IN-VIVO FORMATION OF 25-HYDROXYCHOLESTEROL FROM ENDOGENOUS CHOLESTEROL AFTER A SINGLE MEAL, DIETARY-CHOLESTEROL CHALLENGE

The role of oxysterols as regulatory molecules in the suppression of 3 -hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity was investigated in the intact rat in response to an acute dietary cholest erol challenge. When rats were fed highly purified cholesterol as a si ngle meal at a level of 5% of the diet, maximal inhibition of enzyme a ctivity (66%) occurred 120 min after the completion of the meal. Furth ermore, when nonsaponifiable liver extracts were chromatographically r eserved and analyzed by high performance liquid chromatography (HPLC) and capillary gas chromatography-mass spectrometry (GC-MS), 25-hydroxy cholesterol was identified in the livers of rats 120 min after the com pletion of the single cholesterol meal. Significantly, only barely det ectable amounts of 25-hydroxycholesterol were observed in the livers f rom control rats fed a sterol-free diet. The biosynthetic origin of 25 -hydroxycholesterol was investigated with the use of deuterated water. Rats were fed deuterium oxide (33%) ad libitum for 3 days and then ki lled 120 min after the completion of a single cholesterol meal. As bef ore, 25-hydroxycholesterol was detected in the livers from cholesterol -fed rats, but not to a significant extent in livers from control-fed rats receiving a sterol-free diet. Isotope ratio mass spectrometry rev ealed that the fractional incorporation of deuterium into 25-hydroxych olesterol (21%) was less than that observed for cholesterol(24%) isola ted from the same livers, indicating that 25-hydroxycholesterol was pr oduced endogenously from exogenous cholesterol and not from autoxidati on of cholesterol. In a separate experiment it was also shown that [H- 3]mevalonate was incorporated into 25-hydroxycholesterol after a singl e meal cholesterol challenge, but was barely detected in the livers of control rats. I The evidence obtained in the present article supports the hypothesis that 25-hydroxycholesterol is endogenously produced fr om cholesterol at early time intervals after an acute dietary choleste rol challenge. In addition, rat liver HMG-CoA reductase was inhibited by the administration of a single intragastric dose (1 mu g/kg) of an aqueous solution of 25-hydroxycholesterol. Thus, the results provide s trong support for the conclusion that 25-hydroxycholesterol plays a si gnificant role in the in vivo regulation of rat liver cholesterol bios ynthesis after an acute dietary cholesterol challenge.