Abstract
Abstract: :
Purpose: Aging human lens crystallins accumulate postsynthetic modifications and crosslinks which decrease their solubility. The process is greatly accelerated during cataractogenesis, and can become extreme in brunescent cataracts. Unequivocal evidence now points to a major role of glycation reactions in these modifications. Although protein crosslinks from glucose and methylglyoxal (MG) have been identified, those from ascorbic acid are yet unknown. Below we present evidence for the formation of lysine–histidine crosslinks from dehydroascorbic acid (DHA) and its degradation products. Methods: A mixture of each 50 mM Z–L–lysine, Z–L–arginine and Z–L–histidine was incubated for 30 days with 100 mM DHA in 100 mM Na/PO4 (pH 7.4) and analyzed by RP–HPLC with UV and fluorescence detection, and iontrap LC/MS/MS. A methanol extract was fractionated with preparative chromatography and the crosslinked material was isolated by repetitive RP–chromatography. Similar reactions were carried out with combinations of amino acids, in presence of various sugars such as glucose, ribose, fructose, erythrulose, glyceraldehyde, MG, threose and glycolaldehyde. Results: Reverse phase HPLC of the Z–lys/Z–arg/Z–his mixture revealed several UV active and fluorescent peaks in the "crosslink" region. LC/MS/MS analysis showed one of the major peaks with m/z = 720.1. These chromatographic, UV and MS data were duplicated by only by Z–lys/Z–his with DHA, threose, erythrulose, glycolaldehyde or glyceraldehyde, but not the other sugars or aminoacids. Conclusions: Preliminary structural analysis (NMR data pending) strongly suggests a lysine–histidine crosslink can form from ascorbic acid and with proteins. A tentative structure involves a pyridinium ring with substituted imidazole ring and hydroxylated aliphatic side chains. This is to our knowledge the first evidence for the implication of histidine in protein crosslinks by the Maillard reaction. Interestingly, carnosine (benzoyl–histidine) has been found to have anticataract properties suggesting potential scavenging of reactive ascorbylation species by this reactive amino acid
Keywords: aging • cataract • diabetes