The protective effect of glucose on soluble rat lens hexokinase in the presence of oxidative stress

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Abstract
An in vitro animal model was used to characterize the protective effect of glucose on lenses subjected to oxidative stress. Paired rat lenses were incubated in TC-199 medium for six hours in presence of an oxidant (0.06 mM H2O2, superoxide produced from 5 mM purine, or hydroxyl radical) and 2 mM glucose (control) or no glucose (experimental). Soluble hexokinase (HK) specific activity and lactate production were measured. 0.06 mM H2O2 inactivates 48% of the hexokinase in the absence of glucose; with glucose present hexokinase activity is reduced only 26%. Control experiments without oxidants show a statistically insignificant difference between hexokinase activities in the O and 2 mM groups, suggesting that the changes observed are not simply due to the presence or absence of glucose. Hexosemonophosphate shunt activity increases nearly 2.5-fold in the presence of 0.06 mM H2O2 and 2.0, 4.0 or 5.5 mM glucose. This suggests that the loss of hexokinase (a -SH enzyme) in the presence of H2O2 and O mM glucose is due to NAPH production inadequate to offset the oxidative stress on enzyme -SH groups. FPLC analysis suggests that type II HK is more susceptible to oxidative inactivation than type I, and further studies have shown that this inactivation is localized to the capsule/epithelium. Lactate levels were measured and controls (without oxidants) were run, to obtain a baseline value for fresh lenses and assess the contribution of endogenous glucose to lactate production. H2O2 levels in superoxide and hydroxyl radical media were measured, and the protective effects of mannitol and catalase were also determined.