Chemistry and Modern Society - ACS Publications - American

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4 Will Milk Make Them Grow? An Episode in the Discovery of the Vitamins STANLEY L. BECKER Bethany College, General Sciences, Bethany, WV 26032

Following the enunciation of the vitamin hypo­ thesis in 1912, the value of cow's milk as a source of vitamins necessary for normal growth of rats (and, by implication, for normal growth of humans) became a significant issue in animal feeding experiments. While there was general agreement about the quali­ tative value of milk as a vitamin source, a major conflict arose between two pioneering research lab­ oratories concerning the quantitative aspects. Al­ though the researchers involved reported differences in quantitative requirements that varied by as much as 5-8 fold, the disparity between results was never resolved and remains a mystery to this day. Critical

Events

J u n e , 1912: C a s i m i r Funk, a P o l i s h b i o c h e m i s t w o r k i n g i n London, E n g l a n d , w r o t e a r e v i e w a r t i c l e i n w h i c h he e l a b o r a t e d on t h e f a c t t h a t d i s e a s e s such as s c u r v y , b e r i - b e r i , r i c k e t s and p e l l a g r a had l o n g been known t o be a s s o c i a t e d w i t h t h e d i e t a r y . Funk s a i d t h a t t h e s e d i s e a s e s c o u l d be p r e v e n t e d o r c u r e d by a d d i n g c e r t a i n o r g a n i c s u b s t a n c e s t o t h e d i e t , s u b s t a n c e s he c a l l ­ ed v i t a m i n e s . (J_) M i d - J u l y , 1912: F r e d e r i c k Gowland H o p k i n s , a b i o c h e m i s t a t Cambridge U n i v e r s i t y , p u b l i s h e d an e l a b o r a t e p a p e r d e m o n s t r a t i n g t h a t a d d i t i o n s o f s m a l l amounts o f f r e s h , w h o l e m i l k t o o t h e r w i s e d e f i c i e n t d i e t s o f e x p e r i m e n t a l r a t s were f o l l o w e d by p e r i o d s o f normal g r o w t h and d e v e l o p m e n t o f t h e a n i m a l s . (2) J u l y , 1913: Ε. V. M c C o l l u m , a b i o c h e m i s t a t t h e U n i v e r s i t y o f W i s c o n s i n A g r i c u l t u r a l Experiment S t a t i o n p u b l i s h e d a paper i n w h i c h he showed t h a t t h e r e was s o m e t h i n g i n b u t t e r t h a t made r a t s grow; s o m e t i n g t h a t was l a t e r t o be named v i t a m i n A and, l a t e r yet, v i tami ns A and D. (3_) A u g u s t , 1913: T. B. O s b o r n e , a p r o t e i n c h e m i s t a t t h e Con0097-6156/83/0228-0061 $06.75/0 © 1983 A m e r i c a n C h e m i c a l Society

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n e c t i c u t A g r i c u l t u r a l Experiment S t a t i o n , i n c o l l a b o r a t i o n w i t h L. B. M e n d e l , a p h y s i o l o g i c a l c h e m i s t a t Y a l e U n i v e r s i t y , p u b l i s h ­ ed a p a p e r iη w h i c h they d e m o n s t r a t e d t h a t b u t t e r was v e r y e f f e c t i v e i n making r a t s grow, {k) T h i s work had been p r e c e d e d by a mono­ g r a p h i n 1911 w h i c h c o n t a i n e d a w e a l t h o f d a t a on t h e n u t r i t i o n a l value o f i s o l a t e d food substances as r e l a t e d t o the growth o f r a t s . (5) In December 1929, F r e d e r i c k Gowland H o p k i n s s h a r e d t h e Nobel P r i z e i n Medicine o r P h y s i o l o g y , " f o r h i s d i s c o v e r y o f the growthstimulating vitamins." The s t o r y r e l a t e d h e r e w i l l c l a r i f y , t o some e x t e n t , t h e r o l e s o f t h e s e p i o n e e r s i n t h e emergence o f t h e v i t a m i n h y p o t h e s i s . More i m p o r t a n t l y , i t w i l l shed l i g h t on some p e c u l i a r i t i e s o f n u ­ t r i t i o n a l b i o c h e m i s t r y a s i t was p r a c t i c e d i n t h e f i r s t q u a r t e r of the twentieth century. In p a r t i c u l a r , i t w i l l f o c u s upon e x ­ p e r i m e n t a l r e s u l t s t h a t h e l p e d one i n v e s t i g a t o r w i n a Nobel P r i z e , r e s u l t s t h a t c o u l d n o t be r e p r o d u c e d by some o f t h e most competent r e s e a r c h workers i n animal f e e d i n g experiments. Nutrition,

1905 S t y l e

The dominant p r i n c i p l e o f n u t r i t i o n i n 1905 was e n e r g y . Cal­ o r i e s were c o u n t e d f o r p e o p l e i n d i f f e r e n t o c c u p a t i o n s , consuming highly varied diets. G i v e n s u f f i c i e n t c a l o r i e s , n u t r i t i o n a l dogma s a i d , and human b e i n g s w i l l grow and d e v e l o p n o r m a l l y . The c a l ­ o r i e s c o u l d b e s t be o b t a i n e d from f a t s and c a r b o h y d r a t e s . In a d ­ d i t i o n , a minimum amount o f p r o t e i n was r e q u i r e d (and t h i s m i n i ­ mum was h o t l y d e b a t e d ) a l o n g w i t h a few m i n e r a l s and w a t e r . Much has been w r i t t e n a b o u t t h e s e e a r l y t w e n t i e t h c e n t u r y v i e w s o f n u t r i t i o n , b o t h o f t h e r i g o r o u s s c i e n t i f i c v a r i e t y and of the l e s s formal but widespread f o l k concepts. The p r e - h i s t o r y o f t h e e p i s o d e b e i n g r e l a t e d h e r e i s r e a s o n a b l y documented i n a number o f w o r k s such as t h o s e o f M c C o l l u m (6) and B e c k e r (7). The 1 i t e r a t u r e shows c l e a r 1 y t h a t i n t h e f i r s t decade o f t h i s c e n t u r y , c a l o r i e - d o m i n a t e d n u t r i t i o n was b e i n g q u i e t l y b u t v i g o r o u s l y c h a l ­ lenged. In t h e s e c o n d decade t h e c h a l l e n g e became open and even more v i g o r o u s . Ρ ioneers C a s i m i r Funk had come f r o m P o l a n d t o t h e L i s t e r I n s t i t u t e i n London, E n g l a n d t o i n v e s t i g a t e t h e c h e m i c a l n a t u r e o f t h e sub­ stance i n r i c e - p o l i s h i n g s ( r i c e bran) t h a t cured o r prevented polyneuritis i nbirds, a disease closely related to beri-beri i n humans. H i s r e s e a r c h i n t h i s s u b j e c t c o u p l e d w i t h h i s a w a r e n e s s o f what were becoming known a s d i e t - r e l a t e d d i s e a s e s , t h e d e f i ­ c i e n c y d i s e a s e s , l e d t o h i s r e v i e w a r t i c l e o f 1912 i n w h i c h he c o i n e d the term, v i t a m i n e . V i t a m i n e , t o Funk, was a c t u a l l y a v a r ­ i e t y o f chemical substances, o r g a n i c amines, t h a t acted s e l e c t i v e -

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l y i n t h e p r e v e n t i o n o r c u r e o f d i s e a s e s such as b e r i - b e r i , s c u r ­ vy, p e l l a g r a and o t h e r d i e t - a s s o c i a t e d p a t h o l o g i e s . In s h o r t , t h e y were v i t a l a m i n e s , hence v i t a m i n e s . In the mid 1920's, Funk became e m b r o i l e d i n a c o n t r o v e r s y o v e r t h e d i s c o v e r y (and the d i s c o v e r e r ) o f the v i t a m i n e s , o r v i t a ­ mins as they were then known. (S_ 9., 10) He gave some c r e d i t t o H o p k i n s f o r h i s e a r l y e n d e a v o r s but r e f u s e d t o a c k n o w l e d g e Hopkins' priority. T h i s e p i s o d e b e a r s s t r o n g l y upon remarks made by Hop­ k i n s i n h i s Nobel A d d r e s s o f 1929 w h e r e i n he d i s c u s s e d Funk's work as w e l l as M c C o l l u m ' s and t h a t o f Osborne and M e n d e l . F r e d e r i c k Gowland H o p k i n s , a n a l y t i c a l c h e m i s t , p h y s i c i a n and b i o c h e m i s t , had c o n d u c t e d numerous e x p e r i m e n t s i n a n i m a l f e e d i n g p r i o r t o h i s famous comment i n 1906 t h a t no a n i m a l c o u l d l i v e on a d i e t o f p u r e p r o t e i n , f a t , c a r b o h y d r a t e , m i n e r a l s and w a t e r . He c i t e d t h e s i m p l e f a c t t h a t a n i m a l s l i v e upon p l a n t s o r o t h e r a n i ­ mals whose t i s s u e s c o n t a i n many o t h e r s u b s t a n c e s b e s i d e s t h o s e u s u a l l y c o n s i d e r e d a d e q u a t e f o r a normal d i e t , " . . . i t is certain t h a t t h e r e a r e many m i n o r f a c t o r s i n a l l d i e t s o f w h i c h t h e body takes account." (11) These comments were made n e a r t h e end o f a t a l k a t a m e e t i n g o f t h e S o c i e t y o f P u b l i c A n a l y s t s , a g r o u p composed p r i n c i p a l l y o f a n a l y t i c a l c h e m i s t s and m e d i c a l d o c t o r s . During the d i s c u s s i o n w h i c h f o l l o w e d , a l m o s t no r e f e r e n c e was made t o H o p k i n s ' new i d e a s and r e m a r k s , i n i t s e l f a measure o f the newness o f such i d e a s a l o n g w i t h the i n a b i l i t y o r i g n o r a n c e o r l a c k o f a t t e n t i o n p a i d t o such comments by t h e g r o u p i n a t t e n d a n c e . Over t h e n e x t s i x y e a r s H o p k i n s p u r s u e d , i n t e r m i t t e n t l y , t h e s e a r c h f o r t h e o t h e r n u t r i e n t s r e q u i r e d t o keep an a n i m a l a l i v e and i n good h e a l t h . In 1912 came t h e p u b l i s h e d r e s u l t s o f h i s r e ­ s e a r c h and w h i l e he c o n f i r m e d them i n p u b l i c a t i o n s o f 1913 (12) and 1920 ( 1 3 ) , i t a p p e a r s t h a t o t h e r p i o n e e r s i n n u t r i t i o n a l s t u d ­ i e s , Osborne and Mendel i n p a r t i c u l a r , were u n a b l e t o r e p e a t o r reproduce h i s r e s u l t s . Thomas B. Osborne and L a f a y e t t e B. Mendel w o r k e d t o g e t h e r f o r n e a r l y t w e n t y y e a r s t o u n r a v e l the c h e m i c a l and p h y s i o l o g i c a l p r o ­ p e r t i e s o f p r o t e i n s . In t h e i r e f f o r t s t o s u p p l y e v e r more s o p h i s ­ t i c a t e d d i e t s , i . e . , d i e t s made up o f p u r e c h e m i c a l p r o t e i n s , min­ e r a l s , e t c . , they d i s c o v e r e d t h a t d i f f e r e n t p r o t e i n s were marked­ l y d i f f e r e n t i n t h e i r a b i l i t y t o promote g r o w t h o r even t o m a i n ­ t a i n a n i m a l s a t c o n t a n t body w e i g h t . Repeated f a i l u r e s o f such d i e t s , c o n t r a s t e d w i t h s u c c e s s f u l ones on w h i c h a n i m a l s grew n o r m a l l y when powdered w h o l e m i l k was the m a j o r i n g r e d i e n t , f o r c e d Osborne and Mendel t o examine m i l k i t s e l f t o e x p l a i n i t s p e c u l i a r a b i l i t y t o s u s t a i n g r o w t h and m a i n ­ tenance of experimental r a t s over extended p e r i o d s of time. Ulti­ m a t e l y they d i s c o v e r e d t h e g r o w t h - p r o m o t i n g p r o p e r t y o f b u t t e r and b u t t e r f a t . (]k) Not s u r p r i s i n g l y , w i t h so many i d e a s " i n t h e a i r " and w i t h o t h e r r e s e a r c h e r s p r o w l i n g i n r e l a t e d a r e a s , Osborne and Mendel were pre-empted by t h r e e weeks i n p u b l i s h i n g t h e i r d i s ­ covery. I t was Ε. V. M c C o l l u m a t W i s c o n s i n who f i r s t r e p o r t e d t h a t ' b u t t e r makes them grow.' 9

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E l m e r V. M c C o l l u m , w h i l e o b t a i n i n g h i s d o c t o r a t e i n c h e m i s t r y a t Y a l e , took some c o u r s e s i n p h y s i o l o g i c a l c h e m i s t r y w i t h Mendel as h i s i n s t r u c t o r . McCollum a l s o worked i n Osborne's l a b o r a t o r y a t t h e C o n n e c t i c u t A g r i c u l t u r a l E x p e r i m e n t S t a t i o n f o r a few months and was h i r e d by t h e U n i v e r s i t y o f W i s c o n s i n A g r i c u l t u r a l E x p e r i m e n t S t a t i o n a s a c h e m i s t t o a n a l y z e t h e i n t a k e and o u t g o o f c a t t l e on r e s t r i c t e d r a t i o n s , i . e . , c a t t l e b e i n g g i v e n f o o d o b t a i n e d from a s i n g l e crop p l a n t . Not c o n t e n t w i t h such a l a b o r i o u s and t i m e consuming e n t e r p r i s e , M c C o l l u m s t a r t e d , on h i s own i n i t i a t i v e i n 1908, t h e f i r s t r a t c o l o n y i n t h e U n i t e d S t a t e s t o be d e v o t e d e x c l u s i v e l y t o t h e study o f n u t r i t i o n . More than f i v e y e a r s l a t e r he was a b l e t o r e port that a s u c c e s s f u l d i e t f o r a r a t required the i n c l u s i o n o f an e t h e r e x t r a c t o f egg o r b u t t e r . (15) C o n f u s i o n , U n c e r t a i n t y and D i s a g r e e m e n t A l t h o u g h t h e w h i t e o r a l b i n o r a t was t o become t h e a n i m a l o f c h o i c e i n n u t r i t i o n a l i n v e s t i g a t i o n s , c h i c k e n s , p i g e o n s , cows, p i g s , g u i n e a p i g s , r a b b i t s , m i c e and dogs (and p r o b a b l y a few c a t s ) were a l s o used i n an a t t e m p t t o b e t t e r u n d e r s t a n d t h e p h y s i o l o g i c a l p r o p e r t i e s o f foods. Species' d i f f e r e n c e s i n dietary needs were o n l y s l o w l y r e c o g n i z e d , a s i t u a t i o n t h a t l e d t o much c o n f u s i o n , u n c e r t a i n t y and d i s a g r e e m e n t o v e r t h e r e s u l t s and s i g n i f i c a n c e o f e x p e r i m e n t a l work. What k i n d o f s e n s e , e . g . , c o u l d be made o f t h e f a c t t h a t g u i n e a p i g s and human b e i n g s c o u l d develop scurvy w h i l e r a t s c o u l d not? Why was i t s o d i f f i c u l t t o induce d e f i c i e n c y d i s e a s e s i n c a t t l e ? Was p e l l a g r a c o n f i n e d t o humans a l o n e ? These were some o f t h e q u e s t i o n s t h a t f a c e d n u t r i t i o n r e s e a r c h e r s i n t h e p e r i o d o f p r i m a r y c o n c e r n i n t h i s p a p e r , 19101925. O t h e r p r o b l e m s added t o t h e h i g h l e v e l o f doubt and s k e p t i c i s m t h a t p r e v a i l e d . C a s i m i r Funk, e . g . , i n i s o l a t i n g what he t h o u g h t t o be t h e amine n e c e s s a r y f o r t h e c o n t r o l o f b e r i - b e r i , a l s o o b t a i n e d n i c o t i n i c a c i d f r o m r i c e p o l i s h i n g s and f r o m y e a s t , b o t h e x c e l l e n t c u r a t i v e s / p r e v e n t i v e s o f b e r i - b e r i . (16) Even though Funk had s u g g e s t e d t h a t e a c h d e f i c i e n c y d i s e a s e was t h e r e s u l t o f t h e l a c k o f a s p e c i f i c c h e m i c a l e n t i t y , he n e v e r r e a l i z ed i n 1911-1935 t h a t t h e p e l l a g r a c u r e / p r e v e n t i v e , n i c o t i n i c a c i d , was s i t t i n g i n a c h e m i c a l l y p u r e s t a t e i n h i s t e s t t u b e s . H o p k i n s was l a t e i n r e a l i z i n g t h a t one o f h i s ' a c c e s s o r y f o o d s u b s t a n c e s ' , l a t e r c a l l e d v i t a m i n s , was c o n t a i n e d i n t h e f a t o f m i l k as w e l l a s i n t h e f a t s o f o t h e r a n i m a l s and i n c e r t a i n p l a n t t i s s u e s as w e l l . ( 1 7 , 18) Osborne and Mendel a c t u a l l y r e a c h e d a p o i n t i n t h e i r r e s e a r c h i n 1912 t h a t made them c o n f i d e n t t h a t f a t s as f a t s were n o t n e c e s s a r y a t a l l i n t h e d i e t s o f e x p e r i m e n t a l r a t s . (19) M c C o l l u m went s o f a r as t o s t a t e a t one t i m e t h a t a s i d e f r o m t h e a n t i - s c o r b u t i c s u b s t a n c e ( l a t e r t o be known a s v i t a m i n C ) , t h e r e were o n l y two new s u b s t a n c e s t o be c o n c e r n e d

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with in rat n u t r i t i o n : ' f a t - s o l u b l e A' and w a t e r - s o l u b l e B'. (20) M c C o l l u m made i t v e r y c l e a r i n t h i s 1916 p a p e r t h a t he d i d n o t l i k e t h e word v i t a m i n e ( w h i c h , n e v e r t h e l e s s , became v i t a m i n i n 1920). (2J_) The c o n f l i c t i n g r e s u l t s and v a r i o u s i n t e r p r e t a t i o n s o b t a i n e d f r o m a n i m a l f e e d i n g e x p e r i m e n t s w e r e , o f c o u r s e , r e l a t e d t o many f a c t o r s , a p o i n t t h a t c a n be i l l u s t r a t e d i n v a r i o u s ways. The a u t h o r has s e l e c t e d what he c a l l s t h e 'mi 1 k - i s - g o o d - f o r - y o u - b u t h o w - m u c h - i s - e n o u g h ? ' p r o b l e m t h a t emerged, f o r t h e most p a r t , i n t h e i n v e s t i g a t i o n s o f H o p k i n s a t Cambridge and Osborne and Mendel a t t h e C o n n e c t i c u t A g r i c u l t u r a l E x p e r i m e n t S t a t i o n i n New Haven d u r i n g t h e i n t e r v a l , 1911-1922. Milk

i s Good f o r You B u t . . .

In J u l y 1911, Osborne and Mendel c o m p l e t e d t h e second p a r t o f t h e monograph c i t e d e a r l i e r , (22) p u b l i s h e d by t h e C a r n e g i e I n s t i t u t i o n w h i c h had p a r t i a l l y s u p p o r t e d O s b o r n e ' s r e s e a r c h e s f o r a number o f y e a r s . T h i s monograph o f 138 p a g e s , c o n t a i n i n g many t a b l e s and 129 c h a r t s , was t h e most c o m p r e h e n s i v e document publ i s h e d t o d a t e on a n i m a l f e e d i n g e x p e r i m e n t s . I t r e p r e s e n t e d t h e d a t a , r a t i o n a l e s and c o n c l u s i o n s o f n e a r l y two y e a r s and i l l u s t r a t e d v i v i d l y Osborne and M e n d e l ' s m a j o r p r e m i s e : d i f f e r e n t prot e i n s , e i t h e r i n d i v i d u a l l y o r i n c o m b i n a t i o n , were r e m a r k a b l y d i f ferent i n t h e i r a b i l i t i e s to e i t h e r maintain a r a t at constant w e i g h t o r t o a l l o w m a r g i n a l o r even normal g r o w t h t o o c c u r i n these animals. Osborne and Mendel were a b l e t o make t h i s p r e m i s e i n p a r t bec a u s e they had d e v e l p e d two d i e t s t h a t a l l o w e d r a t s t o grow n o r mally: t h e m i x e d f o o d (dog b i s c u i t , s u n f l o w e r and o t h e r s e e d s , f r e s h v e g e t a b l e s and s a l t ) and t h e m i l k f o o d (whole m i l k powder, s t a r c h , a m i x t u r e o f m i n e r a l s a l t s and l a r d ) . I t was t h e l a t t e r d i e t a r y t h a t , when c o m p r i s i n g a l l o r o n l y a p a r t o f an e x p e r i m e n t a l r a t i o n , w o u l d p r o v e t o be a s o u r c e o f much c o n f u s i o n a b o u t the n u t r i t i o n a l v a l u e o f m i l k . In J u l y 1912, H o p k i n s had p u b l i s h e d h i s f i r s t p a p e r c o n c e r n ed w i t h f e e d i n g s m a l l amounts o f w h o l e m i l k t o r a t s on e x p e r i m e n tal diets. In A u g u s t he w r o t e s e p a r a t e l e t t e r s t o Osborne and M e n d e l . The f o l l o w i n g i s f r o m t h e l e t t e r t o O s b o r n e : I t r u s t t h a t n e i t h e r y o u n o r P r o f . Mendel w i l l t h i n k t h a t my p a p e r w h i c h has j u s t a p p e a r e d i n t h e J o u r n a l o f P h y s i o l o g y ( o f w h i c h I am now s e n d i n g you a r e p r i n t ) i n t r u d e s u n d u l y i n t o y o u r domain. As a m a t t e r o f f a c t I have engaged i n such f e e d i n g e x p t s . f o r a good many y e a r s , and t h e g r e a t p l e a s u r e w i t h w h i c h I read y o u r work...was (human n a t u r e b e i n g what i t i s ) somewhat s p o i l t by t h e f a c t t h a t i t t a k e s t h e w i n d o u t o f t h e s a i l s o f many s c o r e s o f e x p e r i m e n t s o f my own! (23)

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H o p k i n s s t a t e d f u r t h e r t h a t he was a b l e t o o b t a i n growth i n r a t s on d i e t s composed o f amino a c i d m i x t u r e s o n l y when s m a l l amounts of " y e a s t - e x t r a c t e t c . were a d d e d . " He s a i d n o t h i n g w h a t s o e v e r a b o u t t h e m i l k s u p p l e m e n t s w h i c h u n d e r l i e h i s e n t i r e 1912 p a p e r . T h i s b e i n g summer, Osborne and Mendel o f t e n went t o t h e i r v a c a t i o n r e t r e a t s from where they c a r r i e d on an e x t e n s i v e c o r r e spondence. In one o f t h e s e l e t t e r s , Mendel remarked t o O s b o r n e : F. G. H o p k i n s w r o t e me a p l e a s a n t n o t e , a s he p r e s u m a b l y d i d t o you. I have not y e t seen t h e r e p r i n t w h i c h he s t a t e s he has s e n t , but I s h a l l r e p l y t h a t we do not r e g a r d o u r s e l v e s a s the s o l e s c i e n t i s t s e n t i t l e d t o f e e d rats. E v i d e n t l y he i s i m p r e s s e d w i t h the i d e a o f a " g r o w t h s u b s t a n c e " o r something o f t h a t s o r t , a s i d e from the o r d i n a r y n u t r i e n t s . (2M) N e a r l y two weeks l a t e r , a f t e r wrote a g a i n to Osborne:

reading Hopkins'

paper,

Mendel

The H o p k i n s paper was v e r y i n t e r e s t i n g . . . . O f c o u r s e h i s f o o d s a r e a l l c o m p a r a t i v e l y c r u d e ; and o u r p o s i t i v e e x p ' t w i t h f a t - f r e e a r t . p. f . m. i s h a r d t o u n d e r s t a n d e x c e p t on the a s s u m p t i o n o f a s t o r e o f t h e growth f a c t o r o f t h e body. ... The e n e r g y p r o p o s i t i o n g e t s a g r e a t s e t back by H o p k i n s work and o u r g r o w t h p a p e r . ...However, t h e s e 'acc e s s o r y ' e x t r a c t s may be an i n v a l u a b l e a i d t o us i n s t u d y ing t h e c o m p a r a t i v e r o l e o f p r o t e i n s i f t h e y p e r m i t us t o keep o u r a n i m a l s i n b e t t e r form. The p o i n t i n o u r paper which Hopkins q u e s t i o n s i s very important i . e . , t h e q u a n t i t y o f p. f . m. t h a t i s s u f f i c i e n t , and i t needs t o be r e p e a t e d p e r h a p s . (25) E x p l a n a t i o n o f two terms m e n t i o n e d above i s i n o r d e r . Osborne and Mendel had been s u c c e s s f u l i n g r o w i n g r a t s on t h e m i l k f o o d diet. In o r d e r t o have a l l t h e a d v a n t a g e s o f m i l k w i t h o u t t h e p r o t e i n c o n t e n t , t h e y d e v e l o p e d what t h e y c a l l e d p r o t e i n - f r e e m i l k or p. f . m. S i n c e t h e i r p r i m a r y r e s e a r c h c o n c e r n l a y w i t h p r o t e i n s and b e c a u s e m i l k was a c o m p l e t e f o o d by i t s e l f , t h e y assumed t h a t i f the w a t e r and p r o t e i n s ( m o s t l y c a s e i n ) were removed from m i l k the r e s u l t a n t p r o d u c t w o u l d have a l l t h e c h a r a c t e r i s t i c s o f m i l k w h i l e a l l o w i n g the e x p e r i m e n t e r s t o i n t r o d u c e o t h e r p r o t e i n s i n t o animal d i e t s . Osborne and Mendel w o u l d t h e r e b y be a b l e t o e v a l u a t e the n u t r i t i v e v a l u e o f any p r o t e i n when i t was f e d w i t h p. f . m. (26) A r t . p. f . m. o r a r t i f i c i a l p r o t e i n - f r e e mi 1k, was a l o g i c a l n e x t s t e p i n t h e i r work. S i n c e they were a t t e m p t i n g t o grow r a t s on t o t a l l y s y n t h e t i c d i e t s , i . e . , d i e t s composed o f pure c h e m i c a l s , Osborne and Mendel a t t e m p t e d t o d u p l i c a t e the c o m p o s i t i o n o f p.f.m. u s i n g l a b o r a t o r y o r c o m m e r i c a l g r a d e c h e m i c a l s a l t s and l a c t o s e i n c o m b i n a t i o n s t h a t a p p r o x i m a t e d as c l o s e l y a s p o s s i b l e the compos i t i o n o f n a t u r a l p. f. m. (27)

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The C o n n e c t i c u t r e s e a r c h e r s had g r e a t s u c c e s s u s i n g t h e s e p r o t e i n - f r e e a d j u v a n t s b u t o n l y up t o a p o i n t . While extensive g r o w t h m i g h t o c c u r e s p e c i a l l y w i t h t h e n a t u r a l p. f . m., s o o n e r or l a t e r the r a t s stopped growing. Some c o u l d be m a i n t a i n e d a t n e a r l y c o n s t a n t w e i g h t f o r months b u t i n e v i t a b l y a l l r a t s w o u l d b e g i n t o d e c l i n e on d i e t s t h a t c o n t a i n e d p. f . m. o r a r t . p. f . m. As Osborne w r o t e t o M e n d e l : " I t i s becoming more and more e v i d e n t t h a t they [the r a t s j g e t some e s s e n t i a l s u b s t a n c e f r o m t h e m i l k , t h e n a t u r e o f w h i c h we do n o t y e t know." (28) ...How Much i s Enough? A l t h o u g h Osborne and Mendel were coming t o terms w i t h t h e p r o b a b i l i t y t h a t 'some e s s e n t i a l s u b s t a n c e ' was p r e s e n t i n m i l k , t h e y had a l r e a d y t h a t y e a r (1912) p u b l i s h e d two a r t i c l e s w h i c h l e d r e a d e r s t o b e l i e v e t h a t Osborne and Mendel had d e v e l o p e d a t o t a l l y s y n t h e t i c and s u c c e s s f u l d i e t . In e a r l y 1913, H o p k i n s and N e v i l l e q u e s t i o n e d t h e v a l i d i t y o f some o f t h e e x p e r i m e n t a l r e s u l t s obt a i n e d by Osborne and M e n d e l . They s a i d t h a t t h e A m e r i c a n s ' r e s u l t s seemed t o i n d i c a t e t h a t young r a t s c o u l d grow on p u r e l y a r t i f i c i a l d i e t s and, t h e r e f o r e , that accessory f a c t o r s o r v i t a mines were d i s p e n s a b l e . " B u t . . . t h e y c o n t r a d i c t what i s now a c o n s i d e r a b l e body o f e x p e r i e n c e , and t h e e x p e r i m e n t s w h i c h y i e l d e d them seem t o c a l l f o r r e p e t i t i o n . " (29) H o p k i n s and N e v i l l e went on t o s a y t h a t t h e y had f e d r a t s on the d i e t used by Osborne and Mendel w i t h one s i g n i f i c a n t d i f f e r ence: they e x t r a c t e d t h e p r o t e i n and s t a r c h w i t h a l c o h o l and r e c r y s t a l l i z e d t h e l a c t o s e ( m i l k s u g a r ) f r o m an aqueous s o l u t i o n by a d d i t i o n o f a l c o h o l . The methods o f f e e d i n g were t h e same as t h o s e d e s c r i b e d by H o p k i n s i n h i s 1912 p a p e r . The r e s u l t s ? Rats c e a s e d t o grow, a l l b e f o r e 15 days on t h e e x t r a c t e d d i e t . After w e i g h t d e c l i n e s e t i n some o f t h e r a t s were g i v e n 2 c c o f m i l k p e r day p e r r a t f o l l o w i n g w h i c h g r o w t h resumed and h e a l t h was ma i n t a i n e d . The a u t h o r s a l s o s t a t e d t h a t t h e i r r a t s behaved d i f f e r e n t l y f r o m t h e t h r e e a n i m a l s f e d by Osborne and Mendel b u t t h a t t h e d i f f e r e n c e c o u l d n o t be e x p l a i n e d . H o p k i n s had f o u n d i n e a r l i e r e x p e r i m e n t s t h a t v e r y s m a l l amounts o f s u b s t a n c e s e x t r a c t e d w i t h h o t a l c o h o l f r o m f o o d s w o u l d , when added t o d e f i c i e n t d i e t s , r e n d e r them c a p a b l e o f some g r o w t h p o t e n t i a l . I t was a p p a r e n t , t o Hopk i n s a t l e a s t , t h a t h o t a l c o h o l i c e x t r a c t i o n s o f f o o d s removed s m a l l amounts o f e f f e c t i v e g r o w t h p r o m o t e r f r o m t h e d i e t . Ether e x t r a c t i o n as c a r r i e d o u t by Osborne and Mendel i n t h e i r f a t - f r e e e x p e r i m e n t s was c o n s i d e r e d by H o p k i n s t o be i n f e r i o r t o a l c o h o l i c extraction. "The p u r p o s e o f t h e p r e s e n t n o t e i s t o i n d i c a t e t h a t there i s s t i l l reason f o r a c o n t i n u a n c e o f t h e search f o r s p e c i a l a c c e s s o r y s u b s t a n c e s o f p o t e n t i n f l u e n c e upon g r o w t h . I t should be p o i n t e d o u t t h a t Osborne and Mendel t h e m s e l v e s a d m i t t h a t s u c h s u b s t a n c e s may e x i s t . " (30)

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In l a t e F e b r u a r y o f 1913, Osborne and Mendel r e s p o n d e d t o t h e note i n the Biochemical Journal i n a l e t t e r t o Hopkins i n which they s t a t e d t h a t t h e y had been s u r p r i s e d by t h e outcomes o f t h e e x p e r i m e n t s on p u r e l y a r i f i c i a l d i e t s and t h a t newer e x p e r i m e n t s d e m o n s t r a t e d e i t h e r p o o r g r o w t h o r none a t a l l on f a t - f r e e a r t i f i c i a l d i e t s . They a l s o s a i d i n t h i s l e t t e r t h a t t h e y b e l i e v e d t h e y had a c l u e as t o t h e c a u s e o f t h e d i f f e r e n c e s i n r e s u l t s . Furthermore : A l l o f us a r e d e a l i n g w i t h p r o b l e m s w h i c h i n v o l v e e x t r e m e l y c o m p l i c a t e d c o n d i t i o n s , and we must be p r e p a r ed t o be c o n f r o n t e d w i t h u n e x p e c t e d r e s u l t s f o r some t i m e t o come. F o r e x a m p l e , o u r e x p e r i e n c e i n s t u d y i n g the e f f e c t o f small a d d i t i o n s o f m i l k t o v a r i o u s a r t i f i c i a l d i e t s has r a r e l y been t h a t w h i c h we e x p e c t e d f r o m y o u r r e p o r t . . . . W e have no i n t e n t i o n o f i n t r u d i n g on y o u r f i e l d i n making t h e s e e x p e r i m e n t s , n o r do we expect t o p u b l i s h o u r r e s u l t s as evidence that your experiments i n v o l v e e r r o r , o r t h a t your p u b l i s h e d conc l u s i o n s a r e i n c o r r e c t . ...We a r e c o n v i n c e d t h a t t h e p u r s u i t o f t h e g r o w t h s u b s t a n c e i n v o l v e s some o f t h e most i m p o r t a n t p r o b l e m s o f b i o c h e m i s t r y , and t h a t i t w i l l n o t be l o n g b e f o r e t h e combined e f f o r t s o f t h o s e who engage i n i t w i l l s o l v e a t l e a s t some o f them. (31) No m e n t i o n was made o f t h e a l c o h o l v. e t h e r e x t r a c t i o n o f foods but i n t h e i r paper r e p o r t i n g t h e r e s u l t s o f f e e d i n g f a t f r e e d i e t s Osborne and Mendel s t a t e d c l e a r l y t h a t none o f t h e f o o d s had been e x t r a c t e d w i t h h o t a l c o h o l . F u r t h e r m o r e , t h e y f o u n d i t d i f f i c u l t t o b e l i e v e ( t h i s was i n mid-1912) t h a t skimmed m i l k c o u l d c o n t a i n an i m p o r t a n t l i p o i d ( f a t - l i k e s u b s t a n c e s s u c h as l e c i t h i n , e.g.) i n any a d e q u a t e amount, " w h i l e . . . b u t t e r which must c o n t a i n some compounds o f t h i s t y p e C i s ] i n a d e q u a t e . " (32) Here we c a n s e e a m a j o r d i f f e r e n c e i n t h e t h i n k i n g o f H o p k i n s and t h a t o f Osborne and M e n d e l . For Hopkins, very small q u a n t i t i e s o f unknown o r g a n i c s u b s t a n c e s , s o l u b l e i n h o t a l c o h o l , were necessary f o r growth. Osborne and Mendel were s t i l l c l i n g i n g t o t h e b e l i e f t h a t e f f e c t i v e n u t r i e n t s w o u l d have t o be p r e s e n t i n q u a n t i t i e s g r e a t e r than t h e ' t r a c e s ' i m p l i e d i n Hopkins' e x p e r i ments. S t i l l , O s b o r n e and Mendel r e c o g n i z e d t h a t o n l y a d d i t i o n a l research would r e s o l v e the c o n f l i c t i n g r e s u l t s . H o p k i n s r e p l i e d t o t h e i r l e t t e r on March 1*4, 1913- (33) He a p o l o g i z e d f o r t h e t o n e o f t h e B i o c h e m i c a l J o u r n a l n o t e : "The f a c t i s I have done so much work i n t h i s ( n o t v e r y s u c c e s s f u l ) endeavo u r t o s e p a r a t e t h e unknown s u b s t a n c e s w h i c h a f f e c t g r o w t h , t h a t when y o u r p a p e r ...came o u t I s u f f e r e d f r o m an a t t a c k o f n e r v e s . " (3*0 H o p k i n s went on t o d i s c u s s h i s s u c c e s s w i t h f e e d i n g s m a l l q u a n t i t i e s o f m i l k t o g r o w i n g r a t s , and s a i d t h a t t h e s t o r y was c l e a r under h i s e x p e r i m e n t a l conditions. F u r t h e r m o r e , he commented t h a t t h e o r g a n i c c o n s t i t u e n t s w h i c h he l a b e l e d , 'exogenous f

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1

g r o w t h hormones, were n o t t h e same as Funk's v i t a m i n e s . Hopkins was most e m p h a t i c i n p o i n t i n g o u t t h e need t o t h o r o u g h l y e x t r a c t f o o d s o as t o remove t h e g r o w t h f a c t o r s . O n l y when t h e b a s a l d i ­ e t a r y was w e l l e x t r a c t e d , he c o n c l u d e d , c o u l d t h e e f f e c t s o f s m a l l addenda (such as m i l k ) make t h e m s e l v e s v i s i b l e . "That such e x t r a ­ o r d i n a r y s m a l l amounts o f t h e 'χ-factors' c a n a c t , r a t h e r d i m i n ­ i s h e s t h e p r a c t i c a l i m p o r t a n c e o f t h e phenomenon; b u t n o t , I t h i n k , i t s t h e o r e t i c a l i n t e r e s t . " (35.) E i g h t years l a t e r Hopkins would e x p r e s s a d i f f e r e n t a t t i t u d e toward t h e p r a c t i c a l importance o f t h e phenomenon; by then v i t a m i n s were 'very b i g ' i n d i e t e t i c s . Osborne and Mendel p r e p a r e d a d r a f t l e t t e r r e p l y t o H o p k i n s and i t c a n be assumed t h a t a f i n a l v e r s i o n was s e n t , most p r o b a ­ b l y i n A p r i l 1913· (36) T h i s l e t t e r r e - e m p h a s i z e d t h e p r o b l e m s o f a p p a r e n t l y c o n f l i c t i n g r e s u l t s and t h e methods used t o s o l v e such p r o b l e m s . They s a i d t h a t t h e y had come t o a g r e e w i t h H o p k i n s ' c o n c l u s i o n t h a t some o r g a n i c s u b s t a n c e , i n s m a l l q u a n t i t y , was n e c e s s a r y f o r g r o w t h . The p. f . m. d i e t s seemed t o be a d e q u a t e f o r a b o u t 80 d a y s ; t h e a r t . p. f . m. d i e t s were h i g h l y v a r i a b l e i n t h e i r r e s u l t s ; t h e s t a n d a r d m i l k f o o d was a l w a y s a d e q u a t e , "...we now t h i n k t h a t m i l k c o n t a i n s s o m e t h i n g e s s e n t i a l f o r g r o w t h w h i c h i s e i t h e r p a r t l y o r w h o l l y d e s t r o y e d o r removed by o u r p r o c e s s o f making t h e p r o t e i n f r e e m i l k . " (37) Osborne and Mendel a l s o s t a t e d t h e i r b e l i e f t h a t t h e young r a t i s b o r n w i t h a s u r p l u s o f t h i s s u b s t a n c e , upon w h i c h i t c a n grow f o r a p e r i o d o f t i m e . Fur­ t h e r m o r e , they c o n f i r m e d t h a t a d d i t i o n o f s m a l l q u a n t i t i e s o f a l ­ c o h o l i c e x t r a c t s o f some f o o d s d i d i n d e e d c o n t a i n a g r o w t h p r o ­ m o t i n g s u b s t a n c e a l t h o u g h t h e y d i d n o t know w h e t h e r i t was o r g a n ­ i c , inorganic o r a combination. And y e t , t h e y s a i d f u r t h e r on t h a t a l c o h o l i c e x t r a c t i o n o f t h e p r o t e i n s c o u l d n o t be s i g n i f i c a n t i n t h e i r work s i n c e t h e i r r e s u l t s were c o n t r o l l e d by u s i n g t h e same p r o t e i n s i n d i e t s w h i c h were i n c a p a b l e o f i n d u c i n g g r o w t h i n rats. I nter i m L e s s than f o u r months a f t e r t h e w r i t i n g o f t h e l e t t e r t o Hop­ k i n s , M c C o l l u m a s w e l l a s Osborne and Mendel had o b s e r v e d and pub­ l i s h e d t h e f a c t t h a t b u t t e r c o n t a i ned someth i ng t h a t made r a t s grow. In t h i s same y e a r , 1913, H o p k i n s became i n v o l v e d w i t h t h e a d m i n i ­ s t r a t i o n o f t h e M e d i c a l R e s e a r c h C o m m i t t e e , (M. R. C ) , an arm o f t h e new N a t i o n a l I n s u r a n c e P r o g r a m i n G r e a t B r i t a i n . As a c o n ­ s e q u e n c e , H o p k i n s was n o t a b l e t o c a r r y o u t any s i g n i f i c a n t r e ­ s e a r c h on h i s 'growth p r o m o t i n g a c c e s s o r y f a c t o r s ' i n m i l k . N e v e r ­ t h e l e s s , h i s a s s o c i a t i o n w i t h t h e M. R. C. u l t i m a t e l y b r o u g h t h i m i n t o i n t e r n a t i o n a l p r o m i n e n c e and p r o v i d e d h i m (and h i s a s s o c i ­ a t e s ) w i t h a f o r u m f r o m w h i c h he c o u l d speak t o a l a r g e r a u d i e n c e a b o u t t h e g r o w t h f a c t o r s , b e t t e r known a s v i t a m i n s . W i t h t h e o u t b r e a k o f W o r l d War I i n 191**, t h e need f o r p r a c ­ t i c a l d i e t e t i c s consumed t h e t i m e and l a b o r s o f many a B r i t i s h n u t r i t i o n i s t s o t h a t w h i l e b a s i c r e s e a r c h i n t h e v i t a m i n s was c o n -

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d u c t e d t o a l i m i t e d e x t e n t i n G r e a t B r i t a i n , i t was i n t h e U n i t e d S t a t e s t h a t such r e s e a r c h a l m o s t l i t e r a l l y " t o o k o f f . " McCollum i n W i s c o n s i n and Osborne and Mendel i n C o n n e c t i c u t p u r s u e d t h e i r b r e a k t h r o u g h s w i t h many s i g n i f i c a n t c o n t r i b u t i o n s . M c C o l l u m , i n 1915, showed t h a t t h e r e were two g r o w t h s u b s t a n c e s needed by r a t s , one s o l u b l e i n f a t s , t h e o t h e r s o l u b l e i n w a t e r o r a l c o h o l . (.38, 39) In t h e s e same p a p e r s McCollum a l s o showed t h a t n o n - e x t r a c t e d l a c t o s e ( o r u n r e c r y s t a 1 1 i z e d l a c t o s e ) was c a p a b l e o f s u p p o r t i n g some growth i n r a t s . F u r t h e r m o r e , he p o i n t e d o u t t h a t c o m m e r c i a l l a c t o s e o r even some l a b o r a t o r y g r a d e l a c t o s e c o n t a i n e d s m a l l q u a n t i t i e s o f a g r o w t h - p r o m o t i n g s u b s t a n c e o r s u b s t a n c e s , an o b s e r v a t i o n c o n f i r m e d by Drummond i n 1SΊ6- (40) Osborne and Mendel c o n t i n u e d t h e i r e x t e n s i v e s t u d i e s o f t h e n u t r i t i v e values o f p r o t e i n s w h i l e extending t h e l i s t o f foods known t o c o n t a i n v i t a m i n e s . k2) They a t t e m p t e d t o i s o l a t e the growth p r o m o t e r i n b u t t e r w i t h no s u c c e s s b u t t h e y made s i g ­ n i f i c a n t p r o g r e s s i n t h e i r q u e s t f o r a t o t a l l y s y n t h e t i c d i e t by d e v e l o p i n g an e x c e l l e n t s a l t m i x t u r e f o r t h e b a s a l d i e t a r i e s o f t h e i r r a t s . (43) Thus, a t t h e war's end i n 1918, v i t a m i n e s o r v i t a m i n s o r g r o w t h - p r o m o t i n g a c c e s s o r i e s , b o t h f a t - s o l u b l e A and w a t e r - s o l u b l e Β were p a r t o f t h e l e x i c o n o f n u t r i t i o n a l s c i e n c e . Even though i t w o u l d r e q u i r e more than a n o t h e r decade b e f o r e t h e f i r s t v i t a m i n was c h e m i c a l 1 y i n d e n t i f i e d , t h e need f o r v i t a m i n s i n r a t g r o w t h was no l o n g e r i n d o u b t . What i s b o t h r e m a r k a b l e and f a s c i n a t i n g h e r e i s t h a t t h e r a t s ' needs were i m m e d i a t e l y t r a n s l a t e d i n t o human needs. The war had evoked h i g h l e v e l s o f i n t e r e s t i n human n u ­ trition. How much o f w h i c h f o o d s were r e q u i r e d f o r good h e a l t h ? The d i s c o v e r i e s made i n t h e n u t r i t i o n a l r e q u i r e m e n t s o f r a t s o n l y served t o heighten t h i s i n t e r e s t . In s h o r t , i f i t was good f o r r a t s i t was good f o r humans. Y e s , b u t How Much M i l k

i sReally

Enough?

W h i l e b o t h t h e f a t - s o l u b l e and w a t e r - s o l u b l e v i t a m i n s had been f o u n d i n a v a r i e t y o f f o o d s , t h e p r o b l e m o f g r o w t h on m i l k d i e t s had n o t y e t been r e s o l v e d , a t l e a s t n o t i n the minds o f Osborne and M e n d e l . In a 1918 p u b l i c a t i o n t h e y s a i d t h a t t h e y were im­ p r e s s e d by t h e " a p p a r e n t d i s c r e p a n c i e s i n the q u a n t i t a t i v e r e l a ­ t i o n s o f the amounts o f m i l k r e q u i r e d t o f u r n i s h t h e v i t a m i n e f a c ­ t o r i n o u r e x p e r i m e n t s i n c o n t r a s t w i t h t h o s e o f H o p k i n s . " (kk) They went on t o c l a i m (and j u s t l y s o ) t h a t a b e t t e r u n d e r s t a n d i n g o f t h e q u a n t i t a t i v e r e l a t i o n s was h i g h l y d e s i r a b l e . "In view o f the r e s u l t s o f H o p k i n s ' e x p e r i m e n t s i t has become g e n e r a l l y be­ l i e v e d t h a t m i l k i s one o f t h e r i c h e s t s o u r c e s o f the w a t e r s o l u b l e v i t a m i n e s among o u r f o o d p r o d u c t s . " (45_) The e x p e r i m e n t a l r e s u l t s shown i n t h i s p u b l i c a t i o n were c e r t a i n l y n o t i n a c c o r d w i t h t h o s e o f H o p k i n s i n h i s 1912 p a p e r . Osborne and Mendel t r i e d , and a p p a r e n t l y f a i l e d , i n two a d ­ d i t i o n a l e x p e r i m e n t s t o r e c o n c i l e t h e i r v a l u e s f o r the q u a n t i t i e s

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WillMilkMakeThemGrow?

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o f m i l k needed w i t h the q u a n t i t i e s c l a i m e d by H o p k i n s . (46, hj) What were t h e s e i r r e c o n c i l a b l e v a l u e s ? H o p k i n s c l a i m e d t h a t 2 cc o f f r e s h w h o l e m i l k w o u l d s u f f i c e f o r a young r a t o f 50-100 g w e i g h t when g i v e n d a i l y . Osborne and M e n d e l ' s d e t e r m i n a t i o n s i n ­ d i c a t e d t h a t 10-16 cc o f f r e s h w h o l e m i l k w o u l d be needed on a daily basis. I f one makes a c r u d e e s t i m a t e o f the e q u i v a l e n t q u a n t i t i e s n e c e s s a r y f o r young humans, u s i n g body w e i g h t as t h e m a j o r c r i t e r i o n , then a baby o f 5 kg w o u l d r e q u i r e a b o u t 200 c c p e r day a c c o r d i n g t o H o p k i n s and 1000 t o l 6 0 0 c c p e r day a c c o r d i n g t o Osborne and M e n d e l , j u s t t o s u p p l y t h e n e c e s s a r y water-soluble vitamin(s). (Of c o u r s e , such c a l c u l a t i o n s assume t h a t no o t h e r f o o d s c o n t a i n i n g the v i t a m i n ( s ) were b e i n g s u p p l i e d . ) D u r i n g t h e w i n t e r o f 1918/19, w h i l e Mendel was a member o f the A l l i e d Food Commission i n E u r o p e , he v i s i t e d H o p k i n s i n Cam­ bridge. Upon h i s r e t u r n the two r e s e a r c h g r o u p s c o r r e s p o n d e d on the s u b j e c t o f the m i l k minimum n e c e s s a r y f o r a g r o w i n g r a t . In December 1919, Osborne and Mendel s e n t H o p k i n s a copy o f a p a p e r they were g o i n g t o p u b l i s h , " . . . p r e s e n t i n g o u r newest e x p e r i e n c e w i t h m i l k p r o d u c e d i n the summer months." (48) H e r e , Osborne and Mendel were d e a l i n g w i t h the p o s s i b i l i t y t h a t summer m i l k was r i c h e r i n v i t a m i n e than w i n t e r m i l k , a p l a u s i b l e h y p o t h e s i s s i n c e d a i r y c a t t l e a t e v e r y d i f f e r e n t r a t i o n s i n the two s e a s o n s . Hop­ k i n s was c e r t a i n l y a l e r t t o t h i s p o s s i b i l i t y and i n a d d i t i o n , i t can be s a i d t h a t e x t e n s i v e r e s e a r c h by a number o f p e o p l e i n v o l v ­ ed w i t h a g r i c u l t u r e and d a i r y i n g i n d i c a t e d t h a t w h i l e f a t - s o l u b l e A was s i g n i f i c a n t l y v a r i a b l e w i t h r e s p e c t t o s e a s o n , the w a t e r s o l u b l e Β a p p e a r e d t o be f a i r l y c o n s t a n t i n i t s c o n c e n t r a t i o n i n m i l k t h r o u g h o u t the y e a r . In t h i s same l e t t e r t o H o p k i n s , Osborne and Mendel s u g g e s t e d t h a t H o p k i n s comment on t h e i r new p a p e r o r p e r h a p s even p u b l i s h jointly. We s h a l l a l s o be g l a d t o have s u g g e s t i o n s as t o the b e s t way t o p r e s e n t o u r combined r e s u l t s t o t h e p u b l i c . Our c h i e f c o n c e r n i s t o a v o i d a d d i n g t o the c o n f u s i o n a 1 ready e x i s t i n g in respect to t h i s important s u b j e c t . Perhaps w i t h the h e l p o f o u r newest e x p e r i m e n t s you can now put the e n t i r e m a t t e r o f the g r o w t h - p r o m o t i n g e f f e c t o f m i l k i n i t s p r o p e r r e l a t i o n s . (49) H o p k i n s r e p l i e d i n F e b r u a r y 1920, p o l i t e l y d e c l i n i n g any j o i n t p u b l i c a t i o n and u r g e d Osborne and Mendel t o p u b l i s h t h e i r work i n ­ dependently of h i s r e s u l t s . L e t me say a t once t h a t my e x p e r i e n c e d u r i n g the p a s t y e a r has c o n v i n c e d me t h a t you a r e p e r f e c t l y r i g h t i n y o u r c o n c l u s i o n t h a t t h e s m a l l q u a n t i t i e s o f mi l k u s e d i n the e x p e r i m e n t s p u b l i s h e d i n my p a p e r o f 1912 are n o t , under a v e r a g e c o n d i t i o n s , c a p a b l e o f m a i n t a i n i n g g r o w t h i n the a n i m a l . N e v e r t h e l e s s I have, w i t h c e r -

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t a i n precaution, succeeded, i n three experiments i n r e ­ p r o d u c i n g t h e r e s u l t s o f the p a p e r i η q u e s t i o n . . . . CI e a r 1 y however, c o n s i d e r a b l y more m i l k i s r e q u i r e d f o r c o n t i n ­ ued g r o w t h a t a h e a v i e r w e i g h t . ...You w i l l u n d e r s t a n d t h a t i n my J o u r n a l o f P h y s i o l o g y p a p e r I had no i d e a o f d e c i d i n g t h e a b s o l u t e amount o f m i l k r e q u i r e d t o s u p p l y the demands f o r v i t a m i n e s , b u t r a t h e r o f e m p h a s i s i n g , g e n e r a l l y , the importance o f accessory f a c t o r s i n d i e t . (50) Hopkins a l s o recounted t h e e x p e r i e n c e s that led him i n t o t h e a c c e s s o r y f a c t o r s p r o b l e m i n g e n e r a l , a g a i n p l a c i n g e m p h a s i s upon e x t r a c t i o n o f f o o d s w i t h h o t a l c o h o l (80%) i n o r d e r t o d e m o n s t r a t e the l o s s o f n u t r i t i v e power o f such f o o d s when so e x t r a c t e d . He a l s o s a i d t h a t he w i s h e d he had had t h e i n s i g h t i n t h o s e days t o have r e c o g n i z e d the e x i s t e n c e o f t h e two g r o w t h f a c t o r s d e m o n s t r a ­ t e d by Osborne and Mendel and by M c C o l l u m . ( T h i s w r i t e r w o u l d l i k e t o know what ' c e r t a i n p r e c a u t i o n s ' H o p k i n s t o o k i n o r d e r t o r e p r o d u c e h i s 1912 r e s u l t s b u t we s h a l l r e t u r n t o t h o s e r e s u l t s s h o r t l y t o s e e what t h e y meant i n t h e c o n ­ t e x t o f n u t r i t i o n a l s c i e n c e o f the day.) In l a t e F e b r u a r y 1920, Osborne and Mendel r e p l i e d t o H o p k i n s ' e p i s t l e i n a manner t h a t b e l i e s t h e i r a c t i v i t i e s : I t seems t o us t h a t y o u a t t a c h too much i m p o r t a n c e t o the q u a n t i t a t i v e r e l a t i o n s o f m i l k a s a s o u r c e o f t h e water-soluble vitamine. Even though l a r g e r q u a n t i t i e s o f m i l k may u l t i m a t e l y be d e m o n s t r a t e d t o be n e c e s s a r y than y o u r o r i g i n a l e x p e r i m e n t s i n d i c a t e d , we a r e s u r e t h a t no one w i l l e v e r t h i n k o f c r i t i c i z i n g y o u r e a r l y work on t h a t s c o r e . The e s s e n t i a l i m p o r t a n c e o f y o u r d i s c o v e r y i s i n no way a f f e c t e d . . . . P i o n e e r s i n such f i e l d s o f i n v e s t i g a t i o n a r e not expected to s e t t l e q u a n t i t a t i v e l i m i t s a t the o u t s e t o f such d i s c o v e r i e s . ...Our work was s i m p l y d e s i g n e d t o e l a b o r a t e y o u r p i i o n e e r i n v e s t i g a t i o n s and d e f i n e t h e c o n d i t i o n s more e x a c t l y t h a n was p o s s i b l e e i g h t o r n i n e y e a r s ago. (51 ) N o t e t h a t Osborne and Mendel b e l i e v e d t h a t l a r g e r q u a n t i t i e s o f m i l k w o u l d p r o b a b l y be needed t o f u r n i s h s u f f i c i e n t vitamine. They may have s a i d t h a t the q u a n t i t a t i v e r e l a t i o n s were n o t t o o important but they e x e r t e d s i g n i f i c a n t e f f o r t s t o determine those relations. The p a p e r t h e y had s e n t t o H o p k i n s was p u b l i s h e d i n 1920 and t h e i r l a s t a t t e m p t t o d i s c o v e r t h e q u a n t i t a t i v e r e l a t i o n s was p u b l i shed i η 1922. In t h e s p r i n g o f 1921, H o p k i n s went t o t h e U n i t e d S t a t e s where he v i s i t e d , among o t h e r p l a c e s , t h e l a b o r a t o r i e s o f Osborne and Mendel a s w e l l a s M c C o l l u m ' s now l o c a t e d i n t h e S c h o o l o f Pub­ l i c H e a l t h a t Johns H o p k i n s U n i v e r s i t y i n B a l t i m o r e . Shortly af­ t e r H o p k i n s r e t u r n e d t o E n g l a n d he r e c e i v e d a n o t e f r o m Mendel i n -

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WillMilkMakeThemGrow?

73

f o r m i n g him t h a t a J a p a n e s e r e s e a r c h g r o u p had o b t a i n e d s i g n i f i ­ c a n t g r o w t h o f r a t s g i v e n 2-3 c c o f m i l k on an o t h e r w i s e v i t a m i n Β d e f i c i e n t d i e t . (52) In 1921 came c o n f i r m a t i o n o f a s o r t i n v a l i d a t i n g Osborne and M e n d e l ' s c l a i m t h a t 10-16 c c o f m i l k were needed p e r day by the rat. D u t c h e r r e p o r t e d t h a t 10 c c o f m i l k o b t a i n e d f r o m cows on vitamine-rich rations usually s u f f i c e d in f u r n i s h i n g s u f f i c i e n t A and Β f o r normal g r o w t h , w h i l e l a r g e r q u a n t i t i e s o f m i l k p r o d u c ­ ed on v i t a m i n e - p o o r r a t i o n s were v e r y d e f i c i e n t i n b o t h f o o d a c ­ cessories. However, w a t e r - s o l u b l e Β d i d not f l u c t u a t e t o any g r e a t e x t e n t s i n c e most d a i r y a n i m a l s r e c e i v e d some g r a i n . (53) On the o t h e r s i d e o f the A t l a n t i c i n E n g l a n d , A. D. Stammers r e p o r t e d i n 1922 t h a t 2 c c o f m i l k added t o a b a s a l d i e t o f r a t s r e s u l t e d i n normal d e v e l o p m e n t . A l t h o u g h the e x p e r i m e n t s were not s t r i c t l y c o m p a r a b l e t o e i t h e r t h o s e o f Osborne and Mendel o r o f H o p k i n s , they d i d show a s i g n i f i c a n t change i n t h e g r o w t h o f r a t s t h a t had been on an Α-deficient d i e t f o r more than t h r e e months. E i g h t o f t e n r a t s e x h i b i t e d a l l the symptoms o f x e r o p h t h a l m i a (an eye d i s e a s e b r o u g h t on by l a c k o f v i t a m i n A ) . After receiving 2 cc o f m i l k per r a t per day f o r more t h a n t h r e e months on a d i e t t o t a l l y d e f i c i e n t i n v i t a m i n s , the x e r o p h t h a l m i a d i s a p p e a r e d and the r a t s a l l i n c r e a s e d i n w e i g h t . (54) A t a b o u t t h i s t i m e ( 1 9 2 2 ) , the c o n f l i c t o v e r q u a n t i t a t i v e r e ­ l a t i o n s o f m i l k seems t o have d i s a p p e a r e d from t h e s c i e n t i f i c l i t ­ erature. So f a r , no s a t i s f a c t o r y o r p l a u s i b l e e x p l a n a t i o n o f t h e d i f f e r e n c e s between H o p k i n s ' r e s u l t s and t h o s e o f Osborne and Mendel has a p p e a r e d i n the h i s t o r y o f n u t r i t i o n a l b i o c h e m i s t r y , and t h e r e i s a v e r y r e a l p o s s i b i l i t y t h a t no e x p l a n a t i o n w i l l e v e r be f o u n d f o r the s i m p l e r e a s o n t h a t the need t o d i s c o v e r a s o l u ­ t i o n has v a n i s h e d . (For the moment we w i l l i g n o r e t h e p o s s i b l e s o l u t i o n by a f u t u r e h i s t o r i a n who w i l l move heaven and e a r t h t o o b t a i n an answer t o what was o n c e a v e x i n g p r o b l e m . ) In o r d e r t o make s e n s e o u t o f the remark t h a t t h e need t o know has v a n i s h e d , we w i l l t u r n t o some s p e c i f i c a s p e c t s o f t h o s e e x p e r i m e n t s t h a t c o u l d not be r e c o n c i l e d , d e r i v e a b i t o f u n d e r ­ s t a n d i n g o f the f o r m i d a b l e n a t u r e o f the p r o b l e m and then r e t i r e q u i e t l y , h a v i n g a s s u r e d t h e r e a d e r t h a t t h e a u t h o r has indeed found the " b e s t " o f a l l p o s s i b l e a n s w e r s . H o p k i n s ' 1912 p a p e r (550 i s , at f i r s t glance, s u p e r l a t i v e , s p e c i f i c i n d e t a i l , s t a t i s t i c a l l y v a l i d and graphîcal 1 y p e r s u a s i v e . A more t h o r o u g h s t u d y r e v e a l s g l a r i n g o m i s s i o n s , numerous a r i t h m e t i c a l e r r o r s , a l a c k o f some c r i t i c a l d e t a i l s and a s e r i e s o f g r a p h s so c o n s t r u c t e d as t o be m i s l e a d i n g ( f i v e d i f f e r e n t g r a p h s c a l e s t o r e p r e s e n t seven g r a p h s . ) The s t a t i s t i c a l p r e s e n t a t i o n o f the p r o t o c o l s i n the Append i x i s o f enormous v a l u e t o the r e a d e r . That i t s h o u l d be m a r r e d by a minimum o f 25 a r i t h m e t i c a l e r r o r s i s a n n o y i n g and makes one wonder who d i d the c h e c k i n g o f d a t a , b u t , as i t t u r n s o u t , t h e o v e r a l l c o n c l u s i o n s o f t h e p a p e r a r e not n e g a t e d by t h e s e e r r o r s . ( U n l e s s , o f c o u r s e , a r e a d e r i s a c o m p l e t e s k e p t i c and c y n i c t o

74

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b o o t , b e l i e v i n g t h a t such a r i t h m e t i c a l f l a w s a r e i n d i c a t i v e o f e x p e r i m e n t a l f l a w s and h e n c e , t h e e n t i r e p a p e r becomes s u s p e c t . ) H o p k i n s was c a r e f u l t o a p p l y t h e g o v e r n i n g r u l e o f n u t r i t i o n to h i s experiments: a l l t h e d i e t a r i e s were more t h a n a d e q u a t e w i t h r e s p e c t to t h e i r energy c o n t e n t . With r a r e e x c e p t i o n s the r a t s used i n h i s s t u d i e s consumed s u f f i c i e n t f o o d , f a t s and c a r b o h y d r a t e s i n p a r t i c u l a r , t o s u p p l y them w i t h t h e c a l o r i e s deemed n e c e s s a r y f o r growth. The q u a n t i t i e s o f p r o t e i n and o f m i n e r a l s a l t s were a l s o a d e q u a t e . W i t h a l l t h i s i n m i n d , a t t e n t i o n can now be f o c u s e d on t h e s p e c i f i c d i e t s , e x p e r i m e n t a l p r o c e d u r e s and r e sults. The

two d i e t a r i e s employed by H o p k i n s a r e l i s t e d as Pure Casein M i x t u r e (A)

Protein Starch Cane s u g a r Lard Salts

22.0% 42.0 21.0 12.4 2.6

follows:

"Protene" Mixture (B) 21.3% 42.0 21.0 12.4 3-3

The p r o t e i n i n b o t h f o o d s was c a s e i n , t h e p u r e c a s e i n b e i n g a l a b o r a t o r y g r a d e o b t a i n e d from t h e Merck Co. w h i l e " P r o t e n e " was a c o m m e r c i a l p r e p a r a t i o n o f c a s e i n , not a s p u r i f i e d a s t h e Merck's. The s a l t s were o b t a i n e d by c o m b u s t i o n o f t h e normal f o o d supp l i e d t o r a t s when n o t under e x p e r i m e n t a 1 s t u d y , namely e q u a l p a r t s o f o a t s and d o g - b i s c u i t s . The n u t r i e n t c o m p o s i t i o n o f t h e dog b i s c u i t s o r t h e o a t s was not g i v e n i n t h e p a p e r but i t can be a s sumed t h a t the f o o d was a d e q u a t e f o r normal g r o w t h and d e v e l o p ment o f r a t s . B o t h o f the above d i e t s s u p p l i e d a l m o s t e x a c t l y 5 C a l / g o f energy. Hopkins r e p o r t e d the food consumption o f e x p e r i m e n t a l r a t s i n terms o f c a l o r i e s p e r 100 g o f l i v e w e i g h t o f t h e a n i m a l s , and i t i s a r e l a t i v e l y s i m p l e t a s k t o work f r o m t h i s d a t a and obt a i n the a c t u a l f o o d c o n s u m p t i o n o f t h e a n i m a l s . R a t h e r than r e p r o d u c e t h e g r a p h s o r p r o t o c o l s e x a c t l y a s publ i s h e d , the a u t h o r has s e l e c t e d some o f the d a t a i n r e c a l c u l a t e d form i n o r d e r t o b r i n g o u t the more s a l i e n t a s p e c t s o f t h e e x p e r i ments a s T a b l e I i l l u s t r a t e s . Observations, Anomalies,

Comments,

Interpretations

The p u r i f i c a t i o n o f f o o d s w i t h h o t a l c o h o l was s p e c i f i e d f o r some o f the e x p e r i m e n t s . H o p k i n s s a i d t h a t when he used e x t r a c t ed d i e t s he a l s o employed s p e c i a l l y p u r i f i e d l a r d but he d i d n o t e x p l a i n how the f a t was p u r i f i e d . A s i d e f r o m e x p e r i m e n t 7» a l l t h e o t h e r s were o f s h o r t d u r a tion. F u r t h e r m o r e , the r a t s used i n e x p e r i m e n t 7 were a l l i n i -

6 6 8 8 6 6 8 8 6 6 6 4 4

36.4 36.6 44.4 44.6 41.3 41.3 41.4 40.0 38.5 37-7 38.5 114.0 113-7 g

g

56.9 2.1 30.2 3-5 25.5 3-9 50.7 20.1 84.8 74.5 47.0 105-3 51.3

Ga i η Avg.

g

36 23* 18 18 19 19 25 25 34 34 34 61 61

Time Days

1.58 0.09 1.68 0.19 1.34 0.20 2.03 0.80 2.49 2.19 1.47 1.73 0.84

g

Avg. Ga i η Per Day P e r Rat

E f f e c t s o f S m a l l M i l k S u p p l e m e n t s on Final Avg. Wt.

93-3 38.7 74.6 48.1 66.8 45-2 92.1 60.1 123.3 112.2 85-5 219-3 165.0

Paper.

Initial Avg. Wt.

1912

Rat

ca. ca. ca. ca. ca. ca. ca.

8.2 g 4.3 ΝΑ ΝΑ 6.8 5-3 6.7 5.0 8.4 7-4 6.4 13 11

Avg. Food Intake, Per Rat P e r Day

Growth

A f t e r the 2 3 r d day t h e s e r a t s began t o f a i l . F i v e were dead by t h e 3 1 s t day. Unextr. = unextracted with a l c o h o l ; ext. = e x t r a c t e d with alcohol P a r t e x t . means p a r t i a l e x t r a c t i o n o f the p r o t e n e . H o p k i n s d i d not e x p l a i n what he meant by par tial extraction. Not A v a i l a b l e . No d a t a p r o v i d e d i n t h e p r o t o c o l . M i l k addendum shown i n c c and measured as c c p e r r a t p e r day. A s i d e f r o m the m i l k f e d r a t s , n e a r l y a l l o t h e r s showed some g r o w t h d u r i n g t h e f i r s t 7 t o 12 days

A, u n e x t . * * +2 c c A, u n e x t . A, e x t . * 3 c c A, e x t . A, e x t . +2 c c A, e x t . B, p a r t . e x t . + 3cc B, p a r t , e x t . * * * B, u n e x t . + 5 c c B, u n e x t . + 2 c c B, u n e x t . B, u n e x t . + 5 c c B, u n e x t .

No. o f Rats

Summary o f H o p k i n s '

Di e t a r y

Table I .

76

CHEMISTRY A N D MODERN SOCIETY

t i a l l y much h e a v i e r t h a n most o f t h e o t h e r s i n t h e r e m a i n i n g e x ­ periments. In e x p e r i m e n t 7, r a t s r e c e i v i n g m i l k g a i n e d more p e r day d u r i n g t h e f i r s t h a l f o f t h e t i m e p e r i o d t h a n i n t h e s e c o n d , w h i l e t h o s e on " p r o t e n e " a l o n e g a i n e d more t h a n t w i c e a s much p e r day i n t h e s e c o n d h a l f o f t h e e x p e r i m e n t a l p e r i o d t h a n i n t h e first. W h i l e he was n o t a b l e t o s u p p l y e q u a l numbers o f male a n d f e m a l e r a t s f o r t h e s e s t u d i e s , t h e r e were s u f f i c i e n t r a t s o f b o t h sexes t o warrant Hopkins' s t a t i s t i c a l l y d e r i v e d c o n c l u s i o n s t h a t a s m a l l m i l k addendum i s a g r o w t h p r o m o t e r a t a r a t e f a r i n e x c e s s o f what m i g h t be e x p e c t e d f r o m t h e a d d i t i o n o f t h e m i l k f a t , p r o ­ t e i n s , c a r b o h y d r a t e and s a l t s c o n t a i n e d i n 2-5 c c o f w h o l e m i l k . The p r o b l e m s o f i n t e r p r e t a t i o n a r e , a s m e n t i o n e d e a r l i e r , multi-factored. The s e x , a g e , w e i g h t a n d g e n e r a l h e a l t h o f t h e r a t s have t o be t a k e n i n t o a c c o u n t a s do t h e i r p e d i g r e e s o r g e n e t ­ i c makeup. The e n e r g y s u p p l y o f t h e n u t r i e n t s must be s u f f i c i e n t and t h e amino a c i d s i n terms o f t h e p r o t e i n s used must be q u a l i t a ­ t i v e l y adequate. Temperature, r e l a t i v e humidity, caging tech­ n i q u e s and h a n d l i n g a r e a l s o i m p o r t a n t f a c t o r s . Then t h e r e a r e t h e c h a r a c t e r i s t i c s o f t h e cows f r o m w h i c h m i l k was o b t a i n e d . The l i s t c a n be e x t e n d e d b u t i n s o f a r a s he was a b l e , H o p k i n s a p p e a r s t o have t a k e n many o f t h e s e f a c t o r s i n t o a c c o u n t . Most impor­ t a n t l y , he used a l a r g e number o f r a t s t o make h i s r e s u l t s s t a t i s ­ t i c a l l y s i g n i f i c a n t by a v e r a g i n g o u t i n d i v i d u a l d i f f e r e n c e s , l e a d ­ i n g t o t h e c o n c l u s i o n t h a t m i l k c o n t a i n s " a c c e s s o r y g r o w t h sub­ stances." In somewhat l a t e r t e r m i n o l o g y , H o p k i n s ' r a t s were a p p a r e n t l y r e c e i v i n g b o t h f a t - s o l u b l e A and w a t e r - s o l u b l e Β i n t h e i r d i e t s . The q u e s t i o n i s : W h e r e d i d t h e y g e t t h e s e v i t a m i n e s i n h i s e x p e r i ­ ments? A l i k e l y s o u r c e i n young r a t s i s t h e ' s t o r e ' , p o s t u l a t e d by Osborne and M e n d e l , o b t a i n e d d u r i n g t h e s u c k l i n g p e r i o d . How­ e v e r , b a s e d upon p r e s e n t - d a y k n o w l e d g e o n l y f a t - s o l u b l e A i s r e ­ t a i n e d i n c e r t a i n f a t t y d e p o s i t s o f t h e body. Young r a t s , j u s t weaned (as most o f H o p k i n s ' r a t s a p p e a r t o have b e e n , j u d g i n g f r o m t h e i r w e i g h t s ) w o u l d have v a r i a b l e q u a n t i t i e s o f A i n t h e i r t i s ­ s u e s and an e x t r e m e l y l i m i t e d s o u r c e o f w a t e r - s o l u b l e Β w h i c h i s s t o r e d p o o r l y i n most a n i m a l s . S i n c e t h e p r e m i s e was t h a t r a t s c o u l d n o t grow w i t h o u t b o t h A and Β ( a l t h o u g h H o p k i n s w o u l d n o t have known t h i s i n 1 9 1 2 ) , then any g r o w t h w h a t s o e v e r i n d i c a t e d t h a t some o f t h e e s s e n t i a l A and Β were p r e s e n t i n h i s most s u c c e s s f u l d i e t s . I t s h o u l d a l s o be s a i d a g a i n t h a t c a s e i n , t h e c o m p l e t e p r o ­ t e i n ( i n terms o f i t s amino a c i d c o n t e n t ) , was o b t a i n e d f r o m milk. Whether h i g h l y p u r i f i e d by t h e Merck Co. o r n o t ( a s i n " p r o t e n e " ) , we have n o t e d H o p k i n s ' i ns i s t e n c e t h a t o n l y h o t a l c o h o l c o u l d e x t r a c t the growth substances c o m p l e t e l y . C a s e i n , Hopkins b e l i e v e d , c o u l d a l s o a d s o r b t r a c e amounts o f t h e s e s u b s t a n c e s . M c C o l l u m , Drummond and Osborne and Mendel w o u l d a l l d e m o n s t r a t e t h a t l a c t o s e , a l s o o b t a i n e d f r o m cow's m i l k , a d s o r b e d v a r i a b l e q u a n t i t i e s o f g r o w t h p r o m o t i n g m a t e r i a l s . (Anyone who has l e f t a

4.

BECKER

Will Milk Make Them Grow?

11

q u a n t i t y o f m i l k i n a r e f r i g e r a t o r i s aware t h a t m i l k ' p i c k s up' o d o r s and f l a v o r s o f o t h e r f o o d s . Most o f t h e s e a d s o r b e d s m e l l s and t a s t e s a r e f o u n d i n company w i t h l a c t o s e a l t h o u g h c a s e i n can a l s o adsorb q u i t e w e l l . ) A n o t h e r p o t e n t i a l s o u r c e o f f a t - s o l u b l e A i n t h e s e d i e t s was the l a r d . H o p k i n s s a i d t h a t i t was p u r i f i e d s o m e t i m e s , but how? And, i n t h e c a s e o f u n p u r i f i e d l a r d , d i d he use l e a f l a r d f r o m t h e back o f t h e p i g , l a r d known ( l a t e r ) t o be l a c k i n g i n A o r d i d he use " k e g " l a r d , a m i x t u r e o f a l l p i g f a t s , some o f w h i c h c o n ­ t a i n f a t - s o l u b l e A? We s i m p l y do n o t know. K e e p i n g a l l t h i s i n m i n d , T a b l e I can be i n t e r p r e t e d a d i f ­ f e r e n t way. E x p e r i m e n t s 2 and 3 showed some g r o w t h i n r a t s n o t receiving milk. T h e i r l i m i t e d s u c c e s s can be a t t r i b u t e d t o a s m a l l s t o r e o f b o t h A and Β w i t h t h e Β d e p l e t e d most r a p i d l y . T h e r e i s a l s o t h e p o s s i b l e a v a i l a b l i 1 i t y o f v e r y s m a l l amounts o f A and Β not e x t r a c t e d by H o p k i n s ' method. E x p e r i m e n t s 5, 6 and 7 a l l i l l u s t r a t e d t h a t commerical " p r o t e n e " c o n t a i n e d both the A and Β f a c t o r s . Why, t h e n , were Osborne and Mendel u n a b l e t o v a l i d a t e Hop­ k i n s ' r e s u l t s ? Why c o u l d n ' t t h e y o b t a i n s i g n i f i c a n t g r o w t h u n t i l t h e y added 10-16 c c o f w h o l e m i l k p e r day p e r r a t t o t h e i r d i e t s ? Were A m e r i c a n r a t s so d i f f e r e n t f r o m E n g l i s h o n e s ? Were E n g l i s h d a i r y c a t t l e much more p r o d u c t i v e i n n u t r i e n t s t h a n A m e r i c a n o n e s ? Careful s c r u t i n y of t h e i r p u b l i c a t i o n s , d r a f t manuscripts, c o r r e s p o n d e n c e , r e c o r d b o o k s , i d e a and p l a n books a l o n g w i t h mar­ g i n a l n o t e s made on t h e i r c o p i e s o f v a r i o u s j o u r n a l a r t i c l e s has r e s u l t e d i n an h y p o t h e s i s t h a t i s t o o l o n g t o be e l a b o r a t e d i n i t s entirety. A few b i t s and p i e c e s w i l l s u f f i c e f o r now. F i r s t of a l l , Osborne and Mendel b e l i e v e d t h e i r own e x p e r i m e n t a l r e s u l t s and c o n c l u s i o n s t o be more c o r r e c t t h a n o t h e r s u n l e s s t h e o t h e r s a g r e e d w i t h them c o m p l e t e l y ( n o t an u n u s u a l phenomenon). Second, t h e y were c o n v i n c e d t h a t H o p k i n s e x p e r i m e n t s w e r e o f t o o s h o r t a d u r a t i o n t o t r u l y compare h i s r e s u l t s w i t h t h e i r s i n w h i c h p. f . m. had been used as a s u c c e s s f u l s u p p l e m e n t f o r up t o 80 d a y s . T h i r d , Osborne and Mendel were c o n v i n e e d o f t h e p u r i t y o f t h e d i e t s t h e y u s e d . They p r e p a r e d most o f t h e p r o t e i n s i n t h e i r own labs i n a d d i t i o n t o t h e p. f . m. I t must have been w i t h some r e l u c ­ t a n c e t h a t t h e y f i n a l l y a d m i t t e d t h a t c o m m e r i c a l l a c t o s e o r even some o f t h e i r 'home-made' l a c t o s e c a r r i e d g r o w t h p r o m o t e r s . The m a t t e r o f e x p e r i m e n t a l d u r a t i o n v e x e d them g r e a t l y . They had m a i n t a i n e d r a t s w i t h o u t s i g n i f i c a n t g r o w t h f o r months on d i e t s w h i c h , as f a r as t h e y c o u l d s e e , were e q u i v a l e n t t o t h o s e o f Hop­ k i n s on w h i c h h i s r a t s d i d n o t s u r v i v e v e r y l o n g . W i t h t h e U n i t e d S t a t e s e v e n t u a l l y e n t e r i n g the war i n E u r o p e i n 1917 came the impetus t o d e v e l o p b e t t e r f o o d s t h a t had l o n g shelf lives. By t h i s d a t e t h e v i t a m i n e s were f i r m l y e n t r e n c h e d i n n u t r i t i o n and i t i s not s u r p r i s i n g t h a t g r e a t e m p h a s i s was p l a c e d upon t h e p r o d u c t i o n o f m i l k and o t h e r d a i r y p r o d u c t s s i n c e e v e r y b o d y knew t h a t m i l k was good f o r y o u . Osborne and Mendel t o o k up the i s s u e o f t h e v i t a m i n e content 1

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o f m i l k once a g a i n i n 1918 and a t t e m p t e d t o r e p r o d u c e H o p k i n s ' r e ­ sults. A t t h i s d a t e t h e y were c e r t a i n t h a t t h e ' m i s s i n g ' v i t a mine was w a t e r - s o l u b l e Β s o t h e y p r e p a r e d d i e t s t o i n c l u d e about 3% b u t t e r f a t t o s u p p l y t h e n e c e s s a r y A f a c t o r . In t h e i r p u b l i ­

c a t i o n o f 1918 i t i s e v i d e n t t h a t t h e d i e t s were n o t e x a c t

rep­

l i c a s o f Hopkins' d i e t a r i e s ; n e v e r t h e l e s s , the c r i t i c a l n u t r i e n t s were a v a i l a b l e t o t h e r a t s i n ample q u a n t i t y , o r so i t a p p e a r e d . Again, t o s i m p l i f y matters, s i g n i f i c a n t data a r e presented in t a b u l a r form ( T a b l e I I ) w h i l e t h e d i e t s a r e d e s c r i b e d b e l o w . Osborne and Mendel d i d n o t p r e s e n t t h e l a r g e b o d i e s o f s t a t i s t i c a l i n f o r m a t i o n as H o p k i n s d i d s o r a t w e i g h t s and t i m e s o f e x p e r i m e n t must be i n t e r p o l a t e d f r o m t h e g r a p h s o f r a t g r o w t h . Because t h e a u t h o r s c o m p l i c a t e d t h e g r a p h s by making many changes i n t h e d i e t s , t h e g r o w t h c u r v e s a r e n o t s i m p l e . There­ f o r e , i n T a b l e ΙΓ w i l l be f o u n d o n l y t h o s e p o r t i o n s o f t h e e x p e r i ­ ments i n w h i c h m i l k was g i v e n a s an addendum i n a p p r o x i m a t e l y t h e same q u a n t i t i e s a s i n H o p k i n s ' e x p e r i m e n t s . Two o t h e r p o i n t s need t o be m e n t i o n e d . A l l t h e r a t s used by Osborne and Mendel were males and none o f t h e f o o d s were e x t r a c t e d w i t h a l c o h o l . Note t h a t t h e s a l t s i n d i e t 6 were p r e p a r e d i n a f a s h i o n s i m ­ i l a r t o t h e t e c h n i q u e used by H o p k i n s : i n c i n e r a t i o n o f equal p a r t s o f o a t s and d o g b r e a d . A l s o note that t h e percentages i n D i e t 7 add up t o 105- The e r r o r was p r o b a b l y i n t h e f i g u r e f o r starch. Osborne and Mendel were c o r r e c t i n b e l i e v i n g t h a t i f cow's m i l k p r o v i d e d t h e n e c e s s a r y v i t a m i n e s i n s m a l l q u a n t i t i e s such a s H o p k i n s had found t o be e f f e c t i v e , t h e i r e x p e r i m e n t s ought t o have shown t h i s u n e q u i v o c a l l y even i f t h e methods and b a s a l d i e t s were somewhat d i f f e r e n t . Y e t , they c o u l d n o t o b t a i n growth i n r a t s as H o p k i n s d i d . Why n o t ? A c a r e f u l s t u d y o f T a b l e s I and I I r e v e a l s t h a t Osborne and M e n d e l ' s r a t s d i d n o t e a t a s much t o t a l f o o d p e r day a s d i d H o p k i n s ' r a t s . Osborne and Mendel were aware o f t h i s phenomenon a s was H o p k i n s , p r e s u m a b l y . But t h i s o n l y adds t o t h e m y s t e r y . Exami n a t i o n o f t h e g r o w t h c u r v e s o f t h e 1922 p a p e r i n p a r t i c u l a r shows t h a t r a t s b e i n g g i v e n a m i l k supplement on a B - d e f i c i e n t d i e t grew p o o r l y i f a t a l l . Y e t , a s H o p k i n s had f o u n d y e a r s e a r l i e r and as Osborne and Mendel had d i s c o v e r e d on t h e i r own, t h e a d d i t i o n o f s m a l l amounts o f y e a s t o r even s m a l l e r q u a n t i t i e s o f a y e a s t e x t r a c t t o t h e d i e t s o f such r a t s c o i n c i d e d w i t h an i n c r e a s e d f o o d i n t a k e and a r e l a t ­ ed g r o w t h s p u r t . At t h i s p o i n t i n 1922 we s h a l l t a k e l e a v e o f t h e c o n f l i c t i n g , a n n o y i n g and c o n f u s i n g r e s u l t s o f such a n i m a l f e e d i n g e x p e r i m e n t s . Let i t s u f f i c e t o s a y t h a t i f t h e a v e r a g e d a i l y g a i n i n w e i g h t i s used as a c r i t e r i o n , t h e n H o p k i n s ' r a t s d i d v e r y w e l l i n d e e d . B u t , by way o f c o m p a r i s o n , t o d a y ' s l a b o r a t o r y r a t s (males) w i l l r o u t i n e l y g a i n from 5-0 t o 5-7 g p e r day d u r i n g g r o w t h from a w e i g h t o f about 45 g t o 220 g w h i l e consuming 9 t o 21 g o f f o o d p e r day. (56)

cc cc cc cc cc

»"

*

1922 1922

6 6

2 1 2 1 3 3

4 3 1 3 3

No. o f Rats

Final Avg. Wt.

Ga i η Avg.

Time Days

Avg. G a i n P e r Day P e r Rat

Avg, . Food I n t a k e , Per Rat P e r Day

40.8 40.8 53-7 59.0

12.9 18.2

10 20

1.29 0.91

c a . 3.6 c a . 3.6

g g**

NA 14 0.46 g 72.0 g 6.5g 78.5 g 0.62 NA 40 25.0 85-3 60.3 NA 20.0 98.0 78.0 0.43 47 NA 21 15.6 0.74 74.7 90.3 Two o f the r a t s d e c l i n e d i m m e d i a t l e y ; t h e o t h e r showed a smal1 g a i n f o r a few days b e f o r e d e c l i η i ng. 0.10 64.0 35 60.5 3.5 3.3 g, 14 d* 0.12 42 70.0 5.0 65.0 5.7 g, 14 d* 18 64.0 0.47 8.5 72.5 3.7 g, 14 d* 4.1 g, 35 d* 33.0 70.0 103.0 0.67 49 66.0 38 0.95 c a . 5 g,, 14 d* 102.7 36.7 N e g l i g i ble growth w i t h e i t h e r q u a n t i t y o f m i l k .

Initial Avg. Wt

Summary o f Osborne and Mendel P a p e r s , 1918-1922 E f f e c t s o f S m a l l M i l k S u p p l e m e n t s on Rat Growth

The a u t h o r s p r o v i d e d i n t a k e d a t a f o r o n l y s p e c i f i c p e r i o d s . The v a l u e s g i v e n h e r e a r e the i n t a k e s per day p e r r a t o v e r the t i m e g i v e n , i n d a y s , d. The 1922 e x p e r i m e n t used t h e same 6 r a t s . For c o m p a r i s o n p u r p o s e s t h e d a t a has been s e p a r a t e d i n ­ t o t h e f i r s t 10 days and t h e t o t a l e l a p s e d t i m e o f 20 d a y s . M i l k addendum shown i n c c and measured as c c p e r r a t p e r day.

D i e t 5 + 2 cc D i e t 5 + 2 cc D i e t 5 + 5 cc Diet 5 + 5 cc Diet 5 + 5 cc D i e t 6 + 2 cc f o r 21 d ther 1 5 c c f o r \28 d Diet 7 + 2 cc Diet 7 + 2 cc

2 2 2 2 4

1920 1920 1920 1920 1920 1920

+ + + + +

Diet Diet Diet Diet Diet

1 2 2 3 4

Dii e t a r y

1918 1918 1918 1918 1918

Paper

Table I I .

80

CHEMISTRY A N D M O D E R N SOCIETY

Addendum t o T a b l e II Composition of Diets Diet i

Diet 2

Diet 3

E d e s t i n 18.0%

Salts 4.5 S t a r c h 50.5 B u t t e r f a t 9-0 Lard 18.0

Salts 0.0-4.5 P. f . m. 28.0-0.0 Starch 25-48.5 B u t t e r f a t 9-0 Lard 20.0

Casein o r E d e s t i n 18.0% P. f . m. 0.0-22.4 Salts 4.5-1.0 Starch 47-7-31.6 B u t t e r f a t 9-0 Lard 18-20.0

Diet 4

Diet 5

Diet 6

C a s e i n 18.0% M i l k Pwdr. 0.0-48.0 Salts 4.5-1.0 Starch 50-18.0 B u t t e r f a t 9-0 Lard 18.0

Casein Salts 4 Starch 49 Butterfat 9 Lard 20

Casein 18.0% Salts 4.5 S t a r c h 50.5 B u t t e r f a t 9-0 Lard 18.0

Casein

18.0%

Diet 7 Casein 18% Salts 4 Starch 54 Butterfat 9 Lard 20

Nevertheless, i t i s remarkable that the pioneers o f the v i t a min h y p o t h e s i s were a b l e t o do so much when f o r t w e n t y y e a r s o r more t h e y had no c l e a r c o n c e p t i o n o f t h e c h e m i c a l e n t i t i e s w i t h w h i c h t h e y were d e a l i n g . C o n t r a d i c t o r y o r n o t , those animal feedi n g e x p e r i m e n t s opened up new v i s t a s i n n u t r i t i o n and i n a l l t h e i n t e r v e n i n g y e a r s s i n c e t h e v i t a m i n h y p o t h e s i s came i n t o b e i n g t h e r e has n o t been any s i n g l e ' b r e a k t h r o u g h ' i n n u t r i t i o n o f comp a r a b l e m a g n i t u d e and o f s u c h p e r v a s i v e i n f l u e n c e . The

A u t h o r Speaks b u t H o p k i n s has t h e L a s t Word, A l m o s t

In f o l l o w i n g t h e c o n t r o v e r s y o v e r v i t a m i n s i n m i l k , I have been a i d e d by h a v i n g many o f Osborne and M e n d e l ' s r e c o r d s , i d e a s , m a n u s c r i p t s and c o r r e s p o n d e n c e a t hand, t h a n k s t o t h e h i s t o r i c a l t r a d i t i o n a t t h e C o n n e c t i c u t A g r i c u l t u r a l E x p e r i m e n t S t a t i o n . Perhaps t h e H o p k i n s ' p a p e r s (such a s e x i s t ) w o u l d e n a b l e me t o s o l v e the ' q u a n t i t a t i v e r e l a t i o n s ' i s s u e b u t , a s s t a t e d e a r l i e r , I do not t h i n k i t w i l l e v e r be s o l v e d . In t h e e a r l y 1920's enough r e l i a b l e i n f o r m a t i o n a b o u t v i t a mins had been o b t a i n e d t o r e n d e r t h e p r o b l e m u n i m p o r t a n t . Many

4.

BECKER

Will Milk Make Them Grow?

81

d i f f e r e n t f o o d s were f o u n d t o c o n t a i n one o r more v i t a m i n s and the r o l e o f m i l k i n t h e d i e t s o f humans, a t l e a s t as a s u p p l i e r o f v i ­

t a m i n B, f e l l i n t o r e l a t i v e u n i m p o r t a n c e . A l t h o u g h d a i r y i n t e r e s t s c o n t i n u e t o a d v e r t i s e m i l k as t h e ' p e r f e c t ' o r h e a r p e r f e c t ' f o o d , t h e f a c t r e m a i n s t h a t cows' m i l k i s n o t a v e r y good s o u r c e o f Β v i t a m i n s i n terms o f p e r c e n t com­ p o s i t i o n by w e i g h t . A f t e r a l l , m i l k s o l i d s c o n s t i t u t e o n l y 12-13% o f f l u i d m i l k and t h a t means a r a t h e r l a r g e i n t a k e r a t i o o f w a t e r t o s o l i d s ( a b o u t 7 / 1 ) , n o t e x a c t l y i d e a l f o r o b t a i n i n g t r a c e nu­ t r i e n t s s u c h as t h e Β v i t a m i n s . As f a r as H o p k i n s ' Nobel P r i z e i s c o n c e r n e d , he d e s e r v e d i t even though h i s e x p e r i m e n t a l " p r o o f s " c o u l d n o t be r e p l i c a t e d i n a consistent fashion. He d e s e r v e d t h e p r i z e f o r one r e a s o n above a l l others: p e r s i s t e n c e i n p r o m u l g a t i n g an i d e a . A t a t i m e when t h e e n e r g y d o c t r i n e c o n t r o l l e d n u t r i t i o n , H o p k i n s was questioning its absolute v a l i d i t y . And w h i l e he was not t h e f i r s t t o do s o , he was the o n l y one o f the e a r l y q u e s t i o n e r s t o p r o c l a i m h i s be­ l i e f s i n p r i n t and t o p e r s i s t i n a s e a r c h f o r t h e e l u s i v e 'ac­ c e s s o r y g r o w t h f a c t o r s ' , a s e a r c h t h a t r e q u i r e d not o n l y e x p e r i ­ mental work o f h i s own but a l s o a commitment t o u r g e and t r a i n others to f o l l o w in h i s f o o t s t e p s . E a r l i e r , I m e n t i o n e d t h a t C a s i m i r Funk had r a i s e d t h e i s s u e o f p r i o r i t y i n t h e d i s c o v e r y o f the v i t a m i n s . H o p k i n s d i d not e n t e r i n t o t h e d e b a t e a t t h e t i m e but he d i d d e v o t e a s i g n i f i c a n t p a r t o f h i s Nobel A d d r e s s t o Funk and h i s c o n t r i b u t i o n s t o t h e vitamin hypothesis. With respect to the o t h e r p r i n c i p a l s i n v o l v e d i n the e a r l y e p i s o d e s o f v i t a m i n r e s e a r c h , i t i s a p p r o p r i a t e t o see what H o p k i n s s a i d a b o u t them i n S t o c k h o l m on t h a t December day i n 1929. H i s comments a f f o r d one more i l l u s t r a t i o n o f b o t h the c l a r i t y and c o n f u s i o n t h a t d o m i n a t e d h i s 1912 p a p e r . In o r d e r t o make s e n s e o f t h i s vague s t a t e m e n t , l e t me r e m i n d the r e a d e r t h a t i n 1917 M c C o l l u m had moved t o J o h n H o p k i n s i n Baltimore. Osborne s p e n t h i s e n t i r e p r o f e s s i o n a l c a r e e r a t t h e C o n n e c t i c u t A g r i c u l t u r a l E x p e r i m e n t S t a t i o n w h i l e Mendel had been a f a c u l t y member o f the S h e f f i e l d S c h o o l o f P h y s i o l o g i c a l C h e m i s t r y at Yale U n i v e r s i t y . T h e i r j o i n t p u b l i c a t i o n s always recognized the two i n s t i t u t i o n s but somehow, o v e r t h e y e a r s , i t was Y a l e t h a t a p p e a r e d t o be t h e s p a w n i n g g r o u n d o f t h e i r n u t r i t i o n a l r e s e a r c h e s and d i s c o v e r i e s . At the c l o s e o f h i s Nobel A d d r e s s , H o p k i n s s a i d t h i s a b o u t M c C o l l u m and Osborne and M e n d e l : So p r o m i n e n t i n d e e d was t h e A m e r i c a n work a t t h i s t i m e CWorld War IJ and so l a r g e a p r o p o r t i o n d i d i t f o r m o f the t o t a l o u t p u t f r o m 1912 t o n e a r the end o f the war t h a t , i f I w i s h e d t o c l a i m t h a t my own p u b l i c a t i o n s ex­ e r t e d any r e a l and e f f e c t i v e i n f l u e n c e i n s t a r t i n g a new movement i n the s t u d y o f d i e t e t i c s , I s h o u l d have t o c o n v i n c e m y s e l f t h a t t h e y h e l p e d t o d i r e c t the t h o u g h t s o f the H a r v a r d and B a l t i m o r e investigators.(57)

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W e l l , H o p k i n s may have been c o n f u s e d i n h i s Y a l e / H a r v a r d g e ­ o g r a p h y and h i s 1912 p a p e r showed t h a t he had p r o b l e m s w i t h s i m p l e a d d i t i o n and d i v i s i o n b u t he c e r t a i n l y knew how t o promote m u l t i pi i c a t i o n i η r a t s . When a l l i s s a i d and done I c a n r e s p o n d t o t h e t i t l e o f t h i s p a p e r , W i l l M i l k Make Them Grow? by s a y i n g y e s , s o m e t i m e s ; n o , f r e q u e n t l y ; and maybe, u n d e r c e r t a i n c o n d i t i o n s . Such a d i f f u s e and q u a l i f i e d r e s p o n s e i s i l l u s t r a t i v e o f t h e d i f f i c u l t i e s i n ­ herent i n o b t a i n i n g c o n s i s t e n t r e s u l t s w i t h animal f e e d i n g e x p e r i ­ ments d u r i n g t h e f i r s t two d e c a d e s o f t h e t w e n t i e t h c e n t u r y .

Literature Cited All correspondence items are located in the Thomas B. Osborne Files, Department of Biochemistry, The Connecticut Agricultural Experiment Station, New Haven, Connecticut. 1. 2. 3. 4.

Funk, C. J. State Med. 1912, 20, 341-368. Hopkins, F. G. J. Physiol. 1912, 44, 425-460. McCollum, Ε. V.; Davis, M. J. Biol. Chem. 1913, 15, 167-175. Osborne, T. B.; Mendel, L. B. J. Biol. Chem. 1913, 15, 311326. 5. Osborne, T. B.; Mendel, L. B. "Feeding Experiments with Isolated Food-Substances"; Carnegie Institution Publication 156, Washington, D. C. 1911; Part I, 1-53; Part I I , 55-138. 6. McCollum, Ε. V. "A History of Nutrition"; Houghton Mifflin, Boston, 1957, Chapters 1-14, 18, 20, 21. 7. Becker, S. L. "The Emergence of a Trace Nutrient Concept through Animal Feeding Experiments"; Unpublished Doctoral Dissertation, University of Wisconsin, 1968, Chapters 1-3. 8. Funk, C. Science. Apr. 30, 1926, 63, 455-456. 9. van Leersum, E. C. Science. Oct. 8, 1926, 64, 357-358. 10. Needham, J.; Baldwin, E. (eds.) "Hopkins and Biochemistry": W. Heffer and Sons, Ltd., Cambridge, 1949, 198-199. 11. Hopkins, F. G. The Analyst. 1906, 31, 395-396. 12. Hopkins, F. G.; Neville, A. Biochem. J. 1913, 7, 97-99. 13. Hopkins, F. G. Biochem. J. 1920, 14, 721-724. 14. Becker, S. L. "Butter Makes them Grow"; Bulletin 767 of Connecticut Agricultural Experiment Station. 1977, 1-21. 15. See citation 3. 16. Funk, C. J. Physiol. 1913, 46, 173-179. 17. Hopkins, F. G. J . Indust. Engr. Chem. 1922, 14, 67-68. 18. See citation 10, 197. 19. Osborne, T. B.; Mendel, L. B. J . Biol. Chem. 1912, 12, 8189. 20. McCollum, Ε. V.; Kennedy, C. J . Biol. Chem. 1916, 24, 491502. 21. Drummond, J . C. Biochem. J . 1920, 14, 660. 22. See citation 5. 23. Letter, Hopkins, F. G. to Osborne, T. B., August 4, 1912.

4.

24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57.

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Letter, Mendel, L. B. to Osborne, T. B., August 17, 1912. Letter, Mendel, L. B. to Osborne, T. B., August 30, 1912. See citation 5, 80-85. Osborne, T. B.; Mendel, L. B. Proceed. Soc. Exper. Biol. Med. April 17, 1912, 9 , 72-73. Letter, Osborne, T. B. to Mendel, L. Β., July 18, 1912. See citation 12, 97. Ibid., 99. Letter, Osborne, T. B.; Mendel, L. B. to Hopkins, F. G., Feburary 28, 1913. See citation 19, 83-84. Letter, Hopkins, F. G. to Osborne, T. B.; Mendel, L. Β., March 14, 1913. Ibid., 1. Ibid., 6. Letter, Osborne, T. B.; Mendel, L. B. to Hopkins, F. G., undated draft manuscript, but probably late March to midApril, 1913. Ibid., 9. McCollum, Ε. V.; Davis, M. J. Biol. Chem. 1915, 23, 184. McCollum, Ε. V.; Davis, M. J. Biol. Chem. 1915, 23, 234. Drummond, J. C. Biochem. J . 1916, 10, 89-102. Osborne, T. B.; Mendel, L. B. J. Biol. Chem. 1917, 31, 149163. Osborne, T. B.; Mendel, L. B. J . Biol. Chem. 1919, 37, 187200. Osborne, T. B.; Mendel, L. B. J. Biol. Chem. 1919, 37, 572. Osborne, T. B.; Mendel, L. B. J . Biol. Chem. 1918, 34, 539. Ibid., 543-4. Osborne, T. B.; Mendel, L. B. J. Biol. Chem. 1920, 41, 515523. Osborne, T. B.; Mendel, L. B. Biochem. J. 1922, 16, 363-367. Letter, Osborne, T. B.; Mendel, L. B. to Hopkins, F. G., December 8, 1919, 2. Ibid. Letter, Hopkins, F. G. to Osborne, T. B.; Mendel, L. Β., February 2, 1920, 2-3. Letter, Osborne, T. B; Mendel, L. B. to Hopkins, F. G., February 26, 1920, 1-2. Letter, Mendel, L. B. to Hopkins, F. G., July 1, 1921. Dutcher, R. A. J. Indust. Engr. Chem. 1921, 13, 1103. Harrow, B. Chem. Abst. 1923, 17, 411. Original article: Stammers, A. D. Biochem. J. 1922, 16, 659-667. See citation 2. Altman, P. L . ; Dittner, D. S. (eds.) "Biology Data Book"; Fed. Amer. Soc. Exper. B i o l . , Bethesday, MD, 1974, 3, 1457. See citation 10, 200.

RECEIVED

March 8, 1983