Serum lipidomics reveals early differential effects of gastric bypass compared with banding on phospholipids and sphingolipids independent of differences in weight loss.

BACKGROUND/OBJECTIVES: Circulating phospholipids and sphingolipids are implicated in obesity-related comorbidities such as insulin resistance and cardiovascular disease. How bariatric surgery affects these important lipid markers is poorly understood. We sought to determine whether Roux-en-Y gastric bypass (RYGB), which is associated with greater metabolic improvement, differentially affects the phosphosphingolipidome compared with adjustable gastric banding (AGB). SUBJECTS/METHODS: Fasting sera were available from 59 obese women (body mass index range 37-51 kg m-2; n=37 RYGB and 22 AGB) before surgery, then at 1 (21 RYGB, 12 AGB) and 3 months follow-up (19 RYGB, 12 AGB). HPLC-MS/MS was used to quantify 131 lipids from nine structural classes. DXA measurements and laboratory parameters were also obtained. The associations between lipids and clinical measurements were studied with P-values adjusted for the false discovery rate (FDR). RESULTS: Both surgical procedures rapidly induced weight loss and improved clinical profiles, with RYGB producing better improvements in fat mass, and serum total cholesterol, low-density lipoprotein-cholesterol (LDL-C) and orosomucoid (FDR <10%). Ninety-three (of 131) lipids were altered by surgery-the majority decreasing-with 29 lipids differentially affected by RYGB during the study period. The differential effect of the surgeries remained statistically significant for 20 of these lipids after adjusting for differences in weight loss between surgery types. The RYGB signature consisted of phosphatidylcholine species not exceeding 36 carbons, and ceramides and sphingomyelins containing C22 to C25 fatty acids. RYGB also led to a sustained increase in unsaturated ceramide and sphingomyelin species. The RYGB-specific lipid changes were associated with decreases in body weight, total and LDL-C, orosomucoid and increased HOMA-S (FDR <10%). CONCLUSIONS: Concomitant with greater metabolic improvement, RYGB induced early and sustained changes in phosphatidylcholines, sphingomyelins and ceramides that were independent of greater weight loss. These data suggest that RYGB may specifically alter sphingolipid metabolism, which, in part, could explain the better metabolic outcomes of this surgical procedure.

[The biological activity of high-density lipoprotein fractions and their role in the development of cardiovascular diseases]. / Biologicheskaia aktivnost' fraktsii lipoproteidov vysokoi plotnosti i ikh rol' v razvitii serdechno-sosudistykh zabolevanii.

Increasing the human plasma concentration of high-density lipoproteins (HDL) may be part of strategy for control of cardiovascular diseases (CVD). HDL particles vary in their structure, metabolism, and biological activity. The review describes major HDL fractions (subpopulations) and discusses new findings on the antiatherogenic properties of HDL particles. The whole spectrum of HDL fractions, small, dense, protein-rich lipoproteins, has atheroprotective properties that are determined by the presence of specialized groups of proteins and lipids; however, this activity may be decreased in atherogenic lesion. Comprehensive structural and compositional analysis of HDL may provide key information to identify the fractions that have characteristic biological properties and lose their functionality in CVD. These fractions may be also biomarkers for the risk of CVD and hence represent pharmacological targets.

An overview of the new frontiers in the treatment of atherogenic dyslipidemias.

Cardiovascular diseases (CVDs) are the leading cause of morbidity/mortality worldwide. Dyslipidemia is a major risk factor for premature atherosclerosis and CVD. Lowering low-density-lipoprotein cholesterol (LDL-C) levels is well established as an intervention for the reduction of CVDs. Statins are the first-line drugs for treatment of dyslipidemia, but they do not address all CVD risk. Development of novel therapies is ongoing and includes the following: (i) reduction of LDL-C concentrations using antibodies to proprotein convertase subtilisin/kexin-9, antisense oligonucleotide inhibitors of apolipoprotein B production, microsomal transfer protein (MTP) inhibitors, and acyl-coenzyme A cholesterol acyl transferase inhibitors; (ii) reduction in levels of triglyceride-rich lipoproteins with ω-3 fatty acids, MTP inhibitors, and diacylglycerol acyl transferase-1 inhibitors; and (iii) increase of high-density-lipoprotein (HDL) cholesterol levels, HDL particle numbers, and/or HDL functionality using cholesteryl ester transfer protein inhibitors, HDL-derived agents, apolipoprotein AI mimetic peptides, and microRNAs. Large prospective outcome trials of several of these emerging therapies are under way, and thrilling progress in the field of lipid management is anticipated.

Relationship between alcohol intake, health and social status and cardiovascular risk factors in the Urban Paris-Ile-de-France Cohort: is the cardioprotective action of alcohol a myth?

BACKGROUND/OBJECTIVES: Observational studies document the inverse relationship between cardiovascular disease (CVD) and moderate alcohol intake. However, the causal role for alcohol in cardioprotection remains uncertain as such protection may be caused by confounders and misclassification. The aim of our study was to evaluate potential confounders, which may contribute to putative cardioprotection by alcohol. SUBJECTS/METHODS: We evaluated clinical and biological characteristics, including cardiovascular (CV) risk factors and health status, of 149,773 subjects undergoing examination at our Center for CVD Prevention (The Urban Paris-Ile-de-France Cohort). The subjects were divided into four groups according to alcohol consumption: never, low (30 g/day); former drinkers were analyzed as a separate group. RESULTS: After adjustment for age, moderate male drinkers were more likely to display clinical and biological characteristics associated with lower CV risk, including low body mass index, heart rate, pulse pressure, fasting triglycerides, fasting glucose, stress and depression scores together with superior subjective health status, respiratory function, social status and physical activity. Moderate female drinkers equally displayed low waist circumference, blood pressure and fasting triglycerides and low-density lipoprotein-cholesterol. Alcohol intake was strongly associated with plasma high-density lipoprotein-cholesterol in both sexes. Multivariate analysis confirmed that moderate and low drinkers displayed better health status than did never drinkers. Importantly, few factors were causally related to alcohol intake. CONCLUSIONS: Moderate alcohol drinkers display a more favorable clinical and biological profile, consistent with lower CV risk as compared with nondrinkers and heavy drinkers. Therefore, moderate alcohol consumption may represent a marker of higher social level, superior health status and lower CV risk.

A machine-learning approach to the prediction of oxidative stress in chronic inflammatory disease.

Oxidative stress is implicated in the development of a wide range of chronic human diseases, ranging from cardiovascular to neurodegenerative and inflammatory disorders. As oxidative stress results from a complex cascade of biochemical reactions, its quantitative prediction remains incomplete. Here, we describe a machine-learning approach to the prediction of levels of oxidative stress in human subjects. From a database of biochemical analyses of oxidative stress biomarkers in blood, plasma and urine, non-linear models have been designed, with a statistical methodology that includes variable selection, model training and model selection. Our data demonstrate that, despite a large inter- and intra-individual variability, levels of biomarkers of oxidative damage in biological fluids can be predicted quantitatively from measured concentrations of a limited number of exogenous and endogenous antioxidants.

Perturbation of hyaluronan metabolism predisposes patients with type 1 diabetes mellitus to atherosclerosis.

AIMS/HYPOTHESIS: Cardiovascular disease contributes to mortality in type 1 diabetes mellitus, but the specific pathophysiological mechanisms remain to be established. We recently showed that the endothelial glycocalyx, a protective layer of proteoglycans covering the endothelium, is severely perturbed in type 1 diabetes, with concomitantly increased plasma levels of hyaluronan and hyaluronidase. In the present study, we evaluated the relationship between hyaluronan and hyaluronidase with carotid intima-media thickness (cIMT), an established surrogate marker for cardiovascular disease. SUBJECTS AND METHODS: Non-smoking type 1 diabetes patients without micro- or macrovascular complications and matched controls were recruited and cIMT of both carotid arteries was measured. To evaluate the relationship between cIMT and hyaluronan and hyaluronidase as well as other parameters, uni- or multivariate regression analyses were performed. RESULTS: We included 99 type 1 diabetes patients (age 10-72 years) and 99 age- and sex-matched controls. Mean cIMT, HbA(1c), high sensitivity C-reactive protein, hyaluronan and hyaluronidase were significantly increased in type 1 diabetes vs controls. Plasma hyaluronan and hyaluronidase were correlated in type 1 diabetes. In univariate regression analyses, mean IMT was associated with plasma hyaluronan, age and male sex, whereas after multivariate analysis only age and sex remained statistically significant. CONCLUSIONS/INTERPRETATION: We conclude that type 1 diabetes patients show structural changes of the arterial wall associated with increased hyaluronan metabolism. These data may lend further support to altered glycosaminoglycan metabolism in type 1 diabetes as a potential mechanism involved in accelerated atherogenesis.

Defective antioxidative activity of small dense HDL3 particles in type 2 diabetes: relationship to elevated oxidative stress and hyperglycaemia.

AIMS/HYPOTHESIS: Elevated oxidative stress, hyperglycaemia, and dyslipidaemia involving low levels of HDL particles are key proatherogenic factors in type 2 diabetes mellitus. We examined the relationship of oxidative stress, and the degree of glycaemia and triglyceridaemia, to antioxidative function of HDL particle subspecies in type 2 diabetes. SUBJECTS AND METHODS: Five HDL subfractions (2b, 2a, 3a, 3b, 3c) were isolated by density gradient ultracentrifugation from well-controlled type 2 diabetic subjects (n=20) and normolipidaemic, non-diabetic controls (n=10). Specific antioxidative activity (capacity to protect LDL from oxidation on a unit particle mass or on a particle number basis), chemical composition and enzymatic activities were measured in each subfraction. Systemic oxidative stress was assessed as plasma levels of 8-isoprostanes. RESULTS: Specific antioxidative activity of small dense HDL3b and 3c particles in diabetic patients was significantly diminished (up to -47%, on a particle mass or particle number basis) as compared with controls. Plasma 8-isoprostanes were markedly elevated (2.9-fold) in diabetic patients, were negatively correlated with both specific antioxidative activity of HDL3 subfractions and plasma HDL cholesterol (HDL-C) levels, and were positively correlated with glycaemia and triglyceridaemia. Paraoxonase 1 activity was consistently lower in diabetic HDL subfractions and was positively correlated with HDL3 antioxidative activity. The altered chemical composition of diabetic HDL3 subfractions (core cholesteryl ester depletion, triglyceride enrichment) was equally correlated with diminished antioxidative activity. CONCLUSIONS/INTERPRETATION: Antioxidative activity of small dense HDL is deficient in type 2 diabetes, is intimately linked to oxidative stress, glycaemia and hypertriglyceridaemia and primarily reflects abnormal intrinsic physicochemical properties of HDL particles.

Amyloid-beta: an antioxidant that becomes a pro-oxidant and critically contributes to Alzheimer's disease.

Elevated production of amyloid-beta (A beta) as a preventive antioxidant for brain lipoproteins under the action of increased oxidative stress in aging is postulated to represent a major event in the development of Alzheimer's disease (AD). Increase in A beta production is followed by chelation of transition metal ions by A beta, accumulation of A beta-metal lipoprotein aggregates, production of reactive oxygen species and neurotoxicity. Chelation of copper by A beta is proposed to be a most important part of this pathway, because A beta binds copper stronger than other transition metals and because copper is a more efficient catalyst of oxidation than other metals. This amyloid-binds-copper (ABC) model does not remove A beta peptide from its central place in our current thinking of AD, but rather places additional factors in the center of discussion. Most importantly, they embrace pathological mechanisms known to develop in aging (which is the major risk factor for AD), such as increased production of reactive oxygen species by mitochondria, that are positioned upstream relative to the generation of A beta.

Influence of vitamin E and C supplementation on lipoprotein oxidation in patients with Alzheimer's disease.

Because increased oxidation is an important feature of Alzheimer's disease (AD) and low concentrations of antioxidant vitamins C and E have been observed in cerebrospinal fluid (CSF) of AD patients, supplementation with these antioxidants might delay the development of AD. Major targets for oxidation in brain are lipids and lipoproteins. We studied whether supplementation with antioxidative vitamins E and C can increase their concentrations not only in plasma but also in CSF, and as a consequence decrease the susceptibility of lipoproteins to in vitro oxidation. Two groups, each consisting of 10 patients with AD, were for 1 month supplemented daily with either a combination of 400 IU vitamin E and 1000 mg vitamin C, or 400 IU vitamin E alone. We found that supplementation with vitamin E and C significantly increased the concentrations of both vitamins in plasma and CSF. Importantly, the abnormally low concentrations of vitamin C were returned to normal level following treatment. As a consequence, susceptibility of CSF and plasma lipoproteins to in vitro oxidation was significantly decreased. In contrast, the supplementation with vitamin E alone significantly increased its CSF and plasma concentrations, but was unable to decrease the lipoprotein oxidizability. These findings document a superiority of a combined vitamin E + C supplementation over a vitamin E supplementation alone in AD and provide a biochemical basis for its use.

The role of vitamin E in atherogenesis: linking the chemical, biological and clinical aspects of the disease.

Atherosclerosis is a disease involving both oxidative modifications and disbalance of the immune system. Vitamin E, an endogenous redox-active component of circulating lipoproteins and (sub)cellular membranes whose levels can be manipulated by supplementation, has been shown to play a role in the initiation and progression of the disease. Recent data reveal that the activities of vitamin E go beyond its redox function. Moreover, it has been shown that vitamin E can exacerbate certain processes associated with atherogenesis. In this essay we review the role of biology of atherosclerosis, and suggest that these two facets decide the clinical manifestation and outcome of the disease.

The effect of pharmacological doses of different antioxidants on oxidation parameters and atherogenesis in hyperlipidaemic rabbits.

The oxidation hypothesis of atherosclerosis implies that antioxidants are able to inhibit lipoprotein oxidation in the arterial wall and thereby retard atherogenesis. Since most of the animal studies performed have used very high doses of antioxidants, it is to date unknown whether antioxidants are effective antiatherosclerotic agents when given in pharmacological doses. Here we addressed this question using homozygous Watanabe heritable hyperlipidaemic (WHHL) rabbits as an animal model of atherosclerosis. The rabbits were divided into four groups, each consisting of ten animals. They received either a standard diet or a diet containing 4.3 mg ubiquinone-10, or 4.3 mg vitamin E or 15 mg probucol/kg body weight daily. After 12 months, the extent of aortic atherosclerosis was assessed as the intima thickness, media thickness and intima-to-media ratio in 14 cross sections equally distributed over the whole aorta. To evaluate the antioxidant effects of the diet, lipophilic and hydrophilic antioxidants, lipids, fatty acids and plasma oxidizability were measured after 0, 3 and 6 months of feeding. We found that supplementation with probucol significantly decreased aortic intima-to-media ratio compared to controls. The antiatherosclerotic action of probucol was accompanied by its beneficial action on plasma oxidizability and some plasma antioxidants. No decrease in aortic atherosclerosis was measured in ubiquinone-10- and vitamin E-supplemented rabbits, despite the fact that both antioxidants decreased plasma oxidizability and ubiquinone-10 increased the plasma levels of antioxidants. Taken together, these data suggest that pharmacological doses of probucol retard atherogenesis in WHHL rabbits by an antioxidant mechanism, while ubiquinone-10 and vitamin E at these dosages are ineffective in this highly hyperlipidaemic model. The measurement of some oxidation-related parameters in plasma, such as lipophilic antioxidants, polyunsaturated fatty acids and lipoprotein oxidizability, may be useful in assessing the risk of atherogenesis in humans.

Amyloid-beta is an antioxidant for lipoproteins in cerebrospinal fluid and plasma.

Amyloid-beta (Abeta) peptide, a major constituent of senile plaques and a hallmark of Alzheimer's disease (AD), is normally secreted by neurons and can be found in low concentrations in cerebrospinal fluid (CSF) and plasma, where it is associated with lipoproteins. However, the physiological role of Abeta secretion remains unknown. Here we show that at the concentrations measured in biological fluids (0.1-1.0 nM), Abeta(1-40) strongly inhibits autooxidation of CSF lipoproteins and plasma low density lipoprotein (LDL). At higher concentrations of the peptide its antioxidant action was abolished. Abeta(1-40) also inhibited copper-catalyzed LDL oxidation when added in molar excess of copper, but did not influence oxidation induced by an azo-initiator. Other Abeta peptides also possessed antioxidant activity in the order Abeta(1-40) > Abeta(1-42) > Abeta(25-35), whereas Abeta(35-25) was inactive. These data suggest that Abeta(1-40) may act as a physiological antioxidant in CSF and plasma lipoproteins, functioning by chelating transition metal ions.

[Lipid peroxidation as a common pathomechanism in coronary heart disease and Alzheimer disease]. / Lipoproteinoxidation als gemeinsamer Pathomechanismus der koronaren Herzerkrankung und der Alzheimer Krankheit.

Oxidative processes are involved in aging as well as the pathogenesis of different degenerative diseases. In the last few years the role of low density lipoprotein oxidation in the development of artherosclerosis and coronary heart disease has become evident. Lipoprotein oxidation in plasma is used as a marker for disease progression. We were interested in the role of lipoprotein oxidation in Alzheimer's disease. For this purpose we developed methods to determine the in vitro oxidizability of cerebrospinal fluid and plasma lipoproteins of Alzheimer patients. In addition we measured the lipophilic and hydrophillic antioxidants, alpha-tocopherol (vitamin E) and ascorbate (vitamin C). Cerebrospinal fluid and plasma lipoprotein oxidation was found to be increased in Alzheimer's patients compared to controls and a corresponding decrease of antioxidant vitamins was found. In a pilot study, in vitro lipoprotein oxidation in cerebrospinal fluid of Alzheimer patients could be delayed by vitamin E and C supplementation. In conclusion these data show that increased lipoprotein oxidation could play an important role in Alzheimer's disease and possibly provide a rationale for the treatment of this disease with antioxidant drugs. The clinical effect of this therapeutical approach remains to be proved in long-term studies.

Alzheimer's amyloid-beta as a preventive antioxidant for brain lipoproteins.

1. Increased production of A beta in a form of lipoprotein antioxidant under the action of increased oxidative stress in aging with subsequent chelation of transition metal ions by A beta, accumulation of toxic A beta-metal lipoprotein complexes, production of reactive oxygen species, and neurotoxicity are reviewed and postulated to form the temporal sequence of events in the development of Alzheimer's disease (AD). 2. Since (i) A beta binds copper stronger than iron and other transition metals, and (ii) copper is a more efficient catalyst of oxidation than other transition metals, chelation of copper by A beta is proposed to be a most important part of this pathway. 3. Whereas this amyloid-binds-copper (ABC) model does not remove A beta peptide from its central place in our current thinking of AD, it places additional factors in the center of discussion. 4. Most importantly, they embrace pathological mechanisms known to develop in aging (which is the most important risk factor for AD), such as increased production of reactive oxygen species by mitochondria, that can be positioned upstream relative to the generation of A beta.

Resistance of human cerebrospinal fluid to in vitro oxidation is directly related to its amyloid-beta content.

Amyloid-beta (A beta) peptide, a major constituent of senile plaques and a hallmark of Alzheimer's disease (AD), is normally secreted by neurons and can be found in low concentrations in cerebrospinal fluid (CSF) and plasma where it is associated with lipoproteins. However, the physiological role of A beta secretion remains unknown. We measured the resistance to in vitro oxidation of CSF obtained from 20 control subjects and 30 patients with AD, and correlated it with CSF levels of antioxidants, lipids and A beta. We found that the oxidative resistance, expressed as a duration of the oxidation lag-phase, was directly related to CSF levels of A beta 1-40, A beta 1-42 and ascorbate and inversely to levels of fatty acids. These data suggest that, besides ascorbate, A beta is another major physiological antioxidant for CSF lipoproteins.

Increased lipoprotein oxidation in Alzheimer's disease.

Oxidation has been proposed to be an important factor in the pathogenesis of Alzheimer's disease (AD) and amyloid beta is considered to induce oxidation. In biological fluids, including cerebrospinal fluid (CSF), amyloid beta is found complexed to lipoproteins. On the basis of these observations, we investigated the potential role of lipoprotein oxidation in the pathology of AD. Lipoprotein oxidizability was measured in vitro in CSF and plasma from 29 AD patients and found to be significantly increased in comparison to 29 nondemented controls. The levels of the hydrophilic antioxidant ascorbate were significantly lower in CSF and plasma from AD patients. In plasma, alpha-carotene was significantly lower in AD patients compared to controls while alpha-tocopherol levels were indistinguishable between patients and controls. In CSF, a nonsignificant trend to lower alpha-tocopherol levels among AD patients was found. Polyunsaturated fatty acids, the lipid substrate for oxidation, were significantly lower in the CSF of AD patients. Our findings suggest that (i) lipoprotein oxidation may be important in the development of AD and (ii) the in vitro measurement of lipid peroxidation in CSF might become a useful additional marker for diagnosis of AD.

Time-course of oxidation of lipids in human cerebrospinal fluid in vitro.

Oxidative mechanisms play an important role in the pathogenesis of Alzheimer's disease, Parkinson's disease and other neurodegenerative diseases. To assess whether the oxidation of brain lipoproteins plays a role in the development of these pathologies, we investigated whether the lipoproteins of human cerebrospinal fluid (CSF) are susceptible to oxidative modification in vitro. We studied oxidation time-course for up to 100 h of human CSF in the absence (autooxidation) or presence of exogenous oxidants. Autooxidation of diluted CSF was found to result in a slow accumulation of lipid peroxidation products. The time-course of lipid hydroperoxide accumulation revealed three consecutive phases, lag-phase, propagation phase and plateau phase. Qualitatively similar time-course has been typically found in human plasma and plasma lipoproteins. Autooxidation of CSF was accelerated by adding exogenous oxidants, delayed by adding antioxidants and completely inhibited by adding a chelator of transition metal ions. Autooxidation of CSF also resulted in the consumption of endogenous ascorbate, alpha-tocopherol, urate and linoleic and arachidonic acids. Taking into account that (i) lipid peroxidation products measured in our study are known to be derived from fatty acids, and (ii) lipophilic antioxidants and fatty acids present in CSF are likely to be located in CSF lipoproteins, we conclude that lipoproteins of human CSF are modified in vitro during its autooxidation. This autooxidation appears to be catalyzed by transition metal ions, such as Cu(II) and Fe(III), which are present in native CSF. These data suggest that the oxidation of CSF lipoproteins might occur in vivo and play a role in the pathogenesis of neurodegenerative diseases.

Alpha- and beta-carotenes in low density lipoprotein are the preferred target for nitric oxide-induced oxidation.

Whereas low plasma levels of carotenes are strongly associated with the elevated risk of atherosclerosis, the reason for this is still unknown. We hypothesized that lipoprotein oxidation in the arterial wall might selectively deplete carotenes, thus explaining the observed effects. In order to assess this hypothesis, we incubated plasma low density lipoprotein (LDL) with different oxidants and measured the consumption of carotenes and tocopherols. We found that when LDL oxidation was induced by nitric oxide, both alpha- and beta-carotene were consumed at a significantly higher relative rate than alpha- or gamma-tocopherol. In contrast, superoxide, peroxynitrite, hypochlorite or transition metal ions were unable to induce selective consumption of carotenes in LDL. These data suggest that the decreased plasma levels of alpha- and beta-carotene frequently measured in atherosclerosis may be related to their preferred consumption by reactive nitrogen species in the arterial wall.

Plasma ubiquinol-10 as a marker for disease: is the assay worthwhile?

Ubiquinol-10 and ubiquinone-10 were measured in plasma of patients with several pathologies known to be associated with increased oxidative stress. Plasma ubiquinol-10, expressed as a percentage of total ubiquinol-10 + ubiquinone-10, was found to be significantly lower in hyperlipidaemic patients and in patients with liver diseases than in age-matched control subjects. In contrast, no decrease in ubiquinol-10 was detected in plasma of patients with coronary heart disease and Alzheimer's disease. Except for ubiquinol-10, no other lipophilic antioxidant was found to be decreased in patients with liver diseases. These data suggest that the level of ubiquinol-10 in human plasma may serve as a marker for liver dysfunction, reflecting its diminished reduction by the liver rather than increased consumption by oxidants.

Lipophilic antioxidants in blood plasma as markers of atherosclerosis: the role of alpha-carotene and gamma-tocopherol.

Oxidative theory of atherosclerosis implies that plasma levels of lipophilic antioxidants might serve as indicators of lipoprotein oxidation in the arterial wall and as markers of the development of atherosclerosis. However, it is unknown whether the measurement of plasma antioxidants is able to reflect atherogenesis or its risk. In order to assess whether the levels of lipophilic antioxidants in human plasma can discriminate between subjects with and without atherosclerosis, we measured the lipophilic antioxidants alpha-tocopherol, gamma-tocopherol, alpha-carotene, beta-carotene and ubiquinol-10 in plasma of 34 patients with coronary heart disease (CHD) and in 40 control subjects. We found that alpha-carotene and gamma-tocopherol were significantly lower in plasma of CHD patients compared to controls. This decrease was significantly independent of whether the antioxidants were expressed as its absolute amounts in plasma (P < 0.001 for alpha-carotene, and P = 0.001 for gamma-tocopherol) or normalized to plasma lipids (P < 0.001 for both). In contrast, beta-carotene was only lower in plasma of CHD patients in comparison to controls, when normalized to the lipids (P = 0.02). Independent contributions of different parameters to the variation in these plasma antioxidants were estimated using multiple regression approach. The analysis showed that both the decrease in alpha-carotene and the decrease in gamma-tocopherol were significantly associated only with the presence of CHD (P < 0.001 for each regression), while the decrease in beta-carotene was significantly related to the presence of hyperlipidaemia (P < 0.001). In striking contrast, no decrease in plasma alpha-tocopherol and ubiquinol-10 was detected in the patient group independently of how these antioxidants were expressed. These data suggest that plasma levels of alpha-carotene and gamma-tocopherol may represent markers of atherosclerosis in humans. Measuring these antioxidants may be of clinical importance as a practical approach to assess atherogenesis and/or its risk.