Novel cell culture medium for use in oxidation experiments provides insights into mechanisms of endothelial cell-mediated oxidation of LDL.
Though one prominent theory of atherogenesis involves free-radical oxidation of low-density lipoprotein (LDL) within the vessel wall by one of the vascular cell types, the mechanism for cell-mediated LDL oxidation remains unclear[sn1]. In these studies we examined the effects of media phenols, thiols, and metals on endothelial cell-mediated oxidation. We found that cell culture media such as Dulbecco modified Eagle medium and minimal essential medium are unable to support cell-mediated oxidation of LDL because they contain high concentrations of phenol red (PR) and tyrosine, both of which strongly inhibit cell-mediated oxidation. Ham's F-10, a commonly used medium for cell-mediated oxidation experiments, is also not entirely appropriate, as it contains both PR and cysteine. Cysteine is not critical for endothelial cell-mediated oxidation, but does increase oxidation of LDL in the absence of cells. Finally, of utmost importance to cell-mediated oxidation was the presence of either micromolar concentrations of Fe(II) or physiological concentrations of holo-ceruloplasmin, the protein which carries copper in plasma. An appropriate culture medium for use in cell-mediated oxidation experiments should thus contain either micromolar concentrations of Fe(II) or physiological concentrations of holo-ceruloplasmin, and should be prepared without PR, cysteine, or large concentrations of tyrosine, all of which are shown here to inhibit endothelial cell-mediated LDL oxidation. These results are consistent with a mechanism of cell-mediated oxidation involving Fenton-type chemistry and redox cycling of the metal.
Dugas
T R
TR
Department of Pathology, University of Texas Medical Branch, Galveston 77555-0609, USA.
Morel
D W
DW
Harrison
E H
EH
D000818:Animals; D002570:Ceruloplasmin / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D003470:Culture Media; D003545:Cysteine / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D005296:Ferrous Compounds / Q000378:metabolism; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D010084:Oxidation-Reduction; D014168:Transferrin / Q000378:metabolism; D014407:Tumor Cells, Cultured
D003470:Culture Media; D005296:Ferrous Compounds; D008077:Lipoproteins, LDL; D014168:Transferrin; D003300:Copper; D008715:Methionine; D002570:Ceruloplasmin; D003545:Cysteine