A simple and sensitive assay of total serum bile acids

Abstract

A simple and sensitive method was developed for the quantification of serum total 3α-hydroxy bile acids. 0.1 ml of serum was mixed with tris(hydroxymethyl) aminomethane hydrochloric acid buffer and heated at 67°C for 30 min. To the solution were added 3α-hydroxysteroid:oxidoreductase (EC 1.1.1.50; 3α-HSD), NAD, diaphorase (EC 1.6.4.3) and resazurin. The mixture was incubated at 20° C for 1 h. The resultant fluorescence of resorfin was measured at 580 nm with the excitation at 560 nm. The blank value was obtained after the same treatment of another 0.1 ml of the same serum without 3α-HSD. A linear relationship was obtained between the amount of bile acids and the fluorescence intensities in the range of 1 to 150 μmol/1. The recovery of bile acids added to the serum was 81,4 ± 2.5 (S.D.) % for cholate, chenodeoxycholate and deoxycholate. The bile acid content in the serum was 48.8 μmol/l with a standard deviation of ±0.42 and a coefficient of variation of ±0.87% in 10 replicate determinations. The mean bile acid content of normal fasting male sera was 8.0 μmol/1 (3.6–12.6 μmol/1, n = 12) and of female sera 6.8 μmol/1 (3.2–12.7 μmol/l, n = 13).