Parenteral use of carotene in the treatment of vitamin A deficiency in albino rats

PARENTERAL

USE

VITAMIN

OF

CAROTENE

A DEFICIENCY

IN IN

THE

TREATMENT

ALBINO

OF

R~TS

Fu-TANG CHU, )/i.D'., AND HARRIET COADY BOSTON~ ~V[ASS.

L T H O U G H it has been well k n o w n t h a t k e r a t o m a l a c i a is due to deficiency of v i t a m i n A in the diet, the oral t r e a t m e n t of the eye condition w i t h substances showing v i t a m i n A p o t e n c y has not. p r o v e d to be u n i f o r m l y effective. A m o n g the f a c t o r s which m a y i n t e r f e r e with the efficacy of v i t a m i n A w h e a a d m i n i s t e r e d orally m a y be mentioned d i a r r h e a l diseases, diseases of liver, gallbladder and pancreas, occlusioa of the bile ducts, and other disorders which result i a a f a u l t y metabolism of fat.. B o t h I-Isa~ and W e e c h 2 m e n t i o n e d the association of k e r a t o m a l a c i a w i t h d i a r r h e a and dysentery. Bloch 8 r e p o r t e d a case of x e r o p h t h a l m i a in which periodic a t t a c k s of d i a r r h e a were a c c o m p a n i e d b y a g g r a v a t i o n of the eye s y m p t o m s . W a g n e r ~ discussed the relation of l i v e r functio~ and x e r o p h t h a l m i a in a series of cases. I n a clinical p a t h o l o g i c report, H i g g i n s a n d 1V[allory '~ p r e s e n t e d the case of an infant in which keratomalacia was associatec~ with fat intoleraace and chronic pancreatitis. Particularly in patients with bi]iary occlusion, evidence has been brought forward by Parsons 7 to show that a very small amount of the ingested fat is absorbed; that is, only about 30 per cent of the fat intake, as compared to 98 per cer~t in the normal, is utilized in these subjects. In a recent article b y B l a c k f a n a n d W o l b a c h G it has been p o i n t e d out t h a t histologie changes c h a r a c t e r i s t i c of ~itamin A deficiency w e r e f o u n d in a number of infants, in w h o m congenital obliteration of the bile ducts, extensive sclerosis of the pancreas, or a c u t e generalized infection were p r o m i n e n t features. F r o m e x p e r i m e n t a l evidence, R o w n t r e e 8 conclv~ded t h a t the loss of ~ i t a m i n A r u n s parallel to t h e elimination of f a t t h r o u g h e x c r e t i o a b y the bowels. F r o m these clinical d a t a and e x p e r i m e n t a l evidence, it can be deduced t h a t a n y m e c h a n i s m which i n t e r f e r e s w i t h the n o r m a l a b s o r p t i o n of fat, w h e t h e r m e c h a n i c a l l y or functionally, m a y h i n d e r the p r o p e r utilization of v i t a m i n A when given b y mouth. The oral use of cod liver oil d u r i n g i h e a d v a a c e d s t a g e of k e r a t o m a l a c i a has b e e n discouraging. I n the experience of t h e writer, large doses, as m u c h as 15 c.c. given t h r e e times a day, h a v e failed to check the p r o g r e s s of the d e s t r u c t i v e eye lesions. I n view of the failure of oral m e t h o d s of t r e a t m e n t , a d m i n i s t r a t i o n b y the p a r e n t e r a l r o u t e is n a t u r a l l y suggested. From the Infants' and Children's Hospitals and the Department v a r d M e d i c a l School, B o s t o n , M a s s a c h u s e t t s .

75

of l % d i a t r i c u , H a r -

76

THE

JOUICI~AL

01~ PEDIATRICS

l~eee~t]y the efficacy of carotene b y m o u t h in the t r e a t m e n t of vitam i n A deficiency has become widely recognized. As f a r as we are aware, however, no m e n t i o n has b e e n m a d e in the l i t e r a t u r e of its p a r e n t e r a l use in this disease. The following e x p e r i m e n t s were und e r t a k e n in an a t t e m p t to t e s t out this m e t h o d of t h e r a p y in r a t s with v i t a m i n A deficiency, w i t h a n d w i t h o u t the a c c o m p a n i m e n t of diarrhea. PI%OCEDURE

A group of female albino rats were placed on a basal synthetic diet low in vitamin ~ at the age of from thirty-five to forty days, at 42 to 61 gin. of weight. The composition of the diet was as follows: PEI~CENTAGE

Casein, purified ( H a r r i s ) S a l t m i x t u r e ( O s b o r n e a n d MendeD~) Vitamin B concentrate* V i o s t e r o l 250D Cornstarch

18.0 4.0 4.0 0.5 73.5

-Basal ration a n d distilled w a t e r were supplied in liberal amounts at a l l times. Separate cages with elevated wire bottoms were used for the individual animals as soon as they were r e a d y for treatment. I n each experiment, rats of nearly corresponding weight and those which came to the condition of depletion at a b o u t the same time were chosen in c o m p a r i n g the effects of the different t y p e s of t r e a t m e n t . W e e k l y records of weight and eye condition, together with other observations, were made t h r o u g h o u t the experiments. I n the experiments the r a t was considered to be in a state of depletion w h e n it h a d shown all of the three e a r l y signs of deficiency; namely, cessation of growth, the persistence of eornified epithelial cells in vaginal smears, and ophthahnia. T r e a t m e n t was sometimes begun soon a f t e r this state of depletion was observed, and sometimes considerably later, d e p e n d i n g on whether we wished to observe the therapeutic effects of the t r e a t m e n t in the early or late stages of the disease. Two b r a n d s of carotene p r e p a r a t i o n s w e r e used f o r the t r e a t m e n t . Carotene No. 1 contained a strength of v i t a m i n A about ten times t h a t of cod liver oil, while No. 2 was a 0.3 p e r cent solution o f p u r e carotene. The technic for the injection of carotene w a s as follows : S t a n d a r d t u b e r c u l i n syringes and h y p o d e r m i c needles, 24 gauge, 5/s inch long, w e r e used. The r a t s were firmly held b y an assistant, one h i n d leg b e i n g held in a n e x t e n d e d position f o r t h e injection. The h a i r was clipped as closely as possible f r o m t h e p o s t e r i o r p o r t i o n of t h e thigh, a n d the clipped a r e a was w a s h e d w i t h alcohol and allowed to dry. I t w a s f o u n d t h a t w h e n the injections w e r e m a d e s u b c u t a n e o u s l y or *:A_eknowledgment is due Mead J o h n s o n & Co. for t he provi s i on of v i t a m i n B conc e n t r a t e a n d a p a r t of the c a r o t e n e p r e p a r a t i o n s us e d i n t he e x p e r i m e n t s :

CI-IU AN'D COADY :

CAt~OTENE II~ VITA1VZIN A D E F I C I E I ~ C Y

77

straight down into the muscle tissue, a large proportion of the material injected leaked out through the site of the puncture. In order to eliminate this loss, the needle was passed through the skin while being held parallel to the skin surface and then sharply turned inward so as to make the injection into the muscle substance. The needle was withdrawn quickly and the site of the puncture was pressed with a small piece of cotton and massaged with a little pressure for a few minutes. Leakage rarely occurred with this technic, but when it did occur the contamination 'of carotene on the skin was washed away with ether until the y e l l o w color was no longer visible. A small drop of collodion was finally applied at the site of the puncture to insure against subsequent oozing. The two thighs were used alternately for the injections.: In the case o f oral administration, the technic consisted simply of giving carotene directiy into the mouth in small drops from the same type of syringe and needle as used for the injections. EXPERIMENT

I

PAt~ENTEI~AL T I ~ E A T M E N T I X T H E ]~ARLY STAGES OF T H E VITAMIN A DEFICIENCY

Treatment was started from two t o six days aft er the development of all three of the deficiency signs mentioned above, when the animals had been on the basal diet for a period of from forty-six to fifty=seven days. Caro.tene No. I was used for one 2Toup of rats, and Carotene No. 2 f or the other. The effects of the intramuscular injections of these preparations were compared to those of oral treatment. Each rat received eleven treatments, Group 2 being given daily treatment and Group 1 being t r e a t e d every ether day. The period of observ~ation lasted for f our weeks for Group 1 and three weeks for Group 2. As f a r as possible all the environmental factors were kept the same for all rats in each group with the exception of the fact that those which received ~he injections were liable to suffer from the local trauma and swelling caused by the injection, from which the other animals were free. In Tables I and II, it is clearly shown that gains in weight and improvement of the eye symptoms followed both forms of treatment. However, the resumption of growth commenced earlier and proceeded more rapidly in the animals being given carotene by mouth than in those receiving the intramuscular injections. It m a y be mentioned ,here that in the course of the treatment, little or no evidence of infection at the site of the injections was obser:ced, in spite of the fact that no particular effort was made to sterilize the

78

THE

JOUI%NAL OF PEDIATRICS

TABL~ I IPAEENTERAL

TI~EATMENT

IN TIIE EAI~LY

~TAGE

WITH

~AI%OTENE- S O L U T I O N

IN-O. ~l

(GEOUP I) WEIGHT I N GM. I~AT

NO.

TOTAL W EIG I~l GAIN

AVE1% VEI%AGE WE ~ 'EE]E K L Y G~AIN AIN IN

IN GM.

G3Z GlV[.

144 129 132 14:-0 126 157

15 22 27 23 32 28

3.8 3.1 5.5 5.~ 6.8 6.1 5.8 8.0 7.0

Negative control

145

-23

-5.8

Negative control

133

T Y P E OF TI~EAT-

DOSAGE

MEET

A T BE,GINNING OP TREAT-

RESUIVfPTION 01~

EYES AT END OF E X F E R I " MEET

GEGWTTI ~

MEET

45 43 44 22 50 24 109 106

Injection Injection Injection Oral 0ral

Oral

---
T 17 14

2

Normal Normal Normal Normal Normal Normal Marked xerophthahnia

Died in 18 days

*Days after the first treatment, when the gain in weight commenced. ~First ~ doses of 0.1 e.e. followed by 7 doses of 0,2 e.c. each.

TABL~ II PARENTE,~AL

T~EATMENT'

IN .T~HE E A R L Y

~TAGE. W I T H

~:AROTENE

SOLUTION

No. 2 (G~our 2) ~VEIGHT

iN GM. IgAT N0,

3~47 ~9 25 104 107

T Y P E 0!~ T~EAT-

D( S A G E

(D ~ILY)

0F

MEET

Injection Injection Oral Oral ~qegative control Negative control

A T BE" INNING

--X-~-~.2 0.2

: TOTAL WEIG~IT GAIN

AVERAGE WEEKLY

IN GM,

GAIN IN GM.

TREATI~[ENT

144 143 130 144 156

23 25 15 28

7.7 8.3 5.0 9.3

' -14

-4.8

172

-30

-10

RESUMPTION OF GROWTH*

10 10

EYES AT END 0N EXFERI ~ NIRNT

Normal Normal Normal Normal ~ a r k e d xerophthalmia, Marked xerophthalmia (Died on 22nd day of 'experiment)

*Days after the first treatment, when the gain in w e i g h t commenced.

carotene preparations injected. In a number of eases, however, swellings in the muscles could be felt for a number of days after the injection. This tendency of the carotene solution to collect at the place of injection and its slow absorption apparently explain the delayed response of this group, of animals to the parenterat administration of carotene.

CHU

AND COADY:

CAROTENE IN VITAMIN

EXPERIMENT PARENTERAL

TREATMENT

A DEFICIENCY

79

II

I N T H E A D V A N C E D S T A G E S OF

VITAMIN A DEFICIENCY

It was pessible to s t u d y the effect of p a r e n t e r a l t r e a t m e n t in the late stages of the disease because of the f a c t t h a t this group of rats was s t a r t e d on the basal diet at an older age t h a n is usually done and was therefore able to survive 9longer. T h a t the disease was well adv a n c e d at the time of t r e a t m e n t is indicated by the long time d u r i n g which these rats h a d been in a state of depletion. These animals h a d shown eessatio,n of g r o w t h for f r o m ten to thirtyseven days and the eye symptoms for from sixteen to thirty-one days before the t r e a t m e n t s were begun. The experiment lasted for two and a half weeks a f t e r the first treatment. As shown in Table I I I , the results of t r e a t m e n t in this g r o u p were essentially similar to those of the preceding experiment. I t m a y be of interest to note t h a t the resumption of g r o w t h s t a r t e d no later t h a n in the g r o u p of animals which were t r e a t e d at an earlier stage of the disease. This is equally true of animals which were given oral administrations or intramuscular injections of carotene.

PARENT]~IZAL

~]]i~EATMENT IN ~ttE AD,VANOED

ST'AGE WITH

CAROTENE

NO. 2

9 o

g~

o

51

28

28

Injection

116

11

4.4

8

53 11 46

30 20 16

30 21 16

Injection Injection Injection

]01 149 133

20 23 11

8.0 9.2 4.4

8 ]0 10.

6 13

24 37

20 24

Oral Oral

140 144

24 22

9.6 8.8

9

14

31

Negative control

120

-14

- 5.6

10

10

25

120

-36

-14.4

L e s s than L e s s than

Slight xeroPhthalmia Normal Normal Normal

7 Normal 7 Normal Marked xeroph~ thalmia Marked xerophthalmia

* E i g h t d o s e s o f 0.2 c.c. e a c h d a i l y w e r e g i v e n in t h e f i r s t e i g h t d a y s of t h e e x p e r i ment which lasted for two and one-half weeks. "~Days a f t e r t h e f i r s t t r e a t m e n t , w h e n t h e g a i n in w e i g h t c o m m e n c e d .

80

TItE

J O U R N A L OF P E D I A T R I C S EXPERIMENT

TItlE INFLUENCE

OF

DIArCI~HEA

ON

III TI:IE PAR, ENTEI~AL

TREATMENT

01~

VITAMIlXT A D E F I C I E N C Y

A c o m p a r i s o n of the effects of oral a n d p a r e n t e r a ! t r e a t m e n t s was m a d e in a n o t h e r g r o u p of r a t s with v i t a m i n A deficiency, in which a s t a t e of defective a b s o r p t i o n in the g a s t r o i n t e s t i n a l t r a c t w a s artificially induced by the administration of 0.5 c.c. of liquid paraffin orally twice a d a y to each rat. The a d m i n i s t r a t i o n of the m i n e r a l oil was s t a r t e d one. d a y before the b e g i n n i n g of the carotene t r e a t m e n t , a n d ia case of r a t s receiving carotene orally; one dose of t h e oil was g i v e n i m m e d i a t e l y following the feeding of carotene. I t was definitely observed d u r i n g the course of the e x p e r i m e n t that. the r a t s which received b o t h liquid paraffin a n d carotene b y m o u t h passed stools of a very o i l y consistency and intense yellow color, indicating that a large a m o u n t of" the carotene was actually carried t h r o u g h the bowels without b e i n g absorbed. The d a i l y dose. of I c.e. of paraffin was chosen because, it was t h o u g h t t h a t a dose s l i g h t l y l a r g e r t h a n the l a r g e s t used h a r m l e s s l y b y R o w n t r e e 9 should be suitable for' our purpose.. C a r o t e n e t r e a t m e n t w a s s t a r t e d f r o m one to two days a f t e r the d e v e l o p m e n t of all three of t h e deficiency signs m e n t i o n e d above, a n d t h e whole e x p e r i m e n t covered a p e r i o d of f o u r weeks, a f t e r the first t r e a t m e n t . As shown in T a b l e IV, t h e p r e s e n c e of diarrhea, caused a m a r k e d reduction in the gain in weight when the carotene was given b y m o u t h b u t did not m a k e a n y a p p r e c i a b l e difference w h e n the t r e a t m e n t w a s p a r e n t e r a l : T h a t 1 e.c. of m i n e r a l oil daily was not deleterious in itself is shown b y the f a c t t h a t r a t s Nos. 51 a n d 53 w e r e able to hold s t a t i o n a r y weights f o r seventeen d a y s a f t e r t h e first dose of oil a n d t h a t t h e i r loss of w e i g h t d u r i n g the entire e x p e r i m e n t was not excessive. I t is p r o b a b l e t h a t if m o r e interference, w i t h t h e absorption of carotene could be p r o d u c e d e x p e r i m e n t a l l y , a m o r e f a v o r a b l e response to the par'enteral t r e a t m e n t m i g h t h a v e followed. DISCUSSION

The relation of carotene to. v i t a m i n A has been a topic of controversy f o r more t h a n a decade. Recently there has appear'ed experimental evidence in f a v o r of the assumption t h a t carotene is a precursor of vitamin A. 1Vfoore1~ showed t h a t carotene in p l a n t material is convertible into v i t a m i n A in the animal body. Olcott and !VfcCann1~ f u r t h e r d e m o n s t r a t e d t h a t carotene could be changed to ~itamin A in v i t r o by incubation with fresh liver tissue or with an aqueous extract of liver; the livers of rats which were raised on a v i t a m i n A free diet were used in p r e p a r a t i o n of the extract. Regardless of the e x a c t relation of carotene to v i t a m i n A, the fact t h a t carotene is a substance h a v i n g

CHU AND COADY:

CAI%OTENE IN VITAMIN A DEFICIENCY

81

TABLE IV THE INFLUENCE

EAT NO.

D

23 27 54 29 32 36 38 48

E

52 51

A

B C

53

OF D I A R R H E A

TYPE

O N T H E E F F E C T S 0F O R A L A N D P A E E N T E R A L

O ~ TREATMENT

(13 DOSES*)

Carotene orally t Carotene orally+ L i q u i d paraffin f Carotene pareaterally Carotene parenterally + Liquid paraffin Liquid paraffin only

W E I G H T IN GIVs A T BEGINNING 01~ TRE,ATMEN~

TOTAL WEIGttT GAIN IN

134 111 119 140 116

TREATMENTS

AVERAGE WEEKLY GAIN IN GIVf.

EYES AT END 0F EXPERIMENT

140 130 126

18 3O 29 7 12 12. 10 10

4.5 7.5 7.3 1.8 3.0 3.0 2.5 2.5

Normal Slight xerophthalmia Normal Normal Slight xerophthMmis Normal Normal Normal

137 125

15 --9

3.8 -2.3

Normal Marked xeroph-

113

--12

-3.0

GIV[.

tha]mla

Marked xerophthalmia Xerophthalmia

N e g a t i v e control 121 -1.8 42 --7 *TWo doses of 0.03 c.e., three doses of 0.05 c.e., and then eight doses of 0.2 e.e. each, all given a t the intervals of every other day. F

vitamin A activities is undisputed. vitamin A in these experiments.

It was therefore used to represent

In this s t u d y the effectiveness of carotene when given by mouth is well confirmed. I n addition it is shown t h a t following intramuscular injections, the sabstance can be absorbed and utilized by the body, although less readily than when given orally. This may imply that the conversion of carotene to vitamin A in vivo is not necessarily through the medium of the digestive system. In a study of the absorption of fat given parenterally, Koehne and Mendel 12 injected b u t t e r fat and cod liver oil subcutaneously and intraperitoneally into a group of rats with vitamin A deficiency and reported fairly favorable results. In our rats the improvement following the parenteral t r e a t m e n t was considerably more marked t h a n that observed by these workers, probably because of the use of a more active substance. Besides the simple factor of containing more substance with vitamin A activity, the c o n c e n t r a t e d f o r m of carotene which we used m a y be of additional advantage in that being bound up in a small amount of lipoid-solvent, the active substance is more r e a d i l y dispersed and utilized. J o r s t a d ' s observations 13 suggest t h a t lipoid solvents when int r o d u c e d subcutaneously tend to remove vitamin A f r o m the surrounding tissue. I t m a y be fttrther deduced t h a t the less the a m o u n t of solvent used, the b e t t e r will be the chance of the injected vitamin's being utilized b y the body. The usefulness of p a r e n t e r a l t r e a t m e n t as compared to oral treatment is best b r o u g h t out b y E x p e r i m e n t III, in which the artificial

82

THE JOURNAI~ OF PEDIATRICS

i n t e r f e r e n c e w i t h n o r m a l a b s o r p t i o n o f f a t is p r o d u c e d to e x e m p l i f y p a t h o l o g i c c o n d i t i o n s w h i c h m a y b e m e t w i t h i n h u m a n cases a n d w h i c h r e s n l t i n a d i s t u r b a n c e of t h e f a t m e t a b o l i s m o f t h e b o d y . I n t h e l i t e r a t u r e , o n l y a f e w r e c o r d s of p a t i e n t s ' r e c e i v i n g p a r e n t e r M t r e a t m e n t w i t h cod l i v e r oil c o n c e n t r a t e s h a v e b e e n r e v i e w e d . 8' 14 A l t h o u g h t h e d a t a i n t h i s s t u d y a r e n o t e x t e n s i v e , t h e r e s u l t s a r e so u n i f o r m t h a t t h e y s e e m to s u p p l y e x p e r i m e n t a l e v i d e n c e f o r t h e v a l u e of s u c h t r e a t m e n t . A s is s u g g e s t e d i n t h e s e e x p e r i m e n t s , t h e i n d i c a t i o n f o r t h e par~enteral t r e a t m e n t w o u l d b e cases o f (a) p r o t r a c t e d k e r a t o m a l a c i a i n a s s o c i a t i o n w i t h c h r o n i c d i a r r h e a , (b) v i t a m i n A d e f i c i e n c y d u e to c o n g e n i t a l d e f e c t s s u c h as t h e t y p e of b i ] i a r y occ l u s i o n w h i c h is a m e n a b l e to s u r g e r y , .(e) v i t a m i n A d e f i c i e n c y in a s s o c i a t i o n w i t h t e m p o r a r y d i s t u r b a n c e of t h e f a t m e t a b o l i s m d u e to i n f e c t i o n s a n d o t h e r causes, a n d (d) a d v a n c e d k e r a t o m a l a c i a i n w h i c h p e r m a n e n t b l i n d n e s s is f r e q u e n t l y a t h r e a t e n i n g c o m p l i c a t i o n a n d w h e r e p a r e n t e r a l t r e a t m e n t s h o u l d b e g i v e n to s u p p l e m e n t t h e o r a l therapy. CO•CLUSI0.NS 1. C a r o t e n e g i v e n i n t r a m u s c u l a r l y is e f f e c t i v e in c u r i n g v i t a m i n A d e f i c i e n c y in r a t s , b o t h in t h e e a r l y a n d l a t e s t a g e s of t h e d i s e a s e . 2. W h e n t h e s u b j e c t ' s c o n d i t i o n is e n t i r e l y d u e to t h e l a c k of v i t a m i n - A - c o n t a i n i n g s u b s t a n c e s in t h e d i e t a n d is n o t c o m p l i c a t e d b y a n y o t h e r f a c t o r , t h e r e s p o n s e to c a r o t e n e i n j e c t i o n is s l o w e r a n d less m a r k e d t h a n the r e s p o n s e t o t h e o r a l a d m i n i s t r a t i o n of c a r o t e n e . 3. I n t h e p r e s e n c e o f a d i s t u r b a n c e w h i c h m a y r e s u l t i n a d e f e c t i v e a b s o r p t i o n of fa~, i n t r a m u s c u l a r i n j e c t i o n of c a r o t e n e is of d i s t i n c t t h e r a p e u t i c v a l u e as a n a d j u n c t to t h e o r a l t r e a t m e n t . I wisl~ to express grateful appreciation to Dr. Kenneth D. Blackfan, Dr. James L. Gamble, and to Dr. Charles F. MeKhann for their constant interest and assistance ]n making this study possible. Acknowledgment is due Mead JohnsorL & Co. for the provision of vitamin B concentrate and a part of the carotene preparations used in the experiments. REFEI~ENCES ]. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Hsu, I~:. L.: China M. J. 41: 825, 1927. Weech, A . A . : Am. J. Dis. Child. 39: 1153, 1930. B~oeh, C . E . : Am. J. Dis. Child. 31: 315, 1930. Wagner, R.: Wien. med. Wehnschr. 74: 2607, 1924. I-Iiggins, It. L.~ and Mallory, T. B.: New England J. ~ed. 20~: 1264, 193]. Blackfan, K. D., and Wolbach, S. ]3.: J. PEDIArIT.3: 678, 1933. Paxsons, L. G.: Am. J. Dis. Child. 43: 1293, :1932. Rowntree, J . I . : J / N u t r i t i o n 3: 265, 1930. Rowntree, J. I.: J. ~utrition 3: 345, 1931. Moore, T.: Biochem. 5. 24: 692, 1930. Oleott, I~. S., and McCann, D. C.: J. Biol. Chem. 94: 185, 193i. ]~oehne, M., ancl Mendel, L. B.: J. ~qutritloa 1: 399, 1929. Jorstad, L. It.: J. Cancer Research 10: 229, 1926. Blegvad, O.: Ugesk. f. laeger ~5: 127, 1923; quoted in the editorial of Brit. M. J. 1: 122, 1924. 15. Osborne, T. B., and Mendel, L . B . : J. Bioh Chem. 37: 557, 1919.