May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Metabolism of 4-Hydroxynonenal in Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • N.H. Ansari
    Human Bio Chem Genetics, University of Texas Med Branch, Galveston, TX, United States
  • S. Choudhary
    Human Bio Chem Genetics, University of Texas Med Branch, Galveston, TX, United States
  • T. Xiao
    Human Bio Chem Genetics, University of Texas Med Branch, Galveston, TX, United States
  • S. Srivastava
    Department of Medicine, Division of Cardiology, University of Louisville, Louisville, KY, United States
  • Footnotes
    Commercial Relationships  N.H. Ansari, None; S. Choudhary, None; T. Xiao, None; S. Srivastava, None.
  • Footnotes
    Support  NIH grant EY13014; Lions Eye Bank
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1620. doi:
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      N.H. Ansari, S. Choudhary, T. Xiao, S. Srivastava; Metabolism of 4-Hydroxynonenal in Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1620.

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

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Abstract

Abstract: : Purpose: Lipid peroxides formed under oxidative stress are degraded to lipid-derived aldehydes (LDAs), which are believed to contribute to the pathogenesis of several diseases. Of the various LDAs, 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE) are the most toxic. Under physiological conditions, cells will detoxify LDAs and thus attenuate their toxicity, but under oxidatively stress, the formation of LDAs overwhelms the detoxifying capacity of the tissue,they can induce associated toxicity. Since retina has high levels of polyunsaturated fatty acids (PUFAs) and is also vulnerable to undergo oxidative stress, we have investigated HNE metabolism in human retinal pigmented epithelial cells (ARPE-19). Methods: ARPE-19 (0.8x106 ) cells were incubated with 30 nmoles of 3H-HNE in 2 ml KH-buffer for 30 min at 37°C. The HNE metabolites in the incubation medium were separated on high performance liquid chromatography (HPLC) using a C-18 ODS reversed phase column. The metabolites were separated using a gradient consisting of solvent A (0.1% aqueous TFA) and solvent B (100% acetonitrile) at a flow rate of 1 ml/min. Fractions containing radioactivity were pooled and analyzed using electrospray-ionization mass spectroscopy (ESI/MS) or gas chromatography-chemical ionization mass spectroscopy (GC/CI-MS). Results: Based upon the retention times of synthesized standards, peak I was tentatively identified to be glutathione conjugates of HNE and 1, 4-dihydroxy-2-nonene (DHN). Peaks II, III and IV were assigned to DHN, 4-hydroxy-2-nonenoic acid (HNA) and unmetabolized HNE, respectively after spiking the tissue extracts with synthetic metabolites and finally confirming by GC/CI-MS. ESI/MS of peak I revealed two prominent metabolites with m/z values corresponding to [MH]+ of GS-HNE and GS-DHN. Sorbinil, an aldose reductase (AR) inhibitor, attenuated GS-DHN levels and cyanamide, an aldehyde dehydrogenase inhibitor, attenuated HNA levels. Pre-incubation with H2O2 altered HNE metabolism. Conclusions: Our results show that the major metabolic transformation of HNE in ARPE-19 cells involves conjugation with glutathione and oxidation to HNA. Further metabolism of GS-HNE conjugate involves AR-mediated reduction to GS-DHN. Thus, RPE has multiple routes to detoxify HNE. However, severe oxidative stress may overwhelm the metabolic capacity of RPE cells to detoxify HNE leading to generation of protein-HNE adducts and cell death by apoptosis which may contribute to Age -Related Macular Degeneration.

Keywords: age-related macular degeneration • apoptosis/cell death • oxidation/oxidative or free radical damage 
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