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F.A. Bettelheim, R. Neal, J.S. Zigler, Jr; Dynamic Light Scattering Studies and Proteomic Analysis of the Topographic Distribution of Components of Human Vitreous . Invest. Ophthalmol. Vis. Sci. 2003;44(13):431.
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Purpose: To determine the topographic distribution of key elements in human vitreous and to examine if cataract surgery alters the distribution. Methods: Human vitreous from phakic and aphakic eyes of the same donors were sampled in 6 areas of the vitreous. Dynamic light scattering measurements were performed on the supernatants after centrifugation. Prior to 2D minigel electrophoresis extensive dialysis with 10 kDa membranes was performed. Phoretix image analysis was performed using gel averaging and mode of non-spot background subtraction on silver stained gels. Proteins were identified by MALDI-TOF-MS or ESI-MS/MS following spot excision, trypsin digestion and peptide extraction. A minimum of 20% protein coverage with 4 peptides identified was required for spot identification. Results: Hyaluronan shows a wide distribution of molecular sizes ranging from 30 to 150 nm in diameter. The size of hyaluronan molecules is largest near the lens vitreous boundary and smallest at the retina/vitreous interface. The concentration of hyaluronan, on the other hand, is highest in the vicinity of the peripheral retina, lower near the foveal area and still lower near the lens/vitreous interface. Vitreous from aphakic eyes exhibits higher concentration of hyaluronans near the lens/vitreous boundary. Similarly small size compounds are also unevenly distributed. A compound with the diameter of 6 nm was present near the lens/vitreous boundary but almost completely absent near the retina/vitreous interface. Over 300 protein spots were visualized on each 2D gel with mass spec identification of 10 dominant protein spots including albumin, transferrin, PEDF and transthyretin whose abundance was a function of location. Conclusions: The topographic distribution of hyaluronan observed in young bovine lenses by Balazs exists also in aging human vitreous. In this study we have also demonstrated spatially dependent protein abundance gradients in the human liquid vitreous which is altered in aphakic eyes. Cataract surgery seems to afflict sufficient trauma to the vitreous to alter the distribution of a number of key components.
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