Vitamin C degradation products and pathways in the human lens

Abstract

Vitamin C and its degradation products participate in chemical modifications of proteins in vivo through non-enzymatic glycation (Maillard reaction) and formation of different products called advanced glycation end products. Vitamin C levels are particularly high in selected tissues, such as lens, brain and adrenal gland, and its degradation products can inflict substantial protein damage via formation of advanced glycation end products. However, the pathways of in vivo vitamin C degradation are poorly understood. Here we have determined the levels of vitamin C oxidation and degradation products dehydroascorbic acid, 2,3-diketogulonic acid, 3-deoxythreosone, xylosone, and threosone in the human lens using o-phenylenediamine to trap both free and protein-bound adducts. In the protein-free fraction and water-soluble proteins (WSP), all five listed degradation products were identified. Dehydroascorbic acid, 2,3-diketogulonic acid, and 3-deoxythreosone were the major products in the protein-free fraction, whereas in the WSP, 3-deoxythreosone was the most abundant measured dicarbonyl. In addition, 3-deoxythreosone in WSP showed positive linear correlation with age (p < 0.05). In water-insoluble proteins, only 3-deoxythreosone and threosone were detected, whereby the level of 3-deoxythreosone was ∼20 times higher than the level of threosone. The identification of 3-deoxythreosone as the major degradation product bound to human lens proteins provides in vivo evidence for the non-oxidative pathway of dehydroascorbate degradation into erythrulose as a major pathway for vitamin C degradation in vivo.

SCHEME 1.

Oxidative and non-oxidative degradation pathways of DHA (1) into reactive dicarbonyls according to Reihl et al. (9), 2,3-DKG (2), 3-deoxythreosone (3), xylosone (4), and threosone (5), and their proposed derivatization products with OPD into corresponding quinoxalines (6–10).

FIGURE 1.

Mass spectrometric detection of OPD derivatives of DHA (1) (A; m/z 247. 0 → m/z 157.0 and m/z 247.0 → m/z 171.0 transitions), 2,3-DKG (2) (B; m/z 265.0 → m/z 161.1 and m/z 265.0 → m/z 185.1 transitions), 3-deoxythreosone (3) (C; m/z 175.0 → m/z 131.9 and m/z 175.0 → m/z 156.9 transitions), xylosone (4) (D; m/z 220.9 → m/z 157.1 and m/z 220.9 → m/z 161.1 transitions), threosone (5) (E; m/z 191.2 → m/z 144.1 and m/z 191.2 → m/z 160.0 transitions) and internal standard (IS) (F; m/z 158.5 → m/z 91.2 and m/z 158.5 → m/z 118.1 transitions) by LC-ESI-MS/MS multiple-reaction-monitoring analysis in human lens.

FIGURE 2.

Level of free dicarbonyl quinoxalines from DHA (1), 2,3-DKG (2), 3-deoxythreosone (3), xylosone (4), and threosone (5) in human lenses measured as OPD derivatives. Error bars, S.D.

FIGURE 3.

Level of quinoxalines from DHA (1; A), 2,3-DKG (2; B), and xylosone (4; C) reversibly bound to water soluble human lens proteins measured as OPD derivatives versus age.

FIGURE 4.

Level of quinoxalines from 3-deoxythreosone (3; A and C) and threosone (5; B and D) reversibly bound to WSP (A and B) and WIP (C and D) human lens proteins measured as OPD derivatives versus age.

FIGURE 5.

Mean ± S.D. levels of quinoxalines from DHA (1), 2,3-DKG (2), 3-deoxythreosone (3), xylosone (4), and threosone (5) reversibly bound to water-soluble (A) and water-insoluble (B) proteins in human lenses (ages 20–80) measured as OPD derivatives. Error bars, S.D.

SCHEME 2.

Summary of the major in vivo pathways of non-enzymatic protein modifications by ascorbic acid degradation products.