Analytical biochemistry, 258(2), 277-284.
Härtel, S., Diehl, H. A., & Ojeda, F. (1998).
ABSTRACT
A variety of methods to incorporate cholesterol into lipid membrane systems have been applied with varying success. We tested an incorporation method based on cholesterol-loaded methyl-β-cyclodextrins and compared it to a method that uses cholesterol-loaded liposomes. With methyl-β-cyclodextrin, we increased the cholesterol content in microsomal membranes to almost the fourfold of the original content. With cholesterol-loaded liposomes instead, we achieved an elevation of 140%. Short incubation times and well-defined carrier properties favor the β-cyclodextrin method. For direct detection of membrane cholesterol, we slightly modified a microenzymatic fluorescence assay originally developed for precise cholesterol detection in serum. Without the need to perform lipid extraction, this assay was reliable for cholesterol detection in liposomes and in microsomes. Additionally, we compared the sensitivity of the fluidity-sensitive fluorescent dyes pyrene, pyrene–methanol, bis-pyrene, 1-6-phenyl-1,3,5,-hexatrien, and1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5,-hexatrien in order to detect cholesterol indirectly by the dynamically relevant changes exerted on lipid matrices. These dyes differ not only in their membrane location but also in their dynamical behavior. We calibrated the dyes in liposomes of defined cholesterol content and used the most suited ones to follow and quantify the cholesterol incorporation into liposomal and microsomal membranes.