Screening of d-amino acid oxidase inhibitor by a new multi-assay method

This paper describes an investigation of the inhibition of a d-amino acid oxidase (DAAO) activity in several kinds of food additives and fruit. To screen for an inhibitor of DAAO, the method employed checked for two kinds of indicators, namely, pyruvic acid (indicator-1) and hydrogen peroxide (indicator-2), both of which are formed by digestion by DAAO of d-alanine (the standard d-amino acid used in this study). For measurement of pyruvic acid, a reliable and authenticated method was employed: the presence of pyruvic acid was determined by converting it into a chromophore derivative by tagging with 2,4-dinitro phenylhydrazine (DNP), followed by measurement of absorbance at 445 nm. The pyruvic acid thus determined is referred to as “(colorimetric) indicator-1”. To measure hydrogen peroxide, a highly sensitive fluorogenic substrate, 3-(p-hydroxyphenyl)propionic acid (HPPA), was employed in the present study. Hydrogen peroxide was detected by measuring fluorescence intensity (Ex. = 320 nm, Em. = 405 nm) of 2,2′-dihydroxybiphenyl-5,5′-dipropionic acid (oxidize-HPPA), which is produced by horseradish peroxidase (HRP)-catalyzed oxidation of HPPA with hydrogen peroxide. The hydrogen peroxide thus determined is referred to as “(flourometric) indicator-2” After optimizing the multi-assay procedures, the inhibition of DAAO activity against d-alanine (used as the standard d-amino acid) in 11 kinds of food additives and five kinds of fruit was evaluated, first in terms of indicator-1. Then, two substrates, potassium sorbate and apple juice, that were screened by indicator-1, were further evaluated by indicator-2 to conform the inhibition of DAAO activity. This is the first demonstration of the inhibition of DAAO activity by potassium sorbate, which is widely used as a synthetic preservative in food and apple juice.