42] Fluorometric and chromatographic methods for measuring microsomal biphenyl hydroxylation

Abstract

Publisher Summary This chapter describes the fluorometric and chromatographic methods for measuring microsomal biphenyl hydroxylation. This method, is the most convenient when biphenyl is used as a routine substrate for the monooxygenase. Biphenyl (I) is a useful model substrate for investigating the cytochrome P-450 (P-448)-mediated, NADPH 2 -, and oxygen-dependent microsomal aryl hydrocarbon hydroxylase. Microsomal hydroxylation of biphenyl is terminated with acid, which also ensures that the hydroxylated metabolites are not ionized and are extracted into n-heptane. The hydroxybiphenyls are then reextracted into alkali, leaving unreacted biphenyl in the organic phase. The fluorescence of the metabolites is measured at pH 5-6, achieved by adding succinic acid to the alkaline extract. Two compounds, 2-hydroxybiphenyl (II) and 4-hydroxybiphenyl (III) constitute more than 90% of the total biphenyl metabolites with liver microsomes of normal rats or hamsters. Small percentages of dihydroxybiphenyls are formed with rodent liver microsomes, and although 3-hydroxybiphenyl is isolated as a metabolite from normal rabbit urine, it is not formed in significant quantities with normal rodent liver microsomes.