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Normal eight month old beagle dogs were fed a diet of 30% galactose for a period of two weeks. One group of dogs was untreated while three other groups were orally dosed with 0.25, 1.0, and 5.0 mg/kg of the aldose reductase inhibitor (ARI), AL01576. No visible changes were observed in the lens but glutathione (GSH) and inositol were depleted while dulcitol was elevated. These biochemical changes closely parallel those found in the (two week) streptozotocin induced diabetic rat. In contrast with the diabetic rat model, retina and nerve inositol was not found to differ from normal in spite of significant dulcitol accumulation. Plasma AL01576 was found to be inversely correlated with lens dulcitol concentration. When plasma AL0P1576 concentration was greater than 1 microgram/mL (5 mg/kg), there was a 95% reduction in dulcitol concentration (relative to untreated), while concentrations of 0.2 to 0.2 mg/mL (1 mg/kg) of AL01576 resulted in a dulcitol reduction of at least 70%. Retina and nerve dulcitol concentrations of galactosemic dogs were similarly diminished by AL01576 treatment. The dog model exhibits a biochemical profile of change and responsiveness to ARI therapy similar to that observed in hyperglycemic rats. Changes in retina morphology in diabetic and galactosemic dogs has been shown to closely resemble those occurring in human diabetics; these early biochemical events may also parallel.

Depletion of lens glutathione (GSH) occurs quickly and drastically following induction of diabetes or galactosemia in rats as well as in lens culture. The explanation for this dramatic loss of GSH has been investigated by many laboratories but the solution has been elusive. There are several possible causes for the change in the reducing power of the lens under hyperglycemia. (a) The enzyme glutathione reductase which reduces oxidized glutathione to GSH is inhibited. (b) The cofactor NADPH which both the aldose reductase of polyol pathway and glutathione reductase require becomes depleted under hyperglycemia to the point that there is an insufficient amount for glutathione reduction. (c) Membrane permeability is increased, due to osmotic-induced lens hydration. We explored all the above possibilities in the mechanism of GSH depletion and studied the effect of aldose reductase inhibitor (ARI) on osmotic change. We found that under hyperglycemic condition, there was no change in the enzyme glutathione reductase activity. There was an initial drop in NADPH level but there was sufficient remaining for glutathione reductase use. Both NADPH and glutathione depletion could be prevented completely by ARI. In addition, ARI could also prevent any hyperglycemic-induced abnormal transport and leakage of amino acids. We have therefore concluded that only the decreased membrane transport of amino acids which are needed for glutathione biosynthesis and the simultaneous loss of GSH through leaky membrane as initiated by the polyol pathway can be responsible for the drastic GSH depletion.

Phosphorylcholine (P-choline) is a precursor of the phospholipids in the lens membrane. A human lens normally contains approx. 1 mM P-choline but this is significantly lowered in some cataractous lenses. A normal rat lens contains a very high concentration (11 mM). We found that rat lens P-choline was depleted drastically when the lenses were exposed to hyperglycemic conditions either in culture, with galactose or xylose, or in vivo by streptozotocin-induced diabetes. The lens P-choline level was measured by fractionating the organic phosphates in the lens homogenate using an ion exchange column, or by quantitating the P-choline 31P NMR intensity in intact lenses. The results of both the chemical method and the noninvasive method agreed remarkably well. Besides the change in P-choline, the choline influx was also drastically reduced both in lenses from diabetic rats and in lenses incubated with 30 mM xylose. In addition, the ATP concentration was greatly diminished under similar conditions. The changes in P-choline, choline, and ATP could all be prevented in the presence of an aldose reductase inhibitor (ARI). It is thus concluded that these changes in phospholipid precursors may result from lenticular membrane defects caused by hyperglycemic stress. The effect of the lowered precursors on lipid biosynthesis was observed, and surprisingly showed a more rapid phospholipid-biosynthesis in the 2-week diabetic rat lens than in the 3-day diabetic rat lens.