GLYCOLYTIC PATHWAY, REDOX STATE OF NAD(P)-COUPLES AND ENERGY-METABOLISM IN LENS IN GALACTOSE-FED RATS - EFFECT OF AN ALDOSE REDUCTASE INHIBITOR

Purpose. The present study was aimed at evaluating early changes in gl ycolysis, the redox state of free cytosolic NAD(P)-couples, and the ad enine nucleotide system in lens in both control and 50% galactose-fed rats, with the possibility of preventing these with an aldose reductas e inhibitor (ARI). Methods. Experiments. were performed on male Spragu e-Dawley rats fed the galactose diet for 2-14 days, The levels of gluc ose, galactose, glycolytic intermediates, alpha-glycerophosphate, mala te, NAD, ATP, ADP, AMP were assayed spectrofluorometrically in individ ual lenses by enzymatic procedures, while galactitol and myo-inositol were quantified by GC-MS. Free cytosolic NAD+/NADH, NADP+/NADPH, and A TP/ADP x P-i (phosphate potential) were estimated from lactate dehydro genase, malic enzyme, and triose phosphate isomerase-glyceraldehyde 3- phosphate dehydrogenase-3-phosphoglycerate kinase systems. Lactate and pyruvate production by lenses of both control and galactose-fed rats was measured in a set of in vitro incubation studies (2 hr, 37 degrees C, Krebs bicarbonate-Hepes buffer. pH 7.45, with 5mM glucose or 5mM g lucose + 30 mM galactose, respectively). Results. Lens galactitol leve ls in 2, 4, 6, 8, 10, and 14-day galactose-fed rats were 48 +/- 8, 58 +/- 9, 68 +/- 8, 73 +/- 5, 81 +/- 20, and 75 +/- 11 mmol/g wet weight (mean +/- SD), respectively. NAD+/NADH ratios were indistinguishable f rom controls after 2-6 days on the galactose diet, but fell dramatical ly between 8 and 10 days, and did not correlate with polyol accumulati on per se. The pattern of glycolytic intermediates (no change in G6P, F6P, and 3-PG, increase in GA3P, decrease in FDP. PEP, pyruvate, and l actate), as well as reduced in vitro lactate and pyruvate production, suggest inhibition of glycolysis at the sites of phosphofructokinase. glyceraldehyde 3-phosphate dehydrogenase, enolase, and pyruvate kinase . ATP levels as well as total ATP/ADP, ATP/ADP x P-i, adenylate charge , and cytosolic phosphate potential were decreased in galactose-fed ra ts, while galactose 1-phosphate and a-glycerophosphate levels as well as NADP+/NADPH ratio were increased, Lens galactitol levels were reduc ed similar to 57% in 10-day galactose-fed rats heated with the ARI (to lrestat, 100 mg/kg bwt/day, 6-day pretreatment): the changes in the lo wer segment of glycolysis, alpha-glycerophosphate levels. redux stale of NAD-couples and energy metabolism were partially prevented while NA DP+/NADPH ratios were unchanged and galactose 1-phosphate levels were further increased. Conclusions. Depressed glycolysis in lens in galact ose-fed rats is consistent with decreased NAD+/NADH and adenine nucleo tide phosphorylation. Early changes in lens glucose utilization, redox state of NAD-couples, and energy metabolism in this model of galactos emia are similar to those in diabetes, are at least in part mediated b y aldose reductase involved mechanisms, and can be partially prevented by an aldose reductase inhibitor.