Low solubility of unconjugated bilirubin in dimethylsulfoxide – water systems: implications for pKa determinations
BackgroundAqueous pKa values of unconjugated bilirubin are important determinants of its solubility and transport. Published pKa data on an analog, mesobilirubin-XIIIα, studied by 13C-NMR in buffered solutions containing 27 and 64 vol% (C2H3)2SO because of low aqueous solubility of mesobilirubin, were extrapolated to obtain pKa values in water of 4.2 and 4.9. Previous chloroform-water partition data on bilirubin diacid led to higher estimates of its pKa, 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 13C-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.ResultsExtensive 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.ConclusionsThe present study strongly supports the validity of the aqueous solubility of bilirubin diacid derived from partition data, and, therefore, the corresponding high pKa values. Many of the mesobilirubin systems in the 13C-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 pKa values of the many soluble reference acids and mesobilirubin, raise doubts regarding the low pKa estimates for mesobilirubin from the 13C-NMR studies.
Validation of infrared spectroscopy for assessment of vinyl polymers of bile-pigment gallstones.
The i.r. spectra of bilirubin isomers that differ in number and position of vinyl groups were examined to verify the assignment of the 988 cm-1 peak of bilirubin (991 cm-1 peak in calcium bilirubinate) to its pendant vinyl groups. There were only small changes in this peak with changes in position of vinyl groups (exo-2- and -18-vinyl versus endo-3- and -17-vinyl), but progressive loss of peaks in this region was observed when vinyl groups were reduced to ethyl groups (dihydrobilirubin and mesobilirubin). Methylvinylmaleimide, a monopyrrole derived from the outer (A and D) rings of bilirubin, has a pendant vinyl group and exhibits a prominent peak at 986 cm-1, but haematinic acid methyl ester derived from the inner (B and C) rings has no vinyl group and shows no peak near 988 cm-1. These observations verify the assignment of the 988 cm-1 peak of bilirubin to its pendant vinyl groups. This supports our previous proposal that a decrease in the peak at 985-995 cm-1 in the i.r. spectra of pigment gallstones, as compared with unconjugated bilirubin or calcium bilirubinate, indicates a consumption of vinyl groups in the process of formation of the polymer in the pigment stones.
Phloroglucinol derivatives from Hypericum japonicum.
The isolation and identification of a new phloroglucinol derivative (2), a diterpenol (4), together with the known compounds flavesone (1) and sarothralen B (3), from the aerial parts of Hypericum japonicum are reported. Their structures were established by extensive spectral analysis and the structure of (3) has also been confirmed by a single crystal X-ray determination.