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Anthranilic Acid 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.
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