Isoextraction Studies of Partition of UCB Between Chloroform and Aqueous Solution

Bilirubin is a major component of gallstones and the solubility of unconjugated bilirubin (UCB) has been shown to play an Important role in gallstone formation. The mechanism of gallstone formation and solubilization of UCB is, however, still unknown. Several investigators have attempted to determine the chemical nature of bilirubin which are thought to be closely related to its solubility. However, there is some controversy over the ionization constants and pK values of carboxyl groups of UCB. In the present study, the effect of pH, the type and concentration of bile salt on UCB solubility were studied and the pK values of bilirubin were calculated. The first ionization censtants of the carboxyl group of UCB (pk(1)) were 7.2 in saline, 6.7 in TC (Taurocholic acid), and 6.6 in TDHC (Taurodehydrocholic acid). The pK(2) was 9.2 in 50 m M TC. Thus, UCB monoanion (BH(−)) accounted for over 90% of the ionized UCB at physiologic pH values. 1. With the isoextraction method of UCB from chloroform, crystal surface effects are avoided and the partition equilibrium could also be attained rapidly. 2. There are more solubilities with higher bile salt concentrations and with increasing pH, and bile salt plays an important role in solubilizing UCB in bile. 3. Bile salt monomers, dimers and micelles are equally effective in solubilizing bilirubin. 4. The bilirubin in bile is almost entirely in the monoanion (BH(−)) form at physiological pH values.