Non chromatographic colorimetric assay for total taurine-conjugated bile acids: application of measurements of glycine: taurine ratio in bile.

A direct colorimetric assay for total conjugated taurine from bile-rich duodenal aspirates is described. The method is based on complete acetylation of the free hydroxyl groups by acetic anhydride at 100 degrees C. of both the tri- and di-hydroxy bile acids in Folch extracted bile samples. Taurine-conjugated bile acids are measured by ion pair formation with Azure A and subsequent extraction of the complex into the organic phase of a biphasic system. Absorption at 645 nm. of this complex directly quantifies total taurine-conjugated bile acid. Total bile acids are then estimated by the 3alpha-hydroxysteroid dehydrogenase assay. The difference between the concentration of the total conjugated bile acid and of the total conjugated taurine determines the concentration of glycine conjugates and the glycine:taurine ratio. Potentially interfering materials such as sulphalipids, certain phospholipids, and unconjugated bile acids are removed by Folch extraction. The 3-hydroxysteroid sulfates (cholesteryl sulfate, lithocholate sulfate, and glycocholate sulfate) are not measurable by heating in acetic anhydride and do not interfere. Taurolithocholate-3-sulfate, under identical conditions, gives a measurable but very low color yield and in normal physiologic concentrations would contribute negligible color. As previously reported, this assay under prescribed conditions is selective for long-chain amphipathic sulfates or sulfonates with no measurable color yield with glycine conjugates, unconjugated bile acids, free fatty acids, or lecithin. Values for glycine:taurine ratios by the above-described method in both normal bile extracts and extracts from patients with either elevated or depressed ratios relate closely to those determined by thin-layer chromatography.