Binding of Low Density Lipoprotein After Incubation With Endothelial Cells in Ham ' s F - 12 Medium and Dulbecco ' s Modified Minimal Essential Medium TABLE 2 . Effect of Gel Medium on Binding of Low Density

It was previously shown that serum low density lipoprotein (LDL) binds to type I collagen gels and that the binding is increased after modification by cultured endothelial cells. It is now demonstrated that when LDL is incubated with cells cultured in Dulbecco's modified minimal essential medium (DMEM), the subsequent binding of LDL to collagen is considerably less than after incubation with endothelial cells cultured in Ham's F-12 medium (F12). To determine the reason for this difference, collagen gels were made with saline containing ingredients of DMEM individually or in groups, and binding of LDL to such gels was measured. Modification of LDL, manifested by a high level of binding to the collagen, by lipid peroxidation (production of thiobarbituric acid-reactive substances), and by increased electrophoretic mobility occurred on exposure to collagen gels made in saline; these changes were almost completely inhibited by the addition of histidine at a concentration equal to that in DMEM. They were also inhibited by butylated hydroxytoluene, desferrioxamine, and EDTA; penicillamine and hydroxyl-radical scavengers inhibited collagen binding but did not inhibit lipid peroxidation or the increase in electrophoretic mobility. Nominally, DMEM contains 270 /iM histidine but no copper, whereas F12 contains 135 /ttM histidine and 10 nM copper; addition of copper (as much as 5 /tiM) to DMEM or of histidine (as much as 2.16 mM) to F12 did not overcome the differences between the media in supporting LDL oxidation by endothelial cells. It is concluded that 1) modification of LDL in saline, which results in increased binding to collagen, is dependent on free radicals generated in the presence of trace amounts of copper; 2) histidine is a potent inhibitor of this process; and 3) although the relative inability of DMEM in comparison to F12 to support such modification of LDL by endothelial cells may be partly due to its lower content of copper and its higher content of histidine, other differences in composition are important factors. It is hypothesized that copper ion binds to histidine residues of the LDL protein and, while bound, catalyzes production of hydroxyl radicals, which degrade the protein, producing or uncovering collagen-binding sites. (Arteriosclerosis and Thrombosis 1991;11:1322-1329)