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The influence of feeding oxythiamine on the thiamine pyrophosphate content of some tissues of the pigeon
44 0
~HORI" COMMUNICATIONS, PRELIMINARY NOTES
VOL.
14 (1954)
REFERENCES 1 R. MARKHAM AND J. D. SMITH, Biochem. J., 52 (1952) 558, 565 . 2 j . D. SMITH AND R. MARKHAM, Nature, 17o (1952) 12o. J. D. SMITH AND R. MARKHAM, Biochim. Biophys. Acta, 8 (1952) 35 o. L. A. HEPPEL, P. R. WHITFIELD AND R. MARKHAM, Biochem. J., 56 (1954) iii. 4 CHONG-Fu LI, W . G. OVEREND AND M. STACEY, Nature, 163 (1949) 538. 5 See also, P. W . KENT, Nature, 166 (195o) 442. 6 D. M. BROWN AND A. R. TODD, J. Chem. Soc., (1952) 52. 7 j . A. L u c Y AND P. W. KENT, Research, 6 (1953) 49S. s C. TAMM, H. S. SHAPIRO, R. LIPSCHITZ AND E. CHARGAFF, J. Biol. Chem., 2o 3 (1953) 673. 9 C. TAMM, M. E. HODES AND E. CHARGAFF, J. Biol. Chem., 195 (1952) 49. R e c e i v e d April 28th, 1954
T H E I N F L U E N C E OF F E E D I N G O X Y T H I A M I N E ON T H E T H I A M I N E P Y R O P H O S P H A T E CONTENT OF SOME TISSUES OF T H E P I G E O N by ELIZABETH
P. S T E Y N - P A R V t ~
Laboratory [or Physiological Chemistry, The University, Utrecht (Netherlands)
T h e r e c e n t p u b l i c a t i o n of a p a p e r b y DE CARe, RINDI AND GRANA 1 on t h e m e t a b o l i c effects of ( n e o ) p y r i t h i a m i n e * a n d t h e t h i a m i n e c o n t e n t s in t h e t i s s u e s of t h e r a t p r o m p t s u s to c o m m u n i c a t e t h e r e s u l t s of s o m e e x p e r i m e n t s , carried o u t in 1952, r e g a r d i n g t h e effect of feeding o x y t h i a m i n e on t h e t h i a m i n e p y r o p h o s p h a t e (TPP) c o n t e n t of t i s s u e s of t h e pigeon. I t w a s originally i n t e n d e d to p u b l i s h t h e s e r e s u l t s as p a r t of m o r e e x t e n s i v e i n v e s t i g a t i o n s on t h e m o d e of a c t i o n of t h i a m i n e a n t a g o n i s t s , b u t c o n s i d e r i n g t h e i n t e r e s t t h i s s u b j e c t evokes in v a r i o u s q u a r t e r s , it s e e m s preferable n o t to d e l a y t h i s i n f o r m a t i o n . Since t h e d i s c o v e r y in 1943 b y WOOLLEY AND WHITE 3 t h a t s m a l l doses of p y r i t h i a m i n e , g i v e n to mice m a i n t a i n e d on a diet c o n t a i n i n g a d e q u a t e a m o u n t s of t h i a m i n e , p r o d u c e d s y m p t o m s closely r e s e m b l i n g t h o s e of t h i a m i n e deficiency, evidence h a s a c c u m u l a t e d p o i n t i n g to t h e fact t h a t s t r u c t u r a l a n a l o g u e s of t h i a m i n e , n o t a b l y p y r i t h i a m i n e a n d o x y t h i a m i n e ~,s, a c t as a n t a g o n i s t s to t h e v i t a m i n in rive. J u d g i n g f r o m c o m p a r a t i v e studies, p e r f o r m e d b y CERECEDO a n d collaboratorse, 7, it w o u l d s e e m t h a t p y r i t h i a m i n e h a s a m o r e p r o n o u n c e d a n t i v i t a m i n effect t h a n o x y t h i a m i n e , as t h e f o r m e r calls f o r t h s y m p t o m s of p o l y n e u r i t i s in mice while t h e l a t t e r fails to do so, a l t h o u g h t y p i c a l s y m p t o m s h a v e b e e n o b s e r v e d in chicks fed o x y t h i a m i n e s. WOOLLEY9 h a s c o m p a r e d t h e T P P c o n t e n t s of t h e livers of 2 g r o u p s of mice, b o t h fed a h i g h l y purified r a t i o n s u p p l e m e n t e d w i t h t h i a m i n e a n d one g r o u p receiving a single dose of 0. 5 m g p y r i t h i a m i n e . Surprisingly, T P P w a s n o t r e d u c e d in t h e livers of t h e l a t t e r g r o u p as c o m p a r e d to t h e former, a l t h o u g h t h e a n i m a l s h a d d e v e l o p e d severe s y m p t o m s of deficiency w h e n t h e y were sacrificed. N o w in a deficiency p r o v o k e d b y d e p r i v a t i o n of t h i a m i n e t h e r e is a l w a y s a m a r k e d decrease of T P P in all t i s s u e s TM. A l t h o u g h t h e precise m o d e of a c t i o n of t h e a n t i v i t a m i n is as y e t u n k n o w n , it is to be e x p e c t e d t h a t it will s o m e h o w interfere w i t h t h e r e p l a c e m e n t of T P P in t h e a p p r o p r i a t e e n z y m e s a n d if c a t a b o l i s m e x c e e d s r e p l a c e m e n t , T P P will g r a d u a l l y d i s a p p e a r . T h e s e c o n s i d e r a t i o n s i n d u c e d u s to i n v e s t i g a t e t h i s point. F o r practical r e a s o n s 11 p i g e o n s were u s e d as t e s t a n i m a l s a n d o x y t h i a m i n e - - m u c h m o r e easily o b t a i n a b l e t h a n p y r i t h i a m i n e - - a s a n t a gonist**. I n t h e first e x p e r i m e n t 2 g r o u p s of 6 p i g e o n s each r e c e i v e d - - b y forced f e e d i n g - - 2 g casein a n d 18 g sucrose daily, s u p p l e m e n t e d w i t h salt a n d v i t a m i n m i x t u r e TM a n d i o o ? t h i a m i n e daily. I n a d d i t i o n o n e g r o u p received I m g o x y t h i a m i n e daily. A f t e r 15 d a y s t h e a n i m a l s were sacrificed a n d T P P w a s d e t e r m i n e d m a n o m e t r i c a l l y TMin e x t r a c t s of liver, h e a r t m u s c l e (left ventricle), b r e a s t m u s c l e (pectoralis major) a n d b r a i n (cerebrum). A t t h i s t i m e t h e a n i m a l s in t h e o x y t h i a m i n e g r o u p did n o t y e t s h o w a n y p r o n o u n c e d s y m p t o m s of t h i a m i n e deficiency. T w o a n i m a l s in t h e control g r o u p receiving t h i a m i n e o n l y h a d died f r o m o t h e r causes. T h e a v e r a g e v a l u e s a n d t h e r a n g e s f o u n d * I n a c c o r d a n c e w i t h WOOLLEY'S s u g g e s t i o n S t h e p u r e p y r i t h i a m i n e will be called p y r i t h i a m i n e and not neopyrithiamine. ** W e w i s h to t h a n k Dr J. LENS of N.V. O r g a n o n , Oss, for k i n d l y s u p p l y i n g u s w i t h a s a m p l e of o x y t h i a m i n e .
voL. 14
(1954)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
44I
TABLE I THIAMINE PYROPHOSPHATE CONTENT (in v[g fresh tissue) oF SOME TISSUES OF PIGEONS RECEIVING THIAMINE AND OXYTHIAMINE, OR THIAMINE ONLY E x p e r i m e n t i : G r o u p A. G r o u p B. E x p e r i m e n t 2 : G r o u p A. G r o u p B. F u r t h e r details in t e x t . Exp. No.
Group of pigeons
4 6 5 7
pigeons, pigeons, pigeons, pigeons,
ioo y IOO ~ IOO 7 IOO ),
Cerebrum A v.
Range
thiamine thiamine thiamine thiamine
daily. -t- i m g o x y t h i a m i n e daily. daily. -t- 2 m g o x y t h i a m i n e daily.
Liver A v.
Range
Hearl muscle A v.
Range
Breast muscle A v.
Range
A B P value
4.15 3.75
(3.80-4.55) (3.oo-4.25) o.215
5.35 4.9o
(3-9°-6.30) (3.oo-5.65) o.352
6.70 3.4 o
(4.70-7.7 o) (2.oo-4.70) o.o14
6.25 4.60
(5.45-7.05) (2.95 --6.1o ) o.o38
A B P value
4.35 3.95
(3.85-4.65) (3.4o--4.5 ° ) o.174
5 .1° 4.7 °
(4.60-5.45) (4.o5-5.55) o.343
5.85 3.85
(5.35-6.15) (3.1°-5.3°) o.ooi
7 .60 6.40
(7 ' I ° - 8 ' 3 ° ) (5.55-7.55) o.o3o
for t h e T P P c o n t e n t s of t h e a f o r e - m e n t i o n e d t i s s u e s in t h e 4 c o n t r o l s a n d t h e 6 pigeons receiving o x y t h i a m i n e are a s s e m b l e d in T a b l e I. As is b o r n e o u t b y t h e P v a l u e s c a l c u l a t e d a c c o r d i n g to WILCOXON t h e r e is a significant decrease of T P P in h e a r t a n d b r e a s t m u s c l e in t h e a n i m a l s receiving o x y t h i a m i n e . T h e slight decrease in liver a n d b r a i n is n o t significant. As is also s h o w n in T a b l e I, a second e x p e r i m e n t , p e r f o r m e d in t h e s a m e way, fully confirmed t h e first results. T h i s t i m e 5 a n i m a l s in t h e c o n t r o l g r o u p a n d 7 a n i m a l s in t h e o x y t h i a m i n e g r o u p were e x a m i n e d . T h e l a t t e r n o w received 2 m g o x y t h i a m i n e daily a n d t h e feeding period lasted 18 d a y s , b u t t h i s m a d e no e s s e n t i a l difference. I t a p p e a r s f r o m t h e s e e x p e r i m e n t s t h a t feeding o x y t h i a m i n e does r e s u l t in a decrease of T P P in s o m e tissues. Of t h e t i s s u e s e x a m i n e d , h e a r t m u s c l e a n d b r e a s t m u s c l e are m o s t s u s c e p t i b l e to t h e influence of o x y t h i a m i n e . T h e liver s h o w s no significant r e s p o n s e to t h e feeding of t h e a n t i v i t a m i n , in a g r e e m e n t w i t h WOOLLEY'S o b s e r v a t i o n s , a l t h o u g h it s e e m s d o u b t f u l w h e t h e r t h e effects of o x y t h i a m i n e a n d p y r i t h i a m i n e m a y be c o m p a r e d directly w i t h one a n o t h e r . I n t h i s r e s p e c t it is i n t r i g u i n g t h a t DE CARO et al. 1 do find a decrease of t o t a l t h i a m i n e in t h e liver a f t e r feeding p y r i t h i a m i n e to rats. H o w e v e r , t h e i r dose c o n s i d e r a b l y e x c e e d s t h a t g i v e n b y WOOLLEY to his mice, also proportionally. I t is n o t s u r p r i s i n g t h a t we o b s e r v e d no deficiency s y m p t o m s . I n h e a r t m u s c l e a n d b r e a s t m u s c l e T P P h a d o n l y declined to t h e level a t t a i n e d a f t e r 4 d a y s of t h i a m i n e d e p r i v a t i o n , u s i n g t h e s a m e b a s a l diet 1°, a n d s y m p t o m s u s u a l l y do n o t occur t h e n before t h e t h i r t e e n t h d a y . T h i s w o r k f o r m s p a r t of i n v e s t i g a t i o n s on t h e m e t a b o l i s m a n d physiological f u n c t i o n of t h i a m i n e carried o u t b y H. G. K. WESTENBRINK a n d collaborators. REFERENCES 1 L. DE CARO, G. RINDI AND E. GRANA, Experientia, IO (1954) 14o. 2 D. W. WOOLLEY, A Study o[ Antimetabolites, Blew York, 1952. 8 D. W. WOOLLEY AND A. G. C. WHITE, J. Biol. Chem., 149 (1943) 285. M. SOODAK AND L. R. CERECEDO,J. A m . Chem. Soc., 66 (1944) 1988; Federation Proc., 6 (1947) 293. 5 C. E. FROHMAN AND H. G. DAY, J. Biol. Chem., 18o (1949) 93. A. J. EUSEBI AND L. R. CERECEDO, Science, IiO (1949) 162. L. R. CERECEDO, M. SOODAK AND A. J. E u s E n I , J. Biol. Chem., 189 (1951) 293. 8 L. J. DANIEL AND L. C. NORRIS, Proc. Soc. Exptl. Biol. Med., 72 (1949) 165. D. W. WOOLLEY, J. Biol. Chem., I91 (1951) 43x0 C. H. MONFOORT, Biochim. Biophys. Acta, 9 (I952) 331. 11 M. GRUBER, Nature, 166 (195 o) 78. 12 M. GRUBER, Acta Physiol. Pharmacol. Neerl., 2 (1951-1952) 411. 18 H. G. K. WESTENBRINK ANn E. P. STEYN-PARv]~,Int. Rev. Vitamin Res., 21 (195 o) 461. R e c e i v e d M a y i 2 t h , 1954
~HORI" COMMUNICATIONS, PRELIMINARY NOTES
VOL.
14 (1954)
REFERENCES 1 R. MARKHAM AND J. D. SMITH, Biochem. J., 52 (1952) 558, 565 . 2 j . D. SMITH AND R. MARKHAM, Nature, 17o (1952) 12o. J. D. SMITH AND R. MARKHAM, Biochim. Biophys. Acta, 8 (1952) 35 o. L. A. HEPPEL, P. R. WHITFIELD AND R. MARKHAM, Biochem. J., 56 (1954) iii. 4 CHONG-Fu LI, W . G. OVEREND AND M. STACEY, Nature, 163 (1949) 538. 5 See also, P. W . KENT, Nature, 166 (195o) 442. 6 D. M. BROWN AND A. R. TODD, J. Chem. Soc., (1952) 52. 7 j . A. L u c Y AND P. W. KENT, Research, 6 (1953) 49S. s C. TAMM, H. S. SHAPIRO, R. LIPSCHITZ AND E. CHARGAFF, J. Biol. Chem., 2o 3 (1953) 673. 9 C. TAMM, M. E. HODES AND E. CHARGAFF, J. Biol. Chem., 195 (1952) 49. R e c e i v e d April 28th, 1954
T H E I N F L U E N C E OF F E E D I N G O X Y T H I A M I N E ON T H E T H I A M I N E P Y R O P H O S P H A T E CONTENT OF SOME TISSUES OF T H E P I G E O N by ELIZABETH
P. S T E Y N - P A R V t ~
Laboratory [or Physiological Chemistry, The University, Utrecht (Netherlands)
T h e r e c e n t p u b l i c a t i o n of a p a p e r b y DE CARe, RINDI AND GRANA 1 on t h e m e t a b o l i c effects of ( n e o ) p y r i t h i a m i n e * a n d t h e t h i a m i n e c o n t e n t s in t h e t i s s u e s of t h e r a t p r o m p t s u s to c o m m u n i c a t e t h e r e s u l t s of s o m e e x p e r i m e n t s , carried o u t in 1952, r e g a r d i n g t h e effect of feeding o x y t h i a m i n e on t h e t h i a m i n e p y r o p h o s p h a t e (TPP) c o n t e n t of t i s s u e s of t h e pigeon. I t w a s originally i n t e n d e d to p u b l i s h t h e s e r e s u l t s as p a r t of m o r e e x t e n s i v e i n v e s t i g a t i o n s on t h e m o d e of a c t i o n of t h i a m i n e a n t a g o n i s t s , b u t c o n s i d e r i n g t h e i n t e r e s t t h i s s u b j e c t evokes in v a r i o u s q u a r t e r s , it s e e m s preferable n o t to d e l a y t h i s i n f o r m a t i o n . Since t h e d i s c o v e r y in 1943 b y WOOLLEY AND WHITE 3 t h a t s m a l l doses of p y r i t h i a m i n e , g i v e n to mice m a i n t a i n e d on a diet c o n t a i n i n g a d e q u a t e a m o u n t s of t h i a m i n e , p r o d u c e d s y m p t o m s closely r e s e m b l i n g t h o s e of t h i a m i n e deficiency, evidence h a s a c c u m u l a t e d p o i n t i n g to t h e fact t h a t s t r u c t u r a l a n a l o g u e s of t h i a m i n e , n o t a b l y p y r i t h i a m i n e a n d o x y t h i a m i n e ~,s, a c t as a n t a g o n i s t s to t h e v i t a m i n in rive. J u d g i n g f r o m c o m p a r a t i v e studies, p e r f o r m e d b y CERECEDO a n d collaboratorse, 7, it w o u l d s e e m t h a t p y r i t h i a m i n e h a s a m o r e p r o n o u n c e d a n t i v i t a m i n effect t h a n o x y t h i a m i n e , as t h e f o r m e r calls f o r t h s y m p t o m s of p o l y n e u r i t i s in mice while t h e l a t t e r fails to do so, a l t h o u g h t y p i c a l s y m p t o m s h a v e b e e n o b s e r v e d in chicks fed o x y t h i a m i n e s. WOOLLEY9 h a s c o m p a r e d t h e T P P c o n t e n t s of t h e livers of 2 g r o u p s of mice, b o t h fed a h i g h l y purified r a t i o n s u p p l e m e n t e d w i t h t h i a m i n e a n d one g r o u p receiving a single dose of 0. 5 m g p y r i t h i a m i n e . Surprisingly, T P P w a s n o t r e d u c e d in t h e livers of t h e l a t t e r g r o u p as c o m p a r e d to t h e former, a l t h o u g h t h e a n i m a l s h a d d e v e l o p e d severe s y m p t o m s of deficiency w h e n t h e y were sacrificed. N o w in a deficiency p r o v o k e d b y d e p r i v a t i o n of t h i a m i n e t h e r e is a l w a y s a m a r k e d decrease of T P P in all t i s s u e s TM. A l t h o u g h t h e precise m o d e of a c t i o n of t h e a n t i v i t a m i n is as y e t u n k n o w n , it is to be e x p e c t e d t h a t it will s o m e h o w interfere w i t h t h e r e p l a c e m e n t of T P P in t h e a p p r o p r i a t e e n z y m e s a n d if c a t a b o l i s m e x c e e d s r e p l a c e m e n t , T P P will g r a d u a l l y d i s a p p e a r . T h e s e c o n s i d e r a t i o n s i n d u c e d u s to i n v e s t i g a t e t h i s point. F o r practical r e a s o n s 11 p i g e o n s were u s e d as t e s t a n i m a l s a n d o x y t h i a m i n e - - m u c h m o r e easily o b t a i n a b l e t h a n p y r i t h i a m i n e - - a s a n t a gonist**. I n t h e first e x p e r i m e n t 2 g r o u p s of 6 p i g e o n s each r e c e i v e d - - b y forced f e e d i n g - - 2 g casein a n d 18 g sucrose daily, s u p p l e m e n t e d w i t h salt a n d v i t a m i n m i x t u r e TM a n d i o o ? t h i a m i n e daily. I n a d d i t i o n o n e g r o u p received I m g o x y t h i a m i n e daily. A f t e r 15 d a y s t h e a n i m a l s were sacrificed a n d T P P w a s d e t e r m i n e d m a n o m e t r i c a l l y TMin e x t r a c t s of liver, h e a r t m u s c l e (left ventricle), b r e a s t m u s c l e (pectoralis major) a n d b r a i n (cerebrum). A t t h i s t i m e t h e a n i m a l s in t h e o x y t h i a m i n e g r o u p did n o t y e t s h o w a n y p r o n o u n c e d s y m p t o m s of t h i a m i n e deficiency. T w o a n i m a l s in t h e control g r o u p receiving t h i a m i n e o n l y h a d died f r o m o t h e r causes. T h e a v e r a g e v a l u e s a n d t h e r a n g e s f o u n d * I n a c c o r d a n c e w i t h WOOLLEY'S s u g g e s t i o n S t h e p u r e p y r i t h i a m i n e will be called p y r i t h i a m i n e and not neopyrithiamine. ** W e w i s h to t h a n k Dr J. LENS of N.V. O r g a n o n , Oss, for k i n d l y s u p p l y i n g u s w i t h a s a m p l e of o x y t h i a m i n e .
voL. 14
(1954)
SHORT COMMUNICATIONS, PRELIMINARY NOTES
44I
TABLE I THIAMINE PYROPHOSPHATE CONTENT (in v[g fresh tissue) oF SOME TISSUES OF PIGEONS RECEIVING THIAMINE AND OXYTHIAMINE, OR THIAMINE ONLY E x p e r i m e n t i : G r o u p A. G r o u p B. E x p e r i m e n t 2 : G r o u p A. G r o u p B. F u r t h e r details in t e x t . Exp. No.
Group of pigeons
4 6 5 7
pigeons, pigeons, pigeons, pigeons,
ioo y IOO ~ IOO 7 IOO ),
Cerebrum A v.
Range
thiamine thiamine thiamine thiamine
daily. -t- i m g o x y t h i a m i n e daily. daily. -t- 2 m g o x y t h i a m i n e daily.
Liver A v.
Range
Hearl muscle A v.
Range
Breast muscle A v.
Range
A B P value
4.15 3.75
(3.80-4.55) (3.oo-4.25) o.215
5.35 4.9o
(3-9°-6.30) (3.oo-5.65) o.352
6.70 3.4 o
(4.70-7.7 o) (2.oo-4.70) o.o14
6.25 4.60
(5.45-7.05) (2.95 --6.1o ) o.o38
A B P value
4.35 3.95
(3.85-4.65) (3.4o--4.5 ° ) o.174
5 .1° 4.7 °
(4.60-5.45) (4.o5-5.55) o.343
5.85 3.85
(5.35-6.15) (3.1°-5.3°) o.ooi
7 .60 6.40
(7 ' I ° - 8 ' 3 ° ) (5.55-7.55) o.o3o
for t h e T P P c o n t e n t s of t h e a f o r e - m e n t i o n e d t i s s u e s in t h e 4 c o n t r o l s a n d t h e 6 pigeons receiving o x y t h i a m i n e are a s s e m b l e d in T a b l e I. As is b o r n e o u t b y t h e P v a l u e s c a l c u l a t e d a c c o r d i n g to WILCOXON t h e r e is a significant decrease of T P P in h e a r t a n d b r e a s t m u s c l e in t h e a n i m a l s receiving o x y t h i a m i n e . T h e slight decrease in liver a n d b r a i n is n o t significant. As is also s h o w n in T a b l e I, a second e x p e r i m e n t , p e r f o r m e d in t h e s a m e way, fully confirmed t h e first results. T h i s t i m e 5 a n i m a l s in t h e c o n t r o l g r o u p a n d 7 a n i m a l s in t h e o x y t h i a m i n e g r o u p were e x a m i n e d . T h e l a t t e r n o w received 2 m g o x y t h i a m i n e daily a n d t h e feeding period lasted 18 d a y s , b u t t h i s m a d e no e s s e n t i a l difference. I t a p p e a r s f r o m t h e s e e x p e r i m e n t s t h a t feeding o x y t h i a m i n e does r e s u l t in a decrease of T P P in s o m e tissues. Of t h e t i s s u e s e x a m i n e d , h e a r t m u s c l e a n d b r e a s t m u s c l e are m o s t s u s c e p t i b l e to t h e influence of o x y t h i a m i n e . T h e liver s h o w s no significant r e s p o n s e to t h e feeding of t h e a n t i v i t a m i n , in a g r e e m e n t w i t h WOOLLEY'S o b s e r v a t i o n s , a l t h o u g h it s e e m s d o u b t f u l w h e t h e r t h e effects of o x y t h i a m i n e a n d p y r i t h i a m i n e m a y be c o m p a r e d directly w i t h one a n o t h e r . I n t h i s r e s p e c t it is i n t r i g u i n g t h a t DE CARO et al. 1 do find a decrease of t o t a l t h i a m i n e in t h e liver a f t e r feeding p y r i t h i a m i n e to rats. H o w e v e r , t h e i r dose c o n s i d e r a b l y e x c e e d s t h a t g i v e n b y WOOLLEY to his mice, also proportionally. I t is n o t s u r p r i s i n g t h a t we o b s e r v e d no deficiency s y m p t o m s . I n h e a r t m u s c l e a n d b r e a s t m u s c l e T P P h a d o n l y declined to t h e level a t t a i n e d a f t e r 4 d a y s of t h i a m i n e d e p r i v a t i o n , u s i n g t h e s a m e b a s a l diet 1°, a n d s y m p t o m s u s u a l l y do n o t occur t h e n before t h e t h i r t e e n t h d a y . T h i s w o r k f o r m s p a r t of i n v e s t i g a t i o n s on t h e m e t a b o l i s m a n d physiological f u n c t i o n of t h i a m i n e carried o u t b y H. G. K. WESTENBRINK a n d collaborators. REFERENCES 1 L. DE CARO, G. RINDI AND E. GRANA, Experientia, IO (1954) 14o. 2 D. W. WOOLLEY, A Study o[ Antimetabolites, Blew York, 1952. 8 D. W. WOOLLEY AND A. G. C. WHITE, J. Biol. Chem., 149 (1943) 285. M. SOODAK AND L. R. CERECEDO,J. A m . Chem. Soc., 66 (1944) 1988; Federation Proc., 6 (1947) 293. 5 C. E. FROHMAN AND H. G. DAY, J. Biol. Chem., 18o (1949) 93. A. J. EUSEBI AND L. R. CERECEDO, Science, IiO (1949) 162. L. R. CERECEDO, M. SOODAK AND A. J. E u s E n I , J. Biol. Chem., 189 (1951) 293. 8 L. J. DANIEL AND L. C. NORRIS, Proc. Soc. Exptl. Biol. Med., 72 (1949) 165. D. W. WOOLLEY, J. Biol. Chem., I91 (1951) 43x0 C. H. MONFOORT, Biochim. Biophys. Acta, 9 (I952) 331. 11 M. GRUBER, Nature, 166 (195 o) 78. 12 M. GRUBER, Acta Physiol. Pharmacol. Neerl., 2 (1951-1952) 411. 18 H. G. K. WESTENBRINK ANn E. P. STEYN-PARv]~,Int. Rev. Vitamin Res., 21 (195 o) 461. R e c e i v e d M a y i 2 t h , 1954