Quantitation of cholesterol-carrying particles in human gallbladder bile

The inter-mixed micellar/vesicular (non-phospholipid-associated) bile salt concentration (IMC) can be rapidly measured in model biles by centrifugal ultrafiltration, thus allowing reliable separation of vesicular and micellar cholesterol carriers by gel filtration with an elution buffer containing bile salts at the correct IMC (Donovan, J. M., and A. A. Jackson. 1993. J. Lipid Res. 34: 1121-1129). We adapted this method to the more complex human gallbladder bile and examined the relationship between cholesterol solubilization and crystallization in gallbladder biles from 10 cholesterol gallstone patients. The IMC (mean +/- SEM) was 9.67 +/- 1.97 (range 3.56-35.02) mM with significant enrichment with hydrophilic bile salt species. Upon gel filtration of these biles with an eluant buffer containing 10 major bile salts at concentrations according to their IMC, cholesterol was found to be solubilized mainly in mixed micelles. Vesicles were detected in all 10 biles after separation by KBr density gradient ultracentrifugation but in only 5 of these biles with the IMC method. Biles without vesicles had a lower CSI (1.15 +/- 0.12 vs. 1.90 +/- 0.28, P < 0.05), a higher total lipid concentration (11.9 +/- 2.3 vs. 5.9 +/- 1.1, P < 0.05), and a higher bile salt/ (bile salt + phospholipid) ratio (0.83 +/- 0.01 vs. 0.74 +/- 0.04, P = 0.07). For both IMC and ultracentrifugation methods, vesicular cholesterol concentration showed a negative correlation with crystal observation time and a positive correlation with cumulative crystal score during 21 days. Our data indicate that methods such as density gradient ultracentrifugation overestimate vesicular cholesterol solubilization in human biles.