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

J Exp Zool. 1995 Feb 15;271(3):190-5. doi: 10.1002/jez.1402710305.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cell Membrane / chemistry
  • Cell Membrane / enzymology
  • Cholesterol / analysis*
  • Cholesterol Oxidase / metabolism*
  • Chromatography, High Pressure Liquid
  • Intestinal Mucosa / metabolism
  • Kinetics
  • Liver / metabolism
  • Oncorhynchus mykiss
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence / methods*

Substances

  • Cholesterol
  • Cholesterol Oxidase