Bharti Mackness

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.

PARAOXONASE : BIOCHEMISTRY, GENETICS AND RELATIONSHIP TO PLASMA LIPOPROTEINS

Human serum paraoxonase is located on an HDL. It has the capacity to retard the accumulation of lipid peroxides in LDL under oxidizing conditions in vitro. Paraoxonase has a genetic polymorphism that results in a single amino acid substitution. Evidence indicates that both the serum concentration of paraoxonase and an individuals genotype are related to plasma lipid and lipoprotein concentrations, and possibly also to coronary heart disease, implicating paraoxonase in the development of atherosclerosis.

Low Paraoxonase Activity Predicts Coronary Events in the Caerphilly Prospective Study

Background—The hypothesis that paraoxonase (PON1) has a role in preventing atherosclerosis is based on experimental, transgenic, and case-control studies but has not previously been tested prospectively. Methods and Results—The Caerphilly Prospective Study is a cohort study of men aged 49 to 65 years observed for coronary heart disease (CHD) events (fatal and nonfatal myocardial infarction) over a mean period of 15 years. Serum PON1 activity toward paraoxon was measured in 1353 participants. PON1 activity was 20% lower in the 163 men who had a coronary event (P =0.039). Men in the highest quintile of PON1 activity had a decreased risk compared with those in the lowest quintile (OR 0.57 [95% CI, 0.33 to 0.96]). The inverse relationship between quintiles of serum PON1 activity and CHD risk was graded, the median change in OR across each quintile being 0.87 (0.77 to 0.98). After adjustment for all other CHD risk factors, including HDL cholesterol, this median value became 0.90 (0.78 to 1.02). PON1 was most predictive of a new CHD event in patients at highest risk by virtue of preexisting CHD (adjusted median OR for each quintile, 0.74 [0.59 to 0.93]; n=313) or the presence of other risk factors. For the highest tertile of CHD risk (n=390) calculated by the Framingham equation, adjusted median OR for each quintile was 0.84 (0.66 to 1.05); n=390. Conclusions—Low serum PON1 activity toward paraoxon is an independent risk factor for coronary events in men at high risk because of preexisting disease or other CHD risk factors.

Effect of the human serum paraoxonase 55 and 192 genetic polymorphisms on the protection by high density lipoprotein against low density lipoprotein oxidative modification

Human serum paraoxonase (PON1) associated with high density lipoprotein (HDL) has been postulated to have a role in protecting low density lipoprotein (LDL) against oxidative modification, which has led to the proposal that PON1 is an anti‐atherogenic, anti‐inflammatory enzyme. PON1 has two genetically determined polymorphic sites giving rise to amino‐acid substitutions at positions 55 (L→M) and 192 (R→Q) and therefore 4 potential alloenzymes. We have examined the effects of these molecular polymorphisms on the ability of HDL to protect LDL from oxidative modification. HDL protected LDL from oxidative modification, whatever the combination of PON1 alloenzymes present in it. However, HDL from QQ/MM homozygotes was most effective at protecting LDL while HDL from RR/LL homozygotes was least effective. Thus after 6 h of co‐incubation of HDL and LDL with Cu2+ PON1‐QQ HDL retained 57±6.3% of its original ability to protect LDL from oxidative modification, while PON1‐QR HDL retained less at 25.1±4.5% (P<0.01) and PON1‐RR HDL retained only 0.75±0.40% (P<0.005). In similar experiments HDL from LL and LM genotypes retained 21.8±7.5% and 29.5±6.6% (P=NS), respectively, of their protective ability, whereas PON1‐MM HDL maintained 49.5±5.3% (P<0.01). PON1 polymorphisms may affect the ability of HDL to impede the development of atherosclerosis and to prevent inflammation.

Human serum paraoxonase

1. Human serum paraoxonase (PON1) is a Ca2+-dependent 45-kDa glycoprotein that is associated with high density lipoprotein (HDL). 2. PON1 hydrolyzes organophosphate (OP) insecticides and nerve gases and is responsible for determining the selective toxicity of these compounds in mammals. 3. PON1 has two genetic polymorphisms giving rise to amino acid substitutions at positions 55 and 192. The position-192 polymorphism is the major determinant of the PON1 activity polymorphism. However, the position-55 polymorphism also modulates activity. 4. Genotyping individuals for both PON1 polymorphisms may provide a method for identifying those most at risk of OP poisoning. The effect of the PON1 polymorphisms on activity may explain why some Gulf War veterans have developed Gulf War syndrome and some have not, despite similar OP exposure. 5. PON1 may also be a determinant of resistance to the development of atherosclerosis by protecting lipoproteins against oxidative modification, perhaps by hydrolyzing phospholipid hydroperoxides. 6. The PON 1 polymorphisms are important in determining the capacity of HDL to protect low density lipoprotein against oxidative modification in vitro, which may explain the relation between the PON1 alleles and coronary heart disease in case-control studies.

Serum Paraoxonase After Myocardial Infarction

HDL has been shown to prevent the oxidative modification of LDL. The antioxidant activity of HDL is believed to reside in its enzymes, particularly paraoxonase. Human serum paraoxonase (PON1) is closely associated with a specific HDL subfraction also containing apoA1 and clusterin. Recently PON1 has been implicated in the pathogenesis of atherosclerosis. We have examined the activity, concentration, and specific activity of PON1 in 50 patients on admission to hospital immediately after acute myocardial infarction (MI) and in 48 age- and gender-matched controls. Serum PON1 activity and concentration were significantly lower in patients with MI than in controls (activity, 221.5 [99.3 to 303.2] nmol. min-1. mL-1 in controls and 130.1 [78.9 to 230.3] nmol. min-1. mL-1 in MI patients [P<0.05]; concentration, 95.7 [73.2 to 135.5] microg/mL in controls and 35.4 [21.6 to 51.3] microg/mL in MI patients [P<0.001]). PON1-specific activity was significantly higher in patients with MI than in controls (1.5 [0.9 to 2.9] versus 3.4 [2.0 to 8.5] nmol. min-1. microg-1 [P<0.001]) due to the much lower PON1 concentration. PON1 activity had risen significantly (P<0.05) to 158.1 (85.4 to 282.0) nmol. min-1. mL-1 at day 42 but was still significantly less than that of controls. No significant variation in PON1 concentration occured in the days after MI or at 6 weeks. Also, no significant variation in specific activity was seen after MI. When the patients were divided into subgroups based on whether or not they received thrombolytic therapy on admission to hospital, no significant difference in PON1 levels was observed. Serum HDL cholesterol in patients with MI on admission was not significantly different than in controls, and the decrease that occurred by the fifth day after MI did not explain the lower PON1 levels. We conclude that low serum PON1 activity in patients with MI may be a consequence of the coronary event itself or could have been present before MI. The low PON1 activity was also not explicable on the basis of PON1 genotypes because the prevalence of genotypes associated with low activity was not sufficient to explain fully the difference in activity levels between patients and controls. The explanation for the low PON1 activity was most likely a decrease in serum PON1 concentration. The importance of PON1 as a predictive risk factor for MI should be assessed in future studies.

Serum paraoxonase (PON1) 55 and 192 polymorphism and paraoxonase activity and concentration in non-insulin dependent diabetes mellitus

Human serum paraoxonase (PON1) is located on high density lipoprotein and has been implicated in the detoxification of organophosphates and possibly in the prevention of low density lipoprotein lipid peroxidation. PON1 has two genetic polymorphisms both due to amino acid substitution, one involving glutamine (A genotype) and arginine (B genotype) at position 192 and the other leucine (L genotype) and methionine (M genotype) at position 55. We investigated the effect of these polymorphisms on serum PON1 activity and concentration in 252 non-insulin dependent diabetes mellitus (NIDDM) individuals and 282 non-diabetic controls. Serum PON1 activity in the controls (214.6 nmol/min per ml (26.3-620.8)) was significantly higher than in NIDDM (158.7 nmol/min per ml (3.6-550.5) (P < 0.001) as was serum PON1 concentration (89.1 microg/ml (16.8-527.4)) compared to 76.7 microg/ml (3.6-443.8) (P < 0.01). In the control population MM homozygotes had significantly lower serum PON1 activity regardless of the 192 polymorphism whereas in NIDDM both LM and MM genotypes had lower serum PON1 activity than LL homozygotes only when the 192 AA genotype was present. Serum PON1 concentration was lower in NIDDM with AA/LM, AA/LL, AB/LL and AB/MM genotypes than in controls. Differences in PON1 activity were the major cause of differences in specific activity between genotypes. Neither the PON1 55 or 192 polymorphisms consistently influenced the serum lipid or lipoprotein concentrations in either population. Low serum PON1 activity in NIDDM may be related to an increased tendency to lipid peroxidation and may also increase susceptibility to toxicity from organophosphate exposure. Our findings thus raise the possibility that PON1 may be of importance in both the genetic and acquired predisposition to premature atherosclerosis and neuropathy in diabetes.

Paraoxonase and coronary heart disease

The antioxidant activity of HDL is largely due to the paraoxonase (PON1) located on it. Experiments with transgenic PON1 knock-out mice indicate the potential for PON1 to protect against atherogenesis. This effect of HDL in decreasing LDL lipid peroxidation is maintained for longer than that of antioxidant vitamins and could thus be more protective. Several important advances in the field of PON research have occurred recently, not least the discovery that two other members of the PON gene family PON2 and PON3 may also have important antioxidant properties. Significant advances have been made in understanding the basic biochemical function of PON1 and the discovery of possible modulators of its activity. Decreased coronary heart disease (CHD) risk associated with polymorphisms of PON1 which are most active in lipid peroxide hydrolysis revealed by meta-analysis is likely to be an underestimate of the true contribution of PON1 to CHD because these polymorphisms explain only a small component of the variation in PON1 activity. However, it is a very important observation because genetic influences are not likely to be confounded by other factors linked with both CHD and diminished PON1 activity. PON1 is extensively researched and strategies will hopefully emerge to increase its activity and provide a more satisfactory test of the antioxidant hypothesis of atherosclerosis than antioxidant vitamins have done.

Effect of the molecular polymorphisms of human paraoxonase (PON1) on the rate of hydrolysis of paraoxon

1 The hydrolysis of organophosphate pesticides (OP) and nerve gases by serum paraoxonase (PON1) is an important factor determining their toxicity to mammals including man. The PON1 gene contains 2 polymorphic sites at amino acid positions 55 (L→M) and 192 (G→A, classically defined as the A and B genotypes) which result in several alloenzymes of PON1 in human serum. 2 The 192 polymorphism has previously been shown to affect PON1 activity. We have investigated the effect of both polymorphisms on the hydrolysis of paraoxon by serum from 279 healthy human subjects. 3 The 55 polymorphism significantly influenced PON1 activity. MM homozygotes had over 50% less activity towards paraoxon compared to the LL and LM genotypes regardless of the 192 genotype (P<0.001). 4 Multiple regression analysis indicated that the 192 polymorphism, 55 polymorphism and serum PON1 concentration were responsible for 46, 16 and 13% of the variation in PON1 activity, respectively (all P<0.001). None of the other parameters investigated significantly affected PON1 activity. 5 Therefore both PON1 polymorphisms affect the hydrolysis of paraoxon. AA/MM and AB/MM individuals may be potentially more susceptible to OP intoxication. 6 Genotyping individuals for both PON1 polymorphisms may provide a method for identifying those individuals at most risk of OP poisoning. The effect of PON1 polymorphisms on activity may also explain why some Gulf War Veterans have developed Gulf War Syndrome and some have not.

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.