Identification and in vivo detection of side-chain hydroxylated metabolites of 4ß-hydroxycholesterol.

ABSTRACT

Oxysterols are oxidized derivatives of cholesterol that are formed by enzymatic processes or through the action of reactive oxygen species. Several of these bioactive lipids have been shown to be affected and/or play a role in inflammatory processes. 4ß-hydroxycholesterol is one of the major oxysterols in mice and humans and its levels are affected by inflammatory diseases. However, apart from its long half-life, little is known about its catabolism. By incubating 4ß-hydroxycholesterol with mouse mitochondria-enriched liver fractions, as well as 25-hydroxycholesterol and 27-hydroxycholesterol with recombinant CYP3A4, we identified 4ß,25-dihydroxycholesterol and 4ß,27-dihydroxycholesterol as 4ß-hydroxycholesterol metabolites. Supporting the biological relevance of this metabolism, we detected both metabolites after incubation of J774, primary mouse peritoneal macrophages and PMA-differentiated THP-1 cells with 4ß-hydroxycholesterol. Across our experiments, the incubation of cells with lipopolysaccharides differentially affected the levels of the 25- and 27-hydroxylated metabolites of 4ß-hydroxycholesterol. Finally, 4ß,27-dihydroxycholesterol was also detected in mice liver and plasma after intraperitoneal administration of 4ß-hydroxycholesterol. To our knowledge, this is the first report of the in vitro and in vivo detection and quantification of 4ß-hydroxycholesterol metabolites.