This report describes a rigorous physical-chemical approach to determine the solubilities of unconjugated bilirubin in model bile systems. In the absence of calcium ions, metastable and equilibrium solubilities can be obtained by acidimetric (HC1) titration of excess unconjugated bilirubin in aqueous solutions of simple and mixed conjugated bile salt micelles. The experiments must be performed with degassed water under argon, in dim light and at constant temperature and ionic strength. This approach provides metastable and equilibrium solubilities of unconjugated bilirubin at a unique pH, i.e., the precipitation pH (pHppt) of bilirubin in the bile salt system. Because high levels of supersaturation can be acidimetrically induced, equilibration is usually complete in less than 20 min. To obtain equilibrium solubilities of bilirubin in simulated bile systems at other pH values (<pHppt>), it is necessary to perform equilibrium dissolution studies of crystalline diacid bilirubin in the presence of a strong buffer (at least 0.2 M). This method must, perforce, be the procedure of choice to estimate micellar bilirubin solubilities in the presence of calcium ions. Each dissolution experiment takes from 2 to 3 days, hence meticulous attention must be paid to the photochemical stability of bilirubin during the equilibration period, and appropriate precautions taken. Bilirubin solubilities obtained by both methods can be validated by titrating predetermined concentrations of unconjugated bilirubin spectrophotometrically in the model bile systems to the pH value at which saturation solubility was obtained. As a check of equilibration, it is imperative that bilirubin solubility values at the pHppt obtained by both dissolution and titra-metric methods display satisfactory agreement. For a complete description of the system, chemical and physical analyses of the excess solid-phase bilirubin that was in equilibrium with the bilirubin-saturated micellar phase should be performed.
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