Measuring the impact of bile acids on the membrane order of primary hepatocytes and isolated mitochondria by fluorescence imaging and spectroscopy

Abstract

Cholestasis is characterized by impaired secretion of bile flow that can result in the accumulation of highly abnormal levels of bile acids causing hepatocyte and biliary injury. As amphipathic molecules, bile acids can intercalate in lipid membranes, and pathophysiologic concentrations of bile acids have the potential to induce marked changes in the biophysical properties of biomembranes, including membrane ordering. These effects, particularly on the mitochondrial and plasma membranes, have been proposed to trigger toxicity of bile acids. This chapter details different fluorescence-based methods (fluorescence polarization, and spectroscopy/imaging of solvatochromic dyes) to evaluate the impact of different bile acids on membrane order. Protocols are described for the application of these methods to biomimetic vesicles, isolated mitochondria, and hepatocytes, following a bottom-up approach.