Quantitation of cholesterol-carrying particles in human gallbladder bile

Erik R.M Eckhardt,Bert J.M Van De Heijning,Karel J Van Erpecum,Willem Renooij,Gerard P Vanberge-Henegouwen

doi:10.1016/s0022-2275(20)33297-1

Abstract

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. —Eckhardt, E. R. M., B. J. M. van de Heijning, K. J. van Erpecum, W. Renooij, and G. P. VanBerge-Henegouwen. Quantitation of cholesterol-carrying particles in human gallbladder bile.

Highlights

The inter-mixed micellar/vesicular bile salt concentration (IMC) can be rapidly measured in model biles by centrifugal ultrafiltration, allowing reliable separation of vesicular and micellar cholesterol carriers by gel filtration with an elution buffer containing bile salts at the correct IMC
Vesicles isolated by the IMC gel filtration method from supersaturated model bile (TLCo 5.7 g/dL, cholesterol saturation index (CSI) 1.4, TC/(TC ϩ Egg-yolk phosphatidylcholine (EYPC)) ϭ 0.7) did not transform into micelles after direct reapplication on the same column with the same eluant buffer
Micelles are the only lipid-containing phase expected to be present in unsaturated model bile (TLCo 10 g/dL, CSI 0.7, TC/(TC ϩ EYPC)ϭ 0.7)

Summary

Introduction

The inter-mixed micellar/vesicular (non-phospholipid-associated) bile salt concentration (IMC) can be rapidly measured in model biles by centrifugal ultrafiltration, allowing reliable separation of vesicular and micellar cholesterol carriers by gel filtration with an elution buffer containing bile salts at the correct IMC Biles without vesicles had a lower CSI (1.15 Ϯ 0.12 vs 1.90 Ϯ 0.28, P Ͻ 0.05), a higher total lipid concentration (11.9 Ϯ 2.3 vs 5.9 Ϯ 1.1, P Ͻ 0.05), and a higher bile salt/ (bile salt ϩ phospholipid) ratio (0.83 Ϯ 0.01 vs 0.74 Ϯ 0.04, P ϭ 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. Two methods have been used for separation of various cholesterol carriers in bile: gel filtration, generally with a constant concentration (often 7.5 or 10 mm) of one bile salt species (usually taurocholate or cholate) in the elution buffer, and density gradient ultracentrifugation.

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Results

Conclusion