Rapid photometric determination of free and esterified 3 alpha-hydroxy fecal bile acids

A rapid, precise, and accurate photometric method for determining free and esterified fecal 3 alpha-hydroxy bile acids is described. Feces are homogenized and (a) extracted with boiling absolute ethanol, or (b) lyophilized and extracted with chloroform:methanol 2:1 (v/v). Hydrolyzed and nonhydrolyzed crude extracts are prepared and aliquots treated with a reagent containing nitro blue tetrazolium (NBT), 3 alpha-hydroxysteroid oxidoreductase, beta-nicotinamide adenine dinucleotide (beta-NAD) and diaphorase. The reagent first oxidizes bile acid 3 alpha-hydroxyls to 3-oxo groups and 3 beta-hydrogen is transferred to beta-NAD yielding beta-NADH. beta-NADH in turn reduces NBT (yellow) to its diformazan (blue). Absorbance is measured at 540 nm and is proportional to the 3 alpha-hydroxy bile acid titer of fecal extract aliquots. Fecal pigments present in crude extracts do not interfere with the assay since they absorb minimally at 540 nm.