Assay of cholesterol autoxidation

The autoxidation of cholesterol (cholest-5-en-3β-ol) by molecular oxygen (³O₂) leads to the formation of a large number of oxysterols of diverse chemistry, biological activities, and biomedical implications. Product analysis by simple one-dimensional thin-layer chromatography (TLC), with high-performance liquid chromatography (HPLC) as adjunct, provides a reliable means to determine autoxidation. In special cases where oxysterol identifications beyond TLC characterizations are crucial, additional HPLC or gas chromatography analyses of trimethylsilyl ethers coupled with mass spectrometry are useful for definitive identifications. Adventitious autoxidation occurring during sample manipulations and analysis must be controlled lest there be misleading results. Sample protection from air, light, and heat is essential. Immediate sample processing by solvent extraction or freeze-drying is recommended, as oxysterols form in frozen tissues stored in contact with air. Operations under an inert atmosphere or vacuum with added antioxidant and with isotopic cholesterol added to assess the extent of adventitious autoxidation during operations are recommended for quantitation. Quantitation of oxysterols is generally achieved by additional chromatography (HPLC or gas chromatography of trimethylsilyl ethers) following preliminary separation of oxysterols from massive amounts of cholesterol.