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

A. Roda, S. Girotti, S. Ghini, B. Grigolo, G. Carrea, R. Bovara

Research output: Contribution to journalArticle

Abstract

We describe a continuous-flow bioluminescence method for measuring primary bile actid in serum, with nylon as the solid support. 7α-Hydroxysteroid dehydrogenase (EC 1.1.1. 159), a bacterial luciferase (EC 1.14.14.3), and NAD+:FMN oxidoreductase (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 7α-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 μL) 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 >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 μmol/L, in agreement with those obtained by other methods.

Original languageEnglish
Pages (from-to)206-210
Number of pages5
JournalClinical Chemistry
Volume30
Issue number2
Publication statusPublished - 1984

ASJC Scopus subject areas

  • Clinical Biochemistry

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