engleski

Lens protein glycation and high molecular weight aggregate formatin in experimental diabetes

Glycation is a post-translational modification which occurs when free amino groups of proteins reacts nonenzymatically with the acylic form of glucose via an Amadori rearrangement. With time, the Amadori product undergoes dehydration, rearrangement and cleavage reactions to give rise to advanced glycation products with brown or fluorescent properties. We have followed the accumulation of glycated products in the lenses of hyperglycaemic Wistar rats during a period of 5 month following alloxan diabetes inducement (D) with coresponding control groups (C). Glycated haemoglobin was measured by fast protein liquid chromatography (C: 1.75ą0.93 vs D:5.3ą0.57%, p<0.001). Relative fluorescence of alkali-soluble lens proteins were determined at 385/335nm. In 5-mo untreated diabetic as compared to control rats, a significant increase was observed in the fluorescence level (3.25ą1.02 vs 1.61ą0.17 FU/mg, p<0.001), while in 1-mo animals the increase was from 1.26ą0.11 FU/mg in controls to 1.80ą0.25 FU/mg in diabetics, <0.001. The amount of glucose bound ketoamine linkage was estimated after acid hydrolysis as released 5-hydroxymethylfurfural (HMF). In 5-mo controls, it was slightly higher than in 1-mo controls (0.57ą0.31vs0.41ą0.20 nmolHMF/mg, respectively).The diabetic group showed a significant increase, however. In 1-mo diabetics, a level of 1.07ą0.36 nmol HMF/mg was found, while in 5-mo animals the glycated protein amount rose to 2.46ą0.79 nmol HMF/mg. In addition to the increases in glycated content with continuing diabetic hyperglycaemia, significant changes in protein composition of alkali-soluble lenses developed. The SDS-PAGE pattern showed an appearance of protein polymers of heterogeneous size (C-1mo: 3.0ą1.1% vs C-5mo:17.9ą2.9% D-1mo:7.3ą2.1% vs D-5mo:19.8ą3.6%) and the proteins of high molecular weight (HMW).Only a small amount of these HMW proteins was present in controls (C-5mo: 2.5ą1.2%) and short-term diabetes (D-1mo: 0.8ą0.2%), whereas in long-term untreated diabetes there was a dramatic increase (D-5mo: 30.5ą3.2%) with a corresponding decrease in other peaks. All diabetic animals from this group had macroscopically detectable cataractous lenses.

Lens protein; Glycation; HMW aggregates

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