Low solubility of unconjugated bilirubin in dimethylsulfoxide – water systems: implications for pK(a) determinations

BACKGROUND: Aqueous pK(a) values of unconjugated bilirubin are important determinants of its solubility and transport. Published pK(a) data on an analog, mesobilirubin-XIIIα, studied by (13)C-NMR in buffered solutions containing 27 and 64 vol% (C(2)H(3))(2)SO because of low aqueous solubility of mesobilirubin, were extrapolated to obtain pK(a) values in water of 4.2 and 4.9. Previous chloroform-water partition data on bilirubin diacid led to higher estimates of its pK(a), 8.12 and 8.44, and its aqueous solubility. A thermodynamic analysis, using this solubility and a published solubility in DMSO, suggested that the systems used to measure (13)C-NMR shifts were highly supersaturated. This expectation was assessed by measuring the residual concentrations of bilirubin in the supernatants of comparable DMSO-buffer systems, after mild centrifugation to remove microprecipitates. RESULTS: Extensive sedimentation was observed from numerous systems, many of which appeared optically clear. The very low supernatant concentrations at the lowest pH values (4.1-5.9) were compatible with the above thermodynamic analysis. Extensive sedimentation and low supernatant concentrations occurred also at pH as high as 7.2. CONCLUSIONS: The present study strongly supports the validity of the aqueous solubility of bilirubin diacid derived from partition data, and, therefore, the corresponding high pK(a) values. Many of the mesobilirubin systems in the (13)C-NMR studies were probably supersaturated, contained microsuspensions, and were not true solutions. This, and previously documented errors in pH determinations that caused serious errors in pK(a) values of the many soluble reference acids and mesobilirubin, raise doubts regarding the low pK(a) estimates for mesobilirubin from the (13)C-NMR studies.