Low density llpoproteins (LDL) collected from 18 fasting humans were subjected to ion exchange chromatography on DEAE Sepharose. By this procedure, a LDL subfraction was isolated with an electric charge more negative than the LDL bulk. This LDL appeared to be mainly characterized by low phospholipld content, high free cholesterol and protein content, low esterlfled/free cholesterol ratio, and a high content of conjugated dlenes, particularly of cholesterol esters. This subtraction, In an amount ranging from 5% to 20% of total LDL, was characterized by the presence of apo B-100 and protein aggregates that were reactive to anti-apo B monoclonal antibodies. Electron microscopy showed the more electronegative LDL to be heterogeneous in size with a tendency to aggregate. This LDL had low binding capacity with high affinity receptors of flbroblasts and low Immunoreactlvlty with the monoclonal antibodies that recognize the receptor binding domain of apo B. Finally, the Incubation of this LDL subtraction with cultured macrophages led to a higher Increase In cellular cholesterol In spite of a lower rate of uptake as compared to the LDL bulk and to acetyl-LDL. The more electronegative LDL subtraction that we Isolated for chemlcophyslcal behavior and conjugated dlene content may represent the peroxldized aliquot of human LDL (Arteriosclerosis 8:79-87, January/February 1988) N umerous studies have demonstrated that the plasma low density lipoproteins (LDL) are a heterogeneous collection of particles that vary in size, 1 density, 2 composition, 3 and electric charge. 4 The metabolic basis and physiological significance of LDL subtractions are unknown, although it now seems possible that some LDL forms are more atherogenic than others.5 Recently a series of in vitro studies proved that modified forms of LDL, being more negatively charged than native LDL, cause accumulation of cholesterol esters in cultured macrophages 6 -7 -8 and induce cytotoxicity in cultured endothelial cells.9 ' 10 The in vivo correlate of modified forms of LDL has not yet been identified although negatively charged LDL have been demonstrated in human atherosclerotic lesions.11 This paper reports data on the isolation and partial characterization of a subtraction of plasma LDL more electronegative than normal LDL. This was accomplished through separation of LDL collected from fasting subjects by ion exchange chromatography. Because modification of LDL mostly involves a free radical-initiated lipoprotein peroxidation, 91 10 ' 12 the extent of lipid peroxidation was also analyzed in the two LDL fractions obtained. Chemical and chemicophysical characterization of the more electronegative LDL, as well as its content of conjugated dienes of cholesterol and triglyceride free fatty acids, supports the From the Regional General Hospital, Regional Center for Atherosclerosis, Venice, Italy.Address for reprints: Pietro Avogaro, Regional General Hospital, 30100 Venice, Italy.This work was partly supported by Grant 84.02179.56 CNR, Consiglio Nazionale delle Ricerche, ...
Oxidative modification of LDL is thought to be a radical-mediated process involving lipid peroxides. The small dense LDL subpopulations are particularly susceptible to oxidation, and individuals with high proportions of dense LDL are at a greater risk for atherosclerosis. An oxidatively modified plasma LDL, referred to as LDL-, is found largely among the dense LDL fractions. LDL- and dense LDL particles also contain much greater amounts of lipid peroxides compared with total LDL or the more buoyant LDL fractions. The content of LDL- in dense LDL particles appears to be related to copper- or heme-induced oxidative susceptibility, which may be attributable to peroxide levels. The rate of lipid peroxidation during the antioxidant-protected phase (lag period) and the length of the antioxidant-protected phase (lag time) are correlated with the LDL- content of total LDL. Once LDL oxidation enters the propagation phase, there is no relationship to the initial LDL- content or total LDL lipid peroxide or vitamin E levels. Beyond a threshold LDL- content of approximately 2%, there is a significant increase in the oxidative susceptibility of nLDL particles (ie, purified LDL that is free of LDL-), and this susceptibility becomes more pronounced as the LDL- content increases. nLDL is resistant to copper- or heme-induced oxidation. The oxidative susceptibility is not influenced by vitamin E content in LDL but is strongly inhibited by ascorbic acid in the medium. Involvement of LDL(-)-associated peroxides during the stimulated oxidation of LDL is suggested by the inhibition of nLDL oxidation when LDL- is treated with ebselen prior to its addition to nLDL. Populations of LDL enriched with LDL- appear to contain peroxides at levels approaching the threshold required for progressive radical propagation reactions. We postulate that elevated LDL- may constitute a pro-oxidant state that facilitates oxidative reactions in vascular components.
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