Sensitive assay for cholesterol in biological membranes reveals membrane‐specific differences in kinetics of cholesterol oxidase

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Abstract
Quantification of cholesterol in biological membranes from a variety of sources is an important step toward understanding cholesterol's roles in membranes function. We extend to biological membranes the fluorometric/enzymatic approach (cholesterol oxidase) to measures cholesterol, originally described for whole cells (Heider and Boyett [1978] J. Lipid Res., 19: 514–518; Gamble et al. [1978] J. Lipid Res., 19: 1068–1070) and serum (Huang et al. [1975] Clin Chem., 21: 1605–1608). This method has a detection limit of 0.3 μg cholesterol. As revealed by comparison with high-performance liquid chromatography, the fluorometric/enzymatic method with biological membranes is accurate (within 4% and 8% for intestinal and hepatic plasma membranes, respectively). The assay may be completed within 3 to 4 hours and requires neither lipid extraction nor chromatographic techniques. Kinetics of the cholesterol oxidase reaction are membrane-specific, and first-order rate constants (k) are positively correlated with membrane order.