Discrete analysis of bile acid in serum and bile with 3?-hydroxysteroid dehydrogenase and diaphorase immobilized onto alkylamine glass beads

摘要

3α-Hydroxysteroid dehydrogenase (3α-HSD) from Pseudomonas testosteronei and diaphorase (lipoyl dehydrogenase) from Clostridium spp were immobilized individually onto alkylamine glass beads through glutaraldehyde coupling. A cost-effective enzymic colorimetric method for determination of bile acid in the serum and bile was developed employing mixture of the immobilized enzymes. The method was based upon measurement of NADH generated from NAD? during oxidation of bile acid by immobilized 3?-HSD with a color reagent consisting of nitrobluetetrazolium (NBT) chloride salt and immobilized diaphorase in 0.065 M sodium phosphate buffer (pH 7.0). The minimum detection limit of the method was 4.8 μmol/L in the serum and 19.5 μmol/L in bile. The per cent recovery of added bile acid in the serum and bile was 89.1 and 95.0, respectively. Within and between batch coefficients of variation (CV) for bile acid determination were 1.0% and 0.2% in the serum and 0.2% and 0.6% in bile, respectively. A good correlation for bile acid in the serum (r?= 0.95) and in bile (r? = 0.93) was obtained by a standard chemical method (a commonly used method in India) and the present method. The mixture of immobilized 3?-HSD and diaphorase lost 30% of its initial activity after 4 months of regular use. The cost of bile acid determination for 100 the serum and bile samples by the present method was found to be lower than by a commercially available method (Sigma kit 450-A)