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 10 pmol 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 (≤40 pmol UCB/g tissue) in non-jaundiced rats.