Enzymatic Microassay of 3-Keto Bile Acids Using 3-Ketosteroid-Δ⁴ Dehydrogenase and Its Application to Fecal Bile Acids

Abstract

A simple and sensitive enzymatic fluorometric microassay is described for the determination of 3-keto bile acids using 3-ketosteroid-Δ⁴-dehydrogenase (Δ⁴ DH) and resazurin.
The present microassay showed high substrate specificity of free and conjugated 3-keto bile acids and the linearity of fluorescence intensity (Emission; 580nm, Excitation; 560nm) to 3-keto bile acids in the range of 2.5 to 80μM with coefficient variation of 2.1% to 3.4%.
The present method was applied to determination of fecal 3-keto bile acids from controls and patients with ileocecal resection or colon resection.
The correlation between HPLC (y) and the present method (X) for determination of fecal 3-keto bile acids was r=0.896 (n=43), and the regression curve was y=0.784x+0.199.
Feces from controls and patients with ileocecal resection or colon resection contained 3-keto bile acids by 35.1±9.1%, 20.5±4.6% and 11.7%±2.5% of total fecal bile acids pergram of dry feces, respectively.