J. Biochem. 84, 687-696 (1978)

The Metabolism of [carboxyl-14C]Anthranilic Acid

I. The Incorporation of Radioactivity into NAD+ and NADP+ 1,

Takashi UEDA,, Hidetsugu OTSUKA ,1- Kiyoshi GODA,* Isao ISHIGURO,** Junko NAITO,** and Yahito KOTAKE*

*Faculty of Nutrition , Kobe-Gakuin University, Arise, Ikawadani-cho, Tarumi-ku, Kobe, Hyogo 673, **Fujita Gakuen University School of Medicine, Kutsukake-cho, Toyoake, Aichi 470-11

Received for publication, February 3, 1978

A new pathway of NAD+ synthesis from anthranilic acid was found in the livers of rats. Starting from [carboxyl-14C]anthranilic acid, radioactive NAD+ and NADP+ were produced as judged by Dowex-1 x 8-formate column chromatography followed by radiochromatography. Several intermediate compounds, such as quinolinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide were also identified with the aid of various chro matographic techniques. In the experiments with liver microsomal hydroxylation systems, anthranilic acid was converted into not only 5-hydroxyanthranilic acid but also 3-hydroxy anthranilic acid.

In the early thirties, anthranilic acid and kynure found a conjugate of anthranilic acid with glycine. nine, the intermediary metabolites of tryptophan, Mitsuba and Ichihara (5) have also demonstrated were discovered by Kotake (1, 2). Kotake and another conjugate of anthranilic acid with glu Honda (3) subsequently observed that L-kynure curonic acid. These authors have proposed that nine was converted stoichiometrically to anthranilic the formation of such conjugated compounds may acid and L-alanine in rabbit liver homogenates. be attributed to the detoxication mechanism. Since then, anthranilic acid has been extensively Based on these observations, it has been generally studied by many investigators. Mason (4) has believed that anthranilic acid is a metabolically inactive and presumably blind alley in the tryp 1 Dedicated to Professor Katashi Ichihara on the occa tophan metabolism in mammals. Nevertheless, sion of his 80th birthday. Kotake and Kotake (6) in 1942 suggested the 2 This paper was presented at the 50th meeting of the hydroxylation of anthranilic acid to produce Japanese Biochemical Society on the 15th of October 5-hydroxyanthranilic acid in mammalian tissues. 1977. In fact, Kotake and Shirai (7) in 1953 isolated Abbreviations: NAD+, nicotinamide adenine dinucleo 5-hydroxyanthranilic acid in a crystalline form tide; NADP+, nicotinamide adenine dinucleotide phos from the urine of rabbits treated with anthranilic phate; NMN, nicotinamide mononucleotide; NaMN, acid. In 1964, Kashiwamata et al. (8) demon nicotinic acid mononucleotide; NaAD, nicotinic acid strated the enzymatic conversion of anthranilic adenine dinucleotide; QA, quinolinic acid; G6P, acid to 5-hydroxyanthranilic acid. Recently, glucose-6-phosphate; EDTA, ethylenediaminetetraacetic acid; DMF, N, N-dimethylformamide. Sutamihardja et al. (9) described a new metabolite

Vol. 84, No. 3, 1978 687 688 T. UEDA, H. OTSUKA, K. GODA, I. ISHIGURO, J. NAITO, and Y. KOTAKE

isolated in a crystalline form from the urine of nilic Acid-Radioactive anthranilic acid, 10.65 rats injected with anthranilic acid; this compound mCi/mmol was prepared as follows: K14CN was has been identified as anthranilamide. On the converted into Cu14CN by the method of Reid other hand, the metabolic conversion of anthranilic et al. (15). o-Iodoaniline and Cu14CN were acid to 3-hydroxyanthranilic acid (10) has been dissolved in DMF and kept at 160•Ž for 1.5 h considered to be questionable in spite of the under N2 gas. After this reaction, DMF was discovery of hydroxylation reactions of anthranilic evaporated off. The residue was refluxed in 1 N acid at the three and five positions in Udenfriend's NaOH for 2h. When the reaction was termi model ascorbic acid system (11). The present nated, the reaction mixture was neutralized with paper describes the enzymatic formation of 3-hy 1 N HCl. [carboxyl-14C]Anthranilic acid obtained droxyanthranilic acid from anthranilic acid in rat was purified by column chromatography. Silica liver microsomes; this reaction participates in the gel G 60 (70-230 mesh) chromatography was relationship between anthranilic acid and NAD+ performed on a 2.5 x 20 cm column, eluting with and NADP+ The biological significance of this chloroform : 96 % acetic acid (95 : 5). The anthra metabolic pathway is under investigation. The nilic acid fraction was concentrated with a rotary biosynthetic pathway of NAD+ from tryptophan evaporator. The concentrated product was applied by way of 3-hydroxykynurenine and 3-hydroxy to the same column and eluted with methyl acetate : anthranilic acid has been established in mammalian isopropanol :25% ammonia (45 :35 :20). The liver (12, 13). purity of radioactive anthranilic acid was deter- mined by means of radiochromatograms of silica

MATERIALS AND METHODS gel G 60 thin-layer plates developed with the following solvent systems; chloroform : 96% acetic Animals-Male albino rats of the Wistar acid (95 : 5), methyl acetate : isopropanol : 25

JCL? strain, weighing 150 g, were fed on rat chow ammonia (45 : 35 : 20), and benzene : methyl OA-2 (Clea Japan, Inc.). The rats were housed acetate : acetic acid (60 : 40 : 1). The radiochro in a room at a constant temperature of 22•Ž with matograms showed only one major peak in each a 12 hour light and dark cycle. The humidity was case. The radiopurity of [carboxyl-14C]anthranilic controlled at 54% saturation. acid was determined to be 98.97% by dilution

Chemicals-All chemicals were of the best analysis. analytical grade obtainable from Nakarai Chemi Procedures for Isotope Study In Vivo-The cals, Ltd. Potassium [14C]cyanide (specific activity specific activity of [carboxyl-14C]anthranilic acid 53.25 mCi/mmol) was supplied by New England was 10.65 mCi/mmol. Each rat was given [car Nuclear. No significant impurity was detected. boxyl-14C]anthranilic acid (7.16 ƒÊCi/100 g body 2,5-Diphenyloxazole (PPO) and 1,4-bis[2-(5- weight) by injection into the tail vein. The rats phenyloxazolyl)]-benzene (POPOP) were purchased were sacrificed by decapitation 3 h later. Their from Packard, Co. Dowex-1 x 8-chloride (200- livers were excised as quickly as possible, weighed, 400 mesh) was purchased from Dow Chemicals frozen, and stored at -20•Ž until analyzed. Their Co. and Dowex-1 x 8-chloride was converted into urine was also stored at -20•Ž. Expired carbon the formate form according to the method of dioxide was trapped by 15 ml of a mixture of Hurbert (14). NAD+, NADP+, G6P, and G6P 2-aminoethanol and 2-methoxyethanol (1 : 2 v/v). dehydrogenase were obtained from Sigma Chemi Radioactive carbon dioxide was assayed with a cal Co. NMN and NaMN were obtained from Packard liquid scintillation spectrometer. Kyowa Hakko Kogyo Co. Quinolinic acid, 3- Extraction of Nucleotides-The liver was hydroxyanthranilic acid, and o-iodoaniline were homogenized in 4 volumes of 5 % perchloric acid. purchased from Nakarai Chemicals, Ltd., Tokyo The homogenate was centrifuged at 10,000 x g for Kasei Kogyo Co. and Wako Pure Chemical 15 min. The precipitate was washed twice with Industries, Ltd., respectively. Anthranilic acid and small amounts of 5 % perchloric acid. The super- 5-hydroxyanthranilic acid were generous gifts from natant fluid and washings were combined and Sonybod Pharmaceutical Co. adjusted to pH 7 with 4 M KOH. Potassium Chemical Synthesis of [carboxyl-14C]Anthra perchlorate was removed by centrifugation, then

J. Biochem. BIOSYNTHESIS OF NADI AND NADP+ FROM ANTHRANILATE 689 the precipitate was washed again and centrifuged in situ with ice-cold 0.15 m NaCl and homogenized at 10,000 x g. The supernatant and washing were in 0.25 m sucrose (4 ml/g of liver). All subsequent combined and used for further analysis . manipulations were carried out at 4°C. The Chromatographic Methods-Dowex-1 x 8-for homogenate was centrifuged at 9,000 x g for 20 mate (200-400 mesh) chromatography was per- min. The resulting supernatant was centrifuged formed on a 2.5 x 25 cm column. Stepwise elution at 105,000 x g for 60 min. The microsomal frac was carried out with 200 ml of distilled water and tion was washed three times with 0.14 m KCl. 150 ml of increasing concentrations of formic acid , such as 0.05 m, 0.1 m, 0.25 M, 0.5 M, 1.0 m, 2.0 m, RESULTS and 4 M ammonium formate in 4 M formic acid . The flow rate of this column was 1.66 ml/min. The metabolic conversion of radioactive anthranilic Fractions of 10 ml were collected. The radio- acid was assayed in exhaled carbon dioxide, urine activity in each fraction was measured by counting and liver. Rats were injected with [carboxyl-14C]- a 0.2 ml aliquot with a Packard liquid scintillation anthranilic acid (10.65 mCi/mmol) into the tail spectrometer. vein and kept in a metabolic cage for three hours. Paper Chromatographic Procedures-Paper The rats were then sacrificed and the distribution chromatography was carried out on Whatman No. 1 filter paper in an ascending fashion with the following solvent systems. 1: 1 M ammonium acetate :95% ethanol (3 :7) 2: isobutyric acid : ammonia : water (66 :1.7 33) pH 3.9 3: upper layer n-butanol : acetone : water (45 : 5 50) 4: pyridine : water (2 : 1) Electrophoresis-The concentrated solution of radioactive compounds was examined by paper electrophoresis, which was conducted at 320 V for 1 h in 0.2 M triethanolamine containing 0.002 M EDTA (pH 7.7) buffer. Whatman No. I filter paper was used in all experiments. Fig. 1. Production of 14CO2 from [carboxyl-14C]an Preparation of Microsome-Rats were fasted thranilic acid in rats. The experimental conditions for 18 h prior to sacrifice. The liver was perfused are described in " MATERIALS AND METHODS."

Fig. 2. Dowex-1 x8-formate column chromatography of 5% perchroric acid extract from rat livers. Substrate: [carboxyl-14C]anthranilic acid. Detailed experimental conditions are described in " MATERIALS AND METHODS."

Vol. 84, No. 3, 1978 690 T. UEDA, H. OTSUKA, K. GODA, I. ISHIGURO, J. NAITO, and Y. KOTAKE of radioactivity was determined. Three per cent (1 : 2 v/v). At this time only 0.38% of the initial of the radioactivity was excreted into the urine in total radioactivity was found in the liver, and most 3 h, and 5.05 % was expired as carbon dioxide. of the radioactivity was still in the body. Figure Radioactive carbon dioxide was collected with a 1 shows the time course of carbon dioxide forma mixture of 2-aminoethanol and 2-methoxyethanol tion. The evolution of carbon dioxide gradually

Fig. 3. Radiochromatograms. A sample from the second peak (Fig. 2) was analyzed by paper chromatography using four different solvent systems as indicated. Radioactivity was measured with a Packard radiochromatoscanner. The peaks obtained were identified by comparison with authentic samples which were developed at the same time. The compounds were visualized with a UV lamp.

Fig. 4. Radiochromatograms. A sample from the fifth peak (Fig. 2) was analyzed by paper chromatography. The details are given in the legend to Fig. 3.

J. Biochem. BIOSYNTHESIS OF NAD+ AND NADP+ FROM ANTHRANILATE 691

reached a p"ateau at three hours. Figure 2 shows the distribution of radioactivity recovered from the the Dowex-1 x 8-formate column chromatographic rat liver after injection of [carboxyl-14C]anthranilic pattern of the liver extract from the rats injected acid into the tail vein. The radioactivity recovered with [carboxyl-14 C]anthranilic acid. One major in hepatic NAD+ and NADP+ fractions was peak and eight minor peaks were found. These approximately 2.6 and 0.5%, respectively, of the fractions were combined separately and then total initial radioactivity. lyophilized. Each radioactive sample was dis solved in a minimum volume of water and analyzed by paper chromatography and paper electro phoresis as described in " MATERIALS AND METHODS. Figures 3 and 4 show the distri bution of radioactivity on paper chromatograms developed with the four different solvent systems and on paper electrophoresis. The radioactive spots coincided with authentic compounds. The second peak from the Dowex-1 x 8-formate column (Fig. 2) was identified as NAD+. The fifth peak was identified as NaMN by the same methods. The other minor peaks were found to be NaAD, NADP+, and QA+X, as indicated in Fig. 2. QA+X means that this peak contained quinolinic acid plus a trace amount of unidentified substance. The R f values of standard compounds were as described by Hagino et al. (16). A sample of the second peak from the Dowex- 1 x 8-formate column was analyzed further by paper electrophoresis. As shown in Fig. 5, the radioactive material again coincided with an authentic sample of NAD+. NaMN and other metabolites, NaAD, NADP+, and QA, were identified by the methods described above. These observations together with the products obtained suggest that anthranilic acid is taken up by the Fig. 6. Radiochromatograms. A sample from the liver and converted to NAD+ and NADP+ by way seventh peak (Fig. 2) was analyzed by paper chro of 3-hydroxyanthranilic acid. Table I summarizes matography. The details are given in the legend to Fig. 3. TABLE I. The metabolites formed from [carboxyl- 14Clanthranilic acid in rat liver. Detailed experimental conditions are described in the text.

Fig. 5. Paper electrophoresis. The compound was detected with a UV lamp, followed by radioactivity scanning with a Packard radiochromatoscanner.

Vol. 84, No. 3, 1978 692 T. UEDA, H. OTSUKA, K. GODA, I. ISHIGURO, J. NAITO, and Y. KOTAKE

The next set of experiments was designed to almost completely separated from 5-hydroxy

show that the liver microsomal fraction was able anthranilic acid. Anthranilic acid and 3-hydroxy

to hydroxylate anthranilic acid. The isolation and anthranilic acid from the ethyl acetate extract were determination of 3-hydroxyanthranilic acid was easily separated by thin-layer chromatography using

conducted as follows. The reaction mixture for the solvent system described above. The 3-

anthranilic acid hydroxylation contained the follow- hydroxyanthranilic acid fraction (the R f 0.20

ing components in 2.0 ml; 10 ƒÊmol of Tris-HCl fraction) contained a trace amount of 5-hydroxy buffer at pH 7.0, 5 ƒÊmol of MgCl2, 4 ƒÊmol of anthranilic acid. The contaminant, 5-hydroxy

anthranilic acid, 4 ƒÊmol of G6P, 0.4 units of G6P anthranilic acid, did not interfere with the quantita dehydrogenase, 0.6 ƒÊmol of NADP+, and 5.0 mg tive determination of 3-hydroxyanthranilic acid at

protein of the microsomal fraction. The concen amounts up to 10 ƒÊg, because of the difference of tration of cytochrome P-450 was determined by fluorescence absorption maximum between 5- the method of Matsubara et al. (17). After incu hydroxyanthranilic acid and 3-hydroxyanthranilic

bation for 60 min at 37°C the reaction was stopped acid. The identification of 3-hydroxyanthranilic

by the addition of 1 N HCl (pH 3), and the mixture acid was carried out with a sample extracted from

was shaken with ether to extract anthranilic and the R f 0.20 fraction with ethyl acetate. Before the 3-hydroxyanthranilic acids. The extracts were extraction, this fraction was suspended in water

subjected to thin-layer chromatography using ethyl and the pH was adjusted to 3. A paper chro

acetate : isopropanol : ammonia : mercaptoethanol matogram of the sample showed faint fluorescent

(45 : 35 : 20 : 5). The R f 0.20 fraction was sus bands at Rf 0.91 and 0.63 and a strong fluorescent pended in a small amount of water adjusted to band at R f 0.88. The former bands were identified

pH 3 and the suspension was extracted with ethyl as anthranilic and 5-hydroxyanthranilic acids. The acetate. Under these conditions, 5-hydroxy latter band coincided with that of standard 3-

anthranilic acid is hardly extracted with ethyl hydroxyanthranilic acid. This suggests that the acetate and ether, so 3-hydroxyanthranilic acid was R f 0.20 fraction contained a small amount of

TABLE II. Parameters of anthranilic acid and its derivatives. These values were obtained by the measurement of standard compounds. The fluorescence spectra were measured in ethyl acetate. Solvent systems TLC, thin-layer chromatography. ethyl acetate : isopropanol : ammonia : mercaptoethanol (45 : 35 : 20 : 5) PPC, paper chromatography. n-butanol : acetic acid : water (4 : 1 : 1) An.A, anthranilic acid; 5-OH.An.A, 5-hydroxyanthranilic acid; 3-OH.An.A, 3-hydroxyanthranilic acid

TABLE III. Hydroxylation at the 3- or 5-position of anthranilic acid by the liver microsomal fraction. Detailed experimental conditions are described in the text. SOD, superoxide dismutase; PF, protein factor

J. Biochem. BIOSYNTHESIS OF NAD+ AND NADP+ FROM ANTHRANILATE 693 contaminants, anthranilic and 5-hydroxyanthranilic acids. The solvent system for paper chromatog raphy was n-butanol : acetic acid : water (4 : 1 : 1). The R f values of anthranilic, 3-hydroxyanthranilic

Fig. 8. The evolution of 14CO2 from [carboxyl-14C)-an thranilic acid with liver postmitochondrial supernatant. The rats were given daily intraperitoneal injections of

phenobarbital (80 mg/kg) for 4 days. The reaction sys- tem contained 80 ƒÊmol of Tris-HCl buffer (pH 7.5), Fig. 7. Fluorescence spectrum of a compound formed 8.0 ƒÊmol of nicotinamide, 5.4 ƒÊmol of [carboxyl 14C]- in the microsomal hydroxylation system. The fluores anthranilic acid (specific activity 0.0788 mCi/mmol), and cence spectrum is that of the compound extracted from 6.7 mg protein of postmitochondrial supernatant. The the incubation mixture of the microsomal hydroxylation other components were as described in the text. The system as shown in Table III. The extracted substance incubation was conducted under an 02 atmosphere. was measured in ethyl acetate. The solid line shows 14CO2 was trapped by 0 .2 ml of a mixture of 2-amino excitation. The dotted line shows emission at 370 nm ethanol and 2-methoxyethanol (1 : 2 v/v). The solution to 460 nm. was adsorbed on a sheet of Whatman No. 1 filter paper.

Fig. 9. Liver microsomal hydroxylation of [carboxyl-14C]anthranilic acid. The incubation was perform- ed on ten times the scale of the previous incubation mixture described in Fig. 8. The pure liver micro somal fraction was used in this hydroxylation system as described in "MATERIALS AND METHODS." The microsomes contained 73.46 nmol of cytochrome P-450. The protein concentration of microsomes used in this system was 57.25 mg. The concentration of cytochrome P-450 in microsomes was assayed by Matsubara's method (17). Watanabe's method (19) was used in system C of thin-layer chro matography.

Vol. 84, No. 3, 1978 694 T. UEDA, H. OTSUKA, K. GODA, I. ISHIGURO, J. NAITO, and Y. KOTAKE and 5-hydroxyanthranilic acids are shown in Table II. This sample (extract of the R f 0.20 fraction) showed the same fluorescence spectrum as standard 3-hydroxyanthranilic acid when it was measured with a fluorospectrophotometer under the condi tions shown in Table II. 5-Hydroxyanthranilic acid was also detected in the aqueous layer of the ether extraction. It was confirmed that the ethyl acetate extract of the aqueous layer contained 5-hydroxy- anthranilic acid by comparison of its R f value and fluorescent spectrum with those of standard 5- hydroxyanthranilic acid. The quantitative anal yses of anthranilic acid and its derivatives were performed by.a fluorospectrophotometric procedure under the conditions in Table II. These experi ments clearly show the formation of 5-hydroxy anthranilic acid and 3-hydroxyanthranilic acid in the microsomal hydroxylation system. In a paper by Ishiguro et al. (18), the hydrox ylation of anthranilamide in rat microsomes was reported to require a protein factor (superoxide dismutase) as an activator. The hydroxylation of anthranilic acid does not require any activating factor. The fluorescence spectrum of 3-hydroxy anthranilic acid formed in microsomes is shown in Fig. 7. This coincides with that of an authentic sample. 5-Hydroxyanthranilic acid was assayed according to Kashiwamata's indophenol method (8). This color reaction is specific for 5-hydroxy Fig. 10. Electrophoresis of the products of microsomal anthranilic acid. hydroxylation. The products of microsomal hydroxy The liver microsomes can convert [carboxyl- lation were developed in solvent system D. Each frac 14C]-anthranilic acid into 5-hydroxyanthranilic and tion was scraped off separately and extracted with 3-hydroxyanthranilic acids. The detection of ethyl acetate. The extracts were subjected to paper 14CO2 and radioactive quinolinate indicates the electrophoresis and compared with authentic samples. degradation of 3-hydroxyanthranilic acid (Figs. The conditions are shown in the figure. 8,9). In order to determine the metabolite of [carboxyl-14C]anthranilic acid, this material was acetate. The metabolites of anthranilic acid incubated in the hydroxylation system on a ten described in Fig. 9 were analyzed again by paper times larger scale. Immediately after terminating electrophoresis. It was confirmed that the com- the reaction, the denatured protein was removed pounds in ethyl acetate had the same mobilities from the reaction mixtures by centrifugation at as standard substances (Fig. 10). In view of these 600 x g. The supernatants were applied to thin- results, it can be concluded that anthranilic acid layer plates. The clearest separations were ob is metabolized to 3-hydroxyanthranilic, 5-hydroxy tained on thin-layer plates using the four solvent anthranilic, and quinolinic acids. systems shown in Fig. 9. The peaks of the radio chromatograms from the hydroxylation system DISCUSSION coincided with standard compounds. Each fraction on thin-layer plate D, using Viollier (20) in 1950 showed that 3-hydroxy benzene : methyl acetate : acetic acid (60 : 40 : 1), anthranilic acid was easily oxidized, both in alkaline was scraped off separately and extracted with ethyl solution and by a liver enzyme. Schweigert (21,

J. Biochem. BIOSYNTHESIS OF NAD+ AND NADP+ FROM ANTHRANILATE 695

22, 23) in 1949 showed that 3-hydroxyanthranilic shown in Figs. 8 and 9. These experiments show acid was readily converted to nicotinic acid via that the liver microsomal fraction can convert quinolinic acid by rat liver slices and homogenates, anthranilic acid into 5-hydroxyanthranilic acid, and this conversion of 3-hydroxyanthranilic acid 3-hydroxyanthranilic acid, and quinolinic acid. to quinolinic acid was equivalent to 86 to 100 The evolution of S4CO2from [carboxyl-14C]anthra on a molar basis. Bokman et al. (24) observed nilic acid also indicates further degradation of the disappearance of 3-hydroxyanthranilic acid 3-hydroxyanthranilic acid by way of the glutarate after prolonged incubation with the formation of pathway of tryptophan, as established by Nishi quinolinic acid. Henderson et al. (25) found that zuka et al. (28). The hydroxylation of anthranilic an active enzyme system was present in cat and acid does not require the protein factor, that is rat livers, which catalyzed the conversion of superoxide dismutase (18). This factor is essential 3-hydroxyanthranilic acid to glutaric acid, in for the microsomal hydroxylation of a carbonyl addition to quinolinic acid and picolinic acid. derivative of anthranilate. Hagino et al. (16) D'Angell (26) reported that anthranilic acid was reported that NAD+ formed from quinolinic acid effective in increasing the urinary excretion of by the isolated perfused liver amounted to 7%, nicotinic acid and quinolinic acid. Iwai and and quinolinic acid is taken up very slowly by rat Taguchi (27) reported that high activities of qui liver. Table II shows that NAD+ formed from nolinate phosphoribosyltransferase were observed anthranilic acid corresponds to 2.6%. Taking in liver and kidney, and this enzyme worked as into consideration the number of steps of the a rate-determining step in the biosynthesis of enzyme reaction, these conversions are not insignifi NAD+. Based on all these observations, one cant. Quinolinate phosphoribosyltransferase (29) might speculate that 3-hydroxyanthranilic acid was was inhibited by ATP and this inhibition was metabolized quickly during the hydroxylation of removed by raising the Mg2+ concentration. Smith anthranilic acid using liver homogenate. The and Gholson (30) suggested that the regulation of present experiments confirm that anthranilic acid NaMN synthesis by ATP was a logical mechanism was hydroxylated at the 3- as well as the 5-position since the next step in the synthesis of NAD+ from by studies using [carboxyl-14C]anthranilic acid, as nicotinic acid required ATP as a reactant and a

Scheme 1. A proposed pathway of NAD+ biosynthesis from tryptophan.

Vol. 84, No. 3, 1978 696 T. UEDA, H. OTSUKA, K. GODA, I. ISHIGURO, J. NAITO, and Y. KOTAKE decrease in the rate of NaMN formation in the 9. Sutamihardja, T.M., Ishikura, A., Naito, J., & presence of a lower level of ATP would help prevent Ishiguro, I. (1972) Chem. Pharm. Bull. 20, 2694- NaMN accumulation. These control mechanisms 2700 would prevent the accumulation of NAD+ and 10. Kotake, Y., Shibata, Y., & Toratani, A. (1956) NADP+ in the tissues. The biosynthetic pathway Proc. Japan Acad. 32, 774-777 11. Udenfriend, S., Clark, C.T., Axelrod, J., & Brodie, of NAD+ from tryptophan was established by B.B. (1954) J. Biol. Chem. 208, 731-739 Nishizuka and Hayaishi (12, 13) and Ikeda et al. 12. Nishizuka, Y. & Hayaishi, O. (1963) J. Biol. Chem. (31). However, the former study was concerned 238,483-484 with showing that NAD+ biosynthesis from 13. Nishizuka, Y. & Hayaishi, O. (1963) J. Biol. Chem. tryptophan occurred via 3-hydroxykynurenine. 238,3369-3377 The de nova biosynthesis of NAD+ and NADP+ 14. Hurbert, R.B., Schmitz, H., Brum, A.F., & Potter, from anthranilic acid was not observed, but the V.R. (1954) J. Biol. Chem. 209, 23-39 present investigation suggests the incorporation of 15. Reid, J.C. & Weaver, J.C. (1951) Cancer Res. 11, radioactivity of [carboxyl-14C]anthranilic acid into 188-194 NAD+ and NADP+ in rat liver. In addition, it 16. Hagino, Y., Lan, S.J., Ng, C.Y., & Henderson, L.M. can be concluded that another biosynthetic pathway (1968) J. Biol. Chem. 243, 4980-4986 17. Matsubara, T., Koike, M., Touchi, A., Tochino, Y., of NAD+ and NADP+ via anthranilic acid operates & Sugeno, K. (1976) Anal. Biochem. 75, 596-603 via the microsomal hydroxylation system. Micro 18. Ishiguro, I., Naito, J., Shinohara, R., Ishikura, A., somal hydroxylation is generally believed to be a & Terai, T. (1974) Biochim. Biophys. Acta 365, nonspecific detoxication process for drugs, but our 148-157 new observations show that the liver microsomal 19. Watanabe, M., Minegishi, K., & Tsutsui, Y. (1972) hydroxylation system participates in the biologically Cancer Research 32,2049-2053 important biosynthesis of NAD+ and NADP+. 20. Viollier, G. & Sullman, H. (1950) Hely. Chim. Acta The physiological significance of this metabolic 33, 776-781 21. Schweigert, B.S. (1949) J. Biol. Chem. 178, 707-708 pathway has not yet been determined. 22. Schweigert, B.S. & Marquette, M.M. (1949) J. Biol.

The authors would like to thank Prof. Yasutomi Chem. 181, 199-205 23. Bokman, A.H. & Schweigert, B.S. (1950) Federation Nishizuka and Prof. Iwao Ueda for helpful discussions Proc.9, 153 and comments during the preparation of this manuscript. 24. Bokman, A.H. & Schweigert, B.S. (1951) Arch. The authors also wish to express their thanks to Mr. Biochem. Biophys. 33, 270-276 Terutoshi Mori and Miss Kyoko Minamikawa for their 25. Henderson, L.M. & Hirsch, H.M. (1949) J. Biol. technical assistance. Chem. 181, 667-675 26. D'Angell, F., Koski, R.E., & Henderson, L.M. REFERENCES (1955) J. Biol. Chem. 214, 781-787 1. Kotake, Y. (1931) Z. Physiol. Chem. 195, 139-166 27. Iwai, K. & Taguchi, H. (1973) J. Nutr. Sri. Vita 2. Kotake, Y. (1939) Osaka Igakukai Zasshi (in Japa minol. 19, 491-499 nese) 38, 579-581 28. Nishizuka, Y., Ichiyama, A., Golson, R.K., & 3. Kotake, Y. & Honda, H. (1942) Osaka Igakukai Hayaishi, O. (1965) J. Biol. Chem. 240, 733-739 Zasshi (in Japanese) 41, 1720-1724 29. Taguchi, H. & Iwai, K. (1976) Agric. Biol. Chem. 4. Mason, M. (1953) J. Biol. Chem. 201, 513-518 40, 385-389 5. Mitsuba, K. & Ichihara, K. (1926) Osaka Igakukai 30. Smith, L.D. & Gholson, R.K. (1969) J. Biol. Chem. Zasshi (in Japanese) 25, 1831-1846 244,68-71 6. Kotake, Y. & Kotake, Y., Jr. (1942) Osaka Igakukai 31. Ikeda, M., Tsuji, H., Nakamura, S., Ichiyama, A., Zasshi (in Japanese) 41, 1028-1031 Nishizuka, Y., & Hayaishi, O. (1965) J. Biol. Chem. 7. Kotake, Y. & Shirai, Y. (1953) Z. Physiol. Chem. 240.1395-1401 295,160-163 8. Kashiwamata, S., Nakashima, K., & Kotake, Y. (1966) Biochim. Biophys. Acta 113, 244-254

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