Elevated Albumin in Retina and Vitreous of Monkeys With Experimental Glaucoma

Abstract

Purpose:: A prominent protein band, between 65 and 70 kDa on SDS-PAGE, is more abundant in experimental glaucoma monkey retinas than in controls. This study established the identity of the prominent protein, the prevalence of the increase, and the location of the protein in the retina. Whether the protein is also increased in the vitreous of monkeys with experimental glaucoma was investigated.

Methods:: Retina and vitreous were collected from five monkeys with unilateral experimental glaucoma and documented visual field defects. Tissues from the untreated fellow eyes served as controls. Retinal homogenates were subjected to 2D-gel separation to identify differentially expressed proteins. The protein spots of interest were excised and digested with trypsin. The extracted peptides were analyzed by LC/MS/MS and identified by sequence determination. The protein identity was confirmed by Western blot. Localization of the protein in retina was investigated by immunohistochemistry and confocal microscopy. After identifying the abundant protein, the quantity and the concentration in vitreous samples were determined in control and experimental eyes by Western blot and BCG assay and compared.

Results:: Proteolytic fragments from in-gel digestion of the unknown proteins were identified by mass spectrometry as fragments of albumin. Western blot analysis, using an antibody to human albumin, confirmed the identity of the protein as albumin. In the five glaucoma eyes examined, a 1.75-5.75 fold increase in albumin was detected over control. Albumin was found outside of blood vessels in the inner retina of glaucoma eyes and frequently along the inner surface of the retina. Western blot analysis of vitreous showed increased levels of albumin in the glaucoma eyes compared to controls. The average concentration of albumin in vitreous of control eyes is 0.01g /dL. Albumin in the vitreous of all the glaucoma eyes was increased: 0.04g/dL-0.25g/dL.

Conclusions:: Albumin is the abundant 67-70 kDa protein found in the retinas of monkey eyes with experimental glaucoma. The increased albumin observed in the retina and vitreous may provide protection, as it does in serum, against oxidative stress and damage known to occur in glaucoma, as well as assist in translocating fatty acids for cell membrane repair.

Keywords: retina • proteomics • vitreous