Determination of serum delta 5-3 beta-hydroxysteroid sulphates by combined high-performance liquid chromatography and immobilized 3 beta,17 beta-hydroxysteroid dehydrogenase in column form.
We studied the use of an immobilized enzyme, covalently bound to aminopropyl-CPG, in the analysis of individual delta 5-3 beta-hydroxysteroid sulphates. A microcolumn with immobilized 3 beta,17 beta-hydroxysteroid dehydrogenase was prepared and used together with high-performance liquid chromatography (HPLC). The reduced nicotinamide-adenine dinucleotide produced from delta 5-3 beta-hydroxysteroids by this enzyme was fluorimetrically determined. The immobilized enzyme was sufficiently stable for at least one month or for 180 tests when used repeatedly. A clinical trial demonstrated that this HPLC-immobilized enzyme method is superior to the soluble enzyme method, giving reliable and reproducible results at a low cost.
D000328:Adult; D002851:Chromatography, High Pressure Liquid; D003687:Dehydroepiandrosterone / Q000032:analysis; D004355:Drug Stability; D004800:Enzymes, Immobilized; D005260:Female; D006801:Humans; D006868:Hydrolysis; D006913:Hydroxysteroid Dehydrogenases; D006914:Hydroxysteroids / Q000032:analysis*; D009243:NAD; D011863:Radioimmunoassay; D013050:Spectrometry, Fluorescence; D013431:Sulfates / Q000032:analysis
D004800:Enzymes, Immobilized; D006914:Hydroxysteroids; D013431:Sulfates; D009243:NAD; D003687:Dehydroepiandrosterone; D006913:Hydroxysteroid Dehydrogenases