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 mumol/l, by fluorometry it is 0.25 mumol/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 mumol/l and of 0.5 to 17.5 mumol/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 -20 degrees C or extraction with dimethylsulfoxide prior to storage are good ways for sample preservation. Twenty stool samples from healthy adults were determined.(ABSTRACT TRUNCATED AT 250 WORDS)
Kotal
P
P
1st Medical Department, Charles University, Prague, Czechoslovakia.
Fevery
J
J
D000328:Adult; D001646:Bile / Q000737:chemistry*; D003124:Colorimetry / Q000379:methods; D005243:Feces / Q000737:chemistry*; D005705:Gallbladder Diseases / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D008107:Liver Diseases / Q000378:metabolism; D008875:Middle Aged; D012016:Reference Values; D012044:Regression Analysis; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry / Q000379:methods; D014558:Urobilinogen / Q000032:analysis* / Q000097:blood / Q000652:urine; D015032:Zinc
D007202:Indicators and Reagents; D014558:Urobilinogen; D015032:Zinc