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A comparative study of the metabolism of nicotinamide and nicotinic acid in normal and germ-free rats
59
BIOCHIM1CA ET BIOPHYSICA ACTA
BBA
26796
A COMPARATIVE STUDY OF T H E METABOLISM OF N I C O T I N A M I D E AND NICOTINIC ACID IN NORMAL AND G E R M - F R E E RATS* YU CHIANG LEE**, ROBERT
M. M c K E N Z I E , R O B E R T
K. G H O L S O N * * *
AXD N I C H O L A S R A I C A
Department of Biochemistry, Agricultural Experiment Station, Oklahoma State University, Stillwater, Okla. 74 074 (U.S.A.) and United States A r m y Medical Research and Nutrition Laboratory, Fitzsimmons General Hospital, Denver, Colo. 8oa4o (U.S.A.) (Received, S e p t e m b e r 27th, i97 I)
SUMMARY
The importance of the gastrointestinal microflora in the metabolism of nicotinic acid and nicotinamide has been investigated using 7-14C-labeled vitamin forms. The CO 2 released from C-7 of the vitamin forms has been shown to result from microbial metabolism. The distribution of label in urinary metabolites was essentially unchanged in germ-free animals, indicating that the tissues of the rat are capable of metabolizing both vitamin forms in a normal manner without microbial assistance.
INTRODUCTION
Since the synthesis of E7-1%]nicotinic acid and E7-14C]nicotinamide in 1947 (ref. i), m a n y papers dealing with the metabolism of these compounds have been published. Roth et al3 found no significant differences in the gross metabolism of these two compounds in mice. Subsequent investigations a-s have established N'methylnicotinamide, nicotinic acid, nicotinamide-'V-oxide, A:-methyl-2-pyridone-5carboxamide, N-methyl-4-pyridone-3-carboxamide and more recently 6-hydroxynicotinic acid and 6-hydroxynicotinamide 9 as the urinary excretion products of E7-14C]nicotinamide and [7-14Clrdcotinic acid. Other studies have been concerned with a comparison of the conversion of nicotinic acid and nicotinamide to hepatic pyridine nucleotides in whole animals 1°-14 and in perfused liver (J. Keller, M. Liersch and H. Grtinicke, personal communication and refs. 15, 16). Some recent evidence suggests that in the whole animal the gastrointestinal flora m a y play an important role in the over-all metabolism of nicotinamide and the pyridine nucleotides in mammals. Ijichi et al. ~° reported that I h after intraportal injection of nicotinamide in mice about 5o % of the total radioactivity injected was found in the lumen of the gastro-intestinal tract. These workers concluded that nicotinamide is deamidated in the gastrointestinal tract to nicotinic acid which is then * J o u r n a l Article of t h e A g r i c u l t u r a l E x p e r i m e n t Station, O k l a h o m a S t a t e U n i v e r s i t y , Stillwater, Okla, U.S.A. ** P r e s e n t a d d r e s s : 3820 I t a s k a No. 8, St. Louis, Mo. 63 116. *** R e p r i n t r e q u e s t s s h o u l d be directed to this a u t h o r .
Biochim. Biophys. Aeta, 264 (1972) 59-64
00
V. C'. I.E]'2 ¢'[ ~l./.
reabsorbed and converted to NAD in the liver. The possible role of gastrointestinal microflora in nicotinamide metabolism is strenghtened by the report of Tanigawa c t a l . ~7 that the nicotinamide deanlidase activity of the pars preventricularis of rat stomach is due to a bacterial deamidase. If microbial deanlidation of nicotinamide plays an important physiological role in nicotinamide and pyridine nucleotide metabolism, one would expect to find considerable differences in the metabolic fate of nicotinamide in germ-free and normal animals. We undertook the studies described below in order to resolve this question as well as to determine if the evolution of x4CO, from injection of L7-14C!nicotinic acid, ~7-14Clnicotinamide 2 and NAD (ITJ4C!nicotinamide) 18, is due to degradation in the animals tissues or by the gastrointestinal microflora, MATERIALS AND METHODS
Nicotinic acid and nicotinamide were obtained from Nutritional Biocliemical Company. [7-14C~Nicotinic acid and 7-1~Cinicotinamide (spec. act. i2. 4 lnCi/mmole) were purchased from Calbiochem Inc. 6-Hydroxynicotinic acid was purchased from Aldrich Chemical Co., Cellulose powder (MN 30o G) from Microchemical Specialities Co., and PPO (2,5-diphenyloxazole) and POPOP (I,4-bis-2-(5-phenyloxazolyl)-benzene) from Packard Instrument Co. The radioactive compounds were repurified betore use by descending paper chromatography using n-butanol saturated with 3 °o ammonia. 6-Hydroxynicotinamide was prepared from 6-hydroxynicotinic acid as reported previously9. All other chemicals were reagent grade. Adult male rats weighing from 2oo to 35o g were used as control animals (Holtzman Co., Madison, Wisc.) and were maintained on Purina Laboratory ('how. Germ-free rats were maintained at the Gnotobiotic Laboratory, Fitzsimons General Hospital, Denver, Colo., by Dr. N. Raica. These animals were checked weekly for gelm-free status by fecal smears on nutrient agar plates. !7-14C]Nicotinamide and 7-14CJnicotinic acid were administered by aseptic intraperitoneal injection at a level of 5.2/~Ci per animal. Sufficient unlabeled nicotinamide or nicotinic acid was injected to bring the dose to 5 or 5oo mg/kg. The animals were housed in sealed glass metabolism cages. The urine was collected over a 24-h period in a I5o-ml Erlenmyer flask surrounded with dry ice. Air was drawn through the cages by a water aspirator. Expired CO 2 was trapped by bubbling the effluent air stream through a series of three traps containing Ioo ml of ethanolamine-nlethvlcellosolve (I: 2, v/v). The urinary metabolites of nicotinic acid and nicotinamide were separated by cellulose thin-layer chromatography (o.5 mm layer on 5 cm × 3o cm plates) using n-butanol saturated with 3 % ammonia as a solvent. 6-Hydroxynicotinamide and nicotinamide-N-oxide were not sufficiently resolved in this system so they were scraped off the plate and counted as a single peak. In a second plate this peak was scraped and eluted with water. The eluant was concentrated and spotted on a strip of Whatman No. I filter paper and separation was accomplished by electrophoresis in o.o 5 M NaaBO a (pH 9.3) at 2ooo V for I h. The distribution of radioactivity between the two compounds was determined and the relative percentage obtained was used to subdivide the value obtained on the previous plate. Biochim. lqiophys. Acta, 2(74 (I972) 59-64
N I C O T I N A M I D E M E T A B O L I S M I N G E R M - F R E E RATS
6I
Radioactivity was measured in a Packard TriCarb Liquid Scintillation Spectrometer. Combined COe traps were assayed for ~4C0~ by addition of 3-ml aliquots to 15 ml of a scintillation fluid containing methylcellosolve-toluene (I : 2, v/v) and 5-5 g of PPO per 1. Radioactive areas on paper chromatograms were located with a Nuclear Chicago 4-~ Actigraph I I I paper strip counter and quantitated by cutting the radioactive areas on the strips into 1-cm bands and counting in a vial containting Io ml of scintillation fluid. Thin layer plates were scanned for radioactivity using a 2-~ thin-layer plate scanning accessory to the Actigraph I I I . The radioactive areas of the plate were scraped into a vial and Io ml of scintillation fluid added. The scintillation solvent used for paper and thin-layer chromatography counting was composed of 6oo ml toluene, 4oo ml absolute ethanol, 4 g of PPO and o.2 g POPOP per 1. Radioactivity in liquid samples was determined using o.I-ml aliquots in lO ml of the above cocktail. RESULTS AND DISCUSSION
Conversion of C- 7 of nicotinamide and nicotinic acid to CO 2 The evolution of ~4CO2 from C-7 of nicotinic acid and nicotinamide was first reported by Roth et al. 2. Intraperitoneal administration of a dose of 28 mg/kg resulted in the release of approx. 1.5 % of the label in I7-14C~nicotinic acid and 3.0 °o of that in ~7-14Clnicotinamide. More recently, Negishi and Ichiyama is reported the release of i4CO2 from NAD ([7-14Clnicotinamide) in mice when injected intravenously or administered by stomach tube. At 4 h, intravenous administration resulted in a loss of 3,46 °o of the dose and oral administration resulted in the loss of 16.5 % of the dose as 14C02. As shown by the data presented in Table I, the germ-free animals release no '4C02, clearly indicating that the C0~ is of microbial origin, as has been suggested by above authors is. TABLE
I
A COMPARISON OF THE EXCRETION OF RADIOACTIVITY IN THE URINE AND EXPIRED CO 2 OF GERM-FREE A N D NORMAL RATS 2 4 h AFTER I N T R A P E R I T O N E A L A D M I N I S T R A T I O N OF ~7-14C~ N I CO T I N I C ACID AND [7-14C~-NICOTINAMIDE
I ~ e s u l t s a r e a v e r a g e s of 2 g e r m - f r e e r a t s p e r e x p e r i m e n t a n d 6 n o r m a l r a t s p e r e x p e r i m e n t .
Type of rat
Treatment (mg/kg)
Radioactivity Expired excreted in the radioactive CO 2 urine (% of dose) ( % of dose)
Nieotinamide G e r m free Normal
500 5 500 5
81.8 62.6 70.4 68.5
o.o o.o z. i 7-7
80.5 48.0 87.7 50.4
o.o o.o 1.2 4.7
Nicotinic acid G e r m free Normal
500 5 500 5
Biochim. Biophys. Acta, 264 (1972) 5 9 - 6 4
ir
I
4a
1.6
33.9 504
Total
I.O
26.6
6.3
T o t a l n i c o t i n i c a c i d a n d its m e t a b o l i t c s
6 - H y d r o x y m i c o t i n i c acid
Nicotinuric acid
N i c o t i n i c acid
16.8
T o t a l n i c o t i n a n l i d e a n d its m e t a b o l i t e s
48.0
35 .'q
I.O
24. 9
9.9
12.2
o. 2
o. 5 --
6-Hydroxynicotinamide
2- a n d 4 - P y r i d o n e
I. 3
o. 5
0. 7 8,4
Nicotinami de-A'-oxide
1 .o 14.8
.Y-Methvlnicotinanlide
Nicotinamide
A'ormal
,z ..a
(;erm-frcc
5 mg/kg
~>
4~
Nicotinic acid
k
87. 7
,qI.2
o.8
19-5
6o. 9
6. 5
o.z
1.8
2.8
i .7
.Vormat
500 mg/kg
80. 5
74.7
o.6
17-3
56.8
5.8
Trace
0.2
2.6
I, 4
i .6
(Term-free
68. 5
3.6
o.3
r. 5
I .S
65.8
0.6
9-9
I. 4
47-I
6.8
Xormal
5 mg/kg
62.6
1.4
o.4
o. 5
o, 5
61.2
0. 5
6-5
3.2
45. I
5-9
( ; e r m free
A'icotinamide
7o.4
I4.5
<).4
8.o
6. I
55.9
0.8
2. 4
6.8
i 1.7
34 .2
Normal
5o0 mg/kg
8~,S
25.2
o-4
9.8
15.0
56.6
0.6
1.5
8.o
8.3
38,2
(;erm-free
G e r m - f r e e figures a r e t h e a v e r a g e of 2 a n i m a l s p e r e x p e r i m e n t . N o r m a l f i g u r e s a r e t h e a v e r a g e t w o e x p e r i m e n t s w i t h 3 a n i m a l s p e r e x p e r i m e n t .
RADIOACTIVITY DISTRIBUTION AMONG TIlE URINARY METABOLITES OF NORMAL AND GI~'RM-FRJEI~2RATS
TABLE
<
ba
NICOTINAMIDE METABOLISM IN GERM-FREE RATS
63
The general relationship between the release of CO 2 from nicotinamide and nicotinic acid reported by Roth et al. ~ holds for these data, about twice as much CO 2 is released from nicotinamide as from nicotinic acid. I t is possible that the evolution of CO~ involves oxidative deamination thus requiring nicotinic acid to be recycled tllrough NAD to nicotinamide before it can be subjected to the loss of C- 7, or perhaps a single enzyme with a markedly different affinity for each of the vitamers is responsible. In line with these findings, the release of CO,, front C- 7 of both vitamers is essentially complete in about 4 h (ref. 2), the time point at which little or no nicotinamide remains. The findings of Negishi and Ichijama ts indicate that the route of administration also has a significant effect on the amount of CO 2 produced. Metabolites of nicotinic acid and nicotinamide in urine The distribution of metabolites in tile urine of normal and germ-free rats is presented in Table II. The data for normal animals are in generally good agreement with those reported previouslyV, 8. In the germ-free rat, the distribution of the metabolites of the two vitamers at either low or high dosages is essentially unchanged. Clearly, the intact rat has the capacity to metabolize both vitamers, including the ability to deamidate nicotinamide without microbial assistance even at dosage levels insufficient to result in an elevation of hepatic NAD. Synthesis of hepatic N A D from nicotinamide Nicotinic acid is more rapidly incorporated into hepatic NAD than nicotinamide; however, the apparent lack of an effective tissue nicotinamide deamidase and the presence of the enzyme capable of converting nicotinamide to NAD via nicotinamide mononucleotide has led to much discussion as to the route by which nicotinamide is utilized in vitro at physiological levels (J. Keller, M. Liersch and H. Gr tinicke, personal communication, and refs. IO, I3, I5, 16, 19). I t is clear that the conversion of nicotinamide to hepatic NAD occurs via nicotinic acid in animals challenged with large doses of nicotinamidea°,15, z). Under these conditions, hepatic NAD concentrations are elevated, nicotinamide phosphoribosyl transferase activity is under very strong feedback inibition and nicotinamide concentrations are increased to levels wh~re th~ synthesis of nicotinic acid via th~ high Km deamidase of liver can be of significance 2°. As a result of its high K m (approx. io -~ M), the significance of hepatic nicotinamide deamidase at physiological nicotinamide concentrations has been strongly questioned (J. Keller, M. Liersch and H. Griinicke, personal communication, and ref. I5). However, with the finding of a low K m deamidase in the stomach of the raW, it could be argued that the gastrointestinal microflora m a y play a significant role in the deamidation of nicotinamide during its conversion to h~patic NAD. Ijichi et al. 1° have shown that label from I7n~Clnicotinamide accumulates in the gut; that the percentage of th~ dose in the gut increases with decreasing dose and that in a short time (2-4 h) essentially all the label is present as nicotinic acid. Tanigawa et al. a7 using low doses of E7-14C]nicotinamide (5.4 #g) administered orally showed that the vitamer was almost completely deamidated in the stomach of rats (2 h, or less) and reported that the resultant nicotinic acid passed into the intestine before being absorbed. However, the data presented here demonstrate the nonessentiality of a deamidation of nicotinamide by gastrointestinal microflora at both a challenge level Biochirn. Biophys. Acta, 264 (i972) 59-64
64
v, c. LI~;E c t a / .
(500 m g / k g ) w h e r e l a r g e i n c r e a s e s in h e p a t i c N A D o c c u r a n d a t a r e l a t i v e l y l o w d o s e l e v e l (5 m g / k g ) w h e r e l i t t l e or n o i n f l u e n c e o n h e p a t i c N A D l e v e l s is o b s e r v a b l e . T h e s e d a t a d o n o t e x c l u d e a m i c r o b i a l c o n t r i b u t i o n t o t h e o v e r a l l m e t a b o l i s m of n i c o t i n a m i d e in t h e n o r m a l a n i m a l a t p h y s i o l o g i c a l c o n c e n t r a t i o n s of t h e v i t a m i n p a r t i c u l a r l y w h e n o n e c o n s i d e r s t h a t i t is n o r m a l l y o b t a i n e d o r a l l y . H o w e v e r t h e d a t a do e x c l u d e t h e p r o c e s s as a n e c e s s a r y r e q u i r e m e n t for t h e c o n v e r s i o n of n i c o t i n a m i d e to n i c o t i n i c acid under our experimental conditions. ACI~'OWS>CDGEM~N'rS T h i s i n v e s t i g a t i o n w a s s u p p o r t e d i n p a r t b y U.S. P u b l i c H e a l t h S e r v i c e g r a n t G M - I o 6 6 6 f r o m t h e N a t i o n a l I n s t i t u t e of G e n e r a l M e d i c a l S c i e n c e s . O n e of t h e a u t h o r s ( R . K . G . ) is a C a r e e r D e v e l o p m e n t A w a r d e e of t h e N a t i o n a l I n s t i t u t e s of G e n e r a l M e d i c a l S c i e n c e s 5 - K 3 - G M - 9 2 5 e .
REFERENCES 1 2 3 4 5 6 7 8 9 io II
12 13 14 15 i6 17 I8 19 2o
A. Murray, W. W. Foreman and W. Langhain, Science, IO6 (1947) 277L. J. Roth, E. Leifer, J. R. Hogness and W. H. Langham, J. Biol. Chem., 176 (I948) 249. E. Leifer, L. J. Roth, D. S. Hogness and M. H. Corsom, J. Biol. Chem., z9o (I95I) 595. M. L. VV. Chang and B. C. Johnson, J. Biol. Chem., 234 (1959) I817. V. Bonavita, S. A. Narrod and N. O. Kaplan, J. Biol. Chem., 236 (196r) 936. S. Chaykin, M. Dagani, L. Johnson and M. Samli, .[. Biol. Chem., 24 ° (i965) 932. B. Petrack, P. Oreengard and H. Kalinsky, J. Biol. Chem., 24i (t966) 2367. P. Greengard, B. Petrack and H. Kalinsky, jr. Biol. Chem., 242 (r967) I52. Y. C. Lee, R. K. Gholson and N. Raica, J. Biol. Chem., 244 (I969) 3277. H. Ijichi, A. [chiyama and O. Hayaishi, .[. Biol. Chem., 241 (I966) 37o~. J. Preiss and P. Handler, J. Biol. Chem., 233 (~958) 488. S. Chaykin, M. Dagani, L. Johnson, M. Samli and J. Battaile, Biochim. Biophys. dcta, lOO (1965) 351 • C. Streffer, \¥. Brauer and J. Benes, in Workshop on Metabolic Effects of Nicotinic Acid and its Derivatives, March 23-25, z97o, Fliins, Switzerland. L. S. Dietrich, L. Fuller and B. Farinas, in Workshop on Metabolic £f/;'cts of Nicotinic Acid and its Derivatives, March 23-25, z97o, Flims, Switzerland. H. Gr/inicke, J. Keller and M. Liersch, in Workshop on Metabolic Effects of Nicotinic Acid and its Derivatives, ~Iarch z 3 25, z97 o, Iqims, Switzerland. Y. Hagino, S. J. Lan, C. Y. Ng and L. M. Henderson, J. Biol. Chem., ~43 (1968) 498o. Y. Tanigawa, M. Shimoyama, R. Murashima, T. Ito, N. Yamaguchi and I. l!eda, Biochim. Biophys. Acta, "2Ol (197 o) 394. T. Negishi and A. Ichiyama, Vitamins (Japan), 4 ° (1969) 38. T, A. Langan, Jr., N. O. Kaplan and L. Shuster, J. Biol. Chem., 234 (I959) 2t6~. L. S. Dietrich, O. Muniz and M. Powanda, J. Vitaminol., r 4 (t968) t23.
Biochim. Biophys. Acta, 264 (J972) 59-64
BIOCHIM1CA ET BIOPHYSICA ACTA
BBA
26796
A COMPARATIVE STUDY OF T H E METABOLISM OF N I C O T I N A M I D E AND NICOTINIC ACID IN NORMAL AND G E R M - F R E E RATS* YU CHIANG LEE**, ROBERT
M. M c K E N Z I E , R O B E R T
K. G H O L S O N * * *
AXD N I C H O L A S R A I C A
Department of Biochemistry, Agricultural Experiment Station, Oklahoma State University, Stillwater, Okla. 74 074 (U.S.A.) and United States A r m y Medical Research and Nutrition Laboratory, Fitzsimmons General Hospital, Denver, Colo. 8oa4o (U.S.A.) (Received, S e p t e m b e r 27th, i97 I)
SUMMARY
The importance of the gastrointestinal microflora in the metabolism of nicotinic acid and nicotinamide has been investigated using 7-14C-labeled vitamin forms. The CO 2 released from C-7 of the vitamin forms has been shown to result from microbial metabolism. The distribution of label in urinary metabolites was essentially unchanged in germ-free animals, indicating that the tissues of the rat are capable of metabolizing both vitamin forms in a normal manner without microbial assistance.
INTRODUCTION
Since the synthesis of E7-1%]nicotinic acid and E7-14C]nicotinamide in 1947 (ref. i), m a n y papers dealing with the metabolism of these compounds have been published. Roth et al3 found no significant differences in the gross metabolism of these two compounds in mice. Subsequent investigations a-s have established N'methylnicotinamide, nicotinic acid, nicotinamide-'V-oxide, A:-methyl-2-pyridone-5carboxamide, N-methyl-4-pyridone-3-carboxamide and more recently 6-hydroxynicotinic acid and 6-hydroxynicotinamide 9 as the urinary excretion products of E7-14C]nicotinamide and [7-14Clrdcotinic acid. Other studies have been concerned with a comparison of the conversion of nicotinic acid and nicotinamide to hepatic pyridine nucleotides in whole animals 1°-14 and in perfused liver (J. Keller, M. Liersch and H. Grtinicke, personal communication and refs. 15, 16). Some recent evidence suggests that in the whole animal the gastrointestinal flora m a y play an important role in the over-all metabolism of nicotinamide and the pyridine nucleotides in mammals. Ijichi et al. ~° reported that I h after intraportal injection of nicotinamide in mice about 5o % of the total radioactivity injected was found in the lumen of the gastro-intestinal tract. These workers concluded that nicotinamide is deamidated in the gastrointestinal tract to nicotinic acid which is then * J o u r n a l Article of t h e A g r i c u l t u r a l E x p e r i m e n t Station, O k l a h o m a S t a t e U n i v e r s i t y , Stillwater, Okla, U.S.A. ** P r e s e n t a d d r e s s : 3820 I t a s k a No. 8, St. Louis, Mo. 63 116. *** R e p r i n t r e q u e s t s s h o u l d be directed to this a u t h o r .
Biochim. Biophys. Aeta, 264 (1972) 59-64
00
V. C'. I.E]'2 ¢'[ ~l./.
reabsorbed and converted to NAD in the liver. The possible role of gastrointestinal microflora in nicotinamide metabolism is strenghtened by the report of Tanigawa c t a l . ~7 that the nicotinamide deanlidase activity of the pars preventricularis of rat stomach is due to a bacterial deamidase. If microbial deanlidation of nicotinamide plays an important physiological role in nicotinamide and pyridine nucleotide metabolism, one would expect to find considerable differences in the metabolic fate of nicotinamide in germ-free and normal animals. We undertook the studies described below in order to resolve this question as well as to determine if the evolution of x4CO, from injection of L7-14C!nicotinic acid, ~7-14Clnicotinamide 2 and NAD (ITJ4C!nicotinamide) 18, is due to degradation in the animals tissues or by the gastrointestinal microflora, MATERIALS AND METHODS
Nicotinic acid and nicotinamide were obtained from Nutritional Biocliemical Company. [7-14C~Nicotinic acid and 7-1~Cinicotinamide (spec. act. i2. 4 lnCi/mmole) were purchased from Calbiochem Inc. 6-Hydroxynicotinic acid was purchased from Aldrich Chemical Co., Cellulose powder (MN 30o G) from Microchemical Specialities Co., and PPO (2,5-diphenyloxazole) and POPOP (I,4-bis-2-(5-phenyloxazolyl)-benzene) from Packard Instrument Co. The radioactive compounds were repurified betore use by descending paper chromatography using n-butanol saturated with 3 °o ammonia. 6-Hydroxynicotinamide was prepared from 6-hydroxynicotinic acid as reported previously9. All other chemicals were reagent grade. Adult male rats weighing from 2oo to 35o g were used as control animals (Holtzman Co., Madison, Wisc.) and were maintained on Purina Laboratory ('how. Germ-free rats were maintained at the Gnotobiotic Laboratory, Fitzsimons General Hospital, Denver, Colo., by Dr. N. Raica. These animals were checked weekly for gelm-free status by fecal smears on nutrient agar plates. !7-14C]Nicotinamide and 7-14CJnicotinic acid were administered by aseptic intraperitoneal injection at a level of 5.2/~Ci per animal. Sufficient unlabeled nicotinamide or nicotinic acid was injected to bring the dose to 5 or 5oo mg/kg. The animals were housed in sealed glass metabolism cages. The urine was collected over a 24-h period in a I5o-ml Erlenmyer flask surrounded with dry ice. Air was drawn through the cages by a water aspirator. Expired CO 2 was trapped by bubbling the effluent air stream through a series of three traps containing Ioo ml of ethanolamine-nlethvlcellosolve (I: 2, v/v). The urinary metabolites of nicotinic acid and nicotinamide were separated by cellulose thin-layer chromatography (o.5 mm layer on 5 cm × 3o cm plates) using n-butanol saturated with 3 % ammonia as a solvent. 6-Hydroxynicotinamide and nicotinamide-N-oxide were not sufficiently resolved in this system so they were scraped off the plate and counted as a single peak. In a second plate this peak was scraped and eluted with water. The eluant was concentrated and spotted on a strip of Whatman No. I filter paper and separation was accomplished by electrophoresis in o.o 5 M NaaBO a (pH 9.3) at 2ooo V for I h. The distribution of radioactivity between the two compounds was determined and the relative percentage obtained was used to subdivide the value obtained on the previous plate. Biochim. lqiophys. Acta, 2(74 (I972) 59-64
N I C O T I N A M I D E M E T A B O L I S M I N G E R M - F R E E RATS
6I
Radioactivity was measured in a Packard TriCarb Liquid Scintillation Spectrometer. Combined COe traps were assayed for ~4C0~ by addition of 3-ml aliquots to 15 ml of a scintillation fluid containing methylcellosolve-toluene (I : 2, v/v) and 5-5 g of PPO per 1. Radioactive areas on paper chromatograms were located with a Nuclear Chicago 4-~ Actigraph I I I paper strip counter and quantitated by cutting the radioactive areas on the strips into 1-cm bands and counting in a vial containting Io ml of scintillation fluid. Thin layer plates were scanned for radioactivity using a 2-~ thin-layer plate scanning accessory to the Actigraph I I I . The radioactive areas of the plate were scraped into a vial and Io ml of scintillation fluid added. The scintillation solvent used for paper and thin-layer chromatography counting was composed of 6oo ml toluene, 4oo ml absolute ethanol, 4 g of PPO and o.2 g POPOP per 1. Radioactivity in liquid samples was determined using o.I-ml aliquots in lO ml of the above cocktail. RESULTS AND DISCUSSION
Conversion of C- 7 of nicotinamide and nicotinic acid to CO 2 The evolution of ~4CO2 from C-7 of nicotinic acid and nicotinamide was first reported by Roth et al. 2. Intraperitoneal administration of a dose of 28 mg/kg resulted in the release of approx. 1.5 % of the label in I7-14C~nicotinic acid and 3.0 °o of that in ~7-14Clnicotinamide. More recently, Negishi and Ichiyama is reported the release of i4CO2 from NAD ([7-14Clnicotinamide) in mice when injected intravenously or administered by stomach tube. At 4 h, intravenous administration resulted in a loss of 3,46 °o of the dose and oral administration resulted in the loss of 16.5 % of the dose as 14C02. As shown by the data presented in Table I, the germ-free animals release no '4C02, clearly indicating that the C0~ is of microbial origin, as has been suggested by above authors is. TABLE
I
A COMPARISON OF THE EXCRETION OF RADIOACTIVITY IN THE URINE AND EXPIRED CO 2 OF GERM-FREE A N D NORMAL RATS 2 4 h AFTER I N T R A P E R I T O N E A L A D M I N I S T R A T I O N OF ~7-14C~ N I CO T I N I C ACID AND [7-14C~-NICOTINAMIDE
I ~ e s u l t s a r e a v e r a g e s of 2 g e r m - f r e e r a t s p e r e x p e r i m e n t a n d 6 n o r m a l r a t s p e r e x p e r i m e n t .
Type of rat
Treatment (mg/kg)
Radioactivity Expired excreted in the radioactive CO 2 urine (% of dose) ( % of dose)
Nieotinamide G e r m free Normal
500 5 500 5
81.8 62.6 70.4 68.5
o.o o.o z. i 7-7
80.5 48.0 87.7 50.4
o.o o.o 1.2 4.7
Nicotinic acid G e r m free Normal
500 5 500 5
Biochim. Biophys. Acta, 264 (1972) 5 9 - 6 4
ir
I
4a
1.6
33.9 504
Total
I.O
26.6
6.3
T o t a l n i c o t i n i c a c i d a n d its m e t a b o l i t c s
6 - H y d r o x y m i c o t i n i c acid
Nicotinuric acid
N i c o t i n i c acid
16.8
T o t a l n i c o t i n a n l i d e a n d its m e t a b o l i t e s
48.0
35 .'q
I.O
24. 9
9.9
12.2
o. 2
o. 5 --
6-Hydroxynicotinamide
2- a n d 4 - P y r i d o n e
I. 3
o. 5
0. 7 8,4
Nicotinami de-A'-oxide
1 .o 14.8
.Y-Methvlnicotinanlide
Nicotinamide
A'ormal
,z ..a
(;erm-frcc
5 mg/kg
~>
4~
Nicotinic acid
k
87. 7
,qI.2
o.8
19-5
6o. 9
6. 5
o.z
1.8
2.8
i .7
.Vormat
500 mg/kg
80. 5
74.7
o.6
17-3
56.8
5.8
Trace
0.2
2.6
I, 4
i .6
(Term-free
68. 5
3.6
o.3
r. 5
I .S
65.8
0.6
9-9
I. 4
47-I
6.8
Xormal
5 mg/kg
62.6
1.4
o.4
o. 5
o, 5
61.2
0. 5
6-5
3.2
45. I
5-9
( ; e r m free
A'icotinamide
7o.4
I4.5
<).4
8.o
6. I
55.9
0.8
2. 4
6.8
i 1.7
34 .2
Normal
5o0 mg/kg
8~,S
25.2
o-4
9.8
15.0
56.6
0.6
1.5
8.o
8.3
38,2
(;erm-free
G e r m - f r e e figures a r e t h e a v e r a g e of 2 a n i m a l s p e r e x p e r i m e n t . N o r m a l f i g u r e s a r e t h e a v e r a g e t w o e x p e r i m e n t s w i t h 3 a n i m a l s p e r e x p e r i m e n t .
RADIOACTIVITY DISTRIBUTION AMONG TIlE URINARY METABOLITES OF NORMAL AND GI~'RM-FRJEI~2RATS
TABLE
<
ba
NICOTINAMIDE METABOLISM IN GERM-FREE RATS
63
The general relationship between the release of CO 2 from nicotinamide and nicotinic acid reported by Roth et al. ~ holds for these data, about twice as much CO 2 is released from nicotinamide as from nicotinic acid. I t is possible that the evolution of CO~ involves oxidative deamination thus requiring nicotinic acid to be recycled tllrough NAD to nicotinamide before it can be subjected to the loss of C- 7, or perhaps a single enzyme with a markedly different affinity for each of the vitamers is responsible. In line with these findings, the release of CO,, front C- 7 of both vitamers is essentially complete in about 4 h (ref. 2), the time point at which little or no nicotinamide remains. The findings of Negishi and Ichijama ts indicate that the route of administration also has a significant effect on the amount of CO 2 produced. Metabolites of nicotinic acid and nicotinamide in urine The distribution of metabolites in tile urine of normal and germ-free rats is presented in Table II. The data for normal animals are in generally good agreement with those reported previouslyV, 8. In the germ-free rat, the distribution of the metabolites of the two vitamers at either low or high dosages is essentially unchanged. Clearly, the intact rat has the capacity to metabolize both vitamers, including the ability to deamidate nicotinamide without microbial assistance even at dosage levels insufficient to result in an elevation of hepatic NAD. Synthesis of hepatic N A D from nicotinamide Nicotinic acid is more rapidly incorporated into hepatic NAD than nicotinamide; however, the apparent lack of an effective tissue nicotinamide deamidase and the presence of the enzyme capable of converting nicotinamide to NAD via nicotinamide mononucleotide has led to much discussion as to the route by which nicotinamide is utilized in vitro at physiological levels (J. Keller, M. Liersch and H. Gr tinicke, personal communication, and refs. IO, I3, I5, 16, 19). I t is clear that the conversion of nicotinamide to hepatic NAD occurs via nicotinic acid in animals challenged with large doses of nicotinamidea°,15, z). Under these conditions, hepatic NAD concentrations are elevated, nicotinamide phosphoribosyl transferase activity is under very strong feedback inibition and nicotinamide concentrations are increased to levels wh~re th~ synthesis of nicotinic acid via th~ high Km deamidase of liver can be of significance 2°. As a result of its high K m (approx. io -~ M), the significance of hepatic nicotinamide deamidase at physiological nicotinamide concentrations has been strongly questioned (J. Keller, M. Liersch and H. Griinicke, personal communication, and ref. I5). However, with the finding of a low K m deamidase in the stomach of the raW, it could be argued that the gastrointestinal microflora m a y play a significant role in the deamidation of nicotinamide during its conversion to h~patic NAD. Ijichi et al. 1° have shown that label from I7n~Clnicotinamide accumulates in the gut; that the percentage of th~ dose in the gut increases with decreasing dose and that in a short time (2-4 h) essentially all the label is present as nicotinic acid. Tanigawa et al. a7 using low doses of E7-14C]nicotinamide (5.4 #g) administered orally showed that the vitamer was almost completely deamidated in the stomach of rats (2 h, or less) and reported that the resultant nicotinic acid passed into the intestine before being absorbed. However, the data presented here demonstrate the nonessentiality of a deamidation of nicotinamide by gastrointestinal microflora at both a challenge level Biochirn. Biophys. Acta, 264 (i972) 59-64
64
v, c. LI~;E c t a / .
(500 m g / k g ) w h e r e l a r g e i n c r e a s e s in h e p a t i c N A D o c c u r a n d a t a r e l a t i v e l y l o w d o s e l e v e l (5 m g / k g ) w h e r e l i t t l e or n o i n f l u e n c e o n h e p a t i c N A D l e v e l s is o b s e r v a b l e . T h e s e d a t a d o n o t e x c l u d e a m i c r o b i a l c o n t r i b u t i o n t o t h e o v e r a l l m e t a b o l i s m of n i c o t i n a m i d e in t h e n o r m a l a n i m a l a t p h y s i o l o g i c a l c o n c e n t r a t i o n s of t h e v i t a m i n p a r t i c u l a r l y w h e n o n e c o n s i d e r s t h a t i t is n o r m a l l y o b t a i n e d o r a l l y . H o w e v e r t h e d a t a do e x c l u d e t h e p r o c e s s as a n e c e s s a r y r e q u i r e m e n t for t h e c o n v e r s i o n of n i c o t i n a m i d e to n i c o t i n i c acid under our experimental conditions. ACI~'OWS>CDGEM~N'rS T h i s i n v e s t i g a t i o n w a s s u p p o r t e d i n p a r t b y U.S. P u b l i c H e a l t h S e r v i c e g r a n t G M - I o 6 6 6 f r o m t h e N a t i o n a l I n s t i t u t e of G e n e r a l M e d i c a l S c i e n c e s . O n e of t h e a u t h o r s ( R . K . G . ) is a C a r e e r D e v e l o p m e n t A w a r d e e of t h e N a t i o n a l I n s t i t u t e s of G e n e r a l M e d i c a l S c i e n c e s 5 - K 3 - G M - 9 2 5 e .
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Biochim. Biophys. Acta, 264 (J972) 59-64