Eur. J. Biochem. 22 (1971) 263-270
Studies on the Biosynthesis of NAD from Nicotinamide and on the Intracellular Pyridine Nucleotide Cycle in Isolated Perfused Rat Liver Jiirgen KELLER,Michael LIERSCH,and Hans GRUNICKE Biochemisches Institut der Universitiit, Freiburg i. Br., Germany (Received May 11/July 14, 1971)
Isolated perfused rat liver incorporates nicotinamide a t a concentration of 10 pM into NAD via nicotinamide mononucleotide. A deamidation of nicotinamide could not be demonstrated. Evidence is presented which indicates that the exchange reaction catalyzed by NAD glycohydrolase is not involved in the uptake of nicotinamide into NAD. At the concentrations used, the rate of the incorporation of nieotinamide into NAD is about 40°/, of the incorporation rate observed with nicotinic acid. Results obtained after perfusion with [14C]nicotinic acid support the concept of an intracellular pyridine nucleotide cycle. However, in contrast to the general opinion, our results demonstrate that in rat liver nicotinamide generated by NAD breakdown is not deamidated but reutilized via nicotinamide mononucleotide. After perfusion with 0.1 mM ['4C]nicotinamide for 2.5 h, less than 2O/, of the total radioactivity is excreted into the bile. The same value is obtained after perfusion with 50 pM [14C]nicotinic acid. From these results it is concluded that the excretion of nicotinamide by the liver and the subsequent deamidation within the intestinal tract which have been considered important in the utilization of nicotinamide by the liver, are without biological relevance a t normal nicotinamide concentrations.
The biosynthesis of NAD in mammalian liver (Fig.l) occurs from tryptophan via quinolinic acid, from nicotinic acid and from nicotinamide (for a review see Chaykin [I]). It is well established that nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide are intermediates in the conversion of quinolinic acid and nicotinic acid to NAD [2-41. However, the biosynthetic route of NAD from nicotinamide in liver has remained subject to question. It has been demonstrated that cell free extracts of mammalian liver have the enzymic capacity to utilize nicotinamide a t biological concentrations for the formation of NAD via nicotinamide mononucleotide [ 5 ] .However, it is widely assumed that this pathEnzymes. NAD glycohydrolase (EC 188.8.131.52); nicotinamide-nucleotide : pyrophosphate phosphoribosyltransferase or nicotinamide phosphoribosyltransferase (EC 184.108.40.206) ; nicotinatenucleotide : pyrophosphate phosphoribosyltransferase or nicotinate phosphoribosyltransferase (EC 220.127.116.11) ; ATP :NMN adenyltransferase or NMN pyrophosphorylase EC 18.104.22.168) ;deamido-NAD pyrophosphorylase (EC 2.7.7.-) ; deamido-NAD : L-glutamine amido-ligase (AMP) or NAD synthetase (EC 22.214.171.124); alcohol : NAD oxidoreductase or alcohol dehydrogenase (EC 126.96.36.199); ATP : 3-phospho-~glycerate 1-phosphotransferase or phosphoglycerate kinase (EC 188.8.131.52); ~-glyceraldehyde-3-phosphate : NAD-oxidoreductase (phosphorylating) or glyceraldehydephosphate dehydrogenase (EC 184.108.40.206).
way is not biologically relevant to mammalian liver, and it is believed that nicotinamide has t o be deamidated to nicotinic acid before entering the biosynthetic
I N'-methyl N ,,
Pig.1. Biosynthesis of NAD in rat liver. Qa: quinolinic acid, Nu: nicotinic acid, Nsm:nicotinamide, NaMN: nicotinic acid mononucleotide,NMN :nicotinamide mononucleotide,NaAD: nicotinic acid adenine dinucleotide, NAD : nicotinamide adenine dinucleotide, 5-P-Rib-1-PP: 5-phosphoribosyl-lpyrophosphate, ADP-Rib : adenosine diphosphate ribose. To simplify description, each reaction has been numbered and these numbers are used in the text to designate a particular Step
Biosynthesis of NAD from Nicotinamide in Perfused Rat Liver
route. This assumption is supported by the appearance of intermediates of the nicotinic acid pathway after application of nicotinamide to mice [6--81, rats  and cats [lo]. However, the formation of nicotinic acid derivatives from injected nicotinamide has been demonstrated only after administration of hyperphysiological concentrations of the vitamin greater than 1 mmole per kg. The studies presented here demonstrate that isolated perfused rat liver incorporates nicotinamide at physiological concentrations (10 pM) into NAD exclusively via nicotinamide mononucleotide. Studies on the fate of the pyridine moiety of NAD suggest that the nicotinamide produced by the degradation of NAD is reutilized for NAD synthesis by an intracellular pyridine nucleotide cycle with nicotinamide mononucleotide as an intermediate. The studies presented here were performed with isolated rat liver perfused with an erythrocyte free medium in order to prevent interference of nonhepatic tissues. MATERIALS AND METHODS
[7-14C]Nicotinic acid (specific activity 59.1 mCi/ mmole) and [7-14C]nicotinamide (specific activity 59.6 mCi/mmole) were obtained from the Radiochemical Centre (Amersham Buckinghamshire, England). The nicotinamide contained traces of impurities which were removed by ion exchange chromatography on a Dowex 1-X2 column, formate form, eluted with 0.01 N HCOOH. Nicotinamide mononucleotide was purchased from Boehringer Mannheim GmbH (Mannheim, Germany). Azaserine (diazoacetylserine) was a gift from Dr. R. R. Engle (Cancer Chemotherapy National Service Center, National Institutes of Health Bethesda, Maryland, U.S.A.). Nicotinic acid adenine dinucleotide was prepared according to Lamborg et al. [ll]. Nicotinic acid mononucleotide was prepared from nicotinic acid adenine dinucleotide using a purified snake venom diesterase as described previously . Rat livers were obtained from female Wistar rats weighing 150-170 g. Perfusion of isolated rat liver was performed by a modification of the procedure described by Teufel et al.  using a n erythrocyte free medium. The liver was perfused with a medium containing 2.5 g bovine serum albumin, 0.55 mmoles glucose, and 4 mg ampicillin in 100 ml of KrebsHenseleit buffer  gassed with carbogen (950/, O,/ 5O/, CO,) v/v. During the entire procedure the liver was kept a t 37 'C. As has been demonstrated, perfusion of isolated rat liver with a hemoglobin free medium yields results which in most metabolic experiments are comparable to those obtained with erythrocyte containing media [15,16]. Radioactive substrates were added 40 min after beginning of the perfusion. At the time points indicated, liver samples (about 0.5 g) were cut off and immediately freeze-stopped by the technique of Wollenberger et al.  and Mat-
Eur. J. Biochem.
schinsky et al. [ls]. The cut was closed with a clamp. The frozen tissue was homogenized in a centrifuge tube with a motor driven teflon pestle in 4 vol. of 0.6 N HC10, a t 0 "C. The perchloric acid was neutralized with solid KHCO, and the KCIO, removed by centrifugation. NAD, nicotinic acid and nicotinamide were isolated from the neutralized perchloric extract by paper chromatography and subsequent electrophoresis in the presence of authentic compounds. Paper chromatography was conducted with the system described by Preiss and Handler  using their solvent C. The chromatogram was then dried and subjected to high voltage electrophoresis 90 'to the direction of the first chromatographic separation. Electrophoresis was performed according to Krijger et al. . The compounds were localized on the chromatogram by their ultraviolet absorption and the radioactivity determined on 1x 6 cm paper strips in a liquid scintillation spectrophotometer. The determination of nicotinamide mononucleotide, nicotinic acid mononucleotide and nicotinic acid adenine dinucleotide was accomplished by ion exchange chromatography on Dowex I-X2 formate (200-400 mesh) columns (1x40 cm) as described by Ijichi et al. . A 2 ml aliquot of the neutralized perchloric acid extract, to which 1.5 mg nicotinic acid, 1.5 mg NAD, 2.5 mg nicotinamide mononucleotide and 1.O mg nicotinic acid adenine dinucleotide were added as carriers was placed on top of the column and eluted with a formic acid gradient according t o Ijichi et al. . 10 ml fractions were collected. The absorbance was recorded by a flow through device and the radioactivity determined in 0.2 or 0.5 ml aliquots as indicated in a scintillation spectrophotometer. The fractions from each radioactive peak were combined and lyophilized. The residues were dissolved in 0.5 ml of water and aliquots subjected t o paper chromatography and paper electrophoresis together with authentic compounds as described above to verify the identity of the peaks. Aliquots of the perchloric acid extract were used for the determination of NAD and ATP. NAD was assayed with alcohol dehydrogenase according to Warburg and Christian . ATP was determined with phosphoglycerate kinase and glyceraldehydephosphate dehydrogenase according to Jaworek etal. . For the determination of radioactive nicotinic acid and nicotinamide in the perfusate, an aliquot was added to an equal volume 0.45 N HC10,. An aliquot of the perchloric acid extract was subjected to paper chromatography on Whatman No. I filter paper in presence of authentic compounds and the chromatogram developed in I-butanol saturated with 3O/, (v/v) aqueous ammonia according to Chaykin et al. . Radioactivity was determined on 1 x 6 cm paper strips in a liquid scintillation spectrophotometer.
J. KELLER,M. LIERSCII,and H. GRUNICEE
VOl. 22, KO. 2, 1971
Table 1. Effect of unlabeled niwtinic acid on the incorporation of [14C]nicotinic acid and [14C]niwtinamide into NAD of isolated perfwed rat liver Livers were perfused for 10 min with media containing either [14C]nicotinicacid or [14C]nicotinamide. Nonlabeled nicotinic acid was added where indicated. Experimental details are described under Materials and Methods. Values obtained from experiments where no unlabeled nicotinic acid was added, are given as means i S.D. The number of experiments is shown in parentheses Addition
counts x min-' x pmole-'
Pig.2. I m p o r a t i o n of [14C]nieotinamide into NAD of isolated perfwed rut liver. 0 , [14C]NAD, each point represents the average of three experiments; A , ['4C]nicotinamide in the medium. The initial concentration of [14C]nicotinamide in t h e medium was 10 pM. Experimental details are described under Methods
Fig.3. Incorporation of ['4C]nicotinic acid into NAD of isolated perfwed rat liver. 0 , [14C]NAD, each value represents the average of three experiments; A , [14C]nicotinic acid in the medium. The initial concentration of [14C]nicotinic acid in the medium was 5 pM. Experimental details are described under Methods
Fig. 2 and 3 demonstrate the uptake of [14C]nicotinamide and [14C]nicotinicacid and the incorporation of these precursors into the NAD of isolated perfused rat liver as a function of time. As can be seen there is a considerable incorporation of nicotinamide although a t the concentrations used, nicotinic acid is more rapidly incorporated than nicotinamide. If the [l4C]nicotinarnideis deamidated to nicotinic acid prior to the incorporation into NAD, addition of unlabeled nicotinic acid together with the radio18 Eur. J. Biochem., Vo1.22
[14C]nicoI tinic acid
3.85 i0.96 (6)
1 mM Nicotinic acid
5 pM [14C]nicotinic acid
10 p M [14C]nicotinamide
1.78 & 0.19 (9)
1mM Nicotinic acid
10 pM [14C]nicotmamide
active nicotinamide should decrease the radioactivity appearing in NAD. Table 1 shows the effects of a n excess of nonlabeled nicotinic acid on the incorporation of radioactivity from [14C]nicotinic acid and [14C]nicotinamideinto NAD. As should be expected, addition of a 200-fold excess of nonlabeled nicotinic acid results in a corresponding decrease in the incorporation of radioactivity from [W]nicotinic acid into NAD to 0.5O/, of the control. Compared t o the drastic effect on the incorporation of [14C]nicotinic acid into NAD which is decreased to 0.5O/, ofthe control, the uptake of radioactivity from labeled nicotinamide is lowered t o only 50°/, by the addition of unlabeled nicotinic acid. These results indicate that a considerable fraction of the nicotinamide is incorporated by a pathway which does not lead through the nicotinic acid pool. As high concentrations of nicotinic acid are known to inhibit the synthesis of NAD from nicotinamide , even the observed decrease in the uptake of radioactivity from nicotinamide does not necessarily reflect a deamidation of the nicotinamide. Azaserine is known to inhibit the nicotinic acid dependent pathway of NAD biosynthesis. If a substantial fraction of the nicotinamide is incorporated without deamidation to nicotinic acid, the uptake of nicotinamide into NAD should be less sensitive to the action of azaserine than the incorporation of nicotinic acid. As Table 2 demonstrates the antibiotic causes a drastic inhibition ofthe uptake of nicotinic acid into NAD. However, as measured
Biosynthesis of NAD from Nicotinamide in Perfused R a t Liver
Table 2. Effect of azaserine on t h imrporation of [14C]niwtinic acid and [14C]niwtinamide into NAD of perfused rat liver Isolated rat livers were perfused for 20 min with media containing either 5 pM labeled nicotinic acid or 10 pM labeled nicotinamide. Where indicated, azaserine was added t o the perfusate t o a final concentration of 2 mM Labeled
counts x min-I PM
3.15 2.99 3.63 2.60
Table 3. Incorporation of ['*C]nicotinM acid and ['*C]niwtinamide into pyridine nucleotides of isolated perfused rat liver Livers were perfused with media containing either 5 p M [Wlnicotinic acid or 10 pM [14C]nicotinamide. At the time points indicated, samples were removed by the freeze-stop technique, the compounds were isolated by ion exchange chromatography and further identified by paper chromatography and electrophoresis as described under Materials and Methods. For abbreviations see legend t o Fig. 1 Precursor
[14C]Nicotinicacid [WINicotinicacid [14C]Nicotinicacid [14C]Nicotinamide [14C]Nicotinamide
5 10 150 5
counts x min-lx mg/iiver-'
236 55 (10