Abstract
A method for quantitating all six (three free forms and three phosphoester forms) vitamin B6 forms and pyridoxine-β-glucoside (PNG) in foods and human-derived samples has been developed. These compounds are specifically converted into the highly fluorescent 4-pyridoxolactone (4-PLA), which is then measured by high-performance liquid chromatography (HPLC). Pyridoxal 4-dehydrogenase (PLDH) converts pyridoxal into 4-PLA. Pyridoxine 4-oxidase and PLDH converts pyridoxine (PN) into 4-PLA. Pyridoxamine-pyruvate aminotransferase (PPAT) and PLDH converts pyridoxamine (PM) into 4-PLA. The three phosphoester forms (PLP, PMP and PNP) are first converted into their free forms by alkaline phosphatase and then converted into 4-PLA; PNG is first converted into PN by β-glucosidase. Thus, seven sets of reaction conditions are used to quantitate the seven compounds. A sample preparation method for this method is also described. The results of food analysis showed that the PN content was lower than those of the other vitamin B6 forms. Thus, this method makes it possible to examine the mechanisms underlying the absorption, circulation, metabolism and excretion of vitamin B6 when animals and human consume usual foods.
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