Rapid analysis of human fecal bile acids.
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 3 alpha-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 3 alpha-hydroxyls are first oxidized to 3-oxo-groups in a reaction catalyzed by 3 alpha-hydroxysteroid dehydrogenase. Resulting protons are transferred to beta-nicotinamide adenine dinucleotide, yielding reduced beta-nicotinamide adenine dinucleotide (beta-NADH). beta-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 3 alpha-sulfates are extracted in the procedure but reduction and/or solvolysis is necessary before quantification.
D001647:Bile Acids and Salts / Q000032:analysis*; D005243:Feces / Q000032:analysis*; D006801:Humans; D006868:Hydrolysis; D008722:Methods; D009243:NAD; D010078:Oxazines; D013050:Spectrometry, Fluorescence; D014966:Xanthenes
D001647:Bile Acids and Salts; D010078:Oxazines; D014966:Xanthenes; D009243:NAD; C005843:resazurin