Paraoxonase prevents accumulation of lipoperoxides in low-density lipoprotein.

Oxidative modification of low-density lipoprotein (LDL) enhances its uptake by macrophages in tissue culture and in vivo may underly the formation of arterial fatty streaks, the progenitors of atheroma. We investigated the possible protection which high-density lipoprotein (HDL) affords against LDL oxidation. The formation of lipoperoxides and thiobarbituric acid reactive substances when LDL was incubated with copper ions was significantly decreased by HDL. The enzyme, paraoxonase (E.C. 3.1.8.1), purified from human HDL, had a similar effect and thus may be the component of HDL responsible for decreasing the accumulation of lipid peroxidation products.     

Paraoxonase 1 (PON1) and its relationship to lipid variables, age and gender in healthy volunteers

Recent studies implied that low-density lipoprotein (LDL) modified predominantly by oxidation or glycation, significantly contributes to the formation of atherosclerotic lesions. In contrast to oxidized LDL (ox-LDL), high-density lipoprotein (HDL) is able to prevent accumulation of ox-LDL in arterial walls. This antiatherogenic property of HDL is attributed in part to several enzymes associated with the lipoprotein, including HDL-associated paraoxonase 1 (PON1). In this study we analyzed PON1 arylesterase/paraoxonase activities in relation to serum lipid profile, gender and age in thirty clinically healthy Slovak volunteers. Our results showed that PON1 arylesterase and paraoxonase activities were lower in citrated plasma than in serum by 16.6% and 27.3%, respectively. Among serum lipoproteins, only HDL-cholesterol level showed significant positive correlation with PON1 arylesterase activity (p = 0.042). Likewise, we found a significant relationship between atherogenic index (AI = total cholesterol/HDL-cholesterol) and PON1 arylesterase activity (p = 0.023). No significant correlation could be demonstrated between PON1 paraoxonase activity and serum lipid profile, age or gender. Furthermore, it was found that PON1 paraoxonase/arylesterase activities were higher in women compared with both investigated activities in men, but these differences were not statistically significant. These results confirmed a positive correlation between HDL-cholesterol and PON1 arylesterase activity. Moreover, it was found out that PON1 paraoxonase activity is not influenced either by gender or by age. PON1 arylesterase activity was however affected by gender to a limited extent.     

4-Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress. Identification of proteasomes as target molecules.

Oxidative stress is associated with important pathophysiological events in a variety of diseases. It has been postulated that free radicals and lipid peroxidation products generated during the process may be responsible for these effects because of their ability to damage cellular components such as membranes, proteins, and DNA. In the present study, we provide evidence that oxidative stress causes a transient impairment of intracellular proteolysis via covalent binding of 4-hydroxy-2-nonenal (HNE), a major end product of lipid peroxidation, to proteasomes. A single intraperitoneal treatment with the renal carcinogen, ferric nitrilotriacetate, caused oxidative stress, as monitored by accumulation of lipid peroxidation products and 8-hydroxy-2'-deoxyguanosine, in the kidney of mice. In addition, transient accumulation of HNE-modified proteins in the kidney was also found by competitive enzyme-linked immunosorbent assay and immunohistochemical analyses. This and the observation that the HNE-modified proteins were significantly ubiquitinated suggested a crucial role of proteasomes in the metabolism of HNE-modified proteins. In vitro incubation of the kidney homogenates with HNE indeed resulted in a transient accumulation of HNE-modified proteins, whereas the proteasome inhibitor significantly suppressed the time-dependent elimination of HNE-modified proteins. We found that, among three proteolytic activities (trypsin, chymotrypsin, and peptidylglutamyl peptide hydrolase activities) of proteasomes, both trypsin and peptidylglutamyl peptide hydrolase activities in the kidney were transiently diminished in accordance with the accumulation of HNE-modified proteins during oxidative stress. The loss of proteasome activities was partially ascribed to the direct attachment of HNE to the protein, based on the detection of HNE-proteasome conjugates by an immunoprecipitation technique. These results suggest that HNE may contribute to the enhanced accumulation of oxidatively modified proteins via an impairment of ubiquitin/proteasome-dependent intracellular proteolysis.     

A novel lecithin-cholesterol acyltransferase antioxidant activity prevents the formation of oxidized lipids during lipoprotein oxidation.

Recent investigations suggest that high-density lipoprotein (HDL) may play an anti-atherogenic role as an antioxidant and inhibit the oxidative modification of low-density lipoprotein (LDL). The antioxidant activity of HDL has been proposed to be associated with several HDL-bound proteins. We have purified one HDL-associated protein, lecithin:cholesterol acyltransferase (LCAT), to apparent homogeneity and have found that LCAT is not only capable of esterifying cholesterol in the plasma, but can also prevent the accumulation of oxidized lipids in LDL. Addition of pure human LCAT to LDL or palmitoyl-linoleoyl phosphatidylcholine/sodium cholate (PLPC) micelles inhibits the oxidation-dependent accumulation of both conjugated dienes and lipid hydroperoxides. LCAT also inhibits the increase of net negative charge that occurs during oxidation of LDL. LCAT has the ability to prevent spontaneous oxidation and Cu2+ and soybean lipoxygenase-catalyzed oxidation of lipids. The antioxidant activity of LCAT appears to be enzymatic, since the enzyme is active for up to 10 h in the presence of mild free-radical generators. The catalytic serine, residue 181, may mediate this activity and act as a reusable proton donor. Chemical modification of the active serine residue with diisopropylfluorophosphate completely inhibits the ability of LCAT to prevent lipid oxidation. Thus, in addition to its well-characterized phospholipase and acyltransferase activities, LCAT can also act as an antioxidant and prevent the accumulation of oxidized lipid in plasma lipoproteins.     

Detection of new epitopes formed upon oxidation of low-density lipoprotein, lipoprotein (a) and very-low-density lipoprotein. Use of an antiserum against 4-hydroxynonenal-modified low-density lipoprotein.

4-Hydroxynonenal (HNE) is a major aldehydic propagation product formed during peroxidation of unsaturated fatty acids. The aldehyde was used to modify freshly prepared human low-density lipoprotein (LDL). A polyclonal antiserum was raised in the rabbit and absorbed with freshly prepared LDL. The antiserum did not react with human LDL, but reacted with CuCl2-oxidized LDL and in a dose-dependent manner with LDL, modified with 1, 2 and 3 mM-HNE, in the double-diffusion analysis. LDL treated with 4 mM of hexanal or hepta-2,4-dienal or 4-hydroxyhexenal or malonaldehyde (4 or 20 mM) did not react with the antiserum. However, LDL modified with 4 mM-4-hydroxyoctenal showed a very weak reaction. Lipoprotein (a) and very-low-density lipoprotein were revealed for the first time to undergo oxidative modification initiated by CuCl2. This was evidenced by the generation of lipid hydroperoxides and thiobarbituric acid-reactive substances, as well as by a marked increase in the electrophoretic mobility. After oxidation these two lipoproteins also reacted positively with the antiserum against HNE-modified LDL.     

Vitamin C preserves the cardio-protective paraoxonase activity of high-density lipoprotein during oxidant stress

HDL-associated paraoxonase (PON) antioxidant enzyme activity is cardio-protective. We investigated whether vitamin C prevented loss of PON activity from HDL during oxidant stress. HDL was incubated with either hydrophilic or lipophilic peroxyl radical initiators in the absence (control) or presence of vitamin C (50 and 100 micromol/L). Regardless of the type of radical, accumulation of lipid oxidation products in HDL was similar in incubations lacking vitamin C. Loss of PON activity was greater in HDL exposed to hydrophilic, in contrast to lipophilic, radicals, but addition of vitamin C maintained enzyme activity. Vitamin C's capacity to attenuate loss of the HDL ability to prevent atherogenic modification of LDL (assessed as electrophoretic mobility) was, however, modest, and appeared limited only to those incubations in which HDL was exposed to lipophilic radicals. Our results indicate that vitamin C may, under some conditions, prevent loss of cardio-protective function from HDL during oxidant stress.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

Kinetics and localisation of haemin-induced lipoprotein oxidation

Abstract

Haemin (iron (III)-protoporphyrin IX) is a degradation product of haemoglobin in circulating erythrocytes. Haemin may play a key oxidising agent for lipoprotein oxidation in patients with haemolytic anaemia. In this study, kinetic changes in chemical composition and target sites of haemin-induced LDL and HDL oxidation were investigated. Haemin initially induced the loss of α-tocopherol, followed by accumulation of lipid hydroperoxide (LP) and alteration of core lipid fluidity. The absence of LP in HDL was explained by the antioxidant activity of PON in addition to α-tocopherol. The target site of haemin was evaluated by ESR spin labelling with 5- and 16-doxyl steric acids. In the presence of t-BuOOH and haemin, ESR signal decay of the doxyl moiety demonstrated the initiation phase and the propagation phase of lipid peroxidation. The results of the lag time and the rate of signal decay indicated that haemin is located near the 16th carbon atom of the fatty acid chain in the phospholipid layer. The analyses of motion parameters, order parameter (S) of 5-DS and rotational correlation time (τ) of 16-DS, supported the observation that the lipid properties changed near the hydrophobic region rather than at the surface region of lipoproteins. Moreover, ESR spin labelling demonstrated that haemin molecules but not iron ions caused lipoprotein oxidation. In conclusion, haemin is a potent inducer of lipoprotein oxidation, and the target site for this oxidation is near the hydrophobic core of the lipoprotein leading to the loss of antioxidant activities and changes in lipid composition and physical properties.     

4-Hydroxy-2(E)-nonenal (HNE) catabolism and formation of HNE adducts are modulated by β oxidation of fatty acids in the isolated rat heart

We previously reported that a novel metabolic pathway functionally catabolizes 4-hydroxy-2(E)-nonenal (HNE) via two parallel pathways, which rely heavily on β-oxidation pathways. The hypothesis driving this report is that perturbations of β oxidation will alter the catabolic disposal of HNE, favoring an increase in the concentrations of HNE and HNE-modified proteins that may further exacerbate pathology. This study employed Langendorff perfused hearts to investigate the impact of cardiac injury modeled by ischemia/reperfusion and, in a separate set of perfusions, the effects of elevated lipid (typically observed in obesity and type II diabetes) by perfusing with increased fatty acid concentrations (1 mM octanoate). During ischemia, HNE concentrations doubled and the glutathione–HNE adduct and 4-hydroxynonanoyl-CoA were increased by 7- and 10-fold, respectively. Under conditions of increased fatty acid, oxidation to 4-hydroxynonenoic acid was sustained; however, further catabolism through β oxidation was nearly abolished. The inhibition of HNE catabolism was not compensated for by other disposal pathways of HNE, rather an increase in HNE-modified proteins was observed. Taken together, this study presents a mechanistic rationale for the accumulation of HNE and HNE-modified proteins in pathological conditions that involve alterations to β oxidation, such as myocardial ischemia, obesity, and high-fat diet-induced diseases.     

Acetylcholine esterase protects LDL against oxidation

Acetylcholine esterase (AChE) and paraoxonase 1 (PON1) are both serum ester hydrolases, which are associated with the prevalence of myocardial infarction. Both genes are located in close proximity on chromosome 7q21-22. As PON1 was suggested to protect against cardiovascular diseases secondary to its ability to break down oxidized lipids and to inhibit LDL oxidation, we examined AChE capacity to protect LDL against oxidation. Preincubation of LDL with AChE retarded the onset of copper ion-induced LDL oxidation in a concentration-dependent manner. AChE significantly reduced the formation of lipid peroxides and TBARS during the course of LDL oxidation, by up to 45%. This effect was associated with AChE-mediated hydrolysis of lipid peroxides, which accounts for the inhibition in the onset of LDL oxidation, the oxidative propagation phase, and aldehyde formation. We conclude that AChE, similar to PON1, can hydrolyze lipid peroxides and thus may prevent the accumulation of oxidized LDL and attenuate atherosclerosis development.     

The kinetics of copper-induced LDL oxidation depend upon its lipid composition and antioxidant content

Copper promotes oxidation of human low-density lipoprotein (LDL) through molecular mechanisms that are still under investigation. We employed native human LDL, phospholipid-containing delipidated LDL ghosts, or trilinolein-reconstituted, phospholipid-containing LDL to investigate both LDL oxidation and the associated process of copper reduction. Both LDL ghosts and trilinolein-reconstituted LDL were devoid of antioxidants and were extremely susceptible to AAPH-induced oxidation but, paradoxically, were rather resistant to copper-mediated oxidation. The dynamic reduction of Cu(II) to Cu(I) was quantitatively decreased in LDL ghosts and in trilinolein-reconstituted LDL, also lacking the initial rapid reduction and the subsequent inhibition phases, due to the absence of endogenous antioxidants. Conversely, the rate of copper reduction was linear and likely due to lipid peroxides, either already present or formed during copper-induced oxidation. We suggest that copper undergoes redox transitions in LDL by utilizing reducing equivalents originating from endogenous antioxidants and/or from lipid peroxides in the LDL lipid core.     

Lipoprotein Oxidation and Induction of Ferroxidase Activity in Stored Human Extracellular Fluids

It was observed that during the storage of human extracellular fluids at – 20°C the azide-inhibitable ferroxidase activity of caeruloplasmin declined, whilst a new azide-resistant ferroxidase activity (ARFA) developed. The literature suggested that storage-induced ARFA might be due to either a poorly defined enzymatic activity of a low density lipoprotein (LDL) or to lipid peroxides formed within the different lipoprotein fractions. To study this further, the major lipoprotein classes were separated from human serum by density gradient centrifugation. After storage of the lipoprotein fractions, it was found that the LDL fraction had the highest specific activity of ARFA and the highest content of lipid peroxidation products, as assessed by diene conjugates. The ARFA of LDL correlated with its content of diene conjugates and TBA reactive material, which initially suggested that the Fe(II) oxidising activity of peroxidised LDL arose from the reduction of peroxides by Fe(II) in the classical reaction between the metal ion and free radical reduction of lipid peroxides. However. steady state kinetic analysis indicated an enzymic role of LDL in Fe(II) oxidation, with lipid peroxides acting as a substrate for the enzyme. These results indicate that LDL may contain a peroxidase activity. catalysing the oxidation of Fe(II) by lipid peroxides, as well as a ferrous oxidase activity where O₂ is the oxidising substrate.     

人血浆低密度脂蛋白亚组分氧化反应敏感性的比较

本文对３种ＬＤＬ亚组分在体外对Ｃｕ＾２＋催化氧化反应敏感性进行了比较。结果表明,随氧化时间延长,各ＬＤＬ亚组分的电泳迁移率均增加。测定脂质过氧化物的含量以及用结合二烯法测定氧化反应的潜伏期,发现较高密度的ＬＤＬ亚组分更易氧化。荧光免疫测定结果显示,较高密度ＬＤＬ中载脂蛋白Ｂ上新生的４－羟壬烯醛抗原决定簇的表达高于较低密度的ＬＤＬ,从而证明较高密度的ＬＤＬ亚组分对氧化反应的敏感性高于较低密度的亚组分     

Does paraoxonase play a role in susceptibility to cardiovascular disease?

Human serum paraoxonase (PON1) is an esterase that is bound to high-density lipoproteins (HDLs). It can hydrolyze organophosphates and its activity is inversely related to atherosclerosis. Some studies also suggest that a relationship exists between polymorphisms of the gene that encodes paraoxonase and coronary heart disease (CHD), whereas other studies, in different populations, have not found such an association. One mechanism by which certain PON1 allozymes might protect against atherosclerosis is by inhibition of the oxidation of HDL and low-density lipoprotein (LDL). Experimental studies suggest that this protection is associated with the ability of PON1 to hydrolyze specific lipid peroxides in oxidized lipoproteins. Interventions that preserve or enhance PON1 activity, as well as manipulations of PON1 polymorphisms, might help delay the onset of CHD.     

Ibuprofen protects low density lipoproteins against oxidative modification

Oxidative modification of LDL by vascular cells has been proposed as the mechanism by which LDL become atherogenic. The effect of ibuprofen on LDL modification by copper ions, monocytes and endothelial cells was studied by measuring lipid peroxidation products. Ibuprofen inhibited LDL oxidation in a dose-dependent manner over a concentration range of 0.1 to 2.0 mM. Ibuprofen (2 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during 2 and 6 h incubation in the presence of copper ions by 52 and 28%, respectively. Weak free radical scavenging activity of ibuprofen was observed in the DPPH test. The protective effect of ibuprofen was more marked when oxidation was induced by monocytes or endothelial cells. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides generated in LDL during monocyte-mediated oxidation by 40%. HUVEC-mediated oxidation of LDL in the absence and presence of Cu2+ was reduced by 32 and 39%, respectively. More lipid peroxides appeared when endothelial cells were stimulated by IL-1beta or TNFalpha and the inhibitory effect of ibuprofen in this case was more pronounced. Ibuprofen (1 mM, 100 microg/ml LDL) reduced the amount of lipid peroxides formed during incubation of LDL with IL-1beta-stimulated HUVEC by 43%. The figures in the absence and presence of Cu2+ for HUVEC stimulated with TNFalpha were 56 and 59%, respectively. To assess the possibility that ibuprofen acts by lowering the production rate of reactive oxygen species, the intracellular concentration of H2O2 was measured. Ibuprofen (1 mM) reduced intracellular production of hydrogen peroxide in PMA-stimulated mononuclear cells by 69%. When HUVEC were stimulated by IL-1beta or TNFalpha the reduction was 62% and 66%, respectively.     

Enhanced lipid oxidation by oxidatively modified myoglobin: role of protein-bound heme

The formation of oxidized low density lipoprotein (LDL) is believed to play a significant role in the pathogenesis of atherosclerosis. Myoglobin in the presence of H(2)O(2) has been shown to catalyze LDL oxidation in vitro. It is established that an oxidatively altered form of myoglobin (Mb-H), which contains a prosthetic heme covalently crosslinked to the apoprotein, is a major product in the reaction of native myoglobin with peroxides. In the current study, we have shown for the first time that Mb-H, in the absence of exogenously added peroxides, oxidizes LDL and purified lipids, as determined by the formation of conjugated dienes, lipid peroxides, and thiobarbituric acid reactive substances. Moreover, the rate of oxidation of pure phosphatidylcholine by Mb-H was found to be at least sevenfold greater than that observed for native myoglobin. The current study strongly suggests a role for Mb-H in the lipid peroxidation observed with myoglobin.     

Lipid profiling of FPLC-separated lipoprotein fractions by electrospray ionization tandem mass spectrometry

Glycerophospholipid and sphingolipid species and their bioactive metabolites are important regulators of lipoprotein and cell function. The aim of the study was to develop a method for lipid species profiling of separated lipoprotein classes. Human serum lipoproteins VLDL, LDL, and HDL of 21 healthy fasting blood donors were separated by fast performance liquid chromatography (FPLC) from 50 microl serum. Subsequently, phosphatidylcholine (PC), lysophosphatidylcholine, sphingomyelin (SM), ceramide (CER), phosphatidylethanolamine (PE), PE-based plasmalogen (PE-pl), cholesterol, and cholesteryl ester (CE) content of the separated lipoproteins was quantified by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Analysis of FPLC fractions with PAGE demonstrated that albumin partially coelutes with HDL fractions. However, analysis of an HDL deficient serum (Tangier disease) showed that only lysophosphatidylcholine, but none of the other lipids analyzed, exhibited a significant coelution with the albumin containing fractions. Approximately 60% of lipoprotein CER were found in LDL fractions and 60% of PC, PE, and plasmalogens in HDL fractions. VLDL, LDL, and HDL displayed characteristic lipid class and species pattern. The developed method provides a detailed lipid class and species composition of lipoprotein fractions and may serve as a valuable tool to identify alterations of lipoprotein lipid species profiles in disease with a reasonable experimental effort.     

Degradation of HNE-modified proteins--possible role of ubiquitin

4-Hydroxynonenal (HNE) is a lipid peroxidation product that is able to modify proteins. HNE-modified proteins are degraded to a considerable extend by the proteasomal system. It is unclear whether the recognition of HNE-modified proteins is mediated by ubiquitin, or whether the ubiquitin-independent proteasomal pathway is involved. In this study we demonstrate that HNE-modified GAPDH is preferentially ubiquitinated in vitro. In an attempt to demonstrate the formation of poly-ubiquitinated HNE-modified proteins in living cells we explored E36 fibroblasts. A clear rise in HNE-protein modification could be demonstrated after HNE treatment of the cells. Using inhibitors, we could show that the ubiquitin-dependent, ubiquitin-independent, and the lysosomal pathways affect the presence of HNE-modified proteins. We conclude that, although several proteolytic pathways exist for the degradation of HNE-modified proteins, there is the possibility of involvement of ubiquitin-dependent degradation.     

Oxidized lipoprotein lipids and atherosclerosis

Plasma lipoproteins contain variable amounts of lipid oxidation products (LOP), which are known to impair normal physiological functions and stimulate atherosclerotic processes. Recent evidence indicates that plasma lipoproteins are active carriers of LOP, low-density lipoprotein (LDL) directing transport toward peripheral tissues, and high-density lipoprotein (HDL) being active in the reverse transport. It has been proposed that the lipoprotein-specific transport of LOP could play a role in atherosclerosis-related effects of LDL and HDL. This article gives an overview of the present knowledge of lipoprotein LOP transport and its association with the risk of atherosclerosis and cardiovascular diseases (CVD). Evidence of the significance of lipoprotein LOP transport comes mainly from studies of physiological oxidative stress and is supported by studies of the functionality apolipoprotein A-1 mimetic peptides. A large body of data has accumulated indicating that lipoprotein LOP transport is connected to the risk of atherosclerosis. While high levels of LOP carried by LDL are indicative of elevated risk, high LOP level in HDL appears to associate with protection. If confirmed, the proposed lipoprotein LOP transport function would affect conception of the etiology of atherosclerosis, but would not conflict current views of the pathophysiological mechanisms. It could open new perspectives, such as the dietary origin of LOP, and the protective function of HDL in clearance of LOP. Focusing on LOP could give additional tools especially for prevention and diagnosis, but would not radically change the management of atherosclerosis and CVD.     

Age-related decrease of dehydroepiandrosterone concentrations in low density lipoproteins and its role in the susceptibility of low density lipoproteins to lipid peroxidation

The incidence of atherosclerosis and related diseases increases with age. The aging process may enhance lipoprotein modification, which leads to an increase in the susceptibility of low density lipoprotein (LDL) and high density lipoprotein (HDL) to oxidation. Dehydroepiandrosterone (DHEA), the most abundant steroid hormone in humans, has been shown to have antiatherogenic effects. This hormone also decreases dramatically with age. In the present study, we were interested in determining the presence of DHEA/DHEAS (dehydroepiandrosterone sulfate) and changes in their concentrations in HDL and LDL lipoproteins with age. Moreover, we studied the susceptibility of LDL to oxidation with age in the presence or absence of vitamin E or DHEA. We demonstrated that vitamin E is unable to restore the decreased resistance to oxidation of LDL from elderly subjects to that of LDL obtained from young subjects. Furthermore, our results provide evidence that DHEA is an integral part of LDL and HDL and disappears to almost nondetectable levels during aging. The DHEA incorporated into the LDL from elderly subjects increased LDL resistance to oxidation in a concentration-dependent manner. The increased resistance provided by DHEA was higher than that with vitamin E. DHEA seems to act either by protecting vitamin E from disappearance from LDL under oxidation or by scavenging directly the free radicals produced during the oxidative process. Our results suggests that DHEA exerts an antioxidative effect on LDL, which could have antiatherogenic consequences. Careful clinical trials of DHEA replacement should determine whether this ex vivo effect could be translated into any measurable antiatherogenic (cardioprotective) action.