The antioxidant properties of high-density lipoproteins in atherosclerosis.

High-density lipoprotein (HDL) is protective against atherosclerosis development. Other than its central role in reverse cholesterol transport, HDL exhibits several other mechanisms by which it is protective. These include antioxidative, anti-inflammatory and antiapoptopic activities and the normalisation of vascular function. In light of the current view that oxidative modification of low-density lipoprotein (LDL) is essential for the initiation and progression of atherosclerosis, the antioxidative properties of HDL may be an important protective mechanism. HDL can retard the oxidation of LDL and limit its atherogenicity. Several proteins are present on HDL and the evidence that some of them metabolise lipid peroxidation products of phospholipids, cholesteryl esters and triglycerides associated with LDL and vascular cell membranes are discussed in this review.

The role of combined assessment of defense against oxidative stress and inflammation in the evaluation of peripheral arterial disease.

Atherosclerosis in symptomatic peripheral arterial disease affects wide portions of numerous arteries in lower extremities. The resulting active inflammation in a considerable amount of arterial tissue facilitates systemic detection via measurement of inflammation-related variables. We reasoned that the combined assessment of defense against oxidative stress, in the form of paraoxonase-1 (PON1), and monocyte migration measured as circulating (C-C motif) ligand 2 (CCL2), may play a role in the evaluation of these patients. Plasma CCL2 and serum PON1-related variables, assessed by their interaction with functional genetic variants, were measured in a cross-sectional study in patients with symptomatic PAD. We found that PON1 activity and concentration were significantly lower and CCL2 concentration higher in PAD patients compared to controls, that the combination of plasma CCL2 and PON1- related values, especially PON1 concentration differentiated, almost perfectly, controls from patients and that the expression of CCL2 and PON1 generally co-localized in the atherosclerotic lesion. Since no association with genetic variants was found, such a relationship is probably the result of the disease. Our data suggest a coordinated role between CCL2 and PON1 that may be detected in blood with simple measurements and may represent an indicator of the extent of atherosclerosis.

Triglycerides and small dense low density lipoprotein in the discrimination of coronary heart disease risk in South Asian populations.

INTRODUCTION: Coronary heart disease (CHD) is exceptionally prevalent amongst globally dispersed migrant groups originating from the Indian subcontinent, but the contribution of dyslipidaemia to their increased risk remains poorly defined. METHODS: Fasting lipids and lipoproteins, apolipoproteins (Apo), low density lipoprotein (LDL) diameter and oxidised LDL were measured amongst rural Indians in India (n=294) and their migrant contemporaries in the UK (n=242). The performance of qualitative and quantitative measures of lipid metabolism were compared in the discrimination of WHO defined metabolic risk and raised Framingham CHD risk scores (>15%) using Receiver Operating Characteristic (ROC) curves. RESULTS: LDL diameter was correlated with triglycerides (R(2)=0.12, P<0.001) and with high density lipoprotein (HDL) cholesterol levels (R(2)=0.15, P<0.001) in both groups. Migrants had less small dense LDL (95% CI: 12.5-14.2%) vs. rural Indians (15.7-17.2, P<0.05). On ROC analysis, triglycerides were the only consistent discriminators of metabolic and CHD risk scores (all P< or =0.001). Apo B was also a strong indicator of raised CHD risk scores. Irrespective of site, individuals with raised triglycerides also had higher total cholesterol and Apo B, denser LDL, lower HDL and more oxidised LDL (all P< or =0.01). DISCUSSION: Fasting triglycerides reflect both qualitative and quantitative aspects of lipid metabolism, and are a comprehensive discriminator of CHD risk in this South Asian population.

Glycation of paraoxonase-1 inhibits its activity and impairs the ability of high-density lipoprotein to metabolize membrane lipid hydroperoxides.

AIMS: High-density lipoprotein (HDL) protects against atherosclerosis development. Defective functioning of HDL in Type 2 diabetes may be one cause of increased cardiovascular disease associated with Type 2 diabetes. HDL modulates low-density lipoprotein and cell membrane oxidation through the action of paraoxonase-1 (PON1), which is one of the major mechanisms by which HDL is anti-atherogenic. METHODS: We have compared the ability of HDL from Type 2 diabetic patients without coronary heart disease (CHD) (n = 36) to metabolize membrane lipid hydroperoxides with HDL from healthy control subjects (n = 19) and people with CHD but no diabetes (n = 37). RESULTS: HDL from subjects with Type 2 diabetes and CHD metabolized 20% less membrane hydroperoxides than HDL from control subjects (P < 0.05). The PON1-192RR was least efficient in all the study groups. PON1 was glycated in vivo: (7.5% control, 12% CHD, 17% Type 2 diabetes P < 0.01) with QQ isoforms most glycated. In vitro glycation of PON1 reduced its ability to metabolize membrane hydroperoxides by 50% (P < 0.001); however, glyoxidation reduced it by 80% (P < 0.001). In the control group only there was a significant negative correlation between PON1 activity and the ability of HDL to metabolize membrane hydroperoxides (r = -0.911, P < 0.001). CONCLUSIONS: HDL from Type 2 diabetic patients without CHD has decreased ability to metabolize membrane lipid hydroperoxides, which could lead to increased susceptibility to cardiovascular disease.

Paraoxonase 1 gene transfer lowers vascular oxidative stress and improves vasomotor function in apolipoprotein E-deficient mice with pre-existing atherosclerosis.

BACKGROUND AND PURPOSE: Transgenesis of human paraoxonase 1 (PON1), a HDL-associated enzyme that destroys lipid peroxides, has been reported to reduce early atherogenesis in mice. The present study explored the therapeutic potential of human PON1 gene transfer in old apolipoprotein E-deficient (apoE(-/-)) mice with advanced atherosclerosis. EXPERIMENTAL APPROACH: ApoE(-/-) mice (18 months, regular chow) were transfected with PON1 adenovirus (AdPON1, n=10) or control adenovirus (AdRR5, n=10). Non-transfected apoE(-/-) (n=9) and C57Bl/6J (WT, n=6) mice served as controls. Three weeks later, plaque size and composition, and endothelial cell (EC) and smooth muscle cell (SMC) function were assessed in the aorta. KEY RESULTS: PON1 gene transfer raised total PON1 serum activity 13-15 fold during the 3-week study period, without affecting hypercholesterolaemia or lesion size. However, PON1 decreased the oxLDL content of the plaque. Plaque-free thoracic aorta rings from apoE(-/-) mice displayed, like rings from WT mice, complete relaxation to acetylcholine (ACh, 86+/-2%), ATP (90+/-2%) or UTP (83+/-3%). In contrast, in plaque-bearing segments amplitude (55+/-7%, 68+/-8%, 52+/-8% respectively) and sensitivity were decreased. EC function was completely (ATP, UTP) or largely (ACh) restored by AdPON1. Furthermore, apoE(-/-) SMCs released less intracellular calcium than WT upon sarco-endoplasmic reticulum calcium ATPase (SERCA) inhibition by cyclopiazonic acid. This defect was also restored by AdPON1 transfection. CONCLUSIONS AND IMPLICATIONS: These data indicate that AdPON1 gene transfer improved vascular wall oxidative stress, EC function, and SMC Ca(2+) homeostasis in segments with pre-existing atherosclerosis, independently of an effect on plaque size.

GST CYP and PON1 polymorphisms in farmers attributing ill health to organophosphate-containing sheep dip.

Previously we reported that in sheep dippers exposed to organophosphates the frequency of paraoxonase (PON1) polymorphisms differed between those with or without self-reported ill health. We have now examined whether polymorphisms in other genes involved in xenobiotic metabolism alter disease risk in this population. There were elevated but non-significant risks associated with the CYP2D6 WT genotype (odds ratio (OR) 1.47, 95% CI 0.83-2.60), or a GSTP1*B or *C allele (OR 1.37, 95% CI 0.88-2.01) or being GSTM1*2/GSTT1*2 homozygous (OR 1.61, 95% CI 0.74-3.48). Similar results were generally obtained after the exclusion of subjects to obtain a more homogenous case-referent population: for double null GSTM1 and GSTT1 homozygotes the OR was 2.06 (95% CI 0.85-2.04). In those also likely to have been exposed to diazinon, risks associated with a GSTP1*B or *C allele (OR 1.82, 95% CI 0.92-3.63) or a GSTM1*2/GSTT1*2 homozygous (OR 2.60, 95% CI 0.72-10.42) were elevated but not to a significant extent. Risk associated with PON1 genotype and phenotype varied with CYP2D6 and GSTP1 genotype but not consistently with a priori hypotheses. Further work is necessary to delineate more clearly pathways of organophosphate activation and non-PON1 pathways of detoxification and to confirm whether CYP and GST polymorphisms alter disease risk in populations exposed to organophosphates.

Impact of migration on coronary heart disease risk factors: comparison of Gujaratis in Britain and their contemporaries in villages of origin in India.

The causes of the excess coronary heart disease (CHD) risk in South Asian migrants from the Indian subcontinent remain unclear. Comparisons of CHD risk factors amongst South Asian migrants living in Britain with those of the general UK population provide only a partial explanation. We compared Gujaratis in Britain with similar, non-migrant Gujaratis in India, to test the hypothesis that differences in CHD risk factors associated with migration would be more informative. Randomly sampled Gujaratis aged 25-79 years living in Sandwell (n = 242) were compared with age-, gender- and caste-matched contemporaries remaining in their villages of origin in Navsari, India (n = 295). Lifestyle indices, food intake and physical activity, were assessed with standardised questionnaires and energy expenditure and metabolic parameters measured. British Gujaratis had higher, mean body mass indices by 6 (4.5-7.4) kg/m(2) mean (95% CI), and greater dietary energy intake, fat intake, blood pressure, fasting serum cholesterol, apolipoprotein B, triglycerides, non-esterified fatty acid (NEFA) and C-reative protein concentrations than Gujaratis in India. Dietary folate and serum folate and Vitamin B(12) were lower and plasma homocysteine was higher in India. Smoking was less prevalent and high-density lipoprotein cholesterol tended to be higher in Britain. Diabetes prevalence was high in both populations and impaired fasting or 2 h post-glucose challenge plasma glucose was even more prevalent in Gujarat. In India, however, where insulin secretion and NEFA were lower diabetes and impaired glucose tolerance were less frequently accompanied by excess metabolic CVD risk factors. In conclusion, exposure to increased fat intake and obesity related to migration is likely to explain the disproportionate combination of established and emerging CHD risk factors prevalent in Gujaratis in Britain. Strategies to improve nutrition and to identify and treat cardiovascular risk factors such as dyslipidaemia and hypertension are urgently required.

The correlation of homocysteine-thiolactonase activity of the paraoxonase (PON1) protein with coronary heart disease status.

Homocysteine (Hcy)-thiolactonase (HTase) activity of the paraoxonase-1 (PON1) protein detoxifies Hcythiolactone in human blood and could thus delay the development of atherosclerosis. We investigated a hypothesis that HTase activity is associated with coronary heart disease. We studied HTase activities and PON1 genotypes in a group of 475 subjects, 42.5% of whom were healthy and 57.5% had coronary heart disease (CHD). We found that HTase activity was positively correlated with total cholesterol (r=0.254, P<0.0001), LDL cholesterol (0.149, P=0.016), ApoB (r=0.167, P=0.006), ApoA1 (0.140, P=0.023), and HDL cholesterol (0.184, P=0.002) in a group of CHD cases (n=270) but not in controls (n=202). Mean HTase activity was significantly higher in CHD cases than in controls (4.57 units vs. 3.30 units, P <10(-5)). The frequencies of the PON1-192 genotypes in CHD cases were similar to those in controls. HTase activity was not different between patients receiving statins and those not treated with statins. Multiple regression analysis shows that CHD status, PON1 genotype, and total cholesterol are determinants of HTase activity in humans. Our results suggest that HTase activity of the PON 1 protein is a predictor of CHD.

The paraoxonase-2-310 polymorphism is associated with the presence of microvascular complications in diabetes mellitus.

OBJECTIVE: To investigate the paraoxonase-2 (PON 2)-C/S 310 polymorphism and its relationship to the presence of diabetic complications and glycaemic control. DESIGN: Case-control study. SETTING: One study centre at University hospital. MATERIALS AND METHODS: The subjects were people with type 2 diabetes (n=252), type 1 diabetes (n=152) and healthy controls (n=282). The PON 2-C/S 310 polymorphism was measured by restriction fragment length polymorphism analysis. Lipids and lipoproteins were measured by standard clinical chemistry methods. Diabetes and diabetic complications were defined by World Health Organization criteria. RESULTS: There was an over-representation of the C/C 310 genotype in those with diabetes and microvascular complications (type 2 diabetes P=0.043, type 1 diabetes P=0.052, both populations combined P=0.014). The PON 2-C/S 310 polymorphism was also associated with glycaemic control. C 310/C 310 homozygotes had the highest HbA(1c) concentration (P=0.020 type 2 diabetes, P=0.065 type 1 diabetes, P=0.035 both populations combined). There was no association between the PON 2-310 polymorphism and lipid and lipoprotein concentrations. CONCLUSIONS: PON 2 could be directly involved in protecting critical enzymes or organelles against oxidative damage; PON2 may thus predispose to the development of microvascular complications.

Paraoxonase polymorphisms and self-reported chronic ill-health in farmers dipping sheep.

BACKGROUND: Serum paraoxonase (PON1) provides protection against organophosphate induced toxicity. Recently we reported that the frequency of paraoxonase polymorphisms in sheep dippers with self-reported chronic ill-health differed from that in dippers with a similar dipping history but no ill-health. As these analyses may have included subjects with conditions unrelated to organophosphate exposure, the aim of this study was to examine whether the risk associated with PON1 polymorphisms varied using a more homogenous case and referent population. METHODS: Each subject completed a detailed symptom questionnaire and their general practitioner was asked whether there was any history of neurological disease that could be confused with the effects of organophosphate poisoning. Subjects were then excluded both on clinical grounds and where identified as atypical on discriminant analysis. RESULTS: Risk associated with the PON1 192 and 55 genotypes altered little with these changes in the population. CONCLUSIONS: These findings are consistent with the hypothesis that organophosphates contribute to the self-reported ill-health of sheep dippers.

Baseline characteristics in the Collaborative AtoRvastatin Diabetes Study (CARDS) in patients with Type 2 diabetes.

AIM: To describe baseline characteristics of patients in the Collaborative AtoRvastatin Diabetes Study (CARDS), a randomized, placebo-controlled trial of lipid lowering with atorvastatin 10 mg daily for the primary prevention of major cardiovascular events in patients with Type 2 diabetes. METHODS: The main eligibility criteria were Type 2 diabetes, age 40-75 years, no previous history of coronary heart disease, stroke or other major cardiovascular events, a documented history of at least one of retinopathy, micro- or macroalbuminuria, hypertension or current smoking, LDL-cholesterol < or = 4.14 mmol/l and triglycerides < or = 6.78 mmol/l. RESULTS: Randomization of 2838 persons (909 women) into CARDS was completed in June 2001. At entry, mean age was 62 years, 12% were over 70 years old and median duration of diabetes was 6 years. Median fasting lipid levels were total cholesterol 5.4 mmol/l, LDL-cholesterol 3.1 mmol/l, HDL-cholesterol 1.4 mmol/l and triglyceride 1.7 mmol/l. There was a documented history of retinopathy in 30% of patients, micro/macroalbuminuria in 11% (additionally 17% had micro/macroalbuminuria based on two elevated pretreatment measurements of albumin-creatinine ratios), hypertension in 79% and 23% were current smokers. CONCLUSION: CARDS will contribute importantly to the evidence for the macrovascular and microvascular benefits of lipid lowering with atorvastatin in patients with Type 2 diabetes. The results are likely to have important implications for the management of patients.

The effect of atorvastatin on serum lipids, lipoproteins and NMR spectroscopy defined lipoprotein subclasses in type 2 diabetic patients with ischaemic heart disease.

The effect of statin therapy on subclasses of LDL, VLDL and HDL lipoproteins is unclear. We compared changes in serum lipids, apolipoproteins and nuclear magnetic resonance (NMR) spectroscopy measured lipoprotein subclass concentration and average particle size over a minimum 6 months treatment period of atorvastatin 10 mg vs. placebo in 122 men and women. All subjects had type 2 diabetes and a modest dyslipidaemia (mean LDL-cholesterol 3.2 mmol/l and median triglycerides 1.8 mmol/l) and had a previous myocardial infarction. Compared with placebo, atorvastatin therapy was associated with a greater decrease in medium VLDL (median within person change -13.4 vs. -5.9 nmol/l, P<0.001 adjusted for baseline level), small VLDL (median change -17.8 vs. -8.1 nmol/l, P=0.002), large LDL (mean within person change -167.9 vs. -48.6 nmol/l, P<0.001) and medium LDL (median within person change -101.8 vs. -22.3 nmol/l, P=0.017). Atorvastatin therapy was also associated with a greater increase in large HDL than placebo (median change 1.40 vs. 0.80 micromol/l, P=0.02) and there was little change in small HDL so that average HDL particle size increased significantly with atorvastatin (P=0.04). In addition to reducing levels of (enzymatically measured) triglyceride, LDL-cholesterol and apolipoprotein B in diabetic patients, atorvastatin significantly reduces NMR measured medium and small VLDL and large and medium LDL, and increases large HDL.

Clinical, biochemical, and immunohistochemical features of necrobiotic xanthogranulomatosis.

AIMS: To describe the clinical features of two patients with paraproteinaemia and necrobiotic xanthogranulomatosis together with detailed immunohistochemistry of the lesions in one. METHODS: The clinical history and results of biochemical investigations of the patients were retrieved from the files. Immunohistochemistry was used to investigate the expression of macrophage and mast cell markers, amyloid A and P, S-100 protein, and apolipoprotein AI and B in xanthogranulomatous skin lesions from patient 2. In addition, protein A-sepharose chromatography was used to separate serum from patient 2 and apolipoprotein B and the IgG paraprotein were measured in the fractions eluted. RESULTS: Monocytes/macrophages comprised the major cellular component of the lesion, and unusually for xanthomata, areas of collagen necrosis were also seen. Activated mast cells were present at the margins of macrophage clusters and adjacent to areas of collagen necrosis. Serum paraprotein was bound to low density lipoproteins as judged by protein A-sepharose chromatography, and was also located within macrophagic foam cells of the lesion on immunohistochemistry. CONCLUSIONS: These observations demonstrate many features similar to atherosclerosis including collagen necrosis and mast cell activation.

Effect of organophosphate intoxication on human serum paraoxonase.

Recently, interindividual variations in serum paraoxonase (PON1) activity and the differences in its metabolic activity towards different organophosphates (OPs) caused by the coding region polymorphisms L55M and Q192R have been found to be important risk factors in susceptibility to OP poisoning. In this study, we investigated the effect of PON1 on the outcome of acute OP intoxication and the effect of acute OP intoxication on PON1. Twenty-eight OP-poisoned patients and 66 healthy volunteers were studied. Patients were evaluated for the clinical manifestations of OP intoxication as well as PON1 activity, PON1 mass and PON1 polymorphisms. Butyrylcholine-esterase (BChE) activity was 50% lower (2,276 +/- 738 U/L versus 5,037 +/- 1,553 U/L, P<0.01) while PON1 activity was 30% lower [114.2 +/- 67.4 nmol/mL/min versus 152.9 +/- 78.9 nmol/mL/ min, P<0.05) in patients than in controls. We observed that the PON1 and BChE activities of eight of the original subjects returned to normal levels when they were reinvestigated six months after exposure. The frequency of the PON192Q allele was significantly higher in patients than controls (85.7% versus 59.7%, chi2=6.745, P=0.034). QQ/ MM individuals had the lowest activity towards paraoxon, while RR/LL individuals had the highest activity. Our data indicate that interindividual differences in PON1 activity and the PON1-55 and -192 polymorphisms are important risk factors in susceptibility to acute OP poisoning; therefore, identifying an individual's PON1 alloenzymes may play an important role in the treatment of patients suffering from OP intoxication.

Serum paraoxonase activity in patients with type 1 diabetes compared to healthy controls.

BACKGROUND: The oxidation of low-density lipoprotein (LDL) is central to current theories on the initiation and progression of atherosclerosis. Type 1 diabetes is associated with an increase in oxidative stress, which may be responsible for the increased susceptibility to coronary heart disease seen in type 1 diabetes. High-density lipoprotein (HDL) associated paraoxonase (PON1) can retard the oxidation of LDL. DESIGN: Paraoxonase activity, concentration and genotype were therefore investigated in 152 people with type 1 diabetes and 282 healthy controls. These parameters were also investigated in the group with type 1 diabetes in relation to the presence of diabetic complications. RESULTS: Both PON1 activity and concentration were significantly lower by 16.7% and 19.2% (both P < 0.05) in the type 1 diabetes group. These differences were independent of the PON1 coding region polymorphisms. The distribution of PON1 activity and mass were the same in both populations, i.e. for the PON1-192 polymorphism RR > RQ > QQ and for the PON1-55 polymorphism LL > LM > MM. There were no differences in either the PON1 polymorphisms, PON1 activity and concentration in people with type 1 diabetes in the presence or absence of micro and macro vascular complications of diabetes. CONCLUSIONS: Low PON1 activity may contribute to the increased atherosclerosis found in type 1 diabetes by reducing the ability of HDL to retard LDL oxidation despite the frequently-found increased HDL in type 1 diabetes when good glycaemic control is established.

Design of the Collaborative AtoRvastatin Diabetes Study (CARDS) in patients with type 2 diabetes.

BACKGROUND: There are few data on the role of lipid lowering in the primary prevention of coronary heart disease (CHD) in diabetic patients. This paper describes the design of a collaborative clinical trial between Diabetes UK, the NHS Research and Development Directorate and Pfizer UK, that addresses this question. METHODS: The Collaborative AtoRvastatin Diabetes Study (CARDS) is a multicentre, randomized, placebo-controlled, double-blind clinical trial of primary prevention of cardiovascular disease in patients with Type 2 diabetes. The primary objective is to investigate whether treatment with the hydroxymethylglutaryl coenzyme A reductase inhibitor, atorvastatin, reduces the incidence of major cardiovascular events. At entry patients have at least one other risk factor for CHD in addition to diabetes, namely current smoking, hypertension, retinopathy, or micro- or macroalbuminuria. At randomization patients have been selected for a serum low-density lipoprotein (LDL) cholesterol concentration < or = 4.14 mmol/l (160 mg/dl) and triglycerides < or = 6.78 mmol/l (600 mg/dl). Randomization was completed in June 2001. Patients will be followed until 304 primary endpoints have accrued (expected date early 2005). The trial includes 2838 men and women aged 40-75 years. This report describes the design and administration of the study and reviews the evidence to date of the effectiveness of lipid-lowering therapy in Type 2 diabetes. CONCLUSIONS: The case for lipid-lowering therapy for the primary prevention of CHD in diabetes has not been demonstrated. CARDS will provide essential information on the extent of any benefits and adverse effects of lipid-lowering therapy in diabetic patients without prior CHD.

Paraoxonase status in coronary heart disease: are activity and concentration more important than genotype?

Human serum paraoxonase (PON1) hydrolyzes oxidized lipids in low density lipoprotein (LDL) and could therefore retard the development of atherosclerosis. In keeping with this hypothesis, several case-control studies have shown a relationship between the presence of coronary heart disease (CHD) and polymorphisms at amino acid positions 55 and 192 of PON1, which we associated with a decreased capacity of PON1 to protect LDL against the accumulation of lipid peroxides, but some other studies have not. However, the PON1 polymorphisms are only 1 factor in determining the activity and concentration of the enzyme. Only 3 of the previous 18 studies directly determined PON1 activity and concentration. Therefore, we studied PON1 activity, concentration, and gene distribution in 417 subjects with angiographically proven CHD and in 282 control subjects. We found that PON1 activity and concentration were significantly lower in subjects with CHD than in control subjects (activity to paraoxon 122.8 [3.3 to 802.8] versus 214.6 [26.3 to 620.8] nmol. min(-1). mL(-1), P<0.001; concentration 71.6 [11.4 to 489.3] versus 89.1 [16.8 to 527.4] microg/mL, P<0.001). There were no differences in the PON1-55 and -192 polymorphisms or clusterin concentration between patients with CHD and control subjects. These results indicate that lower PON1 activity and concentration and, therefore, the reduced ability to prevent LDL lipid peroxidation may be more important in determining the presence of CHD than paraoxonase genetic polymorphisms.