Sensitive assay for cholesterol in biological membranes reveals membrane-specific differences in kinetics of cholesterol oxidase.
Quantification of cholesterol in biological membranes from a variety of sources is an important step toward understanding cholesterol's roles in membrane function. We extend to biological membranes the fluorometric/enzymatic approach (cholesterol oxidase) to measure 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 microgram 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.
Crockett
E L
EL
Department of Zoology, Arizona State University, Tempe 85287-1501.
Hazel
J R
JR
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D002784:Cholesterol / Q000032:analysis*; D002789:Cholesterol Oxidase / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D007413:Intestinal Mucosa / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D017686:Oncorhynchus mykiss; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
D002784:Cholesterol; D002789:Cholesterol Oxidase