Use of an O Inhalation Technique and Mass Isotopomer Distribution Analysis to Study Oxygenation of Cholesterol in Rat EVIDENCE FOR IN VIVO FORMATION OF 7-OXO-, 7β-HYDROXY-, 24-HYDROXY-, AND 25-HYDROXYCHOLESTEROL

Abstract Cholesterol oxidation products (oxysterols) have been detected in many different tissues, often at concentrations 103 to 104 times lower than cholesterol. This constitutes a considerable risk of quantitation errors, since even a minor oxidation of cholesterol during sample processing would yield a substantial increase of oxysterol levels. It has therefore been suggested that some of the oxysterols do not occur in vivo and their detection in tissues merely are artifacts produced in vitro. In the present work, an 18O2 inhalation technique was developed in order to clarify which oxysterols are produced in vivo. Rats were exposed for 3 h to an atmosphere with a composition similar to normal air, except that it contained 18O2 instead of 16O2. Control rats were kept in 16O2-containing atmosphere throughout the experiment. The 18O enrichment of oxysterols in plasma and liver was determined by gas/liquid chromatography-mass spectrometry and mass isotopomer distribution analysis. In vivo formation of oxysterols, indicated by enrichment in 18O, was established for cholest-5-ene-3β,7α-diol, cholest-5-ene-3β,7β-diol, 7-oxocholesterol, cholest-5-ene-3β,24-diol, cholest-5-ene-3β,25-diol, and cholest-5-ene-3β,27-diol. Additionally, it seems likely that cholest-5-ene-3β,4β-diol is formed in vivo. The 18O labeling pattern suggests that there is incomplete equilibration between the liver and plasma pools of cholest-5-ene-3β,27-diol. No evidence for the in vivo formation of 5,6-oxygenated oxysterols was obtained.