Rates of oxidation of bilirubin species were determined spectrometrically using enzymatic oxidation of bilirubin by peroxidase (POD: EC 184.108.40.206) and hydrogen peroxide. Rates of oxidation of conjugated and unconjugated bilirubin (Bc and Bu) were examined with and without serum albumin and compared, and those of bilirubin bound covalently to albumin were determined using naturally occurring delta-bilirubin (Bd). In the presence of serum albumin (Alb), Bc-Alb showed the highest rate of oxidation by POD oxidation followed by Bu-Alb and Bd. Without serum albumin Bu showed a higher rate of oxidation than Bc, despite the fact that Bc exerted more interference than Bu on the enzymatic determination of uric acid (coupled with POD reaction and dye formation).
We describe a new enzymatic assay method to measure sulfated bile acids in urine, in. which the essential components are a novel bile acid sulfate sulfatase (BSS) and β-hydroxysteroid dehydrogenase (β-HSD: EC 220.127.116.11.51). The BSS produced inducibly by Pseudomonas testosteroni was able to efficiently hydrolyze various kinds of bile acid 3α-sulfates to form 3β-hydroxyl bile acids and sulfuric acid. Total sulfated bile acid (TSBA) in urine was determined by the combined reaction of desulfation of bile acid 3α-sulfates by BSS, followed by dehydrogenation of desulfated substrates by β-HSD and conventional colorimetric assay of reduced nicotinamide adenine dinucleotide (NADH) as formazan dye in the presence of nitrotetrazolium blue and diaphorase. The calibration curve for glycolithocholic acid 3-sulfate as the standard was linear up to 250μmol/l. Analytical recovery of various sulfated bile acids in urine averaged 98% excepting the recovery of taurolithocholic acid 3-sulfate which was approximately 80%. The CV for intra-and inter-assay variations was ≤3.2% and ≤3.4%, respectively. This method is accurate and simple, and less time-consuming than those previously reported. We determined the concentration of TSBA in adult urine samples by this method, and observed that urinary TSBA of normal subjects was less than 7.6μmol/g creatinine (mean2.0±1.8μmol/g creatinine), while in patients with acute hepatitis, liver cirrhosis, and intra-and extra-hepatic biliary obstruction urinary TSBA was markedly increased.