Continuous-flow determination of primary bile acids, by bioluminescence, with use of nylon-immobilized bacterial enzymes.

Abstract

Abstract We describe a continuous-flow bioluminescence method for measuring primary bile acid in serum, with nylon as the solid support. 7 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1. 159), a bacterial luciferase (EC 1.14.14.3), and NAD+:FMN oxido-reductase (EC 1.6.8.1) are covalently co-immobilized on a nylon coil (1 m X 1.0 mm i.d.). The assay is highly specific for 7 alpha-hydroxy bile acids. Other bile acids and steroids do not interfere. The continuous-flow light-emitting system, in which the reactor (nylon coil) is placed in front of a photomultiplier tube inside a luminometer, is versatile and simple. The flow is air-segmented, and serum samples (5-50 microL) can be injected directly. Concentration and response are linearly related from 10 to 2500 pmol per assay tube. The precision of the method is satisfactory (CV 5-10%), both inter- and intra-assay. We validated the technique by comparing results with those by RIA, enzyme immunoassay, and "high-performance" liquid chromatography. More than 20 samples an hour can be analyzed, with no carryover. The nylon-immobilized enzymes are stable for more than two months, and greater than 500 samples can be analyzed with use of a few milligrams of enzymes. Normal values for bile acid content of serum ranged from 1 to 2.5 mumol/L, in agreement with those obtained by other methods.