Abstract
Purpose: :
Vitamin C degradation products, particularly dicarbonyls, participate significantly in chemical modifications of lens crystallins that affect their chaperone function and favor aggregation and cataractogenesis. However, quantification of the chemical modifications generated from Vitamin C is limited, mostly due to their instability and reversibility. Here we described a method for the quantification of reversible bound dicarbonyls on proteins generated by Vitamin C.
Methods: :
Quinoxaline derivatives from 1,2-diaminobenzene (OPD) and Vitamin C degradation products (dehydroascorbic acid (DHA, 1), 2,3-diketogulonic acid (DKGA, 2), xylosone (3), threosone (4) and 3-deoxythreosone (5)) were synthesized, isolated by RP-HPLC and identified by NMR and mass spectrometry. These products were quantified by LC-MS/MS in water soluble (WS) and water insoluble (WI) human lens proteins.
Results: :
Levels of 1, 2, 3, 4 and 5 in WS proteins for ages 60-80 years were 13.18±9.54; 27.06 ±19.78; 1.90±2.46; 8.19±2.80 and 123.72±33.97 pmol/mg protein, respectively. In WI only 4 and 5 were detected in levels of 8.52±2.55; 197.50±41.20 pmol/mg protein, respectively. In addition, 5 in WS showed positive linear correlation with age (p<0.05).
Conclusions: :
A simple method for Vitamin C degradation products reversibly bound to proteins was developed and their levels were quantified in vivo. The highest level of 3-deoxythreosone (5) in human lens proteins suggests a non-oxidative pathway as a major pathway for Vitamin C degradation in vivo, confirming thereby the original postulate of Simpson & Ortwerth (Biochim Biophys Acta. 1501;12, 2000).
Keywords: protein modifications-post translational • crystallins • aging