Rapid analysis of human fecal bile acids

Article abstract of DOI:10.1016/0039-128X(81)90064-7

A rapid, accurate, precise method for determining human fecal bile acids is reported. Feces are homogenized and then briefly extracted with boiling absolute ethanol. A portion of the extract is evaporated to dryness and the residue heated with mild alkali to hydrolyze bile acid 3α-hydroxyl esters. Aliquots of hydrolyzed crude extract are treated with resazurin reagent which effects a series of enzyme catalyzed reactions in which bile acid free 3α-hydroxyls are first oxidized to 3-oxo-groups in a reaction catalyzed by 3α-hydroxysteroid dehydrogenase. Resulting protons are transferred to β-nicotinamide adenine dinucleotide, yielding reduced β-nicotinamide adenine dinucleotide (β-NADH). β-NADH then reduces nonfluorescent resazurin to fluorescent resorufin in a reaction catalyzed by diaphorase. Developed fluorescence, which is proportional to the extract aliquots bile acid content, is excited at 565 nm and read at 580 nm, wavelengths which lie in a spectral region in which there is minimal fecal pigment absorption. 3-Oxo-bile acids and bile acid 3α-sulfates are extracted in the procedure but reduction and/or solvolysis is necessary before quantification.