Molecular and micellar associations in the pH-dependent stable and metastable dissolution of unconjugated bilirubin by bile salts.

Unconjugated bilirubin (UCB) is almost insoluble in water at neutral pH, but appears in normal human gallbladder bile at concentrations up to 35 microM. We therefore determined whether conjugated bile salts could increase the dissolved concentration [( Bt]) of UCB over the pH range 3.0-11.0. Using crystalline UCB, [Bt] was higher with less ordered crystals, with increasing pH and bile salt concentration, and with taurocholate (TC) micelles compared to taurodehydrocholate (TDHC) dimers. Plots of [Bt] verus pH from pH 3.0-9.3 fit the equation, [Bt] = A(1 + K'1/[H]+ + K'1.K'2/[H+]2), where A = [Bt] at pH less than 4.0, and K'1 and K'2 are the two apparent ionization constants of UCB. Estimated pK'1 values in NaCl, TC, and TDHC were 6.8, 6.0, and 5.6, respectively; pK'2 was greater than or equal to 9.3 in each system. Acidification of disodium bilirubinate to pH less than 8.5 produced high, metastable [Bt] in 50 mM TC; this was absent in 0.15 M NaCl, and minor in 50 mM TDHC. In all solutions, maximum [Bt] of 60-65 mM was attained at pH greater than or equal to 10.5. This work helps explain the immense variation among reported [Bt] values, indicates that UCB monoanion predominates at the pH range of bile, and suggests that bile salt monomers, dimers, and micelles enhance the solubility of UCB in bile.