URINARY OXALIC ACID EXCRETION BY MAN FOLLOWING INGESTION OF LARGE AMOUNTS OF ASCORBIC ACID1

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1 THE JOURNAL OF VITAMINOLOGY 12, (1966) URINARY OXALIC ACID EXCRETION BY MAN FOLLOWING INGESTION OF LARGE AMOUNTS OF ASCORBIC ACID1 HISASHI TAKIGUCHI, SHUNSUKE FURUYAMA AND NORIO SHIMAZONO*2 Department of Biochemistry, Nihon University School of Dentistry, Chiyoda-ku, Tokyo and *Departjnent of Biochemistry, Faculty of IVledicine, University of Tokyo, Bunkyo-ku, Tokyo (Received July 14, 1966) Oxalic acid has been known to be an oxidation product of AsA (1-4) and glycine (5, 6). Many investigators (6, 7) have found the presence of a considerable amount of radioactive oxalic acid in urine following the administration of AsA-1-C14, while no conversion of AsA-6-C14 to radioactive oxalic acid has been demostrated (8). Burns et al. (9) have reported that urinary oxalic acid is one of the end products of the metabolites of AsA. Lamden et al. (10) have reported that the increase of oxalic acid in urine was not appreciable following the oral administration of 3g of AsA a day, but it was marked after oral administration of 9g of AsA a day. Takenouchi et al. (11) have reported that the urinary excretion of oxalic acid did not increase after oral administration of 3g of AsA a day. Recently, large amounts of AsA are used over a long period of time for the treatments of various diseases. It would be, therf ore, an important problem whether a marked increase of oxalic acid excretion is brought about or not as a result of administration of large amounts of AsA. In the experiments by Lamden et al., the subjects were ingested by large amounts of AsA for a short period of time, and by Takenouchi et al., few subjects were administered AsA, so that a futher investigation of the effect of AsA on the urinary oxalic acid excretion is warranted. It would be, moreover, necessary to put a subject on experimental diet to observe the effect, since a part of urinary oxalic acid would come from glycine. In this paper we have carried out the studies on the urinary metabolites of AsA, especially oxalic acid, which are produced following the administration of large amounts of AsA over a long period. EXPERIMENTAL 1. Materials AsA used was supplied from the Takeda Chemical Industries materials were also available commercially., Ltd. Other 1 Following abbreviations were used: AsA, L-ascorbic acid; DAsA, dehydro-l-ascorbic acid; DKG, 2, 3-diketo-L-gulonic acids. 307

2 308 TAKIGUCHI, FURUYAMA ET AL Assay Methods of Oxalic acid, AsA, DAsA and DKG The determination of oxalic acid in urine was carried out by a slight modifica tion of the method of Powers and Levatin (12). The determination of AsA, DAsA and DKG was carried out with 2, 4-dinitrophenylhydrazine by a slight modification of the method of Roe et al. (13), the incubation period being 1 hour at 50 in half scale of the original method (4). 3. Menu of the Experimental Diet The menu of the experimental diet used is shown in Table 1. TABLE 1 Menu of the Experimental Diet 4. Experiments in Human Subjects Thirty healthy subjects, ranged years of age, were divided into three groups, each of which consisted of ten subjects. Subjects were ingested by 1g of AsA daily for 90days in the first group, and by 2g of AsA daily at breakfast and supper for 180 days in the second group. In the third group, in addition to ingestion of 1 g of AsA each at breakfast and supper (2g/day) for 90 days, the experimental diet was given on the last three days, and the 24-hour urine was collected on the 90th day. The 24-hour urine was collected in a urinal containing 225 ml of stabilizing solution (20% metaphosphoric acid and 3.3% thiourea) ac cording to Lamden et al. (10), and the contents of oxalic acid, AsA, DAsA and DKG were determined. This stabilizing agent was demostrated to be satisfactory by the experiments of the authors. RESULTS 1. Urinary Excretion of Oxalic Acid following the Administration of Large Amounts of AsA In the Cases of Daily Ingestion of 1 g of AsA (First Group).--Ten subjects were given 1g of AsA daily for 90 days, and oxalic acid eliminated in daily urine

3 Vol. 12 URINARY OXALIC ACID 309 was measured before and after administration. In this experiment, any remarkable increase of urinary oxalic acid was not observed as shown in Table 2. TABLE 2 Oxalic Acid Eliminated in Urine Following Ingestion of 1g of AsA (First Group) In the Cases of Daily Ingestion of 2g of AsA (Second Group)-Ten subjects were given 2g of AsA daily for 180 days, and oxalic acid eliminated in daily urine was determined before and after administration. Urinary excretion of oxalic acid was increased about 14% on the 30th, 16% on the 90th and 11% on the 180th day, as shown in Table 3. TABLE 3 Oxalic Acid Eliminated in Urine Following Ingestion of 2g of AsA (Second Group) In the Cases of Daily Ingestion of 2g of AsA in Addition to the Experi mental Diet (Third Group)-In the third group, subjects were, in addition to 1g of AsA ingestion each at breakfast and supper (2g/day) for 90 days, given the experimental diet on the last three days, and the 24-hour urine was collected on the 90th day. Urinary excretion of oxalic acid was only increased about 6% as shown in Table 4.

4 310 TAKIGUCHI, FURUYAMA ET AL TABLE 4 Oxalic Acid Eliminated in Urine Following Ingestion of 2g of AsA in Addition to Experimental Diet (Third Group) 2. Urinary AsA, DAsA and DING Following the Daily Ingestion of 1g of AsA (First Groin) In the first group, AsA, DAsA and DKG eliminated in urine were determined after administration of AsA as in Table 5. TABLE 5 AsA, DAsA and DKG Eliminated in Urine Following Ingestion of 1g of AsA (First Group) After administration of large amounts of AsA, it was eliminated in urine mainly in the form of reduced AsA, and a little was determined as DAsA and DKG. DISCUSSION Oxalic acid is a product of oxidation of AsA and glycine (1-6). A part of urinary oxalic acid is one of the end products of AsA (9). The important problem con cerning this substance might be the excretion as calcium oxalate leading to the formation of urinary caluculi when the substance is produced in large amounts in the body.

5 Vol. 12 URINARY OXALIC ACID 311 Recently, large amounts of AsA are used for the treatments of various diseases, so that the studies on the formation of oxalic acid would warrant. The present paper has described the urinary oxalic acid excretion following the ingestion of large amounts of AsA for a long period of time. In the first group, no significant increase in urinary excretion of oxalic acid was observed following the administration of 1g of AsA daily for a long period of time. High concentrations of AsA in the cells for a long time would not be ex pected following the single administration of 2g of AsA, since AsA is one of the threshold bodies, and the excess is excreted easily in urine after the administration of large amounts. In the second group, therfore, subjects were given 1g of AsA each at breakfast and supper (2g/day) for 180 days, but any remarkable increase of urinary oxalic acid was not observed. In the third group, subjects were, in addition to 1g of AsA administration each at breakfast and supper (2g/day) for 90days, given the experimental diet for the last three days, and the 24-hour urine was collected on the 90th day. No significant increase in urinary oxalic acid was observed. Urinary AsA, DAsA and DKG were determined in the first group. A large part of ingested AsA was excreted in urine as reduced AsA, and urinary DKG was only 22 mg. In vivo, DAsA is easily reduced to AsA (14), but DKG is not (15, 16). DAsA is delactonized enzymatically to DKG but delactonizing enzyme activity in man has been reported to be very low (16, 17), and besides the reduc tion activity of DAsA to AsA is known to be high in red blood cells (18, 19). It should be, therf ore, presumable that urinary DKG is little following the ingestion of large amounts of AsA. Even if all of the urinary DKG in the above experi ment were metabolized to oxalic acid, urinary oxalic acid would be about 40mg, which is the maximum value in the nomal range. SUMMARY Recently, large amounts of ascorbic acid are used for the treatments of various diseases, so that the investigation on the urinary excretion of oxalic acid, which is one of the metabolites of ascorbic acid, was carried out in man following the admin istration of large amounts of the vitamin and the following findings were obtained. 1. No significant increase in urinary oxalic acid was observed following the ingestion of large amounts (1-2g) of ascorbic acid for a long period ( days). 2. After ingestion of large amounts of ascorbic acid, it was eliminated in urine mainly in the reduced form of the vitamin and little was determined as diketogu lonic acid, the precursor of oxalic acid. REFERENCES 1. Flaschentrager, B., and Muller, P. B., Physiol. Ciean. 251, 52 (1938). 2. Ghosh, J. C., and Rakshit, P. C., Biochem. Z. 299, 394 (1938). 3. Rosenfeld,, B., J. Biol. Chem. 150, 281 (1943). 4. Kagawa, Y., J. Biochem. 51, 134 (1962). 5. Nakada, H. I., Friedman, B., and Weinhouse, S., J. Biol. Clzein. 216, 583 (1955).

6 312 TAKIGUCHI, FURUYAMA ET AL Barker, F. M., Sauberlich, H. F., Wolfskill, S. J., Wallace, W. T., and Dean, E. E., Proc. Soc. Exp. Biol. Med. 109, 737 (1962). 7. Hellman, L., and Burns, J. J., J. Biol. Chem. 230, 923 (1958). 8. Dayton, P. G., Eisenberg, F., and Burns, J. J., Arch. Biochem. Biophys. 81, 111 (1959). 9. Burns, J. J., Burch, H. B., and King, C. G., J. Biol. Chem. 191, 501 (1951). 10. Lamden, M. P., and Chrystowski, G. A., Proc. Exp. Biol. Med. 85, 190 (1954). 11. Takenouchi, K., Aso, K., Kawase, K., Ichikawa, H., and Shiomi, T., J. V itarninol. 12, 49 (1966). 12. Powers, H. H., and Levatln, P., J. Biol. Chem. 154, 207 (1944). 13. Roe, J. H., Mills, M. B., Oesterling, M. J., and Damron, C. M., J. Biol. Chem. 174, 201 (1948). 14. Fox, F. W., and Levy, L. F., Biochem. J. 30, 211 (1936). 15. Damron, C. M., Monger, M. M., and Roe, J. H., J. Biol. Chem. 195, 599 (1952). 16. Kagawa, Y., Takiguchi, H., and Shimazono, N., Biochim. Biophys. Acta 51, 413 (1961). 17. Kagawa, Y., and Takiguchi, H., J. Biochem. 51, 197 (1962). 18. Schultze, M. O., Stotz, E., and King, C. G., J. Biol. Chem. 122, 395 (1938). 19. Lloyd, B. B., J. Physol. 112, 49 (1951).