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Metabolism of nicotinic acid under normal conditions and in pantothenate deficiency, studied in the rat
VOL. 22 (I956)
BIOCHIMICA ET BIOPHYSICA ACTA
M E T A B O L I S M O F N I C O T I N I C ACID U N D E R
547
NORMAL CONDITIONS
A N D IN P A N T O T H E N A T E D E F I C I E N C Y , S T U D I E D IN T H E R A T T. K. SUNDA1RAM AND P. S. S A R M A
University Biochemical Laboratory, Madras (India)
Investigations carried out in recent years on the metabolism of nicotinic acid in the rat reveal that nicotinuric acid, the glycine conjugate of nicotinic acid, is probably not a major end product of the metabolism of the vitamin when it is administered in physiological dosage, but is extensively excreted when massive doses of nicotinic acid are given to the animaP. The glycine conjugate thus is to be regarded more as a "detoxication" product of the extra-physiological quantities of nicotinic acid than as a normal metabolite of the vitamin. There appears to be a close parallelism between the conjugation of nicotinic acid and glycine to form nicotinuric acid and the detoxication of benzoic acid by conjugation with glycine to yield hippuric acid. The enzyme catalyzing the latter detoxication process and termed glycine N-acylase 2 has been fairly well investigated and it has been suggested that nicotinuric acid may be formed in vivo by a similar enzymic mechanism 3. Also, conclusive evidence has been forthcoming in recent years to show that coenzyme A and hence the B vitamin, pantothenic acid, is obligatory for the formation of hippuric acid4-e; thus, it has been observed by BRAUNSTEIN 4 that pantothenatedeficient rats excrete significantly lower amounts of hippuric acid than control animals in response to a test dose of benzoic acid, and the in vitro investigations of CHANTRENNE5 and of SCHACHTER AND TAGGART6 with isolated enzyme systems support the same conclusion. A study was therefore undertaken of the comparative excretion of nicotinuric acid by pantothenate-deficient rats in response to a test dose of nicotinic acid as against the excretion by similarly treated control animals. The results of this investigation are reported in the present communication. EXPERIMENTAL
Animals and diet Y o u n g female albino r a t s were divided into two g r o u p s of five each, one b e i n g t h e p a n t o t h e n a t e deficient g r o u p a n d t h e o t h e r s e r v i n g as control. All t h e a n i m a l s were g i v e n ad libitum t h e diet of t h e following c o m p o s i t i o n a n d w a t e r : glucose 73 g, casein v i t a m i n - f r e e i8 g, salts ~ 4 g, vegetable oil 5 g, t h i a m i n e h y d r o c h l o r i d e 40o/zg, riboflavin 8o0/zg, p y r i d o x i n e h y d r o c h l o r i d e 40o/~g, nicotinic acid 2 mg, biotin 4 °/~g, folic acid 20o/~g, inositol Io rag, p - a m i n o b e n z o i c acid IO rag, v i t a m i n B12 3 ~ug, a n d choline chloride ioo rag. T h e r a t s also received each 7 drops of Adexolin (Glaxo Laboratories) p e r week. T h e a n i m a l s of t h e control g r o u p were given in a d d i t i o n each 250 /~g of c a l c i u m p a n t o t h e n a t e daily. In a few cases, p a n t o t h e n a t e deficiency in t h e r a t s of t h e deficient group was a c c e n t u a t e d b y t h e a d m i n i s t r a t i o n of calcium c o - m e t h y l - p a n t o t h e n a t e , w h i c h h a s been s h o w n to possess a n t i p a n t o t h e n a t e p o t e n c y for t h e r a t s. T h e o n s e t of p a n t o t h e n a t e deficiency in t h e a n i m a l s of t h e deficient g r o u p was clearly discernible f r o m t h e f o u r t h week o n w a r d s b y m a r k e d inhibition of g r o w t h a n d e x t e r n a l s y m p t o m s like d e r m a t i t i s a n d g r e y i n g of hair.
Collection o/urine After five to six weeks on t h e r e g i m e n described above, t h e r a t s were t r a n s f e r r e d to i n d i v i d u a l m e t a b o l i s m cages a n d g i v e n a n i n t r a p e r i t o n e a l injection of nicotinic acid in sterile physiological
Re[erences p. 549.
548
T . K . SUNDARAM, P. S. SARMA
VOL. 2 2
(1956)
saline. T h e urines were collected for t h e following 48 h o u r s in acetic acid (3 m l in each case) u n d e r toluene. D u r i n g this collection period, t h e r a t s received ad lib. their respective diets, b u t w i t h o u t nicotinic acid. T h e u r i n e s a m p l e s were each m a d e u p to lOO ml, filtered, t h e first few m l of t h e filt r a t e being discarded, a n d p r e s e r v e d u n d e r t o l u e n e in t h e refrigerator till analysis. A n a l y s i s o[ u r i n e s a m p l e s D e t e r m i n a t i o n o] "'total n i c o t i n i c a c i d " : " T o t a l nicotinic a c i d " in this c o n t e x t r e p r e s e n t s all t h e d e r i v a t i v e s of nicotinic acid w h i c h c a n s u p p o r t t h e g r o w t h of t h e o r g a n i s m L a c t o b a c i l l u s a r a b i n o s u s 17- 5. T h e s e include m a i n l y t h e t e r t i a r y derivatives, nicotinic acid, n i c o t i n a m i d e a n d nicotinuric
acid, since o t h e r d e r i v a t i v e s like c o z y m a s e are n o t excreted b y t h e r a t to a v e r y appreciable ext e n t 8. I n t h e p r e s e n t investigation, t o t a l nicotinic acid was d e t e r m i n e d b y one of t h e modifications r e c o m m e n d e d b y SNELL 9 of t h e original microbiological p r o c e d u r e l° e m p l o y i n g L . a r a b i n o s u s 17- 5. D e t e r m i n a t i o n o[ " # e e n i c o t i n i c a c i d " : T h e t e r m "free nicotinic a c i d " m e a n s t h e nicotinic acid p a r t of " t o t a l nicotinic acid" o b t a i n e d b y t h e L . a r a b i n o s u s m e t h o d . Free nicotinic acid was estim a t e d b y t h e m e t h o d of JOHNSON n u s i n g t h e o r g a n i s m , Leuconostoc m e s e n t e r o i d e s 9135, w h i c h r e s p o n d s specifically to nicotinic acid a n d is insensitive to n i c o t i n a m i d e a n d nicotinuric acid. C a l c u l a t i o n o] the n i c o t i n u r i c acid content o[ Urine s a m p l e : R e c e n t i n v e s t i g a t i o n s u s i n g radioactive nicotinic acid a n d t h e p a p e r c h r o m a t o g r a p h i c a n d r a d i o a u t o g r a p h i c t e c h n i q u e s 1,13 d e m o n s t r a t e t h a t w h e n n i c o t i n i c a c i d is t h e f o r m in w h i c h t h e v i t a m i n is a d m i n i s t e r e d to t h e rat, t h e a m o u n t of n i c o t i n a m i d e e x c r e t e d is v e r y s m a l l a n d u s u a l l y n o t m o r e t h a n 5 %. Therefore, in t h e p r e s e n t s t u d y , t h e difference b e t w e e n t h e " t o t a l nicotinic a c i d " a n d t h e "free nicotinic a c i d " v a l u e s was t a k e n to give, as a fair a p p r o x i m a t i o n , t h e nicotinuric acid c o n t e n t of t h e u r i n e s a m p l e . T h i s simplified differential a s s a y p r o c e d u r e t h u s d i s p e n s e d w i t h t h e n e c e s s i t y of h y d r o l y z i n g t h e s a m p l e u n d e r specified conditions laid d o w n b y JOHNSON et al. TM a n d KREHL et al. 14 in t h e i r inv e s t i g a t i o n s where t h e a m o u n t s of n i c o t i n a m i d e p r e s e n t in t h e a s s a y s a m p l e s were appreciable. RESULTS AND DISCUSSION
The results obtained are summarized in Table I. It is seen that a major portion (about 60 %) of the tertiary derivatives of the vitamin excreted by the control rats is accounted for by the Conjugate, nicotinuric acid. This observation is in good agreement with those of REDDI AND KODICEKTM and of LIN AND JOHNSON1, who employed the paper chromatographic and radioautographic techniques respectively in their experiments with normal rats. TABLE I EXCRETION OF FREE AND CONJUGATED NICOTINIC ACID BY NORMAL AND PANTOTHENATE-DEFICIENT RATS
Rat
Experimental
Weight at start o/ experiment g
Weight at end o/ experiment g
I 2 3 4 5
Normal Normal Normal Normal Normal
63.0 70.0 81.5 48.o 45-5
121.o 121.o 137.5 lO8.5 ioi.5
1122.o lO96.1 1212-7 733-0 964.o
386.6 581.3 395.4 293.0 283.5
735.4 514.8 817-3 440.o 68o.5
64.9 47.0 67-4 6o.0 70.5
Deficient Deficient Deficient Deficient Deficient
68.5 73.o 56.o 48.5 47-5
98.0 lol. 5 82.o 81.o 7o.o
307 .1 528.5 499.5 649.0 4o3.o
195-4 382.o 350.5 382.5 285.o
111.7 146. 5 149.o 266.5 118.o
36. 4 27. 7 29.8 41. I 29.9
No.
6*** 7*** 8 9 IO
condition
Total nicotinic acid excreted A* itg
Free nicotinic acid excreted B* * Itg
Conjugated Conjugated nicotinic acid nicotinic acid excreted as percent total A -- B nicotinic acid #g (A--B)IA × IOO
* Determined by L. arabinosus method. ** D e t e r m i n e d b y L . mesenteroides m e t h o d . *** ~ o - M e t h y l p a n t o t h e n a t e - t r e a t e d R a t Nos. i, 2, 3, 6, 7 received 3 m g nicotinic acid a n d r a t Nos. 4, 5, 8, 9, io 2. 5 m g nicotinic acid i n t r a p e r i t o n e a l l y as t e s t dose. Re[erences p . 549"
VOL. 22 (1956)
METABOLISM OF NICOTINIC ACID
549
The excretory pattern obtained in the case of the pantothenate-deficient rats in the present study is, however, interestingly enough different. The animals of the deficient group are seen to excrete a markedly lower proportion of nicotinic acid in the conjugated form and a correspondigly higher percentage as ]ree nicotinic acid than the control rats. This decreased excretion of the conjugate by the deficient group of rats would clearly point to the involvement of pantothenic acid in the conjugation of nicotinic acid and glycine to form nicotinuric acid. The discovery of the role of coenzyme A in the enzymic synthesis of hippturic acidS, e and certain related compounds 15 and more recently in the biosynthesis of some of the bile salts I8 has widened the range of coenzyme A-dependent reactions and it appears that the coenzyme may be obligatory for other cases of condensation of an amino group and a carboxyl group. It has been further established that coenzyme A mediates the conjugation of benzoic acid and glycine in a manner similar to the mechanism of biological acetylation. It is possible that a similar mechanism operates in the biosynthesis of nicotinuric acid from nicotinic acid and glycine. Interesting in this context is the report s on the enzyme glycine N-acylase, which catalyzes the conjugation of glycine with organic carboxylic acids and is very specific with respect to glycine and relatively not so in the case of the compounds having the carboxyl group. ACKNOWLEDGEMENTS
The authors wish to acknowledge with thanks the generous gift from Prof. B. C. JOHNSON, University of Illinois, of the culture of Leuconostoc mesenteroides 9135, and the award of a post-graduate research scholarship by the Lady Tata Memorial Trust, Bombay, to one of them (T.K.S.). SUMMARY T h e m e t a b o l i s m of nicotinic acid in n o r m a l a n d p a n t o t h e n a t e - d e f i c i e n t r a t s h a s been s t u d i e d w i t h p a r t i c u l a r reference to t h e e x c r e t i o n of nicotinuric acid, t h o glycine c o n j u g a t e of nicotinic acid. It h a s been s h o w n t h a t , w h e r e a s nicotinuric acid c o n s t i t u t e s a b o u t 6o % of t h e t e r t i a r y d e r i v a t i v e s of nicotinic acid e x c r e t e d b y control rats, it a c c o u n t s for a m a r k e d l y lower p r o p o r t i o n of t h e tert i a r y d e r i v a t i v e s in t h e case of t h e p a n t o t h e n a t e - d e f i c i e n t a n i m a l s . T h e r e s u l t s are i n t e r p r e t e d to indicate a n i n v o l v e m e n t of CoA in nicotinuric acid b i o s y n t h e s i s as in t h e e n z y m i c s y n t h e s i s of h i p p u r i c acid f r o m benzoic acid a n d glycine. REFERENCES i PEI-HSINGLIN AND B. C. JOHNSON,J. Am. Chem. Soc., 75 (I953) 2974. 2 D. SCHACHTER AND J. V. TAGGART, J. Biol. Chem., 208 (1954) 263a R. J. WILLIAMS, R. E. EAKIN, E. BEERSTECHER, Jr. ANn W. SHIVE, The Biochemistry o/B-vitamins, R e i n h o l d P u b l i s h i n g Corp., N e w York, 195 o, p. 361. 4 H. E. BRAUNSTEIN AND E. F. EFIMOCHKINA, Doklady Akad. Nauk. S.S.S.R., 71 (195 o) 347. 5 H. CHANTRENNE, J. Biol. Chem., 189 (1951) 227. D. SCHACHTER AND J. V. TAGGART, J. Biol. Chem., 203 (1953) 925. 7 R. E. OLSON AND N. O. KAPLAN, J. Biol. Chem., 175 (1948) 515 . s W . DRXLL AND M. S. DLT~N, Arch. Biochem. Biophys., 33 (1951) IiO. @E. E. SNELL, in P. GYORGY, Vitamin Methods, Vol. I, A c a d e m i c Press, N e w York, I95 o, p. 360. 10 E. E. SNELL AND L. D. WRIGHT, J. Biol. Chem., 139 (1941) 675. n B. C. JOHNSON, J. Biol. Chem., 159 (1945) 227. is K. K. REDDI AND E. KODICEK, Biochem. J., 53 (1953) 286. la B. C. JOHNSON, H. H. MITCHXLL AND T. S. HAMILTON, J. Biol. Chem., I59 (1945) 231. 14 W. A. KREHL, J. DE LA HUERGA AND C. A. ELVEHJEM, J. Biol. Chem., 166 (1946) 53. is T. R. RIGGS AND D. M. HEGSTED, J. Biol. Chem., 172 (1948) 539. IS j . BREMER, Acta Chem. Scand., 9 (1955) lO36.
Received February I9th , 1956 36
BIOCHIMICA ET BIOPHYSICA ACTA
M E T A B O L I S M O F N I C O T I N I C ACID U N D E R
547
NORMAL CONDITIONS
A N D IN P A N T O T H E N A T E D E F I C I E N C Y , S T U D I E D IN T H E R A T T. K. SUNDA1RAM AND P. S. S A R M A
University Biochemical Laboratory, Madras (India)
Investigations carried out in recent years on the metabolism of nicotinic acid in the rat reveal that nicotinuric acid, the glycine conjugate of nicotinic acid, is probably not a major end product of the metabolism of the vitamin when it is administered in physiological dosage, but is extensively excreted when massive doses of nicotinic acid are given to the animaP. The glycine conjugate thus is to be regarded more as a "detoxication" product of the extra-physiological quantities of nicotinic acid than as a normal metabolite of the vitamin. There appears to be a close parallelism between the conjugation of nicotinic acid and glycine to form nicotinuric acid and the detoxication of benzoic acid by conjugation with glycine to yield hippuric acid. The enzyme catalyzing the latter detoxication process and termed glycine N-acylase 2 has been fairly well investigated and it has been suggested that nicotinuric acid may be formed in vivo by a similar enzymic mechanism 3. Also, conclusive evidence has been forthcoming in recent years to show that coenzyme A and hence the B vitamin, pantothenic acid, is obligatory for the formation of hippuric acid4-e; thus, it has been observed by BRAUNSTEIN 4 that pantothenatedeficient rats excrete significantly lower amounts of hippuric acid than control animals in response to a test dose of benzoic acid, and the in vitro investigations of CHANTRENNE5 and of SCHACHTER AND TAGGART6 with isolated enzyme systems support the same conclusion. A study was therefore undertaken of the comparative excretion of nicotinuric acid by pantothenate-deficient rats in response to a test dose of nicotinic acid as against the excretion by similarly treated control animals. The results of this investigation are reported in the present communication. EXPERIMENTAL
Animals and diet Y o u n g female albino r a t s were divided into two g r o u p s of five each, one b e i n g t h e p a n t o t h e n a t e deficient g r o u p a n d t h e o t h e r s e r v i n g as control. All t h e a n i m a l s were g i v e n ad libitum t h e diet of t h e following c o m p o s i t i o n a n d w a t e r : glucose 73 g, casein v i t a m i n - f r e e i8 g, salts ~ 4 g, vegetable oil 5 g, t h i a m i n e h y d r o c h l o r i d e 40o/zg, riboflavin 8o0/zg, p y r i d o x i n e h y d r o c h l o r i d e 40o/~g, nicotinic acid 2 mg, biotin 4 °/~g, folic acid 20o/~g, inositol Io rag, p - a m i n o b e n z o i c acid IO rag, v i t a m i n B12 3 ~ug, a n d choline chloride ioo rag. T h e r a t s also received each 7 drops of Adexolin (Glaxo Laboratories) p e r week. T h e a n i m a l s of t h e control g r o u p were given in a d d i t i o n each 250 /~g of c a l c i u m p a n t o t h e n a t e daily. In a few cases, p a n t o t h e n a t e deficiency in t h e r a t s of t h e deficient group was a c c e n t u a t e d b y t h e a d m i n i s t r a t i o n of calcium c o - m e t h y l - p a n t o t h e n a t e , w h i c h h a s been s h o w n to possess a n t i p a n t o t h e n a t e p o t e n c y for t h e r a t s. T h e o n s e t of p a n t o t h e n a t e deficiency in t h e a n i m a l s of t h e deficient g r o u p was clearly discernible f r o m t h e f o u r t h week o n w a r d s b y m a r k e d inhibition of g r o w t h a n d e x t e r n a l s y m p t o m s like d e r m a t i t i s a n d g r e y i n g of hair.
Collection o/urine After five to six weeks on t h e r e g i m e n described above, t h e r a t s were t r a n s f e r r e d to i n d i v i d u a l m e t a b o l i s m cages a n d g i v e n a n i n t r a p e r i t o n e a l injection of nicotinic acid in sterile physiological
Re[erences p. 549.
548
T . K . SUNDARAM, P. S. SARMA
VOL. 2 2
(1956)
saline. T h e urines were collected for t h e following 48 h o u r s in acetic acid (3 m l in each case) u n d e r toluene. D u r i n g this collection period, t h e r a t s received ad lib. their respective diets, b u t w i t h o u t nicotinic acid. T h e u r i n e s a m p l e s were each m a d e u p to lOO ml, filtered, t h e first few m l of t h e filt r a t e being discarded, a n d p r e s e r v e d u n d e r t o l u e n e in t h e refrigerator till analysis. A n a l y s i s o[ u r i n e s a m p l e s D e t e r m i n a t i o n o] "'total n i c o t i n i c a c i d " : " T o t a l nicotinic a c i d " in this c o n t e x t r e p r e s e n t s all t h e d e r i v a t i v e s of nicotinic acid w h i c h c a n s u p p o r t t h e g r o w t h of t h e o r g a n i s m L a c t o b a c i l l u s a r a b i n o s u s 17- 5. T h e s e include m a i n l y t h e t e r t i a r y derivatives, nicotinic acid, n i c o t i n a m i d e a n d nicotinuric
acid, since o t h e r d e r i v a t i v e s like c o z y m a s e are n o t excreted b y t h e r a t to a v e r y appreciable ext e n t 8. I n t h e p r e s e n t investigation, t o t a l nicotinic acid was d e t e r m i n e d b y one of t h e modifications r e c o m m e n d e d b y SNELL 9 of t h e original microbiological p r o c e d u r e l° e m p l o y i n g L . a r a b i n o s u s 17- 5. D e t e r m i n a t i o n o[ " # e e n i c o t i n i c a c i d " : T h e t e r m "free nicotinic a c i d " m e a n s t h e nicotinic acid p a r t of " t o t a l nicotinic acid" o b t a i n e d b y t h e L . a r a b i n o s u s m e t h o d . Free nicotinic acid was estim a t e d b y t h e m e t h o d of JOHNSON n u s i n g t h e o r g a n i s m , Leuconostoc m e s e n t e r o i d e s 9135, w h i c h r e s p o n d s specifically to nicotinic acid a n d is insensitive to n i c o t i n a m i d e a n d nicotinuric acid. C a l c u l a t i o n o] the n i c o t i n u r i c acid content o[ Urine s a m p l e : R e c e n t i n v e s t i g a t i o n s u s i n g radioactive nicotinic acid a n d t h e p a p e r c h r o m a t o g r a p h i c a n d r a d i o a u t o g r a p h i c t e c h n i q u e s 1,13 d e m o n s t r a t e t h a t w h e n n i c o t i n i c a c i d is t h e f o r m in w h i c h t h e v i t a m i n is a d m i n i s t e r e d to t h e rat, t h e a m o u n t of n i c o t i n a m i d e e x c r e t e d is v e r y s m a l l a n d u s u a l l y n o t m o r e t h a n 5 %. Therefore, in t h e p r e s e n t s t u d y , t h e difference b e t w e e n t h e " t o t a l nicotinic a c i d " a n d t h e "free nicotinic a c i d " v a l u e s was t a k e n to give, as a fair a p p r o x i m a t i o n , t h e nicotinuric acid c o n t e n t of t h e u r i n e s a m p l e . T h i s simplified differential a s s a y p r o c e d u r e t h u s d i s p e n s e d w i t h t h e n e c e s s i t y of h y d r o l y z i n g t h e s a m p l e u n d e r specified conditions laid d o w n b y JOHNSON et al. TM a n d KREHL et al. 14 in t h e i r inv e s t i g a t i o n s where t h e a m o u n t s of n i c o t i n a m i d e p r e s e n t in t h e a s s a y s a m p l e s were appreciable. RESULTS AND DISCUSSION
The results obtained are summarized in Table I. It is seen that a major portion (about 60 %) of the tertiary derivatives of the vitamin excreted by the control rats is accounted for by the Conjugate, nicotinuric acid. This observation is in good agreement with those of REDDI AND KODICEKTM and of LIN AND JOHNSON1, who employed the paper chromatographic and radioautographic techniques respectively in their experiments with normal rats. TABLE I EXCRETION OF FREE AND CONJUGATED NICOTINIC ACID BY NORMAL AND PANTOTHENATE-DEFICIENT RATS
Rat
Experimental
Weight at start o/ experiment g
Weight at end o/ experiment g
I 2 3 4 5
Normal Normal Normal Normal Normal
63.0 70.0 81.5 48.o 45-5
121.o 121.o 137.5 lO8.5 ioi.5
1122.o lO96.1 1212-7 733-0 964.o
386.6 581.3 395.4 293.0 283.5
735.4 514.8 817-3 440.o 68o.5
64.9 47.0 67-4 6o.0 70.5
Deficient Deficient Deficient Deficient Deficient
68.5 73.o 56.o 48.5 47-5
98.0 lol. 5 82.o 81.o 7o.o
307 .1 528.5 499.5 649.0 4o3.o
195-4 382.o 350.5 382.5 285.o
111.7 146. 5 149.o 266.5 118.o
36. 4 27. 7 29.8 41. I 29.9
No.
6*** 7*** 8 9 IO
condition
Total nicotinic acid excreted A* itg
Free nicotinic acid excreted B* * Itg
Conjugated Conjugated nicotinic acid nicotinic acid excreted as percent total A -- B nicotinic acid #g (A--B)IA × IOO
* Determined by L. arabinosus method. ** D e t e r m i n e d b y L . mesenteroides m e t h o d . *** ~ o - M e t h y l p a n t o t h e n a t e - t r e a t e d R a t Nos. i, 2, 3, 6, 7 received 3 m g nicotinic acid a n d r a t Nos. 4, 5, 8, 9, io 2. 5 m g nicotinic acid i n t r a p e r i t o n e a l l y as t e s t dose. Re[erences p . 549"
VOL. 22 (1956)
METABOLISM OF NICOTINIC ACID
549
The excretory pattern obtained in the case of the pantothenate-deficient rats in the present study is, however, interestingly enough different. The animals of the deficient group are seen to excrete a markedly lower proportion of nicotinic acid in the conjugated form and a correspondigly higher percentage as ]ree nicotinic acid than the control rats. This decreased excretion of the conjugate by the deficient group of rats would clearly point to the involvement of pantothenic acid in the conjugation of nicotinic acid and glycine to form nicotinuric acid. The discovery of the role of coenzyme A in the enzymic synthesis of hippturic acidS, e and certain related compounds 15 and more recently in the biosynthesis of some of the bile salts I8 has widened the range of coenzyme A-dependent reactions and it appears that the coenzyme may be obligatory for other cases of condensation of an amino group and a carboxyl group. It has been further established that coenzyme A mediates the conjugation of benzoic acid and glycine in a manner similar to the mechanism of biological acetylation. It is possible that a similar mechanism operates in the biosynthesis of nicotinuric acid from nicotinic acid and glycine. Interesting in this context is the report s on the enzyme glycine N-acylase, which catalyzes the conjugation of glycine with organic carboxylic acids and is very specific with respect to glycine and relatively not so in the case of the compounds having the carboxyl group. ACKNOWLEDGEMENTS
The authors wish to acknowledge with thanks the generous gift from Prof. B. C. JOHNSON, University of Illinois, of the culture of Leuconostoc mesenteroides 9135, and the award of a post-graduate research scholarship by the Lady Tata Memorial Trust, Bombay, to one of them (T.K.S.). SUMMARY T h e m e t a b o l i s m of nicotinic acid in n o r m a l a n d p a n t o t h e n a t e - d e f i c i e n t r a t s h a s been s t u d i e d w i t h p a r t i c u l a r reference to t h e e x c r e t i o n of nicotinuric acid, t h o glycine c o n j u g a t e of nicotinic acid. It h a s been s h o w n t h a t , w h e r e a s nicotinuric acid c o n s t i t u t e s a b o u t 6o % of t h e t e r t i a r y d e r i v a t i v e s of nicotinic acid e x c r e t e d b y control rats, it a c c o u n t s for a m a r k e d l y lower p r o p o r t i o n of t h e tert i a r y d e r i v a t i v e s in t h e case of t h e p a n t o t h e n a t e - d e f i c i e n t a n i m a l s . T h e r e s u l t s are i n t e r p r e t e d to indicate a n i n v o l v e m e n t of CoA in nicotinuric acid b i o s y n t h e s i s as in t h e e n z y m i c s y n t h e s i s of h i p p u r i c acid f r o m benzoic acid a n d glycine. REFERENCES i PEI-HSINGLIN AND B. C. JOHNSON,J. Am. Chem. Soc., 75 (I953) 2974. 2 D. SCHACHTER AND J. V. TAGGART, J. Biol. Chem., 208 (1954) 263a R. J. WILLIAMS, R. E. EAKIN, E. BEERSTECHER, Jr. ANn W. SHIVE, The Biochemistry o/B-vitamins, R e i n h o l d P u b l i s h i n g Corp., N e w York, 195 o, p. 361. 4 H. E. BRAUNSTEIN AND E. F. EFIMOCHKINA, Doklady Akad. Nauk. S.S.S.R., 71 (195 o) 347. 5 H. CHANTRENNE, J. Biol. Chem., 189 (1951) 227. D. SCHACHTER AND J. V. TAGGART, J. Biol. Chem., 203 (1953) 925. 7 R. E. OLSON AND N. O. KAPLAN, J. Biol. Chem., 175 (1948) 515 . s W . DRXLL AND M. S. DLT~N, Arch. Biochem. Biophys., 33 (1951) IiO. @E. E. SNELL, in P. GYORGY, Vitamin Methods, Vol. I, A c a d e m i c Press, N e w York, I95 o, p. 360. 10 E. E. SNELL AND L. D. WRIGHT, J. Biol. Chem., 139 (1941) 675. n B. C. JOHNSON, J. Biol. Chem., 159 (1945) 227. is K. K. REDDI AND E. KODICEK, Biochem. J., 53 (1953) 286. la B. C. JOHNSON, H. H. MITCHXLL AND T. S. HAMILTON, J. Biol. Chem., I59 (1945) 231. 14 W. A. KREHL, J. DE LA HUERGA AND C. A. ELVEHJEM, J. Biol. Chem., 166 (1946) 53. is T. R. RIGGS AND D. M. HEGSTED, J. Biol. Chem., 172 (1948) 539. IS j . BREMER, Acta Chem. Scand., 9 (1955) lO36.
Received February I9th , 1956 36