Highly sensitive method for quantitative determination of bilirubin in biological fluids and tissues.
Unconjugated bilirubin (UCB) exhibits potent antioxidant and cytoprotective properties, but causes apoptosis and cytotoxicity at pathologically elevated concentrations. Accurate measurement of UCB concentrations in cells, fluids and tissues is needed to evaluate its role in redox regulation, prevention of atherosclerotic and malignant diseases, and bilirubin encephalopathy. In the present study, we developed and validated a highly sensitive method for tissue UCB determinations. UCB was extracted from rat organs with chloroform/methanol/hexane at pH 6.2 and then partitioned into a minute volume of alkaline buffer that was subjected to HPLC using an octyl reverse phase (RP) column. Addition of mesobilirubin as an internal standard corrected for losses of UCB during extraction. Recoveries averaged 75+/-5%. The detection limit was 10pmol UCB/g wet tissue. Variance was +/-2.5%. When used to measure UCB concentrations in tissues of jaundiced Gunn rats, this procedure yielded UCB levels directly comparable to published methods, and accurately determined very low tissue bilirubin concentrations (</=40pmol UCB/g tissue) in non-jaundiced rats.
Zelenka
Jaroslav
J
Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Faculty of Medicine, Charles University in Prague, Czech Republic.
Lenícek
Martin
M
Muchová
Lucie
L
Jirsa
Milan
M
Kudla
Michal
M
Balaz
Peter
P
Zadinová
Marie
M
Ostrow
J Donald
JD
Wong
Ronald J
RJ
Vítek
Libor
L
D000818:Animals; D001663:Bilirubin / Q000032:analysis*; D001826:Body Fluids / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D008099:Liver / Q000737:chemistry; D008297:Male; D051381:Rats; D011911:Rats, Gunn; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D001663:Bilirubin