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N. Mast, I. R. Bederman, M. E. Cabrera, I. A. Pikuleva; Cholesterol Metabolism in the Neuronal Retina and RPE. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2352.
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Cholesterol is the major sterol in the retina playing an important role in the vision process. Cholesterol-metabolizing cytochromes P450 (CYP) catalyze the first and rate-limiting step in all pathways of cholesterol elimination from different organs. Three cholesterol-metabolizing P450s (CYP11A1, CYP27A1, and CYP46A1) were found to be expressed in the retina. CYP46A1 catalyzes the conversion of cholesterol to 24(S)-hydroxycholesterol, CYP27A1 to 27-hydroxycholesterol, and CYP11A1 to pregnenolone. Since all these enzymes utilize the same substrate, cholesterol, but convert it to different products, it is not currently clear how cholesterol is metabolized in the retina. The purpose of this work was to develop analytical tools and investigate initial steps in cholesterol degradation in the retina.
At first we extracted lipids from the bovine neuronal retina and retinal pigment epithelium (RPE), saponified them, converted to trimethylsilyl (TMS) derivatives and analyzed by gas chromatography-mass spectrometry (GC-MS) for the presence of hydroxylated cholesterol derivatives. Then, we isolated microsomes and mitochondria from the bovine neuronal retina and RPE and incubated them with NADPH and cytochrome P450 redox partners. Lipids from these incubations were extracted and analyzed by GC-MS.
The only hydroxycholesterol that we could detect in the lipid extracts from the bovine neuronal retina and RPE was 24-hydroxycholesterol, and this sterol was present in the neural retina but not in the RPE. In the incubations with subcellular fractions, we observed increased amounts of 24-hydroxycholesterol (the product of CYP46A1 activity) with the retinal but not RPE microsomes, and increased amounts of 27-hydroxycholesterol (the product of CYP27A1 activity) with the RPE but not neural retina mitochondria. Interestingly, 16-hydroxypregnenolone, metabolite of pregnenolone (the product of CYP11A1 activity), was detected in both microsomal and mitochondrial fractions of the neuronal retina.
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