Measurement of the Ratio of Primary to Total Bile Acids in Serum by Enzymatic Fluorometric Microassay and Its Clinical Significance in Patients with Liver Disease

Abstract

IKAWA, S., KAWASAKI, H., YAMANISHI, Y., MURA, T. and MIYAKE, M. Measurement of the Ratio of Primary to Total Bile Acids in Serum by Enzymatic Fluorometric Microassay and Its Clinical Significance in Patients with Liver Disease. Tohoku J. exp. Med., 1985, 145(2) 185-195-An enzymatic fluorometric microassay using 3α-or 7α-hydroxysteroid dehydrogenase was devised to determine serum total or primary bile acids. The present microassay using 50μl of a serum sample was run as a two-step method; the first step is the reaction of substrates with NAD⁺ and the hydroxysteroid dehydrogenase to produce as much NADH as possible in the incubation system and the second step is that of NADH with resazurin coupled with 1-methoxy phenazinemethosulfate to produce fluorescent resorfin. The assay is specific for 3α-or 7α-hydroxy bile acids, sensitive (a detection limit of 1.5μM with a linear range of 1.5-80μM), reproducible (CV=less than 5%) and inexpensive. As measured by this microassay, the ratio of serum primary to total bile acids (P/T ratio) in 10 normal subjects was 62.1±0.7% and was significantly elevated in the early phase of acute hepatitis (p<0.01), followed by return to the normal level after 4 weeks.