Abstract
Purpose: :
Aging human lens crystallins accumulate damage that includes phosphorylation, deamination, truncation, glycation, oxidation, and kynurenine adduct formation. These modifications have been associated with enhanced risk of crystallin aggregation. We initiated a comprehensive determination of advanced glycation/oxidation products to understand their potential hierarchical role during nuclear sclerosis.
Methods: :
55 lenses (age range 6-79 years), comprising 33 non-diabetics and 22 diabetics, were separated into soluble and insoluble fractions. Both soluble and insoluble fraction were either hydrolyzed by 6N HCl or digested by proteases. The target modifications were quantitated by gas GC-MS, LC-MS, and HPLC.
Results: :
Almost all AGEs except fructose-lysine (FL) and glyoxal-hydroimidazolone G-H1 increased with age. By far the most abundant modification was methylglyoxal-hydroimidazolone (MG-H1), which modified up to 0.4 mol/mol prot (~20,000 pmol/mg crystallin) at 60-70 yrs, as previously reported (Ahmed IOVS 2003). The second level of modifications, 10x less than MG-H1, were carboxyethyl-lysine (CEL), carboxymethyl-lysine (CML) glucosepane, comparable to K2P and argpyrimidine. The least of these were metal catalytic oxidation (MCO) products, i.e. allysine and 2-AAA, with levels are 200 times less than MG-H1 and comparable to pentosidine and vesperlysine A. CEL (r=0.900) and MG-H1 (r=0.864) were among the highest correlated with age followed by K2P (r=0.764) and fluorescence at 335nm/385nm (r=0.818) and 370nm/440nm (r=0.791). Both oxidation related modifications CML (r=0.648) and allysine (r=0.668) also correlated with age. For diabetic lenses, glucosepane had the highest correlation with diabetes(r=0.870, p<0.0001), followed by fluorescence at 370nm/440nm (r=0.710, p<0.001) and 335nm/385nm (r=-.560, p=0.01). Importantly, MG-H1 was not increased by diabetes.
Conclusions: :
These findings confirm the overwhelming modifications by methylglyoxal (MGO) whereby the absence of MG-H1 elevation in diabetes suggests MG primarily originates from ascorbic acid during photooxidation instead via glycolytic metabolism. Protein oxidation during normal lens aging is relatively minor compared to carbonyl stress, confirming thereby the "canned" state of the lens.
Keywords: protein modifications-post translational • aging • cataract