Measurement of bile acid in serum and bile with arylamine-glass-bound 3alpha-hydroxysteroid dehydrogenase and diaphorase.
3Alpha-hydroxysteroid dehydrogenase (3-HSD) from Pseudomonas testosteroni and diaphorase (lipoyl dehydrogenase) from Clostridium spp. have been immobilized individually onto arylamine glass beads through diazotization. A cost-effective enzymic colorimetric method for determination of bile acid in serum and bile employing a mixture of these immobilized enzymes was developed. The method is based on measurement of reduced nicotinamide adenine dinucleotide generated from bile acid in serum/bile by immobilized 3alpha-HSD with a color reagent consisting of nitro blue tetrazolium chloride salt, oxidized nicotinamide adenine dinucleotide, and immobilized lipoyl dehydrogenase in 0.065 M sodium phosphate buffer, pH 7.0. Analytical recovery of added bile acid (50 and 200 micromol/L) was 95.57 and 85.46% in serum and 97.6 and 91.6% in bile, respectively. Within- and between-batch coefficients of variation (CV) for bile acid determination were <1.2 and <0.2% in serum and >0.1 and <0.1% in bile, respectively. Good correlations for bile acid in serum (r1=0.92) and in bile (r2=0.97) were obtained by use of a standard chemical method and the present method. The mixture of immobilized 3alpha-HSD dehydrogenase and lipoyl dehydrogenase lost 50% of its initial activity after 6 months of regular use. The cost of bile acid determination in 100 serum and bile samples by the present method has been compared with that of the Sigma kit method.
Biochemistry Research Laboratory, Department of Bio-Sciences, M.D. University, Rohtak 124001, Haryana, India.
Kirti
K
Rani
P
P
Garg
C S
CS
Pundir
D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) / Q000378:metabolism*; D001646:Bile / Q000378:metabolism*; D001647:Bile Acids and Salts / Q000032:analysis* / Q000097:blood / Q000378:metabolism; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D005898:Glass; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013657:Taurodeoxycholic Acid / Q000032:analysis
D001647:Bile Acids and Salts; D013657:Taurodeoxycholic Acid; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D008058:Dihydrolipoamide Dehydrogenase