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A. K. Junk, M. Goel, T. K. Mundorf, E. J. Rockwood, S. K. Bhattacharya; Decreased Carbohydrate Metabolism Enzyme Activities in the Glaucomatous Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3218.
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To determine whether activity of carbohydrate metabolism enzymes (Aldolase, Pyruvate Kinase and Isocitrate Dehydrogenase) are altered in the glaucomatous trabecular meshwork (TM) compared to controls.
Tissue specimens were obtained from trabeculectomy (n=45 glaucoma; Caucasian, average age 61±8 of both genders) and from cadaver eyes (n= 15 control and n= 5 glaucoma; Caucasian, average age 63±4 of both genders). Protein extracts from TM tissue were prepared in a non-denaturing buffer containing 0.1% genapol. Aldolase activity was measured spectrophotometrically using reaction of 3-phosphoglycerate with hydrazine to form hydrazone that absorbs at 240 nm. Pyruvate Kinase activity was measured by coupling with Lactate Dehydrogenase with NADH and pyruvate. Absorbance was measured at 340 nm. Isocitrate Dehydrogenase activity was measured using reduction of NADP to NADPH resulting in a characteristic absorbance at 340 nm.
Aldolase, Pyruvate Kinase and Isocitrate Dehydrogenase activities in the glaucomatous TM tissue were found to be reduced 70, 50 and 25 percent, respectively. SDS-PAGE analysis suggests presence of 4-hydorxynonenal modified Isocitrate Dehydrogenase protein in the glaucomatous TM tissue compared to controls.
Several Krebs cycle enzyme activities are considerably reduced in glaucomatous TM. HNE modified isocitrate dehydrogenase activity is consistent with reduced inactivated form of the protein.
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