BACKGROUND/AIMS: Our previous studies of ionization and solubility of unconjugated bilirubin (UCB) yielded inappropriately large differences between the two carboxylic pK'a values of UCB. These data, however, were not ideal due to crystal effects, matastability, impurities of the bilirubin, and imprecision of analyses at low UCB. METHODS: The sodium salt of taurocholate (TC) was purified and dissolved in water to 100 mM. Chloroform (CHCl3) was purified by vacuum distillation. Buffers used were: citrate from pH 4 to 6, phosphate from pH 6 to 8, and borate above pH 8. All had an ionic strength of 0.10. The problems were minimized by rapid solvent partition of UCB from CHCl3 into buffered aqueous NaCl, and a new, accurate assay of low UCB in the aqueous phase which was achieved by concentrating the UCB through back extraction into small volumes of CHCl3. RESULTS: In contrast with the crystal dissolution studies, the two pK'a value were similar. H2B0, not HB-, was the dominant UCB species in the pH range of bile (6.0 to 8.0). The aqueous solubilities of UCB were 90 to 98% less. Less than 0.01% of the bile salt partitioned into the CHCl3 phase and self-association of B= was negligible. UCB solubilities in 50 mM TC were 2 to 10% of those obtained by crystal dissolution, and, up to pH 7.9, were below the maximum UCB concentration in normal human bile. CONCLUSIONS: We suggest that the markedly increased binding of UCB with each ionization step is due to the disruption of the internal hydrogen bonds of the ionized carboxyl groups on interaction with the bile salt. We propose to extend the study of partition to determine the activity and the degradation products of calcium salts of unbound bilirubin fractions.
BACKGROUND/AIMS: Our previous studies of ionization and solubility of unconjugated bilirubin (UCB) yielded inappropriately large differences between the two carboxylic pK'a values of UCB. These data, however, were not ideal due to crystal effects, matastability, impurities of the bilirubin, and imprecision of analyses at low UCB. METHODS: The sodium salt of taurocholate (TC) was purified and dissolved in water to 100 mM. Chloroform (CHCl3) was purified by vacuum distillation. Buffers used were: citrate from pH 4 to 6, phosphate from pH 6 to 8, and borate above pH 8. All had an ionic strength of 0.10. The problems were minimized by rapid solvent partition of UCB from CHCl3 into buffered aqueous NaCl, and a new, accurate assay of low UCB in the aqueous phase which was achieved by concentrating the UCB through back extraction into small volumes of CHCl3. RESULTS: In contrast with the crystal dissolution studies, the two pK'a value were similar. H2B0, not HB-, was the dominant UCB species in the pH range of bile (6.0 to 8.0). The aqueous solubilities of UCB were 90 to 98% less. Less than 0.01% of the bile salt partitioned into the CHCl3 phase and self-association of B= was negligible. UCB solubilities in 50 mM TC were 2 to 10% of those obtained by crystal dissolution, and, up to pH 7.9, were below the maximum UCB concentration in normal human bile. CONCLUSIONS: We suggest that the markedly increased binding of UCB with each ionization step is due to the disruption of the internal hydrogen bonds of the ionized carboxyl groups on interaction with the bile salt. We propose to extend the study of partition to determine the activity and the degradation products of calcium salts of unbound bilirubin fractions.