Enzymic fluorimetric determination of sulphated and non-sulphated primary bile acids in urine using a rapid solvolysis technique

Abstract

A simple and rapid enzymic fluorimetric method for the determination of sulphated and non-sulphated primary bile acids in urine has been developed. Octadecylsilane-bonded silica cartridges (Sep-Pak C₁₈) are used for the solid-phase extraction of bile acids (BA) from urine samples. Sulphated BA are solvolysed before measurement with an improved rapid solvolysis procedure. The measurement is based on the reaction of 7α-hydroxylated BA with β-nicotinamide adenine dinucleotide (β-NAD⁺) in the presence of the enzyme 7α-hydroxysteroid dehydrogenase (7α-HSD). The NADH generated is monitored fluorimetrically. The mean relative standard deviation of the method was 8%(n= 32), the detection limit 0.4 µM and the mean recovery of added BA 99%. The solvolysis step in this procedure takes about 4 h and is faster than the conventional procedures which are usually carried out in an 18-h protocol. Sulphated and non-sulphated primary BA were determined in urine from 16 healthy persons and 67 hospitalised patients suffering from various hepatobiliary diseases. The reference range for 7α-hydroxylated urinary BA was in agreement with previously published results. The method is simple and suitable for routine clinical use. About 30 urine samples can be analysed in one working day.