The PON1 M55L gene polymorphism is associated with reduced HDL-associated PAF-AH activity

The platelet-activating factor acetylhydrolase activity associated with high density lipoprotein (HDL-PAF-AH) may substantially contribute to the antioxidant, anti-inflammatory, and overall antiatherogenic effects of HDL. Two enzymes associated with HDL express PAF-AH catalytic activity, PAF-AH itself and paraoxonase-1 (PON1). The relative contribution of these enzymes in the expression of PAF-AH activity on HDL remains to be established. We investigated whether the PON1 polymorphisms (M55L and Q192R) or the PAF-AH polymorphism V379A could affect the PAF-AH activity associated with HDL in both normolipidemic and dyslipidemic (type IIA and IIB) populations. We show for the first time that the PON1 M55L polymorphism significantly affects the HDL-PAF-AH activity in all studied groups, the PON1 L55L individuals having lower enzyme activity compared to those having 1 M and 2 M alleles. No differences in the HDL content concerning the major apolipoprotein and lipid constituents were observed between individuals carrying the PON1 L55L and those with the M55M polymorphism. Our results provide evidence that PON1 significantly contributes to the pool of HDL-PAF-AH activity in human plasma, and suggest that the low PAF-AH activity in HDL carrying the PON1 L alloenzyme may be an important factor contributing to the low efficiency of this HDL in protecting LDL against lipid peroxidation.     

Lipoprotein-associated PAF-acetylhydrolase activity in subjects with the metabolic syndrome

Plasma- and lipoprotein-associated activity of the platelet activating factor acetylhydrolase (PAF-acetylhydrolase, PAF-AH) plays an important role in inflammation and in atherosclerotic process, which are present in the metabolic syndrome (MS). Paraoxonase 1 (PON1) is an esterase associated with high-density lipoprotein (HDL) which contributes to the anti-atherogenic effects of this lipoprotein. We investigated the activities of both enzymes in 60 patients with MS and 110 age- and sex-matched subjects without it (non-MS group). Plasma PAF-AH activity was higher in the MS compared to the non-MS group, while HDL-PAF-AH and serum PON1 activities were lower in the MS compared to the non-MS group. Univariate regression analysis in the MS group showed that plasma PAF-AH activity was positively associated with systolic blood pressure, whereas HDL-PAF-AH activity was inversely associated with the homeostasis model assessments (HOMA) index. Both associations remained significant in the multivariate regression analysis, suggesting that insulin resistance and systolic hypertension are major determinants for the alterations in plasma and HDL-associated PAF-AH activity among those observed in MS patients.     

Structure-function relationships in reconstituted HDL: Focus on antioxidative activity and cholesterol efflux capacity

AimsHigh-density lipoprotein (HDL) contains multiple components that endow it with biological activities. Apolipoprotein A-I (apoA-I) and surface phospholipids contribute to these activities; however, structure-function relationships in HDL particles remain incompletely characterised.MethodsReconstituted HDLs (rHDLs) were prepared from apoA-I and soy phosphatidylcholine (PC) at molar ratios of 1:50, 1:100 and 1:150. Oxidative status of apoA-I was varied using controlled oxidation of Met112 residue. HDL-mediated inactivation of PC hydroperoxides (PCOOH) derived from mildly pre-oxidized low-density lipoprotein (LDL) was evaluated by HPLC with chemiluminescent detection in HDL + LDL mixtures and re-isolated LDL. Cellular cholesterol efflux was characterised in RAW264.7 macrophages.ResultsrHDL inactivated LDL-derived PCOOH in a dose- and time-dependent manner. The capacity of rHDL to both inactivate PCOOH and efflux cholesterol via ATP-binding cassette transporter A1 (ABCA1) increased with increasing apoA-I/PC ratio proportionally to the apoA-I content in rHDL. Controlled oxidation of apoA-I Met112 gradually decreased PCOOH-inactivating capacity of rHDL but increased ABCA1-mediated cellular cholesterol efflux.ConclusionsIncreasing apoA-I content in rHDL enhanced its antioxidative activity towards oxidized LDL and cholesterol efflux capacity via ABCA1, whereas oxidation of apoA-I Met112 decreased the antioxidative activity but increased the cholesterol efflux. These findings provide important considerations in the design of future HDL therapeutics.Non-standard abbreviations and acronyms: AAPH, 2,2′-azobis(-amidinopropane) dihydrochloride; ABCA1, ATP-binding cassette transporter A1; apoA-I, apolipoprotein A-I; BHT, butylated hydroxytoluene; CV, cardiovascular; EDTA, ethylenediaminetetraacetic acid; HDL-C, high-density lipoprotein cholesterol; LOOH, lipid hydroperoxides; Met(O), methionine sulfoxide; Met112, methionine 112 residue; Met86, methionine 86 residue; oxLDL, oxidized low-density lipoprotein; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PL, phospholipid; PCOOH, phosphatidylcholine hydroperoxide; PLOOH, phospholipid hydroperoxide.     

Platelet-activating factor acetylhydrolase,and not paraoxonase-1, is the oxidized phospholipid hydrolase of high density lipoprotein particles

Paraoxonase-1 (PON1), an high density lipoprotein (HDL)-associated organophosphate triesterase, suppresses atherosclerosis in an unknown way. Purified PON1 protects lipoprotein particles from oxidative modification and hydrolyzes pro-atherogenic oxidized phospholipids and the inflammatory mediator platelet-activating factor (PAF). We find human PON1 acted as a phospholipase A(2) but not as a phospholipase C or D through cleavage of phosphodiester bonds as expected. PON1 requires divalent cations, but EDTA did not block the phospholipase A(2) activity of PON1. In contrast, a serine esterase inhibitor abolished phospholipase activity even though PON1 has no active-site serine residues. PAF acetylhydrolase, an oxidized phospholipid phospholipase A(2), is a serine esterase associated with specific HDL particles. Western blotting did not reveal detectable amounts of PAF acetylhydrolase in PON1 preparations, although very low amounts of PAF acetylhydrolase might still account for PON1 phospholipase A(2) activity. We revised the standard PON1 purification by first depleting HDL of PAF acetylhydrolase to find PON1 purified in this way no longer hydrolyzed oxidized phospholipids or PAF. Serum from a donor with an inactivating mutation in the PAF acetylhydrolase gene did not hydrolyze oxidized phospholipids or PAF, yet displayed full paraoxonase activity. We conclude that PAF acetylhydrolase is the sole phospholipase A(2) of HDL and that PON1 has no phospholipase activity toward PAF or pro-atherogenic oxidized phospholipids.     

Fenofibrate induces HDL-associated PAF-AH but attenuates enzyme activity associated with apoB-containing lipoproteins

Human plasma platelet-activating factor acetylhydrolase (PAF-AH) is an enzyme associated mainly with the apolipoprotein B (apoB)-containing lipoproteins and primarily with LDL. A small proportion of enzymatic activity is also associated with HDL. Plasma paraoxonase 1 (PON1) is an esterase exclusively associated with HDL. The effect of fenofibrate on PAF-AH and PON1 activities in patients with dyslipidemias of Types IIA, IIB, and IV were studied. Fenofibrate reduced plasma PAF-AH activity in all patient groups. In Type IIA patients, this reduction was mainly due to a fall in enzyme activity associated with the dense LDL subspecies, whereas in Type IIB and Type IV patients, it was due to the decrease in PAF-AH activity associated with both the VLDL+IDL and dense LDL subspecies. Drug therapy in Type IIB and Type IV patients significantly increased the HDL-associated PAF-AH activity due to the increase in enzyme activity associated with the HDL-3c subfraction. Fenofibrate did not affect serum PON1 activities toward paraoxon and phenylacetate in either patient group. The fenofibrate-induced elevation of HDL-associated PAF-AH activity in dyslipidemic patients of Type IIB and Type IV, as well as the reduction in enzyme activity associated with atherogenic apoB-containing lipoproteins in all patient groups, may represent a new and important antiatherogenic effect of this potent lipid-modulating agent.     

Oxidative inactivation of paraoxonase—implications in diabetes mellitus and atherosclerosis

Human serum paraoxonase (PON1) has been implicated to play an important role in cardiovascular disease and diabetes. Studies in the literature indicate that PON1 has two different enzyme activities, i.e., esterase and hydroperoxide reducing activities. The objective of this study was to establish the importance of these two activities and to distinguish between them. As the addition of copper immediately inactivated the enzyme, we used auto-oxidation as the model system. Auto-oxidation of HDL resulted in more than 80% reduction of the esterolytic activity, which was protected by antioxidants, Vitamin E (50%) and PDTC (95%) and completely by 1 M glucose. In contrast, the hydroperoxide reducing activity, using unesterified hydroperoxides remained unaffected with time. We also used pNPHPODE (novel substrate) to establish that hydrolysis might be a prerequisite for the enzyme to act on the esterified hydroperoxide. The results indicated that the hydrolysis of the substrate was inhibited under oxidizing conditions with no reduction of the hydroperoxide. Overall, our findings suggest that protecting the esterolytic activity of PON1 by antioxidants might be important in preserving its action on phospholipid peroxides and a concerted reaction involving the esterolytic and hydroperoxide reducing activities might be suggested for the action of PON1.     

Paraoxonase-1 does not reduce or modify oxidation of phospholipids by peroxynitrite

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated esterase/lactonase implicated to play a role in protection against atherosclerosis. However, the exact mechanism(s) and substrates for PON1 are still uncertain. In this article, we review some of the evidence for PON1's antioxidant activity, as well as our efforts to identify the actual substrates and products for this activity. We originally reported that PON1 had phospholipase activity toward oxidized phosphatidylcholine (J. Biol. Chem. 276:24473-24481; 2001). Subsequently, Marathe et al. (J. Biol. Chem. 278:3937-3947; 2003) reported that this activity was due to a contaminating lipase. However, that article did not replicate the conditions used in our previous study. To address this controversy, we purified serum PON1 by a modified method that separates the paraoxonase activity from an activity detectable as platelet-activating factor acetyl hydrolase (PAF-AH) (Teiber et al., J. Lipid. Res. 2004; Epub ahead of print, PMID 15342686) and reexamined the oxidation of phosphatidylcholine by peroxynitrite using 3-morpholinosydnonimine as a peroxynitrite generator and apolipoprotein AI-phosphatidylcholine- PON1 complexes. The phosphatidylcholines were studied by electrospray ionization tandem mass spectrometry. PON1 preparations free of PAF-AH activity showed no phospholipase activity when reconstituted into apolipoprotein AI-phosphatidylcholine complexes. We conclude that PON1 does not affect the accumulation of phosphatidylcholine oxidation products. Further, we have no evidence that PON1 has an intrinsic phospholipase A2 activity toward oxidized phospholipids.     

HDL capacity to inhibit LDL oxidation in well-trained triathletes

Physical activity is known to play a cardioprotective role. Nevertheless, a paradox seems to arise when considering that aerobic exercise enhances oxidative stress. In previous works, we showed that free radical formation during physical activity was counteracted by an increase in antioxidant defenses. Low density lipoprotein (LDL) oxidation is a crucial step in atherosclerosis, process that can be inhibited by high density lipoprotein (HDL) through its oxidable components or associated enzymes like paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH). In this study, we evaluated copper-induced oxidation in isolated LDL and HDL fractions, and the effect of HDL on LDL oxidation in samples from well trained amateur athletes who were participating in an ultra-distance triathlon (n=18) in comparison with healthy sedentary controls (n=18). PON and PAF-AH activities and PON phenotype were also evaluated. The oxidability of isolated lipoproteins, as well as HDL antioxidant capacity, was similar in both groups of subjects. After classification by paraoxonase phenotype, only sportsmen belonging to the QR phenotype showed higher HDL susceptibility to in vitro oxidation (thiobarbituric reactive substances, TBARS) than controls (p<0.05). HDL oxidability exhibited a positive correlation with its triglyceride content (r=0.58; p<0.01). Similarly, HDL capacity to inhibit LDL oxidation was increased in athletes (p<0.05) which was positively associated with HDL oxidability (HDL-TBARS: r=0.55, p<0.005; HDL-lag time: r=0.45, p<0.01; HDL-D max: r=0.35, p<0.05). In conclusion, regular aerobic exercise was associated to a more efficient antioxidant function played by HDL from PON-QR carriers, which could constitute an adaptive response to the increased oxidative stress.     

Antioxidant properties of HDL in transgenic mice overexpressing human apolipoprotein A-II

Transgenic mice overexpressing human apolipoprotein A-II (huapoA-II) display high VLDL and low HDL levels. To evaluate the antioxidant potential of huapoA-II enriched HDL, we measured the activities of paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH). Both activities decreased up to 43% in the serum of transgenic mice compared with controls, varied in parallel to HDL levels, but decreased less than HDL levels. The major part of PON and PAF-AH was associated with HDL, except in fed high huapoA-II-expressing mice, in which 20% of PAF-AH and 9% of PON activities were associated with VLDL. PON mRNA levels in the liver, its major site of synthesis, were similar in transgenic and control animals, indicating normal enzyme synthesis. In transgenic mice, the basal oxidation of lipoproteins was not increased, whereas their VLDL were more susceptible to oxidation than VLDL of controls. Interestingly, HDL of transgenic mice protected VLDL from oxidation more efficiently than HDL of controls. In conclusion, the decrease in both PON and PAF-AH activities in huapoA-II transgenic mice is best explained by their lower plasma HDL levels. However, the unchanged basal lipoprotein oxidation in transgenic mice suggests that huapoA-II-rich HDL may maintain adequate antioxidant potential.     

Study of the paraoxonase and platelet-activating factor acetylhydrolase activities with aging

The purpose of this study was to investigate, with aging, the activity of two enzymes associated to HDL and responsible for its anti-atherogenic activity; paraoxonase (PON1) and platelet-activating factor acetylhydrolase (PAF-AH). Ninety-five subjects aged between 26 and 77 years were recruited for the study. The prevalence of phenotype A, AB, and B in our subjects group was 69.47,21.05 and 9.47% respectively. Plasma as well as HDL paraoxonase activity decreased significantly with aging (r =-0.218, P < 0.039) and (r = -0.280, P < 0.006) respectively. PAF-AH activity was unchanged with aging however, we noted a negative correlation between PAF-AH and PON1 activity in HDL (r = -0.243, P < 0.02) and in LDL vs HDL (r =-0.462, P < 0.001).     

Exclusive association of paraoxonase 1 with high-density lipoprotein particles in apolipoprotein A-I deficiency.

Paraoxonase1 (PON1) is a high-density lipoprotein (HDL)-associated protein which removes peroxidized lipids from lipoproteins. It has been proposed that apolipoprotein A-I (apoA-I) is an important determinant for its stabilization on HDL. However, little is known about its existence and activity in an apoA-I-deficient state in humans. To characterize the nature of PON1 in apoA-I deficiency, we investigated PON1 in an apoA-I-deficient patient. When serum was analyzed on fast protein liquid chromatography, PON1 protein was distributed almost exclusively on HDL despite the absence of apoA-I; on the other hand, 38.5% of PON1 protein was found in the lipoprotein-free fraction when the lipoproteins were fractionated through ultracentrifugation. The stability of PON1 activity in the patient serum was almost the same as in the normal control sera throughout incubation at 14 degrees C for 7 days. However, when the sera were incubated at 37 degrees C for 24 h, its activity declined more than those in the normal controls (19% versus 4% reduction of the initial values). Our results demonstrated that PON1 protein possesses a preferential association with HDL even in the absence of apoA-I, although apoA-I is a crucial factor for the maximal activity and stabilization of PON1.     

Myeloperoxidase-induced modification of HDL by isolevuglandins inhibits paraoxonase-1 activity

Reduced activity of paraoxonase 1 (PON1), a high-density lipoprotein (HDL)-associated enzyme, has been implicated in the development of atherosclerosis. Post-translational modifications of PON1 may represent important mechanisms leading to reduced PON1 activity. Under atherosclerotic conditions, myeloperoxidase (MPO) is known to associate with HDL. MPO generates the oxidants hypochlorous acid and nitrogen dioxide, which can lead to post-translational modification of PON1, including tyrosine modifications that inhibit PON1 activity. Nitrogen dioxide also drives lipid peroxidation, leading to the formation of reactive lipid dicarbonyls such as malondialdehyde and isolevuglandins, which modify HDL and could inhibit PON1 activity. Because isolevuglandins are more reactive than malondialdehyde, we used in vitro models containing HDL, PON1, and MPO to test the hypothesis that IsoLG formation by MPO and its subsequent modification of HDL contributes to MPO-mediated reductions in PON1 activity. Incubation of MPO with HDL led to modification of HDL proteins, including PON1, by IsoLG. Incubation of HDL with IsoLG reduced PON1 lactonase and antiperoxidation activities. IsoLG modification of recombinant PON1 markedly inhibited its activity, while irreversible IsoLG modification of HDL before adding recombinant PON1 only slightly inhibited the ability of HDL to enhance the catalytic activity of recombinant PON1. Together, these studies support the notion that association of MPO with HDL leads to lower PON1 activity in part via IsoLG-mediated modification of PON1, so that IsoLG modification of PON1 could contribute to increased risk for atherosclerosis, and blocking this modification might prove beneficial to reduce atherosclerosis.     

Iron-ascorbic acid-induced oxidant stress and its quenching by paraoxonase 1 in HDL and the liver: comparison between humans and rats

Paraoxonase 1 (PON1) is a serum enzyme closely associated with high-density lipoprotein (HDL), which may protect against atherosclerosis by hydrolyzing lipid peroxides and several organophosphorus compounds. The purpose of the present study was to test the hypothesis that lipid peroxidation modifies the activity and protein mass of PON1 in humans and rats. Our findings revealed that the bulk of the activity monitored by the hydrolysis of paraoxon and phenyl acetate was confined to liver intracellular endoplasmic reticulum-derived microsomes and was mostly recovered in circulating HDL3. Confirmation was obtained by the determination of PON1 expression by Western blot. It is noteworthy that PON1 levels were consistently decreased in human sera, HDL, and liver microsomes compared with rat counterparts. Concomitant with iron-ascorbate-mediated lipid peroxidation, there was a decline in PON1 activity and protein in both HDL3 and microsomes, which was attenuated by butylated hydroxytoluene antioxidant treatment. The current data indicate that PON1 localization in microsomes and HDL3 could represent a selective cellular and lipoprotein response to oxidative stress. This was tested by the iron-ascorbate oxygen-radical generating system. It is also proposed that the increased PON1 level may have a function related to the well-known atherosclerosis resistance of rats.     

Lipid complex of apolipoprotein A-I mimetic peptide 4F is a novel platform for paraoxonase-1 binding and enhancing its activity and stability

High density lipoprotein (HDL) associated paraoxonase-1 (PON1) is crucial for the anti-oxidant, anti-inflammatory, and anti-atherogenic properties of HDL. Discoidal apolipoprotein (apo)A-I:1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) complex has been shown to be the most effective in binding PON1, stabilizing it, and enhancing its lactonase and inhibitory activity of low density lipoprotein oxidation. Based on our earlier study demonstrating that apoA-I mimetic peptide 4F forms discoidal complex with 1,2-dimyristoyl-sn-glycero-3-phosphocholine, we hypothesized that lipid complexes of 4F would be able to bind PON1 and enhance its activity and stability. To test our hypothesis, we have expressed and purified a recombinant PON1 (rPON1) and studied its interaction with 4F:POPC complex. Our studies show significant increase, compared to the control, in the paraoxonase activity and stability of rPON1 in the presence of 4F:POPC complex. We propose that 4F:POPC complex is a novel platform for PON1 binding, increasing its stability, and enhancing its enzyme activity. We propose a structural model for the 4F:POPC:PON1 ternary complex that is consistent with our results and published observations.     

The catalytic histidine dyad of high density lipoprotein-associated serum paraoxonase-1 (PON1) is essential for PON1-mediated inhibition of low density lipoprotein oxidation and stimulation of macrophage cholesterol efflux

High density lipoprotein (HDL)-associated paraoxonase-1 (PON1) anti-atherogenic properties in macrophages, i.e. inhibition of cell-mediated oxidation of low density lipoprotein (LDL) and stimulation of cholesterol efflux, were studied using recombinant variants of PON1 and apoA-I expressed in Escherichia coli and reconstituted HDL (rHDL) particles composed of phosphatidylcholine/free cholesterol (PC/FC) and apoA-I. PON1 lactonase activity is stimulated by apoA-I by approximately 7-fold relative to PC/FC particles. Wild-type (WT) PON1 bound to rHDL inhibited macrophage-mediated LDL oxidation and stimulated cholesterol efflux from the cells to 2.3- and 3.2-fold greater extents, respectively, compared with WT PON1 bound to PC/FC particles without apoA-I. We also tested PON1 catalytic histidine dyad mutants (H115Q and H134Q) that are properly folded and that bind HDL in a similar mode compared with WT PON1, but that exhibit almost no lactonase activity. These could not inhibit macrophage-mediated LDL oxidation or stimulate rHDL-mediated cholesterol efflux from the cells. Furthermore, whereas HDL-bound WT PON1 induced the formation of lysophosphatidylcholine (LPC) in macrophages, the His dyad mutants did not, suggesting that the above anti-atherogenic properties of HDL-associated PON1 involve LPC release. Indeed, enrichment of macrophages with increasing concentrations of LPC resulted in inhibition of the cells' capability to oxidize LDL and in stimulation of HDL-mediated cholesterol efflux from the macrophages in an LPC dose-dependent manner. Thus, we provide the first direct indication that the anti-atherogenic properties of PON1 are related to its lipolactonase activity and propose a model in which PON1 acts as a lipolactonase to break down oxidized lipids and to generate LPC.     

Spontaneous transfer of phospholipid and cholesterol hydroperoxides between cell membranes and low-density lipoprotein: assessment of reaction kinetics and prooxidant effects

Under oxidative pressure in the vascular circulation, erythrocytes and phagocytic cells may accumulate membrane lipid hydroperoxides (LOOHs), including cholesterol- and phospholipid-derived species (ChOOHs, PLOOHs). LOOH translocation from cells to low-density lipoprotein (LDL) might sensitize the latter to free radical-mediated oxidative modification, an early event associated with atherogenesis. To test this, we examined the spontaneous transfer kinetics of various ChOOH species (5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH, 7 alpha/7 beta-OOH) and various PLOOH groups (PCOOH, PEOOH, PSOOH, SMOOH) using photoperoxidized erythrocyte ghosts as model donors and freshly prepared LDL as an acceptor. LOOH departure or uptake was monitored by reverse-phase HPLC with reductive electrochemical detection. Mildly peroxidized ghost membranes transferred overall ChOOH and PLOOH to LDL with apparent first-order rate constants approximately 60 and approximately 35 times greater than those of the respective parent lipids. Individual ChOOH rate constants decreased in the following order: 7 alpha/7 beta-OOH > 5 alpha-OOH > 6 alpha-OOH > 6 beta-OOH. Kinetics for reverse transfer from LDL to ghosts followed the same trend, but rates were significantly higher for all species and their combined activation energy was lower (41 vs 85 kJ/mol). PLOOH transfer rate constants ranged from 4- to 15-fold lower than the composite ChOOH constant, their order being as follows: PCOOH approximately PEOOH approximately PSOOH > SMOOH. Similar PLOOH transfer kinetics were observed when LDL acceptor was replaced by unilamellar liposomes, consistent with desorption from the donor membrane being the rate-limiting step. The susceptibility of transfer LOOH-enriched LDL to Cu2+-induced chain peroxidative damage was assessed by monitoring the accumulation of conjugated dienes and products of free radical-mediated cholesterol oxidation. In both cases, transfer-acquired LOOHs significantly reduced the lag time for chain initiation relative to that observed using nonperoxidized ghosts. These findings are consistent with the idea that LDL can acquire significant amounts of "seeding" LOOHs via translocation from various donors in the circulation.     

Coincubation of PON1, APO A1, and LCAT increases the time HDL is able to prevent LDL oxidation

The inhibition of low-density lipoprotein (LDL) oxidation by high-density lipoprotein (HDL) is a major antiatherogenic property of this lipoprotein. This activity is due, in part, to HDL associated proteins. However, whether these proteins interact in the antioxidant activity of HDL is unknown. LDL was incubated with apolipoprotein A1 (apo A1), lecithin:cholesterol acyltransferase (LCAT), and paraoxonase-1 (PON1) alone or in combination, in the presence or absence of HDL under oxidizing conditions. LDL lipid peroxide concentrations were determined. Apo A1, LCAT, and PON1 all inhibit LDL oxidation in the absence of HDL and enhance the ability of HDL to inhibit LDL oxidation. Their effect was additive rather than synergistic; the combination of these proteins significantly enhanced the length of time LDL was protected from oxidation. This seemed to be due to the ability of PON1 to prevent the oxidative inactivation of LCAT. Apo A1, LCAT, and PON1 can all contribute to the antioxidant activity of HDL in vitro. The combination of apo A1, LCAT, and PON1 prolongs the time that HDL can prevent LDL oxidation, due, at least in part, to the prevention LCAT inactivation.     

The determination of Q192R polymorphism of paraoxonase 1 by using non-toxic substrate p-nitrophenylacetate

CONTEXT:

The human serum paraoxonase 1 (PON1) is calcium-dependent esterase and associates with the high density serum lipoproteins. PON1 plays a major role in oxidation of high density lipoprotein and low density lipoprotein and prevention of atherogenesis in coronary heart disease. PON1Q and R allele hydrolyses number of substrates like paraoxon (PO) (diethyl p-nitrophenyl phosphate) and phenylacetate.

AIMS:

The aim of the study is to the determination of Q192R polymorphism of PON1 by using non-toxic substrate p-nitrophenylacetate and compares it with the phenotype determined by using PO as substrate.

MATERIALS AND METHODS:

The study group consists of 60 healthy normal patients. Paraoxonase activity was measured using the procedure described by Eckerson (Reference method) and for phenotyping; the ratio of hydrolysis of PO in the presence of 1 M NaCl (salt-stimulated PON1, SALT) to the hydrolysis of phenylacetate (PA) is calculated. In new method (Haagen et al.) arylesterase activity measured using p-nitrophenylacetate and for phenotyping arylesterase, the ratio of inhibition of enzymatic hydrolysis of p-nitrophenylacetate (substrate) by phenyl acetate to non-inhibited hydrolysis of p-nitrophenylacetate (inhibited arylesterase activity (IA-IA₀)/non-inhibited arylesterase activity (NIA).

RESULTS:

It was found that paraoxonase activity is trimodally distributed in both the methods. There is no significant difference in the distribution of PON1 phenotypes of both reference method and new method being frequencies 0.946 and 0.376 respectively and there was no significant difference for phenotypic polymorphism for an individual by both methods (χ²= 0.15 and P = 0.9262).

CONCLUSION:

The Q192R polymorphism of PON1 by using non-toxic substrate p-nitrophenylacetate showed trimodal distribution of QQ (homozygous), QR (heterozygous), and RR (homozygous) phenotype and it is comparable with reference method. This method can be used for PON1 phenotype in different pathological and complex disease conditions.     

Paraoxonase 1 overexpression in mice and its effect on high-density lipoproteins.

Paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme believed to protect against the early events of atherogenesis by its ability to hydrolyze oxidized phospholipids. A transgenic mouse overexpressing PON1 (mPON1) was developed to address the question of whether overexpression of PON1 is important in protecting HDL function during oxidative stress. Transgenic mice were obtained that have up to a 5-fold increase in mPON1 activity measured as arylesterase activity [52.7 +/- 17.3 U/ml versus 251.7 +/- 25.1 U/ml for wild-type (WT) and mPON1 high expressers, respectively]; this increase in mPON1 activity was reflected by a 5.3-fold increase in relative mass of the enzyme. Excess mPON1 was associated solely with HDL but did not alter HDL composition, size, or charge. Lecithin:cholesterol acyltransferase (LCAT) on HDL is a sensitive indicator of oxidative stress; exposure of plasmas from both WT and mPON1 overexpresser mice to 0.4 mM copper ions for 2 h showed a 30-40% protection of LCAT activity in mPON1 overexpressers compared to WT. Excess mPON1 also inhibited lipid hydroperoxide formation on HDL. These data strongly suggest that overexpression of mPON1 protects HDL integrity and function.