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Witold Karol Subczynski, Marija Raguz, Laxman Mainali, William J O'Brien, ; EPR spin-labeling studies of the fiber-cell plasma membranes of the eye lens: From pooled samples to single donor and single eye lens samples. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5596.
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© ARVO (1962-2015); The Authors (2016-present)
The lateral organization and properties of the lipid bilayer portion of eye lens fiber cell membranes can significantly differ between donors and even between the left and the right eye. To investigate these differences we developed methods which allowed quantitatively evaluate the relative amounts of phospholipids (PL) and cholesterol (Chol) in lipid domains in intact human eye lens membranes. Here we compared results obtained for pooled samples (from ~20 lenses), eye lenses of single donors, and single lenses.
Intact fiber cell membranes from cortical and nuclear regions of human lenses were isolated for pooled, single donor, and single lens samples. The PL analog spin label (12-SASL) was used to evaluate the distribution of PLs and the Chol analog spin label (ASL) was used to evaluate the distribution of Chol between membrane domains.
In intact membranes of all samples lipids were distributed between three distinct domains forming bulk lipids, boundary lipids, and trapped lipids. We were not able to detect pure cholesterol bilayer domains, which were detected in membranes made of the total lipid extracts. The amount of lipids (PL and Chol) in domains uniquely formed due to the presence of membrane proteins was greater in nuclear than in cortical membranes. In nuclear membranes the amount of lipids in these domains increased with age, while in cortical membranes it did not change. The left and the right eye lenses from the same donor showed very similar results, while for different donors of similar age results were more scattered. Surprisingly, the amount of Chol in trapped domains of cortical membranes was extremely low.
Future strategies for slowing cataract formation may depend on a detailed examination of cataractous and age-matched normal lenses, as well as lenses from people who retain clear lenses into their eighth and ninth decades. For such studies, the individual experimental approach is critical because it will allow the presentation of results from the cortex and nucleus of a single lens in relation to the donor age and health history. The single lens approach combined with EPR spin-labeling methods should help to elucidate the biophysical basis of lens transparency on a molecular level and help determine the causes and mechanisms of age-related changes in the lens membrane that lead to cataracts.<br />
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