Abstract
A simple, rapid, and sensitive bioluminescence method for measuring primary bile acids has been developed and validated. The method is based on enzymatic dehydrogenation of bile acids using a bacterial 7 alpha-hydroxysteroid dehydrogenase that is co-immobilized on Sepharose 4B beads with NADH:FMN oxidoreductase and a bacterial luciferase. The assay is specific for 7 alpha-hydroxy bile acids and has a detection limit of 0.5 pmol/tube, with a linear range of 0.5-50 pmol/tube. The assay shows good precision (6-8% intra-assay; 8-10% inter-assay). The values obtained with the bioluminescence assay showed good agreement with those obtained by gas-liquid chromatography, radioimmunoassay, or endpoint enzymatic assays. When applied to the measurement of serum bile acids, there was no interference from serum albumin, and the effect of other dehydrogenase activity in serum could be eliminated by heating the sample prior to assay. Since the method is rapid (1 minute), extremely sensitive (requires only 10 microliters of serum), and specific, it appears to be the best method currently available for the measurement of serum primary bile acids.
Highlights
A simple, rapid, and sensitive bioluminescence method for measuring primary bile acids has been developed and validated
We report here the development, validation, and application of a bioluminescence assay of 7a-hydroxy bile acids based on 7a-hydroxysteroid dehydrogenase that has been co-immobilized with NADH:flavin mononucleotide (FMN) oxidoreductase and a bacterial luciferase
Luciferase, NADH:FMN oxidoreductase, and 7a-hydroxysteroid dehydrogenase were co-immobilized on cyanogen bromide-activated Sepharose according to the method of Ford and DeLuca [26]
Summary
A simple, rapid, and sensitive bioluminescence method for measuring primary bile acids has been developed and validated. Serum bile acids originate from the intestinal absorption of bile acids during enterohepatic cycling and their levels increase postprandially. An increased serum bile acid level in the fasting state or postprandial is considered to be a specific indicator of liver disease, reflecting decreased hepatic uptake or systemic shunting of portal venous blood [3]. Enzymatic methods based on group-specific steroid dehydrogenases isolated from bacteria [11, 12] have been used successfully to measure total Sa-hydroxylated [13] and 7a-hydroxylated [14] serum bile acids. T h e suffix “yl” is used to denote the bile acid moiety of the glycine and taurine conjugates, which are N-acyl ami-
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