c+ \
p.)
:1

c
H.
:‘Y
F
’(o
3
o

g.

m

( a

.

In<ley, C. C. 1915a. Anti-EIGMlG su'ista.m 33 iron liver. Lancet 1: 771.

Ungleg, C C lfi'tt.“1crc1\tic anzmia. Eric. £91. J. : 151,

bngl By C. C. 1919. Vitamin $12 in permic
2

acministration. firit. med. J.

“aadehn, F. 1932, Ver51che UJGF die binwirkung dgs‘iachstmushormons auf
die Mans. diochem. Z. 29;: ch.

hatson J. ard Ca tle ‘E. 5. 19LC. hutriticnal rac ?OCyti
71

.
3 \J
a “arc. Hesyonsc to a sagst; nce 1n liver

, s
es.ec ially in p - other
tlan that effective in pernicious anemia. :mcr. J. 10;. Sci.
211: 513.

'fiatts i. 3,, M38“, 0. b,,'1hitehair C. K. and Eac Vi 3r, A. 1951.

o C
hEZS once 01 crstrateq male are 13m? ls hyperthgrcid rats to vitimin

312. PrOC. Soc. mxp. Biol. 13d. H/ p21.

1:8 '1: D 9 V . - 3.11.3-9 It 2'- ?Lin; 1. \. bye leG, G. C. 1952 . P-sr Jaw?“
n C 8.

faster in 1 ti ants with pcrniciOLS ancmia ir "emission
1

given racicgct'vs Vitiuin 513. rCi. ircc. 11: 30L.

hells, B. B. and Randall E. C. 19b0, Proc. Staff fleet. haho Clinic
1L; 324 (v1 ted b, 1”runs J. 1951. 00:3 t r1ction c‘ CLFtiSODQ

/

ink iciticn oi c913, Mair arc tijmcc growti Lv vitimin 913

(I
a.c {Nirccr5c111. 111m). 630. liq)..;icl. 1191. 7L} <;yz,)

“ 3‘ t__' ‘ o 4-- - ,‘\ o ‘ ‘ '3 - fi -_: P. ‘ ~.». g .1 . _-'
1est, fl. l>cL. n3 lVib; 01 Jitizuin 113 in 31c103111n CthiClodS §£5u1&,

, \

J.
\J
Scicnce 107: 3);.

;inter, C. 1., Siluar. n. I. aha StcC rk H. C. 19,”. P10 ctjcn of
reversible hflpgrairenccortin131 in rats by prolcxi,1 1Hlourau10h

. f—j

5‘ 1' 3 .~~~\- 1 L" . -‘ a ,» 4 « ,
01 cx1zt1ouuc. 1J11QCT1K010;3 M{ c .
F/l‘ ‘1'“ 1" '1'" r‘,— ,3; I) ‘1 ' ‘ T '1¢x,-J _ _‘. ,\ - v .3. : ;wnfi ’ ,1 f
“igncr, l. n. ang AucRCr, u. ,. 1},O. "uleql protein ructur" 311
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Figure 4 1
Self abnorption curve for CoCL labeled vitamin 512

25

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