Comparative hemopoietic effects of vitamin B12, folic acid and iron

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COMPARATIVE HEMOPOIETIC EFFECTS OF VITAMIN

B12,FOLIC

ACID AND IRON

A Thesis Presented to the Faculty of the Department of Pharmacy The University of Southern California

In Partial Fulfillment of the Requirements for the Degree Master of Science in Pharmacy

by Ersilia Feliza de Guia August

1950

UMI Number: EP63470

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Pha.

'5 I

D3al

T h is thesis, w ritte n by

ERSILIA FELIZA DE GUIA under the guidance o f he.r?— . F a c u lty Com m ittee, and a pp ro ved by a l l its members, has been presented to and accepted by the C o u n c il on G raduate S tudy and Research in p a r t ia l f u l f i l l ­ ment o f the requirements f o r the degree o f

..M&s.t'

£ ...Sci. s n c e P h a r m a c y

D ate.A u& usL,...1.95Q

Faculty Committee

Chdtrmati

J

TABLE OF CONTENTS PAGE INTRODUCTION ...................................

1

HISTORY ........................................

9

I r o n .........................................

9

Folic a c i d ...................................

11

Vitamin B - 1 2 .................................

16

Growth factor of vitamin B-12 (APF factor)

26

.

P R O C E D U R E .....................................

29

Vitamin B - 1 2 .................................

31

..........................

32

Folic acid and vitamin B - 1 2 ...............

33

Vitamin B-12 plus i r o n ......................

3^

Folic acid plus i r o n ........................

36

R E S U L T S .......................................

38

D I S C U S S I O N .....................................

11A

S U M M A R Y .......................................

121

BIBLIOGRAPHY ...................................

127

Folic acid

LIST OP TABLES TABLE I.

PAGE Blood Counts and Hemoglobin Levels of Chicks Treated by Parenteral Admin­ istration .................................

II.

Age of Chicks in Anemia and Hate of Recovery (I-B)

III.

(I-D)

(II-3)

(III-B)

.......................

53

(III-D)

.......................

56

(IV-B)

.........................

59

(IV-D)

.........................

62

Age of Chicks in Anemia and Rate of Recovery

XI.

50

Age of Chicks in Anemia and Rate of Recovery

X.

. . . . .

Age of Chicks in Anemia and Rate of Recovery

IX.

...............

Age of Chicks in Anemia and Rate of Recovery

VIII.

47

Age of Chicks in Anemia and Rate of Recovery

VII.

.........................

Age of Chicks in Anemia and Rate of Recovery (II-D)

VI.

44

Age of Chicks in Anemia and Rate of Recovery

V.

41

Age of Chicks in Anemia and Rate of Recovery

IV.

39

(V-B)

65

Age of Chicks in Anemia and Rate of Recovery

(V-D)

68

iv

TABLE XII.

PAGE Blood Counts and Hemoglobin Levels of Chicks Treated by Oral Administration . .

XIII.

Age of Chicks in Anemia and Rate of Recovery (I - A ) ........................

XIV.

........................

........................

........................

92

........................

95

........................

98

Age of Chicks in Anemia and Rate of Recovery (V-A)

XXII.

89

Age of Chicks in Anemia and Rate of Recovery (IV-C)

XXI.

........................

Age of Chicks in Anemia and Rate of Recovery (IV-A)

XX.

86

Age of Chicks in Anemia and Rate of Recovery (III-C)

XIX.

83

Age of Chicks in Anemia and Rate of Recovery (III-A)

XVIII.

80

Age of Chicks in Anemia and Rate of Recovery (II-C)

XVII.

77

Age of Chicks in Anemia and Rate of Recovery (II-A)

XVI.

.

Age of Chicks in Anemia and Rate of Recovery (I-C) ...........................

XV.

75

..........................

101

Age of Chicks in Anemia and Rate of Recovery (V-C)

..........................

104

PAGE

TABLE XXIII.

Weight Data on Chicks Which Received Supplements by Parenteral Administra­ tion as Compared to Untreated Chicks . .

XXIV.

111

Weight Data on Chicks Which Received Supplements by Oral Administration as Compared to Untreated Chicks .

. . .

112

LIST OF GRAPHS PAGE Rate of Red Blood Cell Development in Group I B .............................. Rate of Hemoglobin

42

Development in Group I B . .

43

Rate of Red Blood Cell Development in Group I D .............................. Rate of Hemoglobin

45

Development in Group I D . .

46

Rate of Red Blood Cell Development In Group II B

...........................

Rate of Hemoglobin

48

Development in Group II B

.

49

Rate of Red Blood Cell Development in Group II D

...........................

Rate of Hemoglobin

51

Development in Group II D

52

Rate of Red Blood Cell Development In Group III B ........................... Rate of Hemoglobin

54

Development in Group III B

.

55

Rate of Red Blood Cell Development in Group III D ........................... Rate of Hemoglobin

57

Development in Group III D

.

58

Rate of Red Blood Cell Development in Group IV B

............................

Rate of Hemoglobin

60

Development in Group IV B

.

61

Rate of Red Blood Cell Development in Group IV D

...................................

63

v ii

GRAPH

PAGE

Rate of Hemoglobin Development in Group IV D

64

Rate of Red Blood Cell Development in Group V B .............................. Rate of Hemoglobin

Development in Group V

66 B

. .

67

Rate of Red Blood Cell Development in

69

Group V D .............................. Rate of Hemoglobin

Development in Group V

D

. .

70

Rates of Red Blood Cell Development in Groups I B, I IB, IIIB, IV B and V B .................

71

Rates of Hemoglobin Development in Groups I B, II B, III B, IV B and V B ....................

72

Rates of Red Blood Cell Development in Groups I D, II D, III D, IV D and V D

.............

73

Rates of Hemoglobin Development in Groups I D, II D, III D, IV D and V D ............ Rate of Red Blood Cell Development

74

in

78

Group I A .............................. Rate of Hemoglobin

Development in Group I

A

. .

79

Rate of Red Blood Cell Development in Group I C .............................. Rate of Hemoglobin

Development in

Group I

81 C

. .

82

Rate of Red Blood Cell Development in Group II A

............................

Rate of Hemoglobin

Development in Group II A

84

85

v iii

PAGE

GRAPH Rate of Red Blood Cell Development in Group II C

...................................

Rate of Hemoglobin Development in Group II C

87

88

Rate of Red Blood Cell Development in Group III A ................................... Rate of Hemoglobin Development in Group III A

90 91

Rate of Red Blood Cell Development in Group III C ................................... Rate of Hemoglobin Development in Group III C .

93

94

Rate of Red Blood Cell Development in Group IV A

...................................

96

Rate of Hemoglobin Development in Group IV A Rate of Red Blood Cell Development in Group IV C

...................................

Rate of Hemoglobin Development in Group IV C

99

100

Rate of Red Blood Cell Development In Group V A ........... .......................... Rate of Hemoglobin Development in Group V A . .

102 103

Rate of Red Blood Cell Development in .....................................

105

Rate of Hemoglobin Development in Group V C . .

106

Group V C

Rates of Red Blood Cell Development In Groups I A , II A, III A, IV A and V A

.............

107

ix GRAPH

PAGE

Rates of Hemoglobin Development in Groups I A, II A, III A and IV A and V A ...............

.

108

Rates of Red Blood Cell Development in Groups I C, II C, III C, IV C, and V

C

..............

109

Rates of Hemoglobin Development in Groups I C, II C, III C, IV C, and V C ......................

110

ACKNOWLEDGEMENT The author wishes to thank Dr. Morris M. Wolfred for his advice and criticism on this research undertaking.

Thanks are also due to

Mr. A. G. Hogan, Dr. J. A. Brockmann, Jr., Dr. E. L. H. Stokstad, Dr. J. R. Couch, and Dr. B. C. Johnson for the manuscripts they supplied pertaining to the subject.

INTRODUCTION Vitamins are naturally occurring, essential, organic constituents of the diet,* in minute amounts, they aid in maintaining the normal activities of the tissues of the body.

Most vitamins enter into enzyme

systems in order to effect their physiological functions. On the basis of nutritional requirements, distribution, and relationship to metabolic rate, vitamins fall into two distinct groups.

One group is composed of the B

vitamins which are associated with the fundamental met­ abolic functions of living cells.

A survey of nutri­

tional requirements, blood levels, tissue levels, and metabolic rates shows that these vitamins are associ­ ated in increased amounts with smaller species of an­ imals (1, 2, 3 , 4).

The other group of vitamins does

not show such relationship and appears to have only highly specialized functions for certain tissues and cell groups.

The vitamins involved in this paper are

both members of the B vitamin group: Vitamin B-12.

Folic Acid and

These have been used in the treatment

of pernicious anemia, tropical sprue and nutritional macrocytic anemia in humans

(5* 6).

Anemia is a condition in which the number of red cells or the amount of hemoglobin in a red cell is

2 reduced below normal. blood,

Anemias are due to:

(1) loss of

(2) destruction of blood cells, or (3 ) defective

formation of the blood constituents. includes acute and chronic hemorrhage.

The first group Destruction of

red cells is brought about by hemolytic poisons which may be of bacterial origin, or the result of jaundice, or the absorption of industrial poisons.

Anemias of

the third group include nutritional anemias, pernicious anemia, and aplastic anemias.

In man, an iron-deficient

diet may give rise to hypochromic anemia, in which the hemoglobin content of the blood is reduced to a greater extent than the number of red cells. be reduced in size.

The cells may also

Pernicious anemia is due to an in­

ability to form red cells.

There is a decrease in the

number of red blood cells and as a. consequence, a reduc­ tion in the percentage of hemoglobin. Normal gastric juice contains a factor, termed the "intrinsic factor," which reacts with the "extrinsic factor" found in foods to produce the antianemia or hemo­ poietic principle (7)*

This may be tied with the fact

that another symptom of pernicious anemia is a lack of both hydrochloric acid and pepsin in the gastric mucosa. The intrinsic factor is thermolabile and is thought to be an enzyme.

The extrinsic factor is found in various

foods, especially beef muscle, beef heart, rice polishings

3 and wheat germ.

Muelengracht (7) pictures the normal

process as follows:

The extrinsic factor is present in

the food; the intrinsic factor is secreted by the pyloric portion of the gastric mucosa and to a lesser extent by the duodenal mucosa. An interaction of the two factors occurs in the small intestine, resulting in the formation of an antianemic principle.

The antianemic principle is

absorbed from the small intestine and is carried by the blood to the liver and other organs where it is stored. The function of the antianemic principle is to stimulate the production of red blood cells.

In order

to accomplish this purpose, there has been established a need for required amino acids, iron, copper, and cer­ tain vitamins. Iron is tied up with hemoglobin formation. Hemoglobin is a conjugated protein with a prosthetic group, heme, united with the protein, globin,The pig­ mentary property and respiratory functions are associa­ ted with heme, the iron-containing pigment.

Iron is a

constituent of many tissues besides blood and is es­ sential for catalysts other than hemoglobin, such as ■cytochromes, peroxidases and catalases. C * j t o

F e * *

-/ C ^ i o c k r a T i r v z

Examples: ty tS 'fe ^

4 C y i o c h 't 'O 7rv£ 0

C o io c k r o

t~&

>

-h Cy'fcQ C-k/^O >n& A F t.

C Fo.*4'^ + C w tG c k n o ^ ^ A FeJ* _J /

Cy F o cA rotnG A F ^ O i

eliminated in the feces.

Defective absorption may re­

sult from gastro-intestinal disturbances such as achlorhydria or diarrhea, leading to anemias which readily yield to large doses of iron.

It is therefore necessary

to have an ample supply of available iron whenever new hemoglobin must be produced.

The recommended daily

allowance of iron ranges from 6 mg ms for children underone year of age to 15 mgms for youths of both sexes. In administering iron therapeutically,

the inorganic

salt is probably as useful as the organic; although ferrous iron is preferable,

ferric is usually reduced

to ferrous and is absorbed as such.

It has been claimed

that iron in the heme combination is not as assimilable

5 as salts of the metal (7)* The presence of a series of new vitamins related to folic acid has been noted in enzymatic digests of hog liver.

This has been designated as the Folinic Acid.

Group (8).

Under tested conditions used, a half maximal

response of Lactobacillus easel is obtained in the pres­ ence of 0.002 gamma per cc. of this concentrate, which is somewhat more active than folic acid.

Depending upon

the time of incubation and response at which the compar­ ison is made, the concentrate is from ten to one hundred times as active as folic acid in preventing the toxicity of methyl folic acid (one gamma per cc.) for Strepto­ coccus faecalis R.

On the basis of estimated purity of

the concentrate, it is not apparent that folinic acid is less active than folic acid in promoting the growth of either organism in the absence of an inhibitor.

Since

the folinic acid group is utilized more effectively than folic acid for such organisms, it may be more active than folic acid in the treatment of sprue, nutritional and pernicious anemia and other nutritional deficiencies related to folic acid and vitamin B-12 groups. Another antianemic substance, 5”m ^thyl uracil (thymine), has proven effective when given in large doses in the treatment of nutritional macrocytic anemia, tropical sprue and pernicious anemia (9)*

Thymine is

6 a constituent of nucleic acid, and the nucleic acids have long been known to play an important role in cellu­ lar metabolism.

There is evidence to indicate that the

antianemic factors are linked to nucleic acid synthesis. Microbiological evidence reported by Wright et al (10), indicates that the primary biochemical defect in perni­ cious anemia may be the inability to synthesize certain nucleosides, particularly thymidine, from parent purines or pyrimidines.

Thus it would appear that the curative

effects observed in this disease with folic acid arise from increased thymine synthesis, which by mass action effects yields more thymidine through reduction.

In a

similar manner, the effectiveness of large amounts of thymine in pernicious anemia may be explained.

H - N - f = 0

n

0 ~C C-CH3 i

n

3

t t on. *

~

c h

H-C C -CH5 i, m 3

n

H -M-c-h

N - C H

T k u

Tk y Tn./gL/fxa.

Elvehjem and Hart (11), in 1929 reported that dayold chicks placed on a diet of cow's milk, together with

polished rice, calcium carbonate, and sodium chloride, became anemic.

The addition of ferrous sulfate to the

ration immediately stimulated hemoglobin synthesis. Hogan and his co-workers (12), in 1940 observed that under certain dietary conditions, day-old chicks developed anemia and failed to grow.

This was attributed

to an unidentified member of the B complex.

They iso­

lated the factor later; it is now known as folic acid. Nichol et al (13 )> 1949* observed that a folic acid deficiency in chicks fed a purified ration was ac­ centuated by supplying Vitamin B-12 as the crystalline material in the form of an injection or as a concentrate added to the ration. Nichol, Harper and Elvehjem (14), 1949* induced severe anemia in chicks by an intramuscular injection of phenylhydrazine hydrochloride after a depletion per­ iod on a folic acid deficient purified ration.

Liver

extract alone did not influence the rate of hemoglobin formation.

When the depletion period was twenty-one

days or longer, the combination of liver extract and folic acid caused a more rapid regeneration of hemo­ globin.

Pure Vitamin B-12 completely replaced liver

extract in stimulating the formation of hemoglobin in the presence of folic acid.

8 Schafer et al (15)* 1950^ reported that Vitamin B-12 could replace Folacin (trade name for folic acid) under their experimental conditions; however, slightly higher hemoglobin levels were consistently observed when both nutrients were present in the diet. Vitamin B-12 has been shown to be a growth factor for rats (16 , 17 )* pigs

(18), and chicks (19 * 20).

The evidence presented above indicated a further need for experimentation on the effectiveness of folic acid and Vitamin B-12 on anemic chicks, and for addi­ tional information concerning the hemopoietic value of combinations of folic acid and Vitamin B-12 with iron; no previous work has been done on such combinations. There was a definite need for exploration of varying dosages and modes of administration in an attempt to discover the degree of effectiveness of such prepara­ tions.

In addition, attempts were made to correlate

the growth factor of folic acid and Vitamin B-12 with iron.

9 HISTORY IRON The empirical use of iron in the treatment of anemias dates back to ancient man.

The therapeutics of

this element is found in the writings of Hindu medicine: the Greeks employed it for the cure of weakness, a prom­ inent symptom of anemia, in an attempt to impart to the patient the strength of iron.

The alchemists designated

iron as the symbol of Mars, the god of war, who repre­ sented strength.

Drinking water in which old swords

had rusted was given to patients with pallor (21). Sydenham (22) in l68l employed iron filings in the treatment,of chlorosis; however, it was not until 1713 that Lemery and Goeffy (23 ) showed that iron was present in the blood. In 1746 Menghini

(23) demonstrated that the con­

sumption of foods rich in iron could elevate the amount of iron in the blood of animals;

this discovery laid a

scientific foundation for iron therapeutics. Pierre Blaud (24) in 1831 recognized that the number of failures in the treatment of chlorosis was due to the use of insufficient doses of iron.

He re­

ported rapid cures with the use of large doses of the metal.

The dose of 0.4-0.6 g m s . of ferrous sulfate,

10 used by Blaud is still considered adequate therapy today. In 1832, E^disch reported a deficiency of iron in the blood of chlorotics; in 1842, Andral et al reported that iron therapy caused an increase in the number of red blood cells in anemia (21). The principles of Sydenham and Blaud were employed in the iron therapy of anemia until the nineteenth cen­ tury, when the teachings of Bunge (25), Quincke (26 ), Von Noorden (27) and others caused a breach in these principles.

At this time it was accepted that the metal

was not absorbed in inorganic form and that small doses were necessary.

This in turn decreased the value of

iron therapy and retarded its progress for a quarter of a century. Elvehjem and Hart (11) in 1929 reported that day-old chicks placed on a diet of c o w 1s milk mixed with polished rice, calcium carbonate and sodium chlor­ ide became anemic.

The incorporation of ferrous sulfate

to the ration caused a spontaneous stimulation of hemo­ globin . Gibson and Howard

(27) demonstrated that a diet

with a high iron content in pernicious anemia produced a favorable influence on iron metabolism.

They estab­

lished high iron balances in anemic patients by means of a high iron diet which contained comparatively low

11 caloric and protein values.

They recommended the use

of iron-rich and vitamin-adequate diets in anemia. Today, the important use of iron is in the treat­ ment of iron-deficient- anemias.

When administered to

patients with normal blood values, it serves only to increase the reserves of the body; however, it does not increase the number of red ceils or the amount of hemo­ globin above normal. FOLIC ACID The first use of liver in the treatment of anemias was demonstrated by the experiment of Whipple and his associates

(28 ), whereby a severe anemia in dogs was

produced by repeated bleedings and the influence of in­ gested food was studied upon blood regeneration.

It

was discovered that beef liver was the most effective food for correcting this condition of anemia.

This led

Minot and Murphy (29) in 1926 to administer large amounts of liver and muscle meat to patients with pernicious anemia.

Following the liver and muscle meat diet, all

patients showed a prompt, rapid and distinct remission of their anemia, coincident with marked symptomatic im­ provement, except for pronounced disorders due to spinal cord degeneration.

12 West (30) In 1927 found that the alcohol ether soluble fractions of fresh beef liver were effective in the treatment of five consecutive cases of pernicious anemia.

Ten to fifteen grains of the alcoholic ether

soluble fraction were emulsified with grape juice and fed twice daily. In 1936, Dakin, Ungley and West (31) reported that the hematopoietic substance in liver is a peptide or an associated peptide, possessing many but not all the properties of an albumose.

It was found that ami-

nohexose was not an essential component for hemopoietic activity.

On hydrolysis, the peptide yielded arginine,

leucine, glycine, proline, hydroxy-proline, aspartic acid, and an acid resembling hydrozyglutamic acid. Ultrafiltration experiments on .the peptide with graded membranes indicated a molecular weight greater than 2000, but less than 5000.

The purified products were active

therapeutically in twenty to twenty-five milligram doses. Kidney, brain and salivary gland tissue did not yield a hematopoietic substance. Hogan and his co-workers

(12), 19^0, observed

that under certain dietary conditions day-old chicks developed anemia and failed to grow.

They recognized

the factor as an unidentified member of the B complex

13 and designated it as Vitamin B c .

They succeeded in

isolating and concentrating this factor from crude liver extract.

In the Fall of 19^0, Pfiffner et al (32) iso­

lated this vitamin as the Vitamin B yeast.

conjugate from

At the same time as the work on the antianemia

factor was being undertaken, Mitchell, Snell, and Williams

(33) reported the preparation of a concentrate

from spinach which was very active in stimulating the growth of Streptococcus lactis R. and Lactobacillus easel in comparable dosage.

They termed this micro­

biological growth factor folic acid. It was not until 19*4-1 that the probability of the chick vitamin and the bacterial growth factor being a single substance was postulated by Hutchings and his associates(3*0 * O ’Dell and Hogan { 3 5 ) > 19*43 j found that antipernicious anemia (APA) liver extracts were ineffective for the cure of anemia Induced by a folic acid defi­ ciency in the chick. Angler and his co-workers

(36), 19*45> developed

the chemical structure and synthesis of the Lactobacillus easel factor, folic acid.

It has been extracted from

liver, kidney, bone marrow, yeast, soybeans and green leaves, such as spinach (7)*

Ill-

Folic acid was found to be a water-soluble B com­ plex vitamin.

It is present in minute amounts in a nor­

mally in the intestine by bacterial action.

The liver

Lactobacillus casei factor, fermentation Lactobacillus easel factor, Folic Acid, Vitamin B c , Vitamin M, Factors R, S and U, and Yeast norite eluate factor are all mem­ bers of the same group.

All are chemically related to

the compound now known as Pteroylglutamic Acid (PGA)

(36 ).

Pteroylglutamic acid is composed of three main parts:

(1) a two-ringed nitrogenous compound called a

"pteridine," a yellow pigment first isolated from butter­ flies 1 wings, (2) p-aminobenzoic acid and (3) Glutamic acid. Structure:

H o o c -c -tt-\c ~ £ Z £ ~ h -^ c

J ^ G lL c lc i Y n ic

1^0 % A y * *

* d T H o - r i c i'm e .

15 The related vitamins differ in the number of glutamic acid groups present, the additional glutamic acid mole­ cules being conjugated in peptide linkages. In chicks, a lack of this factor causes anemia as well as decreased resistance to malarial infection and impairment of the response to estrogens.

Curative

effects of pteroylglutamic acid have been shown in anemia, leucopenia and granulocytopenia in humans (7)*

It has

not been found effective for the treatment of pernicious anemia unless administered with liver extracts

(5 )•

Results of the clinical use of synthetic folic acid in humans were first reported by Spies and his associates

(5 ) in 19^5*

They showed that folic acid,

given either in doses of 50 mgrn. twice a day by mouth, or 20 mgrn. daily parenterally, induced a striking hemo­ poietic response similar to that which follows admin­ istration of potent liver extracts. There are strong indications that folic acid provides the complete answer to tropical nutritional macrocytic anemia.

Instances have been reported in

which there was no response (5)*

However, anemias of

this class have several determining causes, all no doubt linked, together.

There are probably some anemias of

different etiology which medical science has been unable to separate from the rest.

16 VITAMIN E-12

It was first noted by Shorb (37) in 1947 that a factor essential for the growth of Lactobacillus lactls Dorner was present in refined liver extract and that the efficiency of the extract varied in direct proportion to the content of this vitamin. A crystalline compound was isolated from liver through purification of various clinically-active liver fractions by Rickes and co-workers

(38) early in 1948.

Its potency was found to be about 11,000,000 LLD units/mg and was identified by name as the LLD factor.

This has

been found to be more effective in micrograrn quantities than folic acid in the treatment of pernicious anemia. West (39)j 1948, giving single injections of 3* 6 and 15 micrograms of this substance which was later called Vitamin B-12, obtained a hematological response in three cases of pernicious anemia. Smith (40), 1948, isolated two highly active red pigments from ox liver which apparently were identical with Vitamin B-12, as evidenced by clinical activity and chromatography. The synonyms for Vitamin B-12 are:

Cow Manure

Factor, Antipernicious anemia (APA) factor, Animal Protein Factor

(APF), Factor X and Zoopherin.

Unlike

17 folic acid it does not occur in green plants, as proven by the post-weaning growth inhibition of the offspring of rats on a well-fortified all-plant ration.

This

condition could be corrected by a liver factor but not by feeding alfalfa, dried grass or young fresh grass

(4l).

Vitamin B-12 is widely distributed in lower animal forms. Ostrea, Nereis, Lumbricus, Rusycon, Drosophila and eggs of Artemla show values comparable with such mammalian tissue as liver.

Its distribution indicates little if

any role in plants but suggests widespread functions in animal life (42). By rat assay methods for Vitamin B-12, fish sol­ ubles , streptomycin "slops,” sheep rumen contents, and glandular meats were found to be excellent sources. Muscle tissue, milk products, and eggs were found to have lesser amounts

(43)•

The definite chemical structure of Vitamin B-12 is not yet known, but preliminary analyses have been made.

The crystalline compound when heated to 300°C

blackens upon melting.

The presence of 4.0 percent

cobalt was found by Smith (43) who noted that, if each molecule of the compound contained one atom of cobalt, the molecular weight would be 1600; this was in good agreement with the value obtained by X-ray crystallographic procedures.

Rickes and co-workers (44) also

noted the presence of cobalt, phosphorus, and nitrogen and the absence of sulfur in B-12. "Half-maximal growth" of L. lactis was supported by 0.013 millimicrogram per milliliter of culture med­ ium.

The microbiological activity of the factor was

o not changed by autoclaving for fifteen minutes at 121 C ., but was slowly inactivated by standing in dilute NaOH or HC1 at room temperatures

(43)•

It has been reported in England that the infra red spectrum of the crystalline APA factor was measured and that the presence of phosphorus to oxygen, nitrogen to hydrogen, and oxygen to hydrogen linkages was indi­ cated together with the absence of an aliphatic carbon to hydrogen linkage and the probable presence of aromatic carbon to hydrogen linkages

(43) •

The existence of more than one molecular species of Vitamin B-12 with similar biological effects is in­ dicated by the chromatographic separation of two pink bands with biological activity and by the non-dialyzability of the factor in cow manure as contrasted with the dialyzability of the similarly-assayed factor pre­ pared from liver (45) •

Vitamin B-12, per unit of weight,

is the most effective antianemic substance known (46). It is the only known pure chemical substance effective

in relieving subacute combined degeneration of the spinal cord in persons with pernicious anemia.

When administ­

ered to suitable patients, under controlled conditions, reticulocytosis occurs and is followed by an increase in red blood cells, platelets and hemoglobin.

The severe

glossitis present in some cases of macrocytic anemia heals spectacularly.

In patients with sprue and nutri­

tional macrocytic anemia, there is a decrease in the number of stools and a tendency for the stools to return to normal. The dosage of Vitamin 3-12 varies from patient to patient.

The average patient will respond maximally

to 100 micrograms or less (4'7) •

Vitamin B-12 has no

effect on the leukopenia of infections or the idiosyn­ crasies due to drugs, idiopathic purpura,

secondary

anemias or leukemia. Spies and his associates

(9)* 19^8, made a com­

parative study of thymine, folic acid and Vitamin B-12 under controlled conditions on selected patients with macrocytic anemia in relapse.

One patient with perni­

cious anemia, one with nutritional macrocytic anemia and one with tropical sprue were each admitted three times to a hospital and given a diet free from meat and meat products.

The three distinct chemical compounds,

20 thymine, folic acid, and B-12, proved effective in each of the three distinct syndromes.

It appeared that sev­

eral thousand times the weight of thymine is required to produce a response similar to that produced by folic acid, and that several thousand times the weight of folic acid is required to produce a response similar to that produced by Vitamin B-12. Smith (40), 1948, reported that incompletely purified material from liver was effective against the spinal cord symptoms of pernicious anemia.

In studies

with impure preparations standardized with L. Lactis Dorner, it was found that single doses corresponding to approximately two to four micrograms gave strong hematological responses Stone and Spies

(48). (49) reported that the mucous

membrane lesions in two patients with pernicious anemia were promptly relieved by either liver extract or Vit­ amin B-12.

The lesions did not respond to thymidine

or folic acid and were thought to be associated with the neurological changes in a combined system disease. Berk and his co-workers

(50), 1948, described

a patient with a combined system disease who showed a hematological response to the daily administration of five micrograms of Vitamin B-12 for eight days.

There

21 was marked improvement during this period.

However,

when the injections were discontinued, the patient showed neurological regression which started, within the remarkably short period of seven days.

Further

treatment led to the resumption of the improvement. The patient was found sensitive to liver but showed no adverse reaction to Vitamin 3-12. Hall and Campbell and Bethell and co-workers

(51 * 52 ), 19^ 8 , presented evidence that patients with a combined system disease showed an over-all response to Vitamin B-12, although one exception was noted. A case of puerperal macrocytic anemia grew worse during treatment with Vitamin B-12, but responded promptly to the administration of PGA.

The response of a pernicious

anemia patient to Vitamin B-12 was apparently blocked by administering 4-amino-PGA which is an antagonist for PGA.

This interesting observation may suggest that

Vitamin B-12 is hemopoietically ineffective in the ab­ sence of PGA. Berk and his co-workers

(53)j 19^8? found that

the hemopoietic activity of orally administered Vitamin B-12, five micrograms daily, was increased by simultan­ eously giving 125-150 c c . of normal human gastric juice. This suggested that the extrinsic factor might be

22 identical with or closely related to 3-12 and relegated the function of the intrinsic factor to facilitating the uptake of B-12 or related factors from the g u t . In addition to L. lactis Dorner, other micro­ organisms have been used for the assay of the APA factor: L. leichmannii 313, ATCC 7630 (5*0 > and Euglena gracilis (55).

Another strain of L. leichmannii, ATCC 4797 (56),

was observed to respond to concentrated preparations of the animal protein factor. Daniel and his co-workers

(57)* 19^8, found that

liver extract was active for L. casei, but stated that it would seem impossible that this animal protein factor activity was identical with the factor required in the treatment of pernicious anemia. A relationship between thymidine and the antipernicious anemia factor was indicated by Shive and his co-workers (58), 1948, and Wright et al (10), 1948, who found that thymidine would replace liver extract in promoting the growth of L. lactic Dorner.

For "half

maximum growth," about 0.5 microgram of thymidine/cc. of medium was required by three lactic acid organisms. These data were interpreted as indicating that Vitamin 3-12 functions as a coenzyme in carrying out reactions concerned with the conversion of thymine to thymidine.

OQ Kaczka et al (5SL 19^9* observed that upon cat­ alytic reduction of Vitamin B-12 with hydrogen, a cryst­ alline product was formed.

They named if Vitamin 3-12^.

This product showed an activity of about 5*2 x 1.0° units/mgm for the growth of L. lactis and 1-3 x 10 units/mgm.

for L. leichmannii and. about one-half the

APP activity of B-12 in rats and 30 per cent ± 15 per cent of B-12 activity in chicks.

West (60) tested 25

micrograms of Vitamin B-12a parenterally in a single pernicious anemia patient and observed 30 per cent of a maximal hematological response. In England, Ellis, Petrow and Snook (6l), 19^9* reported that upon spectroscopic examination, Vitamin B-12 yielded 1:5:6 trisubstituted benziminazole as the gamma component.

Spectroscopic and chemical evidence

leads to the conclusion that only one 5*6 dimethylbenziminazole exists preformed in the nucleus. Brink and Folkers

(62), 19^9* assigned the follow'

ine empirical structure to Vitamin B-12:

CH 3

24 Pierce and his associates

(63)* 1949* found that

a chromatograph of liver extract yielded two pink hands. Fractional precipitation of the first band with acetone gave rod-like red crystals, which they named Vitamin B-12fc).

The crystals contained cobalt and phosphorus.

They were biologically active in the chick assay and in the assay of L. leichmannii 313*

According to Lichtman

et al (64), 1949* Vitamin 3-12^ was effective in treat­ ment of patients with Addisonian pernicious anemia in daily parenteral doses of one to two micrograms. Various investigations in poultry nutrition pro­ vided evidence during 1948, which confirmed and extended previous findings relative to the presence of APF in fish meal

(65 , 66), fish solubles (67 , 68, 69 , 70 ), and

cow manure (55, 56).

The presence of this factor was

reflected by such criteria as improved growth and higher survival rate of chicks and increased hatchability of h e n ’s eggs (72 , 73)* Nichol et al (13 ), 1949, observed that a folic acid deficiency in chicks fed a purified ration was ac­ centuated by supplying Vitamin B-12 as the crystalline material in doses of 0.1 microgram daily by injection, or as a concentrate added to the ration in the proportion of 3 micrograms per hundred grams.

Extruded, quivering

wings, body tremors and paralysis developed in more than fifty per cent of the anemic chicks which received B-12. Treatment with twenty micrograms of folic acid parenterally caused complete disappearance of these symptoms within twenty-four hours.

Injection of twenty micro­

grams of folic acid was more effective than fifty m i ­ crograms administered orally.

None of the above symp­

toms developed in control groups. Nichol, Harper and Elvehjem (14), 19^9* induced severe anemia in chicks by intramuscular injection of phenylhydrazine hydrochloride, following a depletion period on a folic acid deficient purified ration.

Liver

extract alone did not influence the rate of hemoglobin formation.

When the depletion period was twenty-one

days or longer, the combination of liver extract and folic acid caused a more rapid regeneration of hemo­ globin.

Pure Vitamin B-12 completely replaced liver

extract in stimulating the formation of hemoglobin in the presence of folic acid. Schafer et al (15) > 1950, reported that Vitamin B-12 could replace Folacin (trade name for folic acid) under their experimental conditions, but slightly higher hemoglobin levels were consistently observed when both nutrients were present in the diet.

26 Brockmann, Jr., and His co-workers (7^)* 1950* stated that mild acid hydrolysis of Vitamin B-12 gave rise to a substance or substances which possessed chromophoric groups and acidic properties similar to those of B-12^. GROWTH FACTOR OF VITAMIN B-12 (APF FACTOR) Rats on a diet containing yeast and wheat germ as Vitamin B sources were found by Mapson (75) to develop a deficiency which responded to liver.

It was noted by

Cary, Hartman and their co-workers (76) that very small doses of the antipernicious anemia liver extract were effective in promoting the growth of rats on a purified diet containing an alcohol-extracted casein and yeast. The deficiency signs were accentuated by adding soybean meal or more casein to the diet and were attributed to the lack of an unidentified Factor X. Zucker and Zucker (16), 19^8* used various high protein diets for rats and noted that the young born of females on such diets showed a high incidence of mortal­ ity soon after weaning.

The syndrome was marked by

slow growth, high incidence of mortality, high blood urea, low white cell count, kidney hypertrophy and other visceral changes; this syndrome was prevented or cured by a liver fraction,

"fish solubles,” crude casein, or

27 a co nc e n tr at e of the

covj

ma n u r e

factor.

Emerson (17)* 194-9* reported that Vitamin B-12 counteracted the growth retarding effect of thyroid powder when fed in conjunction with a diet devoid of protein.

It is also a growth factor for pigs(l 8 ).

Ott, Rickes and Wood

(20), 1949* reported that

crystalline Vitamin B-12, when fed in conjunction with purified basal diets containing 40-70 per cent soybean meal as the sole source of protein, exhibited APF act­ ivity in day-old chicks.

The amount required for op­

timal growth appeared to be more than six, but less than thirty micrograms/Kgm. of ration.

It was concluded

that Vitamin B-12 is identical with or closely related to the APP because it elicited growth responses com­ parable to those obtained with crude sources of APF as supplement. Nichol et al (13)* 194-95 reported that Vitamin B-12 increased the rate of growth of folic acid defi­ cient chicks but did not produce a great deal of im­ provement in anemia and feathering. Nichol and his co-workers

(77* 14), 1947 and

1949* reported that no change in the blood hemoglobin level accompanied the growth response of chicks to the injection of APA liver extracts or Vitamin B-12 when

2b fed a. corn-soybean meal basal ration containing ade­ quate folic acid. Stokstad and Jukes

193b„ observed that

fermentation products of Stre ptomyces aureofaciens were found to promote growth in depleted chicks on various diets which were adequately supplied with Vit­ amin B-12.

Growth responses in chicks on a corn-soybean

diet were also produced by crystalline aureomycin hydro­ chloride.

Aureornyein hydrochloride produced a growth

response on diets deficient in Vitamin B-12. The evidence to date 3 therefore, indicates that Vitamin B-12 is the catalyst which allows the body to use all the substances that are necessary to counteract the multiple deficiencies in macrocytic anemias.

That

this catalyst is manufactured by the stomach has still to be proven; however,, its absence only in cases of histamine-fast achlorhydria suggests this.

PROCEDURE Day-old white Leghorn chicks with numbered rings on their legs for identification were placed in electri­ cally heated cages.

The chicks were given a special

prepared diet deficient in folic acid and were allowed to indulge in the special feed and water ad libitum until they became anemic.

Blood samples were taken

periodically by wing vein punctures, and red blood cell counts and hemoglobin levels were recorded. The folic acid deficient basal ration consisted of the following formula: Casein

2 p •0 gms

Cerelose

5O .7

Gelatin

1 0 . 0 gms

Soybean oil, q.s. Salts'*" Methionine Cod liver oil

gms

6 . 0 gms 5 . 0 gms 0 . 3 gms 2 0 0 0 . 0 mgins

Thiamine

0 . 4 rrigms

Riboflavin

0 . 8 mgms

Nicotinic acid

5 . 0 mgms

Pyridoxine hydrochloride

0 . 6 mgms

Salts used according to nutritional re­ quirements Journal of Nutrition, 32, 460 (lybo).

30 Calcium Pantothenate

2.0 mgms

Biotin

0.02mgms

Choline chloride

200.0 mgms

Inositol

100.0 mgms

Alpha tocopherol

0.3 mgms

Vitamin

203

mgrn

To make 100 gms ilood counts and hemoglobin levels were taken at weekly intervals until the red blood cell count reached below two million per cubic millimeter of blood and the hemoglobin level was 5 grams per 100 cc. of blood.

This

has been previously determined as the anemic level in chicks

(12).

It usually required twenty-four to thirty

days for the chicks to reach the anemic level. Of the 180 chicks given a folic acid deficient diet, only 132 survived long enough to reach the anemic level; therefore, the mortality was 26.6 per cent.

The

chicks which were affected by the deficiency diet showed body tremors, extended wings and inability to maintain balance, and also signs of paralysis.

The chicks re­

mained in this condition two to three days before dying. The folic acid deficient chicks all showed poor feather­ ing, poor growth, enlarged crops, soft stools and anorexia.

31 Once they became anemic, the chicks were divided into five main groups, according to their mode and manner of treatment.

The chicks were weighed at the peak of

their anemia and their weights recorded weekly during their process of treatment. For every sub-group, three chicks were set as controls. On the basis of dosage and manner of administra­ tion, each main group was divided into four sub-groups as follows: *

Vitamin B ^ *

Group I consisted of treating

a group of twelve anemic chicks by dividing them into groups of three. To Group A were administered three micrograms of Vitamin

Per 100 grams of the basic

folic acid deficient ration. to feed ad libitum.

The chicks were allowed

Blood counts and hemoglobin levels

were taken at one to two day intervals and the chicks were weighed weekly. To Group B, Vitamin B ^

was administered in doses

of three micrograms daily by intramuscular injection in­ to the leg and the chicks maintained on a basic folic acid deficient ration ad libitum.

Blood counts and hem­

oglobin levels were taken at one to two day intervals •jf

,

,

Cobione (Merck and Company, I n c .).

32 and the chicks were weighed weekly. To Group C were administered six micrograms of Vitamin B-^ Per ^00 grams of folic acid deficient diet. The chicks were given feed ad libitum.

Blood counts

and hemoglobin levels were taken at from one to two day intervals and the chicks were weighed weekly. To Group D, Vitamin B-^ was administered in doses of six micrograms daily by intramuscular leg injection. The chicks were fed a folic acid deficient ration ad libitum.

Blood counts and hemoglobin levels were taken

at one to two day intervals and the weights of the chicks were recorded weekly. *

Folic Acid.

Group II consisted of a group of

twelve anemic chicks divided into groups of three.

To

Group A were administered three mgms. of folic acid per 100 gms. of the folic acid deficient ration. were allowed to feed ad libitum.

The chicks

Blood counts and hemo­

globin levels were recorded at one to two day intervals and the chicks were weighed weekly. To Group B, folic acid was administered in doses of three mgms. daily by intramuscular injection into the leg and the chicks maintained on a basic folic acid de­ ficient ration ad libitum.

Blood counts and hemoglobin

Folvite (Lederle Laboratories).

levels were taken at one to two day intervals and the chicks were- weighed weekly. To Group C were administered six mgms. Folic Acid per 100 gms. diet. libitum.

The chicks were given the ration ad

Blood counts and hemoglobin levels were taken

from one to two day intervals and the chicks were weighed weekly. To Group D, Folic Acid was administered in doses of six micrograms daily by intramuscular injection into the leg.

The chicks were fed a folic acid deficient

ration ad libitum.

Blood counts and hemoglobin levels

were taken from one to two day intervals and the weights of the chicks were recorded weekly. Folic Acid and Vitamin

Group III consisted

of treating a group of twelve anemic chicks by dividing them into groups of three. three micrograms Vitamin

To Group A were administered ^ plus three m g m s . Folic Acid

per 100 gms. of the basic folic acid deficient ration. The chicks were allowed to feed ad libitum. a pronounced improvement in feathering.

There was

Blood counts

and hemoglobin levels were taken from one to two day intervals and the chicks were weighed weekly. To Group B, three milligrams folic acid plus three micrograms Vitamin

were administered in daily doses

by intramuscular injection into the leg and the chicks maintained on a folic acid deficient ration ad libitum. Blood counts and hemoglobin levels were taken from one to two day intervals and the chicks were weighed weekly. To Group C were administered six milligrams folic acid plus six micrograms Vitamin 13-^2 folic acid deficient diet. libitum.

1°° gms.

The chicks were fed ad

Blood counts and hemoglobin levels were taken

from one to two day intervals and the chicks were weighed weekly. To Group D, six mgms. folic acid were administered by intramuscular injection in one leg daily, and six micrograms Vitamin B-^2 were administered by intramuscular injection into the other leg daily.

The chicks were fed

a folic acid deficient ration ad libitum.

Blood counts

and hemoglobin levels were taken at one to two day inter­ vals and the weights of the chicks were recorded weekly. Vitamin B^2 plus Iron.

Group IV consisted of

treating a group of twelve anemic chicks by dividing them into groups of three.

To Group A were administered

three micrograms Vitamin B-^2 plus three milligrams fer­ rous gluconate per 100 gms. of the basic folic acid de­ ficient ration.

The chicks were allowed to feed ad

35 libitum.

It was noted that the chicks ate more, became

more active and started to grow.

Blood counts and hemo­

globin levels were taken one to two day intervals and the chicks were weighed weekly. To Group Eg three micrograms of Vitamin

plus

three mgms. ferric ammonium citrate were administered by intramuscular injection Into the leg and the chicks maintained on a basic folic acid deficient ration ad libitum.

Blood counts and hemoglobin levels were taken

from one to two day intervals and the chicks were weighed weekly. To Group C were administered six micrograms of Vitamin B-^g plus six mgms.

ferrous gluconate per 100 gms.

of folic acid deficient diet. ration ad libitum.

The chicks were given the

Blood counts and hemoglobin levels

were taken at one to two day intervals and the chicks were weighed weekly. To Group Dj Vitamin 3-, ^ was administered In doses of six micrograms daily by Intramuscular Injection into one leg and six milligrams ferric ammonium citrate were administered In doses of six milligrams daily by intra­ muscular injection into the opposite leg immediately afterward.

The chicks were fed a folic acid deficient

ration ad libitum.

Blood counts and hemoglobin levels

36 were taken at one to two day intervals and the weights of the chicks were recorded weekly. Folic Acid plus Iron.

Group V consisted of

treating a group of twelve anemic chicks by dividing them into groups of three. three mgms.

To Group A were administered

folic acid plus three mgms. ferrous gluconate

per 100 gms. of the basic folic acid deficient diet. chicks were allowed to feed ad libitum.

The

Blood counts

and hemoglobin levels were taken at one to two day inter­ vals and the chicks were weighed weekly. To Group By three mgms.

folic acid plus three

mgms. ferric ammonium citrate were administered in daily doses by intramuscular injection into the leg* and the chicks were maintained on a basic folic acid deficient ration ad libitum.

Blood counts and hemoglobin levels

were taken from one to two day intervals and the chicks were weighed weekly. To Group C were administered six mgms. folic acid, plus six mgms. ferrous gluconate per 100 gms. of the folic acid deficient ration. ad libitum.

The chicks were given feed

Blood counts and hemoglobin levels were

taken at one to two day intervals and the chicks were weighed weekly.

To Group D, six mgms. folic acid were administered daily by intramuscular injection into one leg and six mgms. ferric ammonium citrate were administered daily by intramuscular injection into the other leg immedi­ ately afterward.

Blood counts and hemoglobin levels

were taken at one to two day intervals and the weights of the chicks were recorded weekly.

R E S U L T S

39 TABLE I BLOOD COUNTS AND HEMOGLOBIN LEVELS OP CHICKS TREATED BY PARENTERAL ADMINISTRATION

Normal Blood

Anemic Blood

R.B.C. H.G. R.B.C. H.G. Count level Count Level No. millions in millions in of /cu mm. Gms/ /cu mm. Gms./ Group No. Chicks (Aver.) 100 (Aver.) 100

Supplement to be Administered

IB

3

4.71

12.8

1.51

4.1

3 microgms Bt o I .M. daily 12

ID

3

4.63

12.6

1.83

4.9

6 microgms B-.p I .M. daily

II B

3

4.26

11.6

1.55

4.2

3 milligms folic acid I.M. daily

II D

3

4.39

11.9

1*68

4.6

6 milligms folic acid I.M. daily

III B

4

4.10

11.1

I .39

3*8

3 microgms B 30 and 3 milligms folic acid I.M. daily

III D

4

4.21

11.4

1.50

4.0

6 microgms Bjo and 6 milligms folic acid I.M. daily

IV B

4

4.58

12.5

1*78

4.8

3 microgms B 12 and 3 milligms Ferric Ammonium Citrate I.M. daily

IV D

4

4.49

12.2

1.69

4.6

6 microgms B ^2 and 6 milligms Ferric Ammonium Citrate I.M. daily

R.B.C. = Red Blood Cells. H.G. * Hemoglobin I.M. s Intramuscular injection

40 TABLE I

(continued)

BLOOD COUNTS AND HEMOGLOBIN LEVELS OP CHICKS TREATED BY PARENTERAL ADMINISTRATION

Normal Blood Anemic Blood R.B.C. H.G. R.B.C. H.G. Count level Count Level No. millions millions in in of /cu mm. Gms/ /cu mm. Gms/ Group 100 Chicks (Aver.) 100 (Aver.) No.

Supplement to be Administered

VB

4

4.35

11.8

1.55

4.2

3 milligms folic acid and 3 milligms ferric ammonium citrate I.M. daily

V D

4

4.46

12.1

1.66

4.5

6 milligms folic acid and 6 milligms ferric ammonium citrate I.M. daily

41 TABLE II AGE OP CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number IB

No. of Chicks 3

Controls ^ (Untreated)

Age in days

Blood upon Treatment R.B.C. Count in mi 11ions/cu. mm

H.G. Level in Gms./lOO cc.

30

1.51

4.1

33

1.64

4.4

36

1.78

4.8

39

2.01

5.4

42

2.40

6.5

45

2.62

7.1

47

2.73

7.4

49

2.77

7.5

30

1.60

4.2

33

1.51

4.1

36

1.44

3.9

39

1.30

3.5

42

1.10

3.0

45

1.00

2.7

47

0.90

2.4

49

O .85

2.3

42. R .A T L

o f

R .L D

b L V L - L O P M L N T

& L O O D m

C £ - L L

C R O U P

I - l b

1.5

07 as.

I a

30

33

4$

A GIL

in

&AYS

R D

l v

a

TL

l l o

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l n

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I- C

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J -IL in

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44 TABLE III AGE OP CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number ID

No. of Chicks 3

Controls 3 (Untreated)

Age in days

Blood upon Treatment R .B .C . Count in H.G. Level in millions/cu.mm. Gms./I00 cc.

30

1.83

4.9

33

2.09

5.6

36

2.20

6.0

39

2.50

6.8

42

2.88

7.8

45

2.90

7.9

47

2.80

7.6

49

2.61

7.1

30

1.80

4.9

33

1.50

4.1

36

1.20

3.2

39

1.00

2.7

42

0.80

2.1

45

0.75

2.0

^7

0.70

1.9

49

0.70

1.9

Hi P ~ a t l

o f

f t c .0

b L V L L O

P M

L N

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B in

l o

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G R O U P

l l l l b

Control

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30

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33

D ays

R A T L

o f

D L V L L O P M L A / r

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4?

TABLE IV AGE OP CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number

No. of Chicks

II B

Controls (Untreated)

3

3

Age in days

Blood upon Treatment R.B.C. Count in H.G. Level ii Gms./I00 cc. millions/cu. mm

24

1.55

4.2

27

1.80

4.9

30

2.09

5.7

33

2.34

6.3

36

2.64

7.2

39

3.22

8.7

41

3.10

8.4

43

3.14

8.5

24

1.50

4.0

27

1.43

3.9

30

1.39

3.7

33

1.36

3.7

36

1.32

3.6

. 39

1.27

3.4

41

1.23

3.2

^3

1.18

3.0

R

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A g il

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Days

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Si R

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53

TABLE VI AGE OP CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number

No. of Chicks

III B

Controls (Untreated)

4

3

Age in days

Blood upon Treatment R.B.C. Count in H.G. Level in Gms./lOO cc. millions/cu. mm

24

1.39

3.8

27

1.71

4.6

30

2.06

5.6

33

2.26

6.1

36

2.49

6.8

39

3-31

9.0

41

4.10

11.1

24

1.40

3.8

27

1.25

3.4

30

1.10

3.0

33

0.95

2.6

36

O .89

2.4

39

0.80

2.1

41

0.75

2.0

5V R a ti,

of

R i

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It

—

1 LS

d

£>l o o d /n

G rout

1 .1 "

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C i l l

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Rate. of /-/l.moguo&in Di_VL.i-OPMk.NT in Group 111-b

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A ge .

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D ays

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56

TABLE VII AGE OP CHICKS IN ANEMIA AND RATE OP RECOVERY

Group Number Ill D

A of Chicks 4

Controls 3 (Untreated)

M in days

Blood upon Treatment R.B.C. Count in H.G. Level in millions/cu. mm Gms./lOO cc. ..____________

-■

. .

24

1.50

4.0

27

2.39

6.5

30

3.74

10.2

32

4.21

11.4

24

1.60

4.3

27

1.40

3.8

30

1.20

3.2

32

0.90

2.4

-____

Logarithmic, 2x

2Cycles.

MA|>f INl) A.

A ge. in

/?££>

Days

& L O O D C L L L

1< A T L _

of

1 -lL .h J O & L . O & I N

in

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r o u p

in

n.s

SI

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59 TABLE VIII AGE OP CHICKS IN ANEMIA AND RATE OF RECOVERY

Blood upon Treatment R.B.C. Count in H.G. Level in millions/cu. mm Gms./lOO cc.

Group Number

No. of Chicks

Age in days

IV B

4

30

1.78

4.8

33

1.94

5.3

36

2.05

5.6

39

2.3

6.2

42

2.23

6.0

45

2.91

7.9

4

2.56

6.9

49

2.18

5-9.

30

1.75

4.8

33

1.70

4.6

36

1.63

4.4

39

1.55

4.2

42

1.49

4.0

45

1.30

3.5

47

1.12

3.0

49

0.97

2.6

Controls (Untreated)

3

60

R D

a t i t v i

.

of

_ l. o

R& C m

p m l

R .n

tc>

[boooo

t

t

Gnoup

IV-D

Coun t

mill i o n s J c u . m.m-

.jGroup 1\Z'£>

33

C tll.

3b

/4g£.

--

40

in

42

Da

ys

■Coniroi.

AS

4-1

SO

61 R

a

T I-

Djlvllo

o f

1 - l L M O G L O t e 1N

r m e -n t

,n

Grour

!V-b>

ii.f

sc

4f

S3

A ga .

sn

D ays

so

62 TABLE IX AGE OF CHICKS IN ANEMIA RATE OF RECOVERY AND :

Group Number

No. of Chicks

IV D

Controls (Untreated)

4

3

Age in days

Blood upon Treatment R.B.C. Count in H.G. Level in millions/cu. mm Gms./lOO cc.

30

1.69

4.6

33

2.22

6.0

36

2.50

6.8

39

2.62

7.1

42

1.92

5.2

45

3-24

8.8

47

3.76

10.2

49

4.01

10.9

30

1.57

4.2

33

1.53

4.1

36

1.46

3.9

39

1.38

3.7

42

1.20

3.2

45

1.00

2.9

47

0.95

2.8

49

0.73

1.9

63 /Z a T £ .

of

/l£ .D

D lv £ - lo f> m £ .n t

REbC

m

/b L O O D in

C l- L L

C ro u p

IVD

Count

mi//*on%f c '

1.0

Gvxuya. .tV ‘£> i

Jo

I

;

!

:

i

4

8

A ge.

.9

4

10

40 in

Days

6

6J+

R ati.

of

U e_

D L V L . L . O F M L . N T

in

G

r o u p

Mb /r*

IV-D

L ^ . vb. l . f /oo «

i.i

45

9b

A c,£.

in

D ays

65 TABLE X AGE OF CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number

No. of Chicks

V B

Controls (Untreated)

4

3

Age in days

Blood upon Treatment R.B.C. Count in H.G. Level in millions/cu. mm Gms./lOO cc.

30

1.55

4.2

33

1.52

4.1

36

1.87

5.1

39

2.31

6.3

42

3.10

8.4

45

3.27

8.9

47

3.34

9.1

49

3.67

10.0

30

1.53

4.1

33

1.48

4.0

36

1.40

3.8

39

1.34

3.6

42

1.24

3.3

45

1.20

3.2

47

1.00

2.7

49

0.85

2.3

66 f l A T L

D

l v l

o f l

R L D

o p m

l n

0 > L O O D

in

f

G

C L L L

r o u p

V -&

Count in

m t //ion S ^ C v . m. /tr.

Group ! l^ZS

S3

3t>

A Gl­

_ ■--

4® ut

44 YS

J CAnJr-atl

45

47

67 R a t i . D l v l l

o

o f p

m

l

. n

I-Il m T

in

o

G

g

l

o

&

r o u p

i n

V -/G

it

HL jn

40

tf

I.F.

40

Agl.

jn

41

D ays

L m. v

ll

qrams / Joe cc

68 TABLE XI AGE OP CHICKS IN ANEMIA AND RATE OP RECOVERY

Group Number

No. of Chicks

V D

Controls (Untreated)

4

3

Age in days

Blood upon Treatment H.G. Level in R.B.C. Count in Gms ./ 1 00 c c . millions/cu. mm

30

1.66

4.5

33

2.09

5.6

36

2.50

6.8

39

3.00

8.1

42

3-48

9.5

43

3.67

10.0

47

3.80

10.3

49

3.98

10.8

30

1.65

4.5

33

1.60

4.3

36

1.54

4.2

39

1.46

4.0

42

1.36

3.7

45

1.28

3.5

47

1.04

2.8

49

0.92

2.5

69

R a TL D

l

V L L O

of P M

/?£_£> L N T

CtLOOD CLLL in

G R O U P

V

b

2.5

JJ

4*

JO

i

A g z.

in

D ays

70 R

a

TL.

o f

b L V L L O P M L N T

l - l L. M O G J L O E> / N ,n

G R O U T

V 'O

iro

IIA

Control \\M

A&L

in

D ays

W

“a -fl?/ fw, r

(JU

rtf

Ol

01 M J>. JL£S££jIn f v,*..~f : a a 3^___

^JiWIHIIllHHIIIII

i*

75 TABLE XII BLOOD COUNTS AND HEMOGLOBIN LEVELS OF CHICKS TREATED BY ORAL ADMINISTRATION

Normal Blood r Tb .c . Count No. millions Group of /cu mm. No. Chicks (Aver.)

Anemic Blood

.g . r .b .c ; level Count Gms/ millions 100 /cu mm. (Av.) (Aver.)

h

h

.'g .' Level Gms/ 100 (Av.)

Supplement to be Administered

I A

3

4.7

12.8

1.5

4.0

3 micrograms Bnp /100 Gms. diet

I C

3

4.6

12.5

1.8

4.9

6 micrograms B-, p /100 Gms. diet

II A

3

4.25

11.0

1.54

4.2

3 milligrams folic acid/100 Gms. diet

II C

3

4.4

12.0

I .69

4.6

6 milligrams folic acid/100 Gms. diet

III A

4

4.00

10.9

1.29

3.5

3 milligrams folic acid and 3 micro­ grams Bio/100 Gms. diet

4.20

11.1

1.49

4.0

6 milligrams folic acid and 6 micrograms B ^ / l Q O Gms. diet

m e

4

IV A

4

4.55

12.4

1.75

4.7

3 mi c rog rams B^ 2 and 3 milligrams ferrous gluconate /lQO Gms. diet

IV C

4

4.49

12.2

1.69

4.6

6 micrograms B j 2 and 6 milligrams ferrous gluconate /100 Gms. diet

76 TABLE XII

(continued)

BLOOD COUNTS AND HEMOGLOBIN LEVELS OF CHICKS TREATED BY ORAL ADMINISTRATION

Normal Blood Anemic Blood R.B.C. H.G. R.B.C. H.G. Count level Count Level No. millions Gms/ millions Gms/ /cu mm. 100 /cu mm. 100 Group of No. Chicks (Aver.) (Av.) (Aver.) (Av.)

Supplement to be Administered

V A

4

4.36

11.9

1.56

4.2

3 milligrams folic acid and 3 milli­ grams ferrous gluconate per 100 Gms. diet

V C

4

4.45

12.1

1.65

4.5

6 milligrams folic acid and 6 milli­ grams ferrous gluconate per 100 Gms. diet

R.B.C. = Red Blood Cells H.G. s Hemoglobin

77

TABLE XIII AGE OF CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number

No. of Chicks

I A

Controls (Untreated)

3

3

Age in days

BLOOD UPON TREATMENT R.B.C. Count in H.G. Level in millions/cu. mm Gms./lOO cc.

24

1.50

^0

27

I .56

4.2

30

1.63

4.4

33

1.79

4.S

24

1.40

3.8

27

1.34

3*6

30

1.26

3.4

33

1.16

3.0

lo

f lA T L .

of

R

ld

bLVLLO F>M LNT

B in

Cl

lo o d

GROUP

fKE)Cm

l l

I-A

Count

mil/fort5 J £-u- rr> m -

3_

t*

1.5

1.0-

-7. ■«.

.S.

.S.

.

« ^Tj *w*\

.

C en/naj

.L

W

36

A

cl.

D ays

79

R A T JL

of

J J L .K /O G L O C M N

Dl.vi_lorma.zut

in Grour 1-A

//£

Lt.Vl.i-

/h tjrom* ^//ao «,

V

i.S

to

Jo

D ays

80

TABLE XIV AGE OP CHICKS IN ANEMIA AND RATE OP RECOVERY

Group Number

No. of Chicks

I C

Controls [Untreated)

3

3

Age in days

Blood upon Treatment R.B.C. Count in millions/cu. mm

H.G. Level in Gms./lOO cc.

24

1.80

4.9

27

1.84

5.0

30

1.90

5.3

33

2.00

5.5

36

2.03

5.8

39

2.10

5.8

24

1.75

4.8

27

1.71

4.6

30

1.63

4.4

33

1.56

4.1

36

1.49

4.0

39

1.44

3.9

81

R

a ta

.

of

D

aval

-O F'm

R

a d

ESl q m

a n t

o d

G

a l a

G r. o u f > I - C

fi.£> C.

Count

tn fT)ito.-* j

A i. O O D

C t-L L .

in G/ioup II-C

t'n

D ays

38 R ATL-

of

Di.VL.LorML.NT

l - l i . h/tOGL-

OZb /A/

in Gnour II-C

o f £>(poc/ 12.*

l|.S

Days

89

TABLE XVII AGE OF CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number

No. of Chicks

III A

Controls ’Untreated)

4

3

Age in days

Blood upon Treatment R.B.C. Count in H.G. Level in Gms./lOO cc. millions/cu. mm

24

1.29

3.5

27

1.40

3.8

30

1.37

4.2

33

1.67

4.5

36

1.79

4.8

39

2.20

6.0

41

2.59

7.3

24

1.30

3.6

27

1.25

3.4

30

1.22

3.3

33

1.12

3.0

36

1.08

2.9

39

0.98

2.6

41

0.80

2.1

90

of RlLD

fl/KTL-

D i - v t . L- o f * m l . n

T

C b i—O O D ;n

G

r o u p

R .& C

2.9

1.5

SO

A

ge.

35

in

D

ays

Cl_LL 1 1 1 -A

Count

91 RATL.

of

U l M O G !.Q & IN

Di^\/£-L.OF>Mi-NT

,n G r o u p ///-A

/-/£

L £. \/£LL.

in y r o m x J /oo

«f,

us

ss

is

A cl.

in

*1

D ays

41

92

TABLE XVIII AGE OF CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number

No. of Chicks

III C

Controls (Untreated)

4

3

Age in days

Blood upon Treatment R.B.C. Count in H.G. Level in millions/cu. mm Gms./lOO cc.

24

1.49

4.0

27

1 *93

5.2

30

2.60

7.0

32

2.84

7.7

24

1.30

3*5

27

1.26

3*4

30

1.23

3.3

33

1.18

3.1

93 H

atl

.

of

R ld

b i-V -L L O P M i-N T

E lo o d in

G ro u p

C lll. J ll- C

/1£>C Count /r> snj///an / / < : - ■ '

3 t.5

I©

IS

so

A cl.

in

D ays

94 R

a t i

.

°f

/ /l

D l w /..of *m e .n r

m o c l

.o & /

i„ G r o u p

_

n

HI-C

!-ib Lsvs.t~ !n

y rc m ( ^ J + o £t.

_ > Group ///-C

11.5

2.5

XJ A

g z

.

in

D

a y s

95

TABLE XIX AGE OF CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number

No. of Chicks

IV A

Controls 'Untreated)

4

3

Age in days

Blood upon Treatment R.B.C. Count in H.G. Level in Gms./lOO c c . millions/cu. mm

30

1.75

4.7

33

1.80

4.9

36

1.84

5.0

39

1.94

5.3

42

1.92

5.2

45

2.19

5.9

47

2.05

5.6

49

1.90

5.1

30

1.75

4.7

33

1.68

4.5

36

1.56

4.2

39

I .50

4.0

42

1.42

3.8

45

1.34

3.6

47

1.30

3.5

49

1.24

3.3

96

H a TL of RLD D l- V L L o p m l n t

& L .O O D C l l l in G r o u p IV -A

RC>C in

Count

//ions

l.o .9 .8 .7 .6

CnoMf* (V-A

.3.

£er*^psi-

2.5

4*

si

A cl.

D ays

fs

^

4f

*

BO

97 R

a

TL.

o f

1 -lT M O G L O IC IN

D l . V i- L O P M i - N T

in

C /i O U P

IV - A

Mb /r*

M

f r * m * Jjoo

«*,

b lo o d

s Group iV ^A

tf

A gl.

9 10

y hi

Da y s

98 TABLE XX AGE OF CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number

No. of Chicks

IV C

Controls Untreated)

4

3

Age In days

Blood upon Treatment R.B.C. Count in H.G. Level in millions/cu. mm Gms./lOO cc.

30

1.69

4.6

33

1.90

5.1

36

2.01

5.4

39

2.06

5.6

42

2.20

6.0

45

2.31

6.3

47

2.52

6.8

49

2.62

7.1

30

1.60

4.3

33

1.52

4.1

36

1.48

4.0

39

1.40

3.8

42

1.34

3.6

45

1.23

3.3

47

1.18

3.1

49

1.00

2.6

99 R .A T L -

Dl v

l

of

R l_ D

-L O R M L -N T

[h L O O D

,n

C i^LL.

G r o u p 1V-C

R/2>C Couni m

?.S

.1*

AGJL

in

D a ys

m> //'on*/ at

100 R

a t i

,

of

I-IL n

i o g l o

&

G a o u r*

n.%

n.j

n

Days

i n

iV - C

101

TABLE XXI AGE OF CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number

No. of Chicks

V A

Controls (Untreated)

4

3

Age in days

Blood upon Treatment R.B.C. Count in H.G. Level in Gms./lOO cc. millions/cu. mm

30

1.56

4.2

33

1.54

4.2

36

1.72

4.7

39

1.92

5.2

42

2.02

5-5

45

2.22

5.8

47

2.30

6.0

49

2.37

6.3

30

1.52

4.1

33

1.42

3.8

36

1.34

3.6

39

1.25

3.4

42

1.13

3.0

45

1.03

2.8

47

0.93

2.5

49

0.75

2.0

102 R .A T L .

o f

R

l

.D

D l .v l - L - O p m l - N T

C iL O O D

C

C r o u p

in

jL

L L

V-A

C Count jin

rrjft///6 aS

2.5

1.5

2.5

A g jl

in

Da y s

a

103 R

a

VL.

of

l-l£_hAOCLO£o/N

bL.vt.L-'OFMi.NT

;«

G nour

V -A

l-!t> Li-V£.Lin

fr&rr** f J O O Ct.

»i.»

4*

51

A c, l .

m

Days

104

TABLE XXII AGE OF CHICKS IN ANEMIA AND RATE OF RECOVERY

Group Number

No. of Chicks

VC

Controls (Untreated)

4

3

Age in days

Blood upon Treatment R.B.C. Count in H.G. Level in millions/cu. mm Gms./lOO cc.

30

1.65

4.5

33

1.86

5.0

36

1.94

5.3

39

2.08

5.7

42

2.16

5.9

45

2.23

6.1

47

2.39

6.5

49

2.49

6.8

30

1.60

4.3

33

1.53

4.1

36

1.45

4.0

39

1.35

3.6

42

1.27

3.4

45

1.20

3.2

47

1.05

2.8

49

0.98

2.6

10?

Ratl

of R.l.d B l o o d

DlVLLOPMLNT

m

G

r

o

Cdll u

p

V-C

fllbC Oouni sa rnsi/iom Jou

ZS

1.5

33

AG£-

in

D ays

106

R n. a

DfVi.

l .o

r M i -

°J a /r

/-il.MOCt I. O t S i N G /z o u / >

/r»

V- C

l-lk> Ll.VS.JL in

14

yr'0/r,s

12.5

Con ire I

11.5

2 .5

1.5

55

D

a y s

10

0) cc-

OQ O -

U

|k (T.

9

117 (1948). Lillie, Biol.

R. J . ,C. A. Denton, and Chem., 176, 1477 (1948).

H. R. Bird,

J.

Kennard, D. C., R. M. Bethke, and V. D. Chamberlin, Poultry, Sci., 27, 477 (1948). Olcese, 0., J. tion, 41, 73

R. Couch, and C. Lyman, J. (1950).

Nutri-

Brockmann, J. A., Jr., J. V. Pierce, E. L. R. Stokstad, H. P. Broquist, and T. H. Jukes, Paper presented at 117th Natl. Meeting of Am. Chem. Soc., Pa., 1950*

133 Mapson, L. W., Biochera. J. , 26 , 970 (1932). Cary, C. A., A. M. Hartman, L. P. Dryden. and G. D. Likely, Federation Proc., 5., 128 (1946). Nichol, C. A., A. H. Robblee, W. W. Cravens, and C. A. Elvehjem, J. Biol. Chem., 170, 419 (1947). Stokstad, E. L. R . , and T. H. Jukes, Paper pre­ sented at 117th Natl. Meeting of Am. Chem. Soc., Pa., 1950. Doyle, L. P., F. P. Matthews, and R. E. Roberts, Poultry Sci., 9 , 6 (1929 ). Kelly, J. W . , and R. S. Dearstyne, North Carolina Agric. Exp. Stat. Bull., 50 (1935). Cook, S. F., Poultry Sci., 16, 291 (1937). Hart, E. B . , C. A. Elvehjem, A. R. Kemmerer, and J. G. Halpin, Pountry Sci., % 92 (1930). Charkey, L. W., H. S. Wilgus, A. R. Patton, and F. X. Gassner, Proc. Soc. Exptl. Biol. Med., 73, 21 (1950 ). Williams, J. N., Jr., C. A. Nichol, and C. A. Elvehjem, J. Biol. Chem., 180, 689 (19^9).