Quantitation of urobilinogen in feces, urine, bile and serum by direct spectrophotometry of zinc complex

Previous methods to quantitate urobilinogen lack precision due to either incomplete reduction of urobilin or to losses of pigment before the use of Ehrlich's aldehyde reaction or due to pigment precipitation, as occurs in Schlesinger's fluorescent assay. The present procedure modifies the latter assay to obviate described problems as it is based on direct spectrophotometry (or spectrofluorometry) of a zinc complex of urobilin in dimethylsulfoxide. The sample is extracted with dimethylsulfoxide to increase recovery of urobilinogen from samples of various origin (feces, urine, bile, serum etc.) and to prevent the precipitation of proteins. After oxidation of urobilinogen with iodine, the concentration of the resulting urobilin is directly determined from the absorption (or fluorescent) spectrum. High sensitivity and high specificity for the procedure result from the high value of absorption coefficient and by the characteristic absorption spectrum of zinc complex of urobilin, respectively. Within-day and day-to-day coefficients of variation of stool and bile samples range from 1.6 to 9.2%. The smallest concentration of urobilinogen measurable by spectrophotometry is approximately 0.5 μ mol/l, by fluorometry it is 0.25 μmol/l. The recovery varies from 82.2 to 93.8% depending on re-extraction of the sample. The method is linear in the range of 1 to 35 μmol/l and of 0.5 to 17.5 μmol/l for spectrophotometric and fluorescent determinations, respectively. The results obtained with the present method correlated well with Ehrlich's determination (r2 = 0.912), but are approximately two-fold higher Storage of the samples at -20oC or extraction with dimethylsulfoxide prior to storage are good ways for sample preservation. Twenty stool samples from healthy adults were determined. The mean concentrations of fecal urobilinogen were 2.61± 1.62 and 1.73 ± 0.65 μmol/g of dry feces for males and females, repectively.