Using gel-permeation chromatography, we studied associative relationships between conjugated bilirubin and various biliary lipid particle species, including lecithin/ cholesterol vesicles, mixed-lipid micelles, and simple bile salt micelles. Five other organic anions were comparably studied: phenol red, Evans blue, sulfobromophthalein, rose bengal, and indocyanine green. For compounds of intermediate hydrophobicity, including conjugated bilirubin, the dominant association was with a bile salt/organic anion hybrid particle of dimensions larger than that of a simple pure bile salt micelle. Vesicular association was found to be dominant only for the most hydrophobic organic anions, indocyanine green and rose bengal; conversely, the most hydrophilic anion, phenol red, showed no vesicular association. Accordingly, a strong positive correlation (P < 0.001) was found between percent vesicular association and degree of hydrophobicity of the organic anion. Alkaline conditions (eluant pH 9) decreased or prevented vesicular hydrophobic interaction with all anions. We conclude that two important particulate mechanisms for transport in bile of conjugated bilirubin and other water-soluble anions are (i) bile salt/organic anion hybrid particles and (it) vesicles. For most organic anions of intermediate hydrophobicity, including conjugated bilirubin, the bile salt/organic anion hybrid particle is the dominant transport vehicle.About 99% of bilirubin found in native bile is in conjugated form, primarily as diglucuronides (1, 2). Physiological studies of bilirubin and other organic anion secretory rates in bile showed a strong correlation with bile acid secretory rates under stimulated conditions but not during fasting (3-8). This observation led to studies that have concluded that a strong relationship exists between biliary mixed-lipid micelles and bilirubin in addition to a variety of other organic anions, although the precise nature of the putative micellar-organic anion interaction has remained unclear (9-15). Thus, a more recent study examining this relationship failed to support the concept of a physicochemical association between biliary lipids and conjugated bilirubin (15). A further complicating factor has been the recent discovery of potential functional roles for vesicles, which have been identified in supersaturated human biles and even in undersaturated but quite dilute rat biles (16)(17)(18)(19)(20)(21)(22)(23)(24). Because of these discrepancies and uncertainties, in the present study three things have been done. First, focusing initially on conjugated bilirubin, we determined preferential distributions between biliary vesicles and micelles for both pure bile salt and bile salt/mixed-lipid types. Interaction with the former micellar species was found to be dominant for conjugated bilirubin. Second, we similarly studied a spectrum of other water-soluble organic anions.Partitioning between vesicles and micelles was found to differ markedly among the various compounds studied. Third, we sought to determine th...