Enzymic measurement of primary bile acids and the primary bile acid ratio in serum with the IL-Multistat III Fluorescence Light-Scattering Centrifugal Analyzer

Enzymic fluorimetric methods are described for the determination of primary bile acids and of chenodeoxycholic acid (CDC) and cholic acid (C) in serum. Bile acids are extracted from 0.3 mL of serum in a simple 5-min step with use of Sep-Pak C cartridges. Total primary bile acids are measured by an equilibrium technique after reaction with beta-NAD in the presence of 7 alpha-hydroxysteroid dehydrogenase. Chenodeoxycholic acid (and its conjugates) is measured by a reaction-rate technique employing the same reaction as above but under different experimental conditions. A small contribution of cholic acid (and its conjugates) to the reaction rate is eliminated by simple calculations. Cholic acid is calculated by difference of the two determinations. In both assays NADH fluorescence is measured with the Multistat centrifugal analyzer. Absolute recovery of bile acids from serum was about 87%. Day-to-day standard deviations for CDC and C were 1.6 and 2.0 mumol/L at serum concentrations of 22.1 and 24.1 mumol/L respectively. Comparison data with a cholylglycine RIA procedure gave the following correlation coefficients (x = RIA, y = proposed method): r = 0.980 (RIA vs total primary bile acids), r = 0.918 (RIA vs CDC) and r = 0.989 (RIA vs C). The methods described appear more practical for use on a routine basis than methods in the literature for the calculation of the primary bile acid ratio.