The determination of 4-Pyridoxic acid in urine ; an investigation into the recovery of 4-pyridoxic acid and the lactone of 4-pyridoxic acid from two Dowex ion exchange resins Public Deposited

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  • 4-Pyridoxic acid is the major metabolite of all forms of vitamin B₆ in mammalian species. The determination of 4-pyridoxic acid, therefore, is important in the investigation of vitamin B₆ metabolism and for the establishment of human requirements for the vitamin. Although 4-pyridoxic acid is itself fluorescent, Huff and Perlzweig (1944) developed a classic method for its determination based on the conversion of the metabolite to the even more highly fluorescent lactone. Sarett in 1951 recognized that there are many substances in urine other than 4-pyridoxic acid which are fluorescent and which may interfere with its determination. His attempts to remove the extraneous fluorescent compounds with Decalso or with charcoal were not entirely satisfactory. He proposed, therefore, the administration of a test dose of 4-pyridoxic acid to subjects whose urine was to be analyzed for the metabolite of vitamin B₆, thus rendering the foreign fluorescence less significant by dilution. In 1955, Fujita and associates employed ion exchange chromatography as a means of separating 4-pyridoxic acid from other fluorescent compounds in urine and in 1958 Reddy, Reynolds and Price developed a chromatographic method using Dowex 1 (Cl⁻), a strongly basic anion exchange resin and Dowex 5OW (H⁺), a strongly acidic cation exchange resin for the separation of 4-pyridoxic acid from extraneous fluorescent materials in urine. Eluates from the latter of the two columns, used in sequence, were subjected to a chemical procedure to oxidize the 4-pyridoxic acid to the more highly fluorescent lactone form. The Reddy, Reynolds and Price procedure has provided the means for achieving more valid results than had been possible previously. Because of the importance of this procedure, the dynamics of the ion exchange method of Reddy and associates was investigated. Standards of 4-pyridoxic acid, or 4-pyridoxic acid lactone, and of urine were subjected to ion exchange chromatography using their procedure. Conversion of the 4-pyridoxic acid to the lactone was accomplished by means of the microprocedure of Woodring, Fisher and Storvick (1964). Effluent, wash, and eluate fractions were collected to determine the pattern of elution of the 4-pyridoxic acid, as well as to determine the effect of interfering fluorescence from the resins and from reagents. Eluates of urine from the second in the series of two Dowex ion exchange resins were subjected to paper and thin layer chromatography along with standards of 4-pyridoxic acid and the lactone of 4-pyridoxic acid. Eluates from influents of hydrolyzed urine produced highly fluorescent zones which did not correspond to those of standards of 4-pyridoxic acid or its lactone. A tryptophan load test is often used to diagnose vitamin B₆ deficiency but some of the metabolites of tryptophan are highly fluorescent and may interfere with the fluorometric measurement of 4-pyridoxic acid even after its conversion to the more fluorescent lactone. To study the effect of the presence of tryptophan metabolites on the determination of 4-pyridoxic acid in urine, a test dose of L-tryptophan was administered to one subject and the subsequent 24-hour urine collection was treated according to the chromatographic procedure of Reddy et al. The final eluate was analyzed for 4- pyridoxic acid using the raicroprocedure of Woodring and associates. Readings of fluorescence indicated that much foreign fluorescence remained even after the lactonization procedure. Recovery of 4- pyridoxic acid was 65.5 percent. Therefore, for the determination of 4-pyridoxic acid following a test dose of L-tryptophan, a recovery curve would have to be used for calculation of 4-pyridoxic acid excretion. It was made emphatically clear that there are highly fluorescent compounds present in the resin itself, even after extensive treatment, which cannot be removed. The results of this study emphasize the great importance of maintaining a constant rate of flow, and thus a constant rate of leaching, during chromatography to minimize the effect of the interfering fluorescence. Dowex 5OW (H⁺) will not retain 4-pyridoxic acid well unless the resin has been rendered strongly acidic. It was ascertained that this procedure should be accomplished just before use. 4-Pyridoxic acid and the lactone of 4-pyridoxic acid are eluted almost at once from the Dowex 1 resin but are released in the middle fractions during elution from freshly activated Dowex 50W, with 50 ml. of elutriant. It was determined that chromatography could be undertaken at a relatively rapid flow rate, thus allowing the entire chromatogtaphic procedure and analysis of eluates to be completed in one day.
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