High galactose levels in vitro and in vivo impair ascorbate regeneration and increase ascorbate-mediated glycation in cultured rat lens.
In contrast to conventional view that glucose is the sole glycating agent, ascorbate has now emerged as a potential precursor of advanced glycation products in lenses during cataractogenesis, owing to the high concentration present in human lens. The effects of high hexose environment in vitro and in vivo on the disruption of redox equilibrium of ascorbate (ASA) to dehydroascorbate (DHA), which is required for ascorbate-mediated crystallin modification by the Maillard reaction during cataractogenesis were examined. Organ culture experiments were performed with rat lenses that were first exposed to high galactose levels in vitro and in vivo and then incubated with 1-14C-labeled ASA, DHA or DKG (2,3-diketogulonic acid). Formation of ASA degradation products as a function of time was assessed by radiometric TLC method. Upon incubation with ASA or DHA, an elevated level of the degradation product, DKG, was detected in lenses exposed to galactose in vivo and in vitro. ASA uptake was significantly enhanced in the galactosemic lenses as compared to controls (P = 0.01). Regeneration of ASA from DHA in both galactose treated and galactosemic lenses was impaired when compared to control lens which completely converted DHA from the medium into ASA. Surprisingly, the galactose exposed lenses showed enhanced permeability to DKG which was picked up readily from the medium in contrast to normal healthy lenses which remained impermeable to DKG. Galactose exposed lenses both in vitro and in vivo showed a 5-9-fold increase in crystallin bound Schiff base-linked radioactivity when incubated with 1-14C-labeled ASA or DHA. As a preamble to the question of whether lens pigmentation predisposes towards ascorbate oxidation, lens homogenate from normal young and old pigmented cataractous lenses were incubated with [1-14C]ASA. After 2 days, ASA levels were found to have decreased by 74% and DKG levels increased by 48% in brunescent lens as compared to the young lens. These data demonstrated that profound abnormalities in ASA metabolism exist in lenses exposed to a high sugar environment suggestive of a breakdown of the redox equilibrium of ASA to DHA and a loss of membrane permeability barrier for DKG. The latter would further contribute toward a ASA-catalysed Maillard reaction in the redox impaired lens.
Saxena
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Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
Saxena
A K
AK
Monnier
V M
VM
D015083:2,3-Diketogulonic Acid / Q000378:metabolism; D000818:Animals; D001205:Ascorbic Acid / Q000378:metabolism* / Q000502:physiology; D002386:Cataract / Q000139:chemically induced / Q000378:metabolism*; D003459:Crystallins / Q000378:metabolism; D003683:Dehydroascorbic Acid / Q000378:metabolism; D005690:Galactose / Q000494:pharmacology*; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D009924:Organ Culture Techniques; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D003459:Crystallins; D015083:2,3-Diketogulonic Acid; D001205:Ascorbic Acid; D005690:Galactose; D003683:Dehydroascorbic Acid