April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Measurement of Dicarbonyls Generated by Vitamin C Degradation in Human Lens Proteins
Author Affiliations & Notes
  • Ina Nemet
    Organic Chemistry and Biochemistry, Rudjer Boskovic Institute, Zagreb, Croatia
    Pathology, Case Western Reserve University, Cleveland, Ohio
  • Vincent M. Monnier
    Patho & Biochem, Case Western Reserve Univ, Cleveland, Ohio
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 4751. doi:
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      Ina Nemet, Vincent M. Monnier; Measurement of Dicarbonyls Generated by Vitamin C Degradation in Human Lens Proteins. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4751.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 
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