By using fast protein liquid chromatography, we isolated from human plasma a minor electronegative LDL subfraction designated LDL(-). After immunoaffinity chromatography against apolipoprotein (apo)(a) and apo A-I, LDL(-) represented 6.7 +/- 0.9% (mean +/- SD; n = 18) of total LDL. Compared with the major LDL subfraction, designated LDL(+), LDL(-) contained similar amounts of thiobarbituric acid-reactive substances, conjugated dienes, and vitamin E and had a similar lipid/protein ratio and mean density. Moreover, the apo B of LDL(-) was not aggregated and its LDL receptor-binding activity was slightly increased. These results were consistent with the nonoxidized nature of LDL(-). LDL(-) showed increased contents of sialic acid (38.1 +/- 5.2 versus 28.9 +/- 3.3 nmol/mg protein; n = 7; P < .01), apo C-III (1.43 +/- 0.21% versus 0.14 +/- 0.04%; n = 7; P < .01), and apo E (1.64 +/- 0.26% versus 0.10 +/- 0.05%; n = 7; P < .0005). Compared with LDL(+), LDL(-) displayed enhanced cytotoxic effects on cultured human umbilical vein endothelial cells, as shown by lactate dehydrogenase assay (P < .003; n = 6), neutral red uptake (P < .02; n = 6), and morphological studies. We also studied the relationship of LDL(-) to age and plasma lipid levels in 133 subjects. The percentage of contribution of LDL(-) to total plasma LDL correlated with age (P < .05), total cholesterol (P < .05), and LDL cholesterol (P < .003). In conclusion, this study shows that LDL(-), a circulating human plasma LDL, is an electronegative native LDL subfraction with cytotoxic effects on endothelial cells. This subfraction, which correlates positively with common atherosclerotic risk factors, might induce atherogenesis by actively contributing to alteration of the vascular endothelium.

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Abstract-We recently showed that sialic acid content of LDL was not a marker of early cardiovascular disease (Arterioscler Thromb Vasc Biol. 1995;15:334 -339). Here, we investigated this parameter in patients with advanced coronary artery disease (CAD). We first examined 100 patients having undergone coronary angiography. The distribution of LDL sialic acid values was very similar in subjects with no coronary stenosis (31.3Ϯ3.7 nmol/mg LDL protein, meanϮSD) and those with Ն75% stenosis in at least one main coronary artery or Ն50% stenosis in at least two main coronary arteries (32.1Ϯ5.5 nmol/mg LDL protein). In contrast, LDL sialic acid content was significantly increased in patients with both coronary stenosis and peripheral arterial atherosclerotic lesions compared with those with either no lesion or only one or the other type of lesion. We then examined LDL sialic acid content in 20 patients with acute myocardial infarction. LDL sialic acid content was significantly higher (35.9Ϯ3.2 nmol/mg LDL protein) than that in the CAD(Ϫ) control group. These data suggest that LDL sialic acid content increases with the extension of atherosclerosis and its progression to acute complications. To explain the discordance with Orekhov and coworkers (Atherosclerosis. 1991;86:153-161), who showed that LDL sialic acid content in patients with advanced CAD was lower than that in healthy subjects, we studied the time courses of sialic acid, TBARS, and vitamin E levels in LDL dialyzed in different experimental conditions. A continuous decrease in both sialic acid and vitamin E levels and an increase in TBARS levels were observed in LDL samples containing less than 1 mmol/L EDTA, the intensity and rapidity of which varied with the EDTA concentration in the buffer. Our data support the idea that desialylation may result from in vitro peroxidation of LDL. (Arterioscler Thromb Vasc Biol. 1998;18:876-883.)

Recent studies have shown that the sialic acid content of LDL isolated from patients with angiographically demonstrated advanced coronary atherosclerosis is lower than that of LDL isolated from healthy subjects. These observations raise the question as to whether LDL sialic acid content could be used as an early marker of atherosclerosis. We screened for carotid, aortic, and femoral plaques by ultrasonography and for coronary calcifications by ultrafast computed tomography in 160 hypercholesterolemic subjects free of cardiovascular disease to investigate the relation between LDL sialic acid content and the prevalence of these early atherosclerotic lesions. LDL sialic acid values varied from 19.6 to 46.6 nmol/mg LDL protein (33.9 +/- 4.4, mean +/- SD) in the whole population, but the distribution was very similar: (1) in subjects with no plaque (34.1 +/- 4.9) relative to those with one or several plaques at one (34.2 +/- 4.4), two (33.0 +/- 3.6), or three (34.8 +/- 3.4) different arterial sites; (2) in subjects with (33.9 +/- 3.7) and without (34.1 +/- 4.8) coronary calcification; and (3) in subjects with both extracoronary and coronary lesions (33.8 +/- 3.9) relative to those with no arterial lesions (34.2 +/- 4.5). LDL sialic acid content was not related to sex, age, body mass index, smoking, blood pressure, or serum total cholesterol and lipoprotein(a) levels but correlated negatively with serum triglyceride levels (P < .001). These results suggest that LDL sialic acid content is not a discriminant marker of early atherosclerosis in asymptomatic hypercholesterolemic subjects.