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.

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.

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.

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.

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.

Paraoxonase and atherosclerosis.

There is considerable evidence that the antioxidant activity of high density lipoprotein (HDL) is largely due to the paraoxonase-1 (PON1) located on it. Experiments with transgenic PON1 knockout mice indicate the potential for PON1 to protect against atherogenesis. This protective effect of HDL against low density lipoprotein (LDL) lipid peroxidation is maintained longer than is the protective effect of antioxidant vitamins and could thus be more important. There is evidence that the genetic polymorphisms of PON1 least able to protect LDL against lipid peroxidation are overrepresented in coronary heart disease, particularly in association with diabetes. However, these polymorphisms explain only part of the variation in serum PON1 activity; thus, a more critical test of the hypothesis is likely to be whether low serum PON1 activity is associated with coronary heart disease. Preliminary case-control evidence suggests that this is indeed the case and, thus, that the quest for dietary and pharmacological means of modifying serum PON1 activity may allow the oxidant model of atherosclerosis to be tested in clinical trials.

Hydrolysis of platelet-activating factor by human serum paraoxonase.

Human serum paraoxonase (human PON1) has been shown to be important in the metabolism of phospholipid and cholesteryl ester hydroperoxides, thereby preventing the oxidation of low-density lipoprotein (LDL) and retarding atherogenesis. However, the exact substrate specificity of PON1 has not been established. In the present study we show that purified PON1 hydrolyses platelet-activating factor (PAF). We could find no evidence for contamination of our preparation with authentic platelet-activating-factor acetylhydrolase (PAFAH) by immunoblotting with a PAFAH monoclonal antibody or by sequencing the purified protein. In addition the specific PAFAH inhibitor SB-222657 did not affect the ability of PON1 to hydrolyse PAF (30.1+/-2.8 micromol/min per mg of protein with no inhibitor; 31.4+/-2.2 micromol/min per mg of protein with 100 nM inhibitor) or phenyl acetate (242.6+/-30.8 versus 240.8+/-31.5 micromol/min per mg of protein with and without inhibitor respectively). SB-222657 was also unable to inhibit PAF hydrolysis by isolated human high-density lipoprotein (HDL), but completely abolished the activity of human LDL. Ostrich (Struthio camelus) HDL, which does not contain PON1, was unable to hydrolyse PAF. These data provide evidence that PON1 may limit the action of this bioactive pro-inflammatory phospholipid.

Low paraoxonase in Persian Gulf War Veterans self-reporting Gulf War Syndrome.

Exposure to organophosphate (OP's) insecticides and nerve gases during the Persian Gulf War has been implicated in the development of Gulf War Syndrome. Paraoxonase (PON1) present in human serum detoxifies OP's. We determined the levels of PON1 in the serum of Gulf War Veterans and compared these to those found in a control population. One hundred fifty-two Gulf War Veterans from the UK who self-reported the presence of Gulf War Syndrome via a questionnaire and 152 age and gender matched controls were studied. PON1 activity, concentration, and genotype were determined. In the Gulf War Veterans, paraoxon hydrolysis was less than 50% of that found in the controls (100.3 (14.8-233.8) vs 214.6 (50.3-516.2) nmol/min/ml, P < 0.001). This low activity was independent of the effect of PON1 genotype. The serum PON1 concentration was also lower in the Gulf War Veterans (75.7 (18.1-351.3) vs 88.2 (34.5-527.4) microg/ml, P < 0.00025), which was again independent of PON1 genotype. There was no difference in the rate of diazoxon hydrolysis between the groups (10. 2 +/- 4.1 micromol/min/ml vs 9.86 +/- 4.4, P = NS). A decreased capacity to detoxify OP insecticides resulting from low serum PON1 activity may have contributed to the development of Gulf War Syndrome.

How high-density lipoprotein protects against the effects of lipid peroxidation.

The protective effect of HDL against the development of atherosclerosis appears to be multifaceted involving a number of mechanisms. One of the major mechanisms is, however, the ability of HDL to decrease, directly or indirectly, the lipid peroxidation of LDL. The hydrolysis of lipid peroxides by PON1 makes a major contribution to this effect of HDL. Evidence is accumulating that the PON1 activity of human serum can be modulated by a variety of natural compounds and that these may increase or decrease the protective ability of PON1 and therefore of HDL on which it is exclusively located. Modulations of PON1 that enhance its activity may help to delay the atherosclerotic process.

Low paraoxonase activity in type II diabetes mellitus complicated by retinopathy.

Human serum paraoxonase 1 (PON1) is located on high-density lipoprotein and has been implicated in the detoxification of organophosphates, and possibly in the prevention of lipid peroxidation of low-density lipoprotein. PON1 has two genetic polymorphisms, both due to amino acid substitutions: one involving glutamine (Q genotype) and arginine (R genotype) at position 192, and the other involving leucine (L genotype) and methionine (M genotype) at position 55. We investigated the effects of these polymorphisms, and of a polymorphism of the PON2 gene at position 310 (Cys/Ser; C and S genotypes respectively), on serum PON1 activity and concentration, plasma lipids and lipoproteins and glycaemic control in 93 individuals with type II diabetes with no complications and in 101 individuals with type II diabetes with retinopathy. Serum PON1 activity in the group with no complications [median 164.1 nmol.min(-1).ml(-1) (range 8.0-467.8)] was significantly higher than in the group with retinopathy [113.4 nmol. min(-1).ml(-1) (3.0-414.6)] (P<0.001), but the serum PON1 concentration was not different between the groups. The gene frequencies of the PON1-55 and PON1-192 polymorphisms and of the PON2-310 polymorphism were not different between the study populations. The PON1-55 and PON1-192 polymorphisms affected PON1 activity in the way described in a previous study of a control group and subjects with type II diabetes. The PON2-310 polymorphism also significantly affected serum PON1. PON1 activity was significantly higher in individuals with the PON2-310 CC genotype in both groups with type II diabetes, and the PON1 concentration was significantly higher in PON2-310 CC homozygotes with no complications than in the group with retinopathy. Neither the PON1-55 nor the PON1-192 polymorphism was correlated with the serum lipid or lipoprotein concentration in either group. In the group with retinopathy (but not the group with no complications), all three PON polymorphisms were correlated with glycaemic control, which was worse for the PON1-55 genotypes in the order MM>LM>LL (P=0.0032), for the PON1-192 genotypes in the order RR>QR>QQ (P=0.011) and for the PON2-310 genotypes in the order CC>CS>SS (P=0.010). Low serum PON1 activity in retinopathy may be related to an increased tendency for lipid peroxidation. Our findings thus raise the possibility that, in retinopathy, the PON2 gene may influence PON1, and that an inter-relationship between the PON1 and PON2 genes may influence glycaemic control in subjects with type II diabetes complicated by retinopathy.

Paraoxonase activity is reduced by a pro-atherosclerotic diet in rabbits.

Serum paraoxonase (PON1) is believed to protect against the development of atherosclerosis because of its ability to retard the oxidation of low-density lipoprotein (LDL) by hydrolysing LDL-associated phospholipid and cholesteryl-ester hydroperoxides. We have examined the relationship between PON1 and atherosclerosis development in transgenic rabbits overexpressing human apolipoprotein (apo) A-I and nontransgenic littermates fed a pro-atherogenic diet. PON1 activity was higher in transgenic (4006.1 +/- 716.7 nmol/min/ml) compared to control (3078.5 +/- 623.3 nmol/min/ml) rabbits (P < 0.01) while high-density lipoprotein (HDL) cholesterol was 1.84 +/- 0.54 mmol/L in transgenic rabbits and 0.57 +/- 0.21 mmol/L in control rabbits (P = 0.0001). After feeding rabbits a high-cholesterol diet for 14 weeks HDL-cholesterol fell by 70% in both transgenic and control rabbits (P < 0.001 compared to week 0) PON1 activity fell by 50% in both groups of rabbits (P < 0. 01 compared to week 0). The amount of thoracic aortic surface area covered by lesions was 29 +/- 16% in the control group and 26 +/- 15% in the transgenic group (P = NS). A pro-atherosclerotic diet reduces PON1 which may exaggerate the effects of the diet on the development of atherosclerosis.

Paraoxonase activity in two healthy populations with differing rates of coronary heart disease.

BACKGROUND: The rate of coronary heart disease is over three-fold greater in Belfast than in Toulouse and the excess risk cannot be totally explained by 'classical' risk factors such as total cholesterol, LDL-cholesterol, smoking, etc. DESIGN: The effect of the human serum paraoxonase (PON1) 192-genetic polymorphism on plasma lipid and lipoprotein concentrations and on PON1 activity and concentration was investigated in 186 randomly selected healthy subjects from Toulouse and 165 from Belfast. RESULTS: The frequency of the R allele of PON1, which has been related to the risk of coronary heart disease, was significantly higher in Belfast (0.33) than in Toulouse (0.24; chi2 = 7.229, P = 0.0072). Subjects from Belfast also had significantly higher serum cholesterol, triglycerides, LDL-cholesterol, and apolipoprotein B, and significantly lower HDL-cholesterol and apolipoprotein A1, but these lipoprotein parameters were independent of the PON1 192-polymorphisms. PON1 activity towards paraoxon was significantly higher in the Belfast population than in Toulouse (median values: 179.7 vs. 129.4 nmol min-1 mL-1 serum, respectively; P < 0.05), which is consistent with our finding of a greater prevalence of the R allele. The median serum concentration of PON1 was 56.3 microgram mL-1 in Belfast, which was significantly lower (P < 0.005) than the level of 71 microgram mL-1 in Toulouse. CONCLUSIONS: Our results thus provide further support for the hypothesis that populations at increased CHD risk have diminished serum PON1 concentration and an increased prevalence of the R allele of PON1. They are also consistent with reports that the ability of PON1 to hydrolyse paraoxon is inversely related to its capacity to hydrolyse lipid-peroxides, and thus to its antiatherogenic action.