Summary A determination method for individual natural vitamin B 6 compounds was developed. The vitamin B 6 compounds were specifically converted into 4-pyridoxolactone (PAL), a highly fluorescent compound, through a combination of enzymatic reactions and HCl-hydrolysis. PAL was then determined by HPLC. Pyridoxal was completely oxidized to PAL with pyridoxal 4-dehydrogenase (PLDH). Pyridoxine and pyridoxamine were totally converted into PAL through a coupling reaction involving pyridoxine 4-oxidase and PLDH, and one involving pyridoxamine-pyruvate aminotransferase and PLDH, respectively. The 5 ¢ -phosphate forms and pyridoxine- -glucoside were hydrolyzed with HCl, and then determined as their free forms. Pyridoxine 5 ¢ -phosphate and pyridoxine- -glucoside were not separately determined here. Three food samples were analyzed by this method. Key Words vitamin B 6 , 4-pyridoxolactone, pyridoxal 4-dehydrogenase, pyridoxine 4-oxidase, pyridoxamine-pyruvate aminotransferase There are six natural forms of vitamin B 6 : pyridoxine, pyridoxal, pyridoxamine, pyridoxine 5 ¢ -phosphate, pyridoxal 5 ¢ -phosphate, and pyridoxamine 5 ¢ -phosphate ( Fig. 1). In addition, plants contain pyridoxine- -glucoside, a storage form of vitamin B 6 ; it is not counted as vitamin B 6 because its nutritional availability as a form of vitamin B 6 in the human body is controversial ( 1 ). The above six vitamin B 6 compounds have identical nutritional functions. On the other hand, recent studies have shown that individual vitamin B 6 compounds exhibit specific biochemical functions; pyridoxamine, in particular, can detoxify active carbonyl products and is undergoing a phase II clinical trial for the treatment of complications of diabetes ( 2 ). Vitamin B 6 compounds are strong quenchers of singlet oxygen ( 3 ), and pyridoxal 5 ¢ -phosphate and pyridoxamine 5 ¢ -phosphate, coenzyme forms of vitamin B 6 , show stronger protection of yeast cells from oxidative death than vitamin C, a representative antioxidative vitamin ( 4 ). Thus, the development of a method for the determination of individual vitamin B 6 compounds plus pyridoxine- -glucoside in foods is required to estimate their functionality.Although many methods have been developed for the determination of individual vitamin B 6 compounds, no method can be satisfactorily applied to the analysis of food samples. Thus, a microbioassay involving a budding yeast, Saccharomyces cerevisiae (former name, S. uvarum ), which was developed about 40 y ago ( 5 ), has been used to determine the total content of vitamin B 6 in food samples; in the figure, the content of pyridoxine- -glucoside is included because food samples are prehydrolyzed with HCl for the microbioassay, and pyridoxine- -glucoside in the samples is converted into pyridoxine. The best method so far developed for individual determination may be cation-exchange HPLC with a fluorescence detection system ( 6 ), which is applicable to biological samples, such as serum, brain, liver, milk and so on, from experimental animals. The seco...
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