Relative sensitivities of plasma lecithin:cholesterol acyltransferase, platelet-activating factor acetylhydrolase, and paraoxonase to in vitro gas-phase cigarette smoke exposure.

In order to identify potential atherogenic properties of gas-phase cigarette smoke, we utilized an in vitro exposure model to determine whether the activities of several putative anti-atherogenic enzymes associated with plasma lipoproteins were compromised. Exposure of heparinized human plasma to gas-phase cigarette smoke produced a dose-dependent reduction in the activity of platelet-activating factor acetylhydrolase (PAF-AH). Reductions of nearly 50% in PAF-AH activity were observed following exposure to gas-phase smoke from four cigarettes over an 8-h period. During this time of exposure, lecithin:cholesterol acyltransferase (LCAT) was rendered almost completely inactive (>80%). In contrast, paraoxonase was totally unaffected by cigarette smoke. Supplementation of plasma with 1 mM reduced glutathione was found to protect both PAF-AH and LCAT from cigarette smoke, suggesting that cysteine modifications may have contributed to the inhibition of these two enzymes. To evaluate this possibility, we blocked the free cysteine residues of these enzymes with the reversible thiol-modifying reagent dithiobisnitrobenzoic acid (DTNB). Reversal of the DTNB-cysteine adducts following cigarette smoke exposures revealed that LCAT, but not PAF-AH, was protected. Moreover, high doses (1.0-10 mM) of acrolein and 4-hydroxynonenal, reactive aldehydic species associated with cigarette smoke, completely inhibited plasma LCAT activity, whereas PAF-AH was resistant to such exposures. Taken together, these results indicate a divergence regarding the underlying mechanism of PAF-AH and LCAT inhibition upon exposure to gas-phase cigarette smoke. While LCAT was sensitive to exposure to volatile aldehydic products involving, in part, cysteine and/or active site modifications, the enzyme PAF-AH exhibited an apparent resistance. The latter suggests that the active site of PAF-AH is in a microenvironment that lacks free cysteine residues and/or is shielded from volatile aldehydic combustion products. Based on these results, we propose that cigarette smoke may contribute to atherogenesis by inhibiting the activities of plasma PAF-AH and LCAT, but the nature of this inhibition differs for the enzymes.

Enhanced oxidative susceptibility and reduced antioxidant content of metabolic precursors of small, dense low-density lipoproteins.

PURPOSE: Elevated plasma concentrations of low-density lipoproteins (LDL) increase risk for coronary heart disease. However, lipoprotein profiles rich in small, dense LDL particles confer greater risk than those that mainly consist of large, buoyant LDL. This may be due, in part, to the greater oxidative susceptibility of small, dense LDL. In the current studies, we tested whether differences in the oxidative behavior of buoyant and dense LDL arise from differences in their immediate metabolic precursors, intermediate-density lipoproteins. SUBJECTS AND METHODS: We compared the properties of intermediate-density lipoproteins and buoyant and dense LDL subfractions in 9 subjects with the large, buoyant LDL phenotype versus 6 with the small, dense LDL phenotype. Oxidative susceptibility was evaluated based on conjugated diene formation and parinaric acid oxidation induced by copper. Antioxidants (ubiquinol-10 and alpha-tocopherol) were measured by high-performance liquid chromatography. RESULTS: Oxidative susceptibility was increased and antioxidant concentrations were decreased with increasing lipoprotein density (intermediate intermediate-density lipoproteins to buoyant LDL to dense LDL). Intermediate-density lipoproteins from subjects with the small, dense LDL phenotype had a greater oxidative susceptibility (by the parinaric acid test) and lower antioxidant concentrations than corresponding particles from subjects with the large, buoyant LDL phenotype. CONCLUSIONS: Differences in oxidative susceptibility between large, buoyant and small, dense LDL particles are apparent in their lipoprotein precursors. These results suggest that lipoprotein oxidative susceptibility may be metabolically programmed and that intermediate-density lipoproteins may contribute to the increased risk associated with the small, dense LDL phenotype.

Increased low density lipoprotein degradation in aorta of irradiated mice is inhibited by preenrichment of low density lipoprotein with alpha-tocopherol.

We previously reported that upper thoracic exposure to ionizing radiation (IR) accelerates fatty streak formation in C57BL/6 mice and that such effects are inhibited by overexpression of the antioxidant enzyme CuZn-superoxide dismutase (SOD). Notably, IR-accelerated lesion formation is strictly dependent on a high fat diet (i.e., atherogenic lipoproteins) but does not involve alterations in circulating lipid or lipoprotein levels. We thus proposed that IR promotes changes in the artery wall that enhance the deposition of lipoprotein lipids. To address this hypothesis, we examined the effects of IR on aortic accumulation and degradation of low density lipoproteins (LDL). Ten-week-old C57BL/6 mice were exposed to a single (8-Gy) dose of (60)Co radiation to the upper thoracic area or were sham irradiated (controls) and were then placed on the high fat diet. Five days postexposure, the mice received either (125)I-labeled LDL ((125)I-LDL) (which was used to measure intact LDL) or (125)I-labeled tyramine cellobiose ((125)I-TC)-LDL (which was used to measure both intact and cell-degraded LDL) via tail vein injection. On the basis of trichloroacetic acid (TCA)-precipitable counts in retroorbital blood samples, > or =95% of donor LDL was cleared within 24 h and there were no differences in time-averaged plasma concentrations of the two forms of LDL among irradiated and control mice. Aortic values increased markedly within the first hour and thereafter exhibited a slow increase up to 24 h. There were no differences between irradiated and control mice at 1 h, when values primarily reflected LDL entry, but a divergence was observed thereafter. At 24 h, (125)I-TC-associated counts were 1.8-fold higher in irradiated mice (P = 0.10). In contrast, (125)I-LDL-associated counts were 30% lower in irradiated mice (P< 0.05), suggesting that most of the retained (125)I-TC was associated with LDL degradation products. Consistent with the proposed involvement of oxidative or redox-regulated events, IR-induced LDL degradation was lower in SOD-transgenic than wild-type mice (P<0.05). The importance of LDL oxidation was suggested by observations that IR-induced LDL degradation was significantly reduced by preenriching LDL with alpha-tocopherol. On the basis of these results, we propose that IR elicits SOD-inhibitable changes in the artery wall that enhance LDL oxidation and degradation leading to the deposition of LDL-borne lipids. These studies provide additional support for the role of oxidation in lipoprotein lipid deposition and atherogenesis and suggest that IR promotes an arterial environment that stimulates this process in vivo.

Associations of HDL, HDL(2), and HDL(3) cholesterol and apolipoproteins A-I and B with lifestyle factors in healthy women and men: the Stanford Five City Project.

BACKGROUND: Measures of the two major high-density lipoprotein (HDL) subfractions, HDL(2) and HDL(3), and the major apolipoproteins of HDL and low-density lipoprotein (LDL), Apo A-I and Apo B, may be etiologically important factors in the development of coronary artery disease. The association of lifestyle factors with these lipoprotein-related variables remains unclear. METHODS: HDL-C, HDL(2)-C, HDL(3)-C, Apo A-I, and Apo B levels were determined in a population-based sample of 1,027 healthy women and men aged 25-64 years, from four California cities who participated in the 1989/1990 survey of the Stanford Five City Project. In this cross-sectional study we examined the independent associations of these lipoprotein-related variables with body mass index (BMI), cigarette smoking, daily energy expenditure, alcohol intake, dietary intake, and hormone use (oral contraceptives and estrogen replacement therapy). RESULTS: In general, BMI and alcohol intake were the strongest independent predictors of the lipoprotein-related variables. The negative association of BMI with HDL-C was attributable primarily to the association with the HDL(2)-C subfraction, while for alcohol intake the positive association with HDL-C was attributable primarily to the association with HDL(3)-C, particularly in men. Among men, but not women, energy expenditure was a significant independent predictor of each of the lipoprotein-related variables, with positive associations observed for HDL-C, HDL(2)-C, HDL(2)-C, and Apo A-I and a negative association observed for Apo B (P < 0.005). CONCLUSIONS: Data from this population-based sample suggest that specific lifestyle factors are more strongly associated with some lipoprotein-related variables than with others, with notable gender differences.

Population frequency distributions of HDL, HDL(2), and HDL(3) cholesterol and apolipoproteins A-I and B in healthy men and women and associations with age, gender, hormonal status, and sex hormone use: the Stanford Five City Project.

BACKGROUND: The purpose of this study is to present population-based frequency distribution data for several lipoprotein-related variables and to examine their associations with gender, age, menopausal status, and sex hormone use. METHODS: High-density lipoprotein cholesterol (HDL-C), HDL(2)-C, HDL(3)-C, apolipoprotein (Apo) A-I, and Apo B were measured in a population-based sample of 1, 027 healthy adults from four California cities who participated in the 1989-1990 survey of the Stanford Five City Project. These data were examined cross-sectionally with sociodemographic and other related variables. RESULTS: Relative to men, all of the HDL-related parameters-HDL-C, HDL(2)-C, HDL(3)-C, Apo A-I-were significantly higher and Apo B levels were significantly lower among women (P < 0. 001). Menopausal status was not associated with HDL-related parameters, but Apo B levels were higher in post- versus premenopausal women (P < 0.001). Among women, HDL-C and Apo A-I levels were higher in oral contraceptive and estrogen replacement therapy users (P = 0.003). Most of the significant findings remained statistically significant after adjusting for age, body mass index, smoking, energy expenditure, and alcohol intake. CONCLUSIONS: These population-based data indicate that gender, menopausal status, and the use of sex hormones among women are associated with differential levels of one or more of HDL-C, HDL(2)-C, HDL(3)-C, Apo A-I, and Apo B, independent of age and a broad set of lifestyle factors.

Ionizing radiation accelerates aortic lesion formation in fat-fed mice via SOD-inhibitable processes.

Ionizing radiation promotes formation of reactive oxygen species, including the superoxide anion (O2-). To evaluate whether O2- or O2--mediated perturbations may contribute to the known atherogenic effects of radiation, we examined aortic lesion formation in irradiated C57BL/6 mice and evaluated the effects of CuZn-superoxide dismutase (CuZn-SOD) overexpression. Ten-week-old mice were exposed to a 2-, 4-, or 8-Gy dose of 250-keV x-rays to the upper thorax and then placed on a high-fat diet for 18 weeks. Based on quantitative lipid staining of serial sections of the proximal aorta, mean lesion area was increased with increasing radiation dose and was 3-fold greater in 8-Gy-irradiated than sham-irradiated mice (7800+/-2140 versus 2635+/-709 micrometer(2), P<0.05). These effects were absolutely dependent on a high-fat diet, which had to be introduced within 1 to 2 weeks of the radiation exposure, suggesting the early involvement of atherogenic lipoproteins that were elevated in response to the diet. The importance of radiation-induced oxidative stress was supported by the observation of a 2-fold lower mean lesion area in irradiated CuZn-SOD transgenic mice than in their irradiated, nontransgenic littermates (3026+/-1590 versus 6102+/-1834 micrometer(2), P<0.05). Lucigenin-enhanced chemiluminescence, used as an index of aortic O2- concentrations, was significantly elevated in the postradiation period, and this response was reduced in CuZn-SOD transgenics. On the basis of these results, we propose that radiation may be a useful tool for initiating oxidative or redox-regulated events that promote atherogenesis and for testing the antiatherogenic properties of antioxidants.

Dissociable and nondissociable forms of platelet-activating factor acetylhydrolase in human plasma LDL: implications for LDL oxidative susceptibility.

Platelet-activating factor acetylhydrolase (PAF-AH) is transported by lipoproteins in plasma and is thought to possess both anti-inflammatory and anti-oxidative activity. It has been reported that PAF-AH is recovered primarily in small, dense LDL and HDL following ultracentrifugal separation of lipoproteins. In the present studies, we aimed to further define the distribution of PAF-AH among lipoprotein fractions and subfractions, and to determine whether these distributions are affected by the lipoprotein isolation strategy (FPLC versus sequential ultracentrifugation) and LDL particle distribution profile. When lipoproteins were isolated by FPLC, the bulk (approximately 85%) of plasma PAF-AH activity was recovered within LDL-containing fractions, whereas with ultracentrifugation, there was a redistribution to HDL (which contained approximately 18% of the activity) and the d>1.21 g/ml fraction (which contained approximately 32%). Notably, re-ultracentrifugation of isolated LDL did not result in any further movement of PAF-AH to higher densities, suggesting the presence of dissociable and nondissociable forms of the enzyme on LDL. Differences were noted in the distribution of PAF-AH activity among LDL subfractions from subjects exhibiting the pattern A (primarily large, buoyant LDL) versus pattern B (primarily small, dense LDL) phenotype. In the latter group, there was a relative depletion of PAF-AH activity in subfractions in the intermediate to dense range (d=1.039-1.047 g/ml) with a corresponding increase in enzyme activity recovered within the d>1.21 g/ml ultracentrifugal fraction. Thus, there appears to be a greater proportion of the dissociable form of PAF-AH in pattern B subjects. In both populations, most of the nondissociable activity was recovered in a minor small, dense LDL subfraction. Based on conjugated dienes as a measure of lipid peroxidation, variations in PAF-AH activity appeared to contribute to variations in oxidative behavior among ultracentrifugally isolated LDL subfractions. The physiologic relevance of PAF-AH dissociability and the minor PAF-AH-enriched oxidation-resistant LDL subpopulation remains to be determined.

Fatty streak formation in fat-fed mice expressing human copper-zinc superoxide dismutase.

Studies in vitro have shown that copper-zinc superoxide dismutase (CuZn-SOD) inhibits a number of events putatively involved in atherogenesis, including cell-mediated oxidation of LDL. To investigate whether increased activity of CuZn-SOD reduces atherogenesis in vivo, we examined diet-induced fatty streak formation in CuZn-SOD transgenic mice (n = 24) as compared with their nontransgenic littermates (n = 28). Transgenic animals were originally created by introduction of an EcoRI-BamHI human genomic DNA fragment containing the CuZn-SOD gene and its regulatory elements into B6SJL zygotes. For the current studies, the transgene was bred for 12 generations into the atherosclerosis-susceptible C57BL/6 background. Animals were fed atherogenic diets (15% fat, 1.25% cholesterol, 0.5% Na cholate) starting at 100 weeks of age and extending for 18 weeks. At the end of the diet period, aortic SOD activity was two-fold higher in transgenics than nontransgenics (mean +/- SE: 46.7 +/- 5.8 versus 20.1 +/- 2.4 units/mg of protein, P < .001). Levels of protein-bound amino acid oxidation products (meta-, ortho-, and dityrosine) were either similar or lower in aorta and heart from transgenics as compared with nontransgenics, suggesting that amplification of CuZn-SOD activity above the normal complement had modest inhibitory effects on basal oxidative stress in these tissues. CuZn-SOD overexpression did not reduce the extent of lesion development as analyzed by quantitative lipid staining of serial sections of the proximal aorta; mean lesion areas (+/- SE) were 997 +/- 478 and 943 +/- 221 mu 2 in transgenics and nontransgenics, respectively. Notably, the range of values for lesion area was 2.2-fold greater in transgenics (0-8403 versus 0-3868 mu 2 in nontransgenics). Moreover, within this group, lesion area showed a significant positive correlation with SOD activity (r = .611, P < .03). These results do not support an antiatherogenic effect of Cu-Zn-SOD over expression and raise the possibility that high tissue SOD activity may potentiate atherogenesis in fat-fed atherosclerosis-susceptible mice [corrected].

Selective resistance of LDL core lipids to iron-mediated oxidation. Implications for the biological properties of iron-oxidized LDL.

Although the nature and consequences of oxidative changes in the chemical constituents of low density lipoproteins (LDLs) have been extensively examined, the physical dynamics of LDL oxidation and the influence of physical organization on the biological effects of oxidized LDLs have remained relatively unexplored. To address these issues, in the present studies we monitored surface- and core-specific peroxidative stress relative to temporal changes in conjugated dienes (CDs), particle charge (an index of oxidative protein modification), and LDL-macrophage interactions. Peroxidative stress in LDL surface and core compartments was evaluated with the site-specific, oxidation-labile fluorescent probes parinaric acid (PnA) and PnA cholesteryl ester (PnCE), respectively. When oxidation was initiated by Cu2+, oxidative loss of the core probe (PnCE) closely followed that of the surface probe (PnA), as indicated by the time to 50% probe depletion (t1/2; 15.5 +/- 7.8 and 30.4 +/- 12 minutes for PnA and PnCE, respectively). Both probes were more resistant in LDL exposed to Fe3+ (t1/2, 53.2 +/- 8.1 and 346.7 +/- 155.4 minutes), although core probe resistance was much greater with this oxidant (PnCE t1/2/PnA t1/2 5.8 vs 2.0 for Cu2+). Despite differences in the rate and extent of oxidative changes in Cu(2+)- versus Fe(3+)-exposed LDLs, PnCE loss occurred in close correspondence with CD formation and appeared to precede changes in particle charge under both conditions. Exposure of LDLs to hemin, a lipophilic Fe(3+)-containing porphyrin that becomes incorporated into the LDL particle, resulted in rapid loss of PnCE and simultaneous changes in particle, charge, even at concentrations that yielded increases in CDs and thiobarbituric acid-reactive substances similar to those obtained with free Fe3+. These results suggest that oxidation of the LDL hydrophobic core occurs in conjunction with accelerated formation of CDs and may be essential for LDL protein modification. In accordance with the known effects of oxidative protein modifications on LDL receptor recognition, exposure of LDLs to Cu2+ and hemin but not Fe3+ produced particles that were readily processed by macrophages. Thus, the physical site of oxidative injury appears to be a critical determinant of the chemical and biological properties of LDLs, particularly when oxidized by Fe3+.

HDL antioxidant effects as assessed using a nonexchangeable probe to monitor particle-specific peroxidative stress in LDL-HDL mixtures.

High density lipoproteins (HDL) have been reported to inhibit oxidation of low density lipoproteins (LDL) based in part on observations that oxidative changes occur more slowly in LDL-HDL mixtures than in LDL alone. In the current studies, we developed an approach to discern particle-specific oxidation kinetics within mixed particle systems using the oxidation-labile fluorescent probe parinaric acid cholesteryl ester (PnCE) and applied this to the study of HDL inhibition effects. PnCE was introduced into acceptor lipoproteins by cholesteryl ester transfer protein (CETP)-mediated transfer from donor microemulsions. Incubation of PnCE-containing LDL and HDL with non-probe-containing HDL and LDL, respectively, followed by measurement of reisolated fractions, indicated that PnCE does not transfer appreciably between lipoprotein fractions. Oxidative loss of lipoprotein-associated PnCE occurred essentially in tandem with changes in conjugated dienes, suggesting that PnCE loss reflects the course of peroxidation of endogenous lipoprotein lipids. Using PnCE to separately monitor LDL- and HDL-specific oxidation within LDL-HDL mixtures, we obtained direct evidence that HDL inhibits both Cu(2+)- and Fe(3+)-induced peroxidation of LDL-associated lipids. Notably, in the presence of Cu2+, loss of HDL-associated PnCE fluorescence also was inhibited in LDL-HDL co-incubations, suggesting that LDL exert an antioxidant effect under these conditions as well. Thus, results obtained using this new methodology are consistent with previously reported antioxidant effects of HDL, but indicate that the behavior of individual lipoprotein particles may be more complicated than can be predicted from the collective behavior of the lipoprotein mixture.

Differing alpha-tocopherol oxidative lability and ascorbic acid sparing effects in buoyant and dense LDL.

The enhanced oxidizability of smaller, more dense LDL is explained in part by a lower content of antioxidants, including ubiquinol-10 and alpha-tocopherol. In the present studies, we also observed greater rates of depletion of alpha-tocopherol (mole per mole LDL per minute) in dense (d = 1,040 to 1,054 g/mL) compared with buoyant (d = 1,026 to 1,032 g/mL) LDL in the presence of either Cu2+ or the radical-generating agent 2-2'-azobis (2-amidinopropane)dihydrochloride. Differences were particularly pronounced at the lowest Cu2+ concentration tested (0.25 mumol/L), with a fivefold greater rate in dense LDL. At higher concentrations (1.0 and 2.5 mumol/L Cu2+), there was a greater dependence of depletion rate on initial amount of alpha-tocopherol, which was reduced in dense LDL, thus resulting in smaller subfraction-dependent differences in depletion rates. Inclusion of ascorbic acid (15 mumol/L), an aqueous antioxidant capable of recycling alpha-tocopherol by hydrogen donation, was found to extend the course of Cu(2+)-induced alpha-tocopherol depletion in both buoyant and dense LDL, but this effect was more pronounced in dense LDL (time to half-maximal alpha-tocopherol depletion was extended 15.6-fold and 21.2-fold in buoyant and dense LDL, respectively, at 2.5 mumol/L Cu2+; P< .05). Thus, dense LDL exhibits more rapid alpha-tocopherol depletion and conjugated diene formation than buoyant LDL when oxidation is performed in the absence of ascorbic acid, but these differences are reversed in the presence of ascorbic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipoprotein oxidation in dyslipidemia: insights into general mechanisms affecting lipoprotein oxidative behavior.

Atherosclerosis susceptibility associated with elevations in specific populations of apolipoprotein-B-containing particles may involve increased oxidation of lipoproteins and associated changes in their biological properties. Lipoprotein oxidation may be potentiated by the greater mass of oxidizable lipoprotein substrates, as well as by a greater intrinsic susceptibility of the specific forms of lipoproteins that arise in these disorders. The atherogenic consequences of increased lipoprotein oxidation may be further enhanced by a greater relative potency or toxicity of the oxidized products of these lipoprotein subpopulations. This review addresses these facets of lipoprotein oxidation in several forms of dyslipidemia, and in view of both differences between and commonalities among these disorders, proposes that lipoprotein oxidative behavior is determined by a complex array of physical, chemical, and metabolic factors.

Reinvestigation of the antioxidant properties of conjugated linoleic acid.

Despite repeated suggestions that antioxidant activity of conjugated linoleic acid (CLA), a collective of conjugated dienoic isomers of linoleic acid, underlies its reported anticarcinogenic and antiatherosclerotic effects, the antioxidant properties of CLA remain ill-defined. Therefore, this study was undertaken to gain more insight into the mechanism of potential CLA antioxidant activity. It was tested whether CLA could protect membranes composed of 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC) from oxidative modification under conditions of metal ion-dependent or -independent oxidative stress. Progress of oxidation was determined by direct spectrophotometric measurement of conjugated diene formation and by gas chromatographic/mass spectrometric analysis of fatty acids. The oxidative susceptibility of CLA was higher than that of linoleic acid, and comparable to arachidonic acid. When oxidation of PLPC (1.0 mM) was initiated using the lipid-soluble 2,2'-azobis(2,4-dimethylvaleronitrile) or the water-soluble 2,2'-azobis(2-amidinopropane) hydrochloride, the radical scavengers vitamin E and butylated hydroxytoluene (BHT) at 0.75 microM efficiently inhibited PLPC oxidation, as evident from a clear lag phase. In contrast, 0.75 microM CLA did not have any significant effect on PLPC oxidation. Inhibition of PLPC oxidation by higher concentrations of CLA appeared due to competition, not to an antioxidant effect. When oxidation of PLPC was initiated by hydrogen peroxide/Fe2+ (500 microM/0.05-20 microM), both vitamin E (1 microM) and ethylene glycol-bis(aminoethyl ether) tetraacetic acid (50 microM) efficiently inhibited PLPC oxidation. However, CLA (1-50 microM) did not show a clear protective effect under any of the conditions tested. We conclude that CLA, under these test conditions, does not act as an efficient radical scavenger in any way comparable to vitamin E or BHT. CLA also does not appear to be converted into a metal chelator under metal-ion dependent oxidative stress, as had previously been suggested. On the basis of our observations, a role for CLA as an antioxidant does not seem plausible.

Greater oxidative susceptibility of the surface monolayer in small dense LDL may contribute to differences in copper-induced oxidation among LDL density subfractions.

We monitored peroxidative stress in the surface monolayer as compared with the outer core of large, buoyant (d 1.025-1.032 g/ml) and small, dense (d 1.040-1.054 g/ml) low density lipoprotein (LDL) subfractions using the oxidation-labile fluorescent probes parinaric acid (PnA) and parinaric acid methyl ester (PnME), which partition preferentially into these respective regions of LDL. Oxidation was initiated either with CuSO4 (5 microM) or the iron (Fe3+)-containing lipophilic complex hemin (1.0 microM) plus cumene hydroperoxide to facilitate heme degradation. In the presence of Cu2+, PnA was depleted significantly more rapidly than PnME in dense (P = 0.039) but not in buoyant LDL, suggesting that surface vulnerability is enhanced in dense LDL particles. With hemin, PnA and PnME were similarly susceptible within both subfractions, although there was a trend toward slower loss of PnA in buoyant LDL (P = 0.10), consistent with the internal site of initiation and a greater surface resistance in buoyant particles. As indicated by conjugated diene lag times, dense LDL was more susceptible than buoyant LDL to oxidation by Cu2+ (P = 0.03) but not hemin (P = 0.68). These results suggest that the increased susceptibility of dense LDL to oxidation by external agents such as Cu2+ is at least partially mediated by an enhanced vulnerability of the surface compartment.