Reactive oxygen metabolites promote cholesterol crystal formation in model bile: Role of lipid peroxidation

In animal models of gallstone disease inflammatory alterations of the gallbladder mucosa are regularly found before the first appearence of cholesterol monohydrate crystals in bile. At sites of inflammation granulocytes generate reactive oxygen metabolites (ROM). The aim of our study was to investigate whether ROM may influence the cholesterol monohydrate crystal formation in supersaturated model bile. Superoxide anions (O2), hydrogen peroxide (H202), and hydroxyl radicals (ʹOH) were generated by the interaction of Fe3+-EDTA with ascorbic acid (Asc). The influence of ROM on cholesterol crystal formation was studied by measurement of the nucleation time. To check whether lipid peroxidation was induced by the ROM generation, production of malondialdehyde equivalents was measured in bile with the thiobarbituric assay. Furthermore, the lipid pattern of bile after ROM exposure was analyzed by thin layer chromatography. Addition of Fe3+-EDTA/Asc to model bile markedly decreased the cholesterol nucleation time (NT) (p < 0.01), caused a significant increase in malondialdehyde equivalents (p < 0.001) and induced the generation of 4-hydroxy-2,3-trans-nonenal (4-HNE). In an attempt to identify a specific oxygen metabolite responsible for the alterations in bile, the effects of various oxygen radical scavengers were tested. Desferal, which prevents ʹOH generation by chelation of ferrous iron, completely protected bile against Fe3+-EDTA/Asc-induced decrease in NT (p < 0.001), increase in lipid peroxidation (p < 0.001) and generation of 4-HNE. Our results indicate that formation of cholesterol crystals in model bile is enhanced by ROM. Hydroxyl radical induced lipid peroxidation appears to be the mechanism responsible for the crystallisation promoting activity of ROM.