Abstract
Purpose::
7-Ketocholesterol (7kCh) is the most toxic naturally occurring oxysterol. This oxysterol is also the most common product found during the photooxidation of cholesterol. The purpose of this study is to detect and unequivocally identify 7kCh and its metabolites in primate retina lipid extracts.
Methods::
Lipids from human and monkey retinas were extracted with methanol:chloroform without saponification and analyzed by reverse phase HPLC using ultraviolet absorption (UV) and mass spectroscopy (MS) with atmospheric pressure chemical ionization (APCi).
Results::
Initial HPLC-UV analyses detected a peak with similar RT and UV spectra to authentic 7kCh (max 238 nm). Another peak was also detected which has similar RT and UV spectra to 7kChSO4 (max 275 nm). HPLC-MS using APCi generated two ions for authentic 7kCh, m/z 401 (M+H)+ and m/z 383 (M-OH)+ and one ion for 7kChSO4, m/z 383 (M-HSO4)+. These same masses where found in the peaks identified by HPLC-UV in the human and monkey retina lipid extracts. Further confirmation of their authenticity was made by generating acetylated derivatives of monkey retina extracts and by MS/MS fragmentation analyses. Analyzes of 15 human samples determined that 7kCh averaged approximately 0.1 nmols per retina while the levels of 7kChSO4 averaged around 0.025 nmols per retina. These values varied greatly from sample to sample. Treatment of cultured human RPE cells with 7kChSO4 demonstrated a loss of cytotoxicity when compared to 7kCh.
Conclusions::
7-Ketocholesterol and its sulfated metabolite 7kChSO4 were unequivocally identified in human and monkey retina samples. The presence of 7kCh in the primate retina suggests it is likely formed by the photooxidation of cholesterol. The lack of toxicity observed with 7kChSO4 suggests sulfation may be used to neutralize any potential 7kCh toxicity.
Keywords: lipids • retina • mitochondria