Enzymatically active paraoxonase-1 is located at the external membrane of producing cells and released by a high affinity, saturable, desorption mechanism.

Paraoxonase-1 (PON1) is a high density lipoprotein (HDL)-associated serum enzyme that protects low density lipoproteins from oxidative modifications. There is a relative lack of information on mechanisms implicated in PON1 release from cells. The present study focused on a model derived from stable transfection of CHO cells, to avoid co-secretion of apolipoprotein (apo) A-I and lipids, which could lead to formation of HDL-like complexes. Our results indicate that, in the absence of an appropriate acceptor, little PON1 is released. The results designate HDL as the predominant, physiological acceptor, whose efficiency is influenced by size and composition. Neither lipid-poor apoA-I or apoA-II nor low density lipoproteins could substitute for HDL. Protein-free phospholipid complexes promoted PON1 release. However, the presence of both apolipoprotein and phospholipid were necessary to promote release and stabilize the enzyme. Immunofluorescence studies demonstrated that PON1 was inserted into the external membrane of CHO cells, where it was enzymatically active. Accumulation of PON1 in the cell membrane was not influenced by the ability of the cell to co-secrete of apoA-I. Release appeared to involve desorption by HDL; human and reconstituted HDL promoted PON1 release in a saturable, high affinity manner (apparent affinity 1.59 +/- 0.3 microg of HDL protein/ml). Studies with PON1-transfected hepatocytes (HuH-7) revealed comparable structural features with the peptide located in a punctate pattern at the external membrane and enzymatically active. We hypothesize that release of PON1 involves a docking process whereby HDL transiently associate with the cell membrane and remove the peptide from the external membrane. The secretory process may be of importance for assuring the correct lipoprotein destination of PON1 and thus its functional efficiency.     

Paraoxonase 1 and homocysteine metabolism

Paraoxonase 1 (PON1), a component of high-density lipoprotein (HDL), is a calcium-dependent multifunctional enzyme that connects metabolisms of lipoproteins and homocysteine (Hcy). Both PON1 and Hcy have been implicated in human diseases, including atherosclerosis and neurodegeneration. The involvement of Hcy in disease could be mediated through its interactions with PON1. Due to its ability to reduce oxidative stress, PON1 contributes to atheroprotective functions of HDL in mice and humans. Although PON1 has the ability to hydrolyze a variety of substrates, only one of them-Hcy-thiolactone-is known to occur naturally. In humans and mice, Hcy-thiolactonase activity of PON1 protects against N-homocysteinylation, which is detrimental to protein structure and function. PON1 also protects against neurotoxicity associated with hyperhomocysteinemia in mouse models. The links between PON1 and Hcy in relation to pathological states such as coronary artery disease, stroke, diabetic mellitus, kidney failure and Alzheimer's disease that emerge from recent studies are the topics of this review.     

HDL subfraction distribution of paraoxonase-1 and its relevance to enzyme activity and resistance to oxidative stress

The subfraction distribution of HDL-associated peptides has implications for their functions and the impact of pathological modifications to lipoprotein metabolism on these functions. We have analyzed the subfraction distribution of paraoxonase-1 (PON1) and the consequences for enzyme activity and stability. HDL subfractions were defined by the presence (LpA-I,A-II) or absence (LpA-I) of apolipoprotein A-II (apoA-II). PON1 was present in both subfractions, although increased concentrations of HDL were associated with significantly increased PON1 in LpA-I. ApoA-II did not modify the capacity of native human HDL or reconstituted HDL to promote PON1 secretion from cells or to stabilize enzyme activity, nor did apoA-II decrease PON1 activity when added to rabbit serum normally devoid of the apolipoprotein. LpA-I,A-II particles isolated from human serum or reconstituted HDL (LpA-I,A-II) showed a significantly greater capacity than HDL(LpA-I) to stabilize secreted PON1 and purified recombinant PON1 added to such particles. PON1 associated with apoA-II-containing particles showed greater resistance to inactivation arising from oxidation. ApoA-I, apoA-II, and LpA-I,A-II, but not LpA-I, were independent determinants of serum PON1 concentration and activity in multivariate analyses. PON1 is at least equally distributed between LpA-I and LpA-II,A-II HDL particles. This dichotomous distribution has implications for PON1 activity and stability that may impact on the physiological role of the enzyme.     

HDL‐paraoxonase and Membrane Lipid Peroxidation: A Comparison Between Healthy and Obese Subjects

High‐density lipoproteins (HDLs) play a key role in the protection against oxidative damage. The enzyme paraoxonase‐1 (PON1) associated at the surface of HDL modulates the antioxidant and anti‐inflammatory role of HDL. Previous studies have demonstrated a decrease of serum PON in obese patients. The aim of this study was to investigate whether modifications of PON1 activity reflect in a different ability to protect and/or repair biological membranes against oxidative damage. Thirty obese patients at different grades of obesity (BMI ranging from 30.4 to 64.0 kg/m²) and 62 age‐matched control subjects (BMI <25 kg/m²) were included in the study. The ability of HDL to protect membranes against oxidative damage was studied using erythrocyte membranes oxidized with 2,2‐azobis(2 amidinopropane)dihydrochloride (AAPH) (ox‐membrane). The membrane lipid hydroperoxide levels were evaluated after the incubation of ox‐membranes in the absence or in the presence of HDL of controls or obese patients. The results confirm that HDL exerts a protective effect against lipid peroxidation. The ability of HDL to repair erythrocyte membranes was positively correlated with HDL‐PON activity and negatively correlated with lipid hydroperoxide levels in HDL. These results suggest that PON modulates the HDL repairing ability. HDL from obese patients repaired less efficiently erythrocyte membranes against oxidative damage with respect to HDL from healthy subjects. A negative relationship has been established between BMI of obese patients and the protective effect of HDL. In conclusion, the decrease of HDL‐PON activity and the lower HDL protective action against membrane peroxidation in obese patients could contribute to accelerate the cellular oxidative damage and arteriosclerosis in obesity.     

Paraoxonase, a cardioprotective enzyme: continuing issues

PURPOSE OF REVIEW: The paraoxonase family consists of three members (PON1, PON2 and PON3) that share structural properties and enzymatic activities, among which is the ability to hydrolyze oxidized lipids in LDL. The exact function of the different family members is not clear although the conservation among the individual family members across species suggests a strong evolutionary pressure to preserve these functional differences. The purpose of this review is to highlight several problems with respect to the mechanism of action of paraoxonase and differences between the family members that merit further study. RECENT FINDINGS: PON1 transgenic mice are at lower risk for atherosclerosis, which is consistent with PON1 gene knockout studies in mice and human genetic polymorphism studies. The exact mechanism by which paraoxonase is cardioprotective is not clear, although it is likely to be related to its antioxidant properties especially on LDL. PON1 levels are influenced by a variety of environmental factors, including statins and cytokines. The preferential association of PON1 with HDL is mediated in part by its signal peptide and by desorption from the plasma membrane of expressing cells by HDL or phospholipid. Apolipoprotein A-I is not necessary for PON1 association with HDL, but its activity is stabilized in the presence of the apolipoprotein. Only in the absence of both lecithin cholesterol acyltransferase and apolipoprotein E is paraoxonase associated with non-HDL lipoproteins. The displacement of paraoxonase by serum amyloid A may explain in part the proinflammatory nature of HDL in the acute phase. The mechanism by which PON3 associates with HDL has not been studied. In addition to the ability to hydrolyze oxidized lipids in LDL, paraoxonase also alters the oxidative state of macrophages. Exogenous PON1 is able to reverse the oxidative stress in macrophages in aged apolipoprotein E deficient and PON1 deficient mice. The increase in oxidative stress in macrophages from PON1 deficient mice occurs despite the expression of PON2 and PON3 in macrophages. PON1 has recently been shown to contain phospholipase A2 activity, with the subsequent release of lysophosphatidylcholine that influences macrophage cholesterol biosynthesis. SUMMARY: PON1 mass and activity in the plasma significantly influence the risk of developing cardiovascular disease. This is likely mediated by its antioxidation properties on LDL and/or macrophages. The precise mechanism by which this HDL associated protein prevents or attenuates oxidation of LDL and the oxidative stress of macrophages remains to be clarified. The role of PON2 and PON3 in atherosclerosis and their antioxidant properties with respect to LDL and macrophages also merit further investigation.     

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.     

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.     

Purified human paraoxonase-1 interacts with plasma membrane lipid rafts and mediates cholesterol efflux from macrophages

Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme thought to make a major contribution to the antioxidant and anti-inflammatory capacities of HDLs. However, the role of PON1 in the modulation of cholesterol efflux is poorly understood. The aim of our study was to investigate the involvement of PON1 in the regulation of cholesterol efflux, especially the mechanism by which it modulates HDL-mediated cholesterol transport. The enrichment of HDL(3) with human PON1 enhanced, in a dose-dependent manner, cholesterol efflux from THP-1 macrophage-like cells and ABCA1-enriched J774 macrophages. Moreover, an additive effect was observed when ABCA1-enriched J774 macrophages were incubated with both PON1 and apo-AI. Interestingly, PON1 alone was able to mediate cholesterol efflux from J774 macrophages and to upregulate ABCA1 expression on J774 macrophages. Immunofluorescence measurement showed an increase in PON1 levels in the cytoplasm of J774 macrophages overexpressing ABCA1. PON1 used an apo-AI-like mechanism to modulate cholesterol efflux from rapid and slow efflux pools derived from the lipid raft and nonraft domains of the plasma membrane, respectively. This was supported by the fact that ABCA1 protein was incrementally expressed by J774 macrophages within the first few hours of incubation with cholesterol-loaded J774 macrophages and that cyclodextrin significantly inhibited the capacity of PON1 to modulate cholesterol efflux from macrophages. This finding suggested that PON1 plays an important role in the antiatherogenic properties of HDLs and may exert its protective function outside the lipoprotein environment.     

Effect of homocysteinylation on high density lipoprotein physico-chemical properties

High density lipoproteins (HDL) exert a protective effect against homocysteinylation due to the activity of the enzyme paraoxonase/thiolactonase associated to the lipoprotein surface. However, a small amount of N-homocysteinylated HDL (N-Hcy-HDL) is present in human plasma, suggesting that homocysteinylation of plasma lipoproteins occurs in vivo.Aim of the present study was to investigate the effect of homocysteinylation on apoprotein structure and physico-chemical properties of HDL using the analysis of the fluorescent emission spectra of tryptophan and Laurdan (6-dodecanoyl-2-dimethyl-aminonaphthalene). Our results demonstrated that the increase in –SH groups in HDL homocysteinylated in vitro (Hcy-HDL) was associated with apoprotein conformational changes and modifications of physico-chemical properties.A significant decrease of paraoxonase and lactonase activity of HDL bound PON1 has also been observed in Hcy-HDL. A significant decrease of the enzyme activity has been observed also in purified PON1 homocysteinylated following the same experimental conditions used for HDL. Moreover, we demonstrated that oxidized HDL were more susceptible to homocysteinylation with respect to control HDL.The modifications of apoprotein conformation and physico-chemical properties in Hcy-HDL and the decrease of paraoxonase-1 activity could affect the protective effect of HDL against oxidative damage and/or homocysteinylation and could contribute to accelerated atherosclerosis in patients affected by diseases associated with oxidative damage, in renal disorders and in patients affected by genetic or nutritional disorders of homocysteine or folate metabolism.     

Paraoxonases (PONs) 1, 2, and 3 are expressed in human and mouse gastrointestinal tract and in Caco-2 cell line: selective secretion of PON1 and PON2

The paraoxonase (PON) family contains three genes (PON1/2/3) that are believed to be involved in the protection against oxidative stress. PON1 and PON3 are circulating in serum attached to high-density lipoprotein fraction (HDL), whereas PON2 is ubiquitously expressed. The intestine is the second major organ that synthesizes lipoproteins; therefore, we examined PON mRNA expression and protein levels in gastrointestinal biopsies from humans, from C57BL6 mice, and from Caco-2 cells, a colon carcinoma-derived cell line that exhibits properties of intestinal epithelium at differentiation. PON 1/2/3 mRNA and proteins were present in human biopsies with variable expression among different gastrointestinal segments. Only PON2 and PON3 were present in mice. All PON mRNA, proteins, and enzymatic activities were present in Caco-2 cells. Oxidation of CaCo-2 cells with ferrum ascorbate had no significant effect on PON mRNA expression, but it increased paraoxonase and lactonase activity, whereas statinase activity was decreased. We showed polarized secretion of PON1 (basolateral) and PON2 (apical) into Caco-2 culture medium, raising the possibility that intestine is capable of producing and releasing PON1 and PON3 to the circulation, whereas PON2 is released at the brush-border membrane to intestinal lumen where it may perform another yet unclear function.     

In vivo administration of BL-3050: highly stable engineered PON1-HDL complexes

Background  

Serum paraoxonase (PON1) is a high density lipoprotein (HDL)-associated enzyme involved in organophosphate (OP) degradation and prevention of atherosclerosis. PON1 comprises a potential candidate for in vivo therapeutics, as an anti-atherogenic agent, and for detoxification of pesticides and nerve agents. Because human PON1 exhibits limited stability, engineered, recombinant PON1 (rePON1) variants that were designed for higher reactivity, solubility, stability, and bacterial expression, are candidates for treatment. This work addresses the feasibility of in vivo administration of rePON1, and its HDL complex, as a potentially therapeutic agent dubbed BL-3050.     

Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein

The oxidation of apolipoprotein B-containing lipoproteins and cell membrane lipids is believed to play an integral role in the development of fatty streak lesions, an initial step in atherogenesis. We have previously shown that two antioxidant-like enzymes, paraoxonase (PON)-1 and PON3, are high density lipoprotein-associated proteins capable of preventing the oxidative modification of low density lipoprotein (LDL) (Reddy, S. T., Wadleigh, D. J., Grijalva, V., Ng, C., Hama, S., Gangopadhyay, A., Shih, D. M., Lusis, A. J., Navab, M., and Fogelman, A. M. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 542-547). In the present study, we demonstrate that PON2 (i) is not associated with high density lipoprotein; (ii) has antioxidant properties; and (iii) prevents LDL lipid peroxidation, reverses the oxidation of mildly oxidized LDL (MM-LDL), and inhibits the ability of MM-LDL to induce monocyte chemotaxis. The PON2 protein was overexpressed in HeLa cells using the tetracycline-inducible ("Tet-On") system, and its antioxidant capacity was measured in a fluorometric assay. Cells that overexpressed PON2 showed significantly less intracellular oxidative stress following treatment with hydrogen peroxide or oxidized phospholipid. Moreover, cells that overexpressed PON2 were also less effective in oxidizing and modifying LDL and, in fact, were able to reverse the effects of preformed MM-LDL. Our results suggest that PON2 possesses antioxidant properties similar to those of PON1 and PON3. However, in contrast to PON1 and PON3, PON2 may exert its antioxidant functions at the cellular level, joining the host of intracellular antioxidant enzymes that protect cells from oxidative stress.     

Single intravenous injection of plasmid DNA encoding human paraoxonase-1 inhibits hyperlipidemia in rats

Paraoxonase-1 (PON1, EC 3.1.8.1) is a high-density lipoprotein (HDL)-associated antioxidant enzyme, and its activity correlates negatively with the level of plasma low-density lipoprotein cholesterol (LDL-C) and triglyceridemia (TG). In this study, we examined the therapeutic effect of plasmid DNA containing the human PON1 gene (pcDNA/PON1) in hyperlipidemic model rats. The rats were fed a high-fat and high-cholesterol diet for 25 days to produce a hyperlipidemic animal model. Single intravenous injection of pcDNA/PON1 into model rats prevented dyslipidemia and hepatic lipid accumulation. The mechanisms of pcDNA/PON1 in treating hyperlipidemia were associated with increases of serum antioxidant PON1 and SOD activities, and with reduction of the levels of total cholesterol (TC), LDL-C and TG. The results suggest the potential therapeutic effect of pcDNA/PON1 on hyperlipidemia.     

One Enzyme,Two Functions: PON2 PREVENTS MITOCHONDRIAL SUPEROXIDE FORMATION AND APOPTOSIS INDEPENDENT FROM ITS LACTONASE ACTIVITY*

The human enzyme paraoxonase-2 (PON2) has two functions, an enzymatic lactonase activity and the reduction of intracellular oxidative stress. As a lactonase, it dominantly hydrolyzes bacterial signaling molecule 3OC12 and may contribute to the defense against pathogenic Pseudomonas aeruginosa. By its anti-oxidative effect, PON2 reduces cellular oxidative damage and influences redox signaling, which promotes cell survival. This may be appreciated but also deleterious given that high PON2 levels reduce atherosclerosis but may stabilize tumor cells. Here we addressed the unknown mechanisms and linkage of PON2 enzymatic and anti-oxidative function. We demonstrate that PON2 indirectly but specifically reduced superoxide release from the inner mitochondrial membrane, irrespective whether resulting from complex I or complex III of the electron transport chain. PON2 left O₂^˙̄ dismutase activities and cytochrome c expression unaltered, and it did not oxidize O₂^˙̄ but rather prevented its formation, which implies that PON2 acts by modulating quinones. To analyze linkage to hydrolytic activity, we introduced several point mutations and show that residues His¹¹⁴ and His¹³³ are essential for PON2 activity. Further, we mapped its glycosylation sites and provide evidence that glycosylation, but not a native polymorphism Ser/Cys³¹¹, was critical to its activity. Importantly, none of these mutations altered the anti-oxidative/anti-apoptotic function of PON2, demonstrating unrelated activities of the same protein. Collectively, our study provides detailed mechanistic insight into the functions of PON2, which is important for its role in innate immunity, atherosclerosis, and cancer.     

Macrophage paraoxonase 1 (PON1) binding sites

Paraoxonase 1 (PON1), an HDL-associated esterase, is known to possess anti-oxidant and anti-atherogenic properties. PON1 was shown to protect macrophages from oxidative stress, to inhibit macrophage cholesterol biosynthesis, and to stimulate HDL-mediated cholesterol efflux from the cells. The aim of the present study was to characterize macrophage PON1 binding sites which could be responsible for the above anti-atherogenic activities.Incubation of FITC-labeled recombinant PON1 with J774 A.1 macrophage-like cell line at 37 °C, resulted in cellular binding and internalization of PON1, leading to PON1 localization in the cell’s cytoplasm compartment. In order to determine whether PON1 uptake is mediated via a specific binding to the macrophage, FITC-labeled recombinant PON1 was incubated with macrophages at 4 °C, followed by cell membranes separation. Macrophage membrane fluorescence was shown to be directly and dose-dependently related to the labeled PON1 concentration. Furthermore, binding assays performed at 4 and at 37 °C, using labeled and non-labeled recombinant PON1 (for competitive inhibition), demonstrated a dose-dependent significant 30% decrement in labeled PON1 binding to the macrophages, by the non-labeled PON1. Similarly, binding assays, using labeled PON1 and non-labeled HDL (the natural carrier of PON1 in the circulation) indicated that HDL decreased the binding of labeled PON1 to macrophages by 25%. Unlike HDL, LDL had no effect on labeled PON1 binding to macrophages. Finally, HDL were pre incubated without or with PON1 or apolipoprotein AI (apoAI) antibodies, in order to block PON1 or apoAI ability to bind to the cells. HDL incubation with antibody to PON1 or to apoAI significantly decreased HDL ability to inhibit macrophages-mediated LDL oxidation (by 32% or by 25%, respectively). A similar trend was also observed for HDL-mediated cholesterol efflux from macrophages, with an inhibitory effect of 35% or 19%, respectively. These results suggest that blocking HDL binding to macrophages through its apo A-I, and more so, via its PON1, results in the attenuation of HDL-PON1 biological activities.In conclusion, PON1 specifically binds to macrophage binding sites, leading to anti-atherogenic effects. Macrophage PON1 binding sites may thus be a target for future cardio protection therapy.     

Cysteine substitutions in apolipoprotein A-I primary structure modulate paraoxonase activity

Paraoxonase (PON) is transported primarily on apolipoprotein A-I (apoA-I) -containing high-density lipoprotein (HDL) and is thought to protect against early atherogenic events including low-density lipoprotein (LDL) oxidation and monocyte migration. It has been proposed that apoA-I may be necessary for PON's association with plasma HDL. On the basis of this, we examined the effect of apoA-I on PON's enzymatic activity and its ability to associate with HDL. Additionally, we examined whether changes in apoA-I primary structure (cysteine substitution mutations) could modulate these effects. Chinese hamster ovary cells stably transfected with human PON1A cDNA were incubated in the presence and absence of recombinant wild-type apoA-I (apoA-I(WT)) and specific Cys substitution mutations. Extracellular accumulation of PON activity in the presence of apoA-I(WT) was 0.095 +/- 0.013 unit/mg of cell protein (n = 7) compared to 0.034 +/- 0.010 unit/mg of cell protein in the absence of apoA-I (n = 7), a 2.79-fold increase in activity when apoA-I was incubated with the cells. Lipid-free apoA-I did not increase PON activity, while preformed nascent HDL increased PON activity only 30%, suggesting that maximal PON activity is lipid-dependent and requires coassembly of PON and apoA-I on nascent HDL. The cysteine mutations R10C, R27C, and R61C significantly increased (p < 0.01) PON activity 32.6% +/- 14.7%, 31.6% +/- 18.9%, and 27.4% +/- 20%, respectively, over that of wild type (WT). No changes in PON activity were observed with apoA-I cysteine substitution mutations in the C-terminal portion of the protein. The data suggest that, for optimal PON activity, coassembly of the enzyme onto nascent HDL is required and that the N-terminal region of apoA-I may be important in the assembly process.     

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.     

Impacts of some antibiotics on human serum paraoxonase 1 activity

Some enzymes are known to be drug target inhibitions of which can be critical for organisms. PON has a critical role to prevent atherogenesis by inhibiting lipid peroxidation. It is well known that paraoxonase 1 (PON1) plays an important function on high-density lipoprotein (HDL) structure to prevent lipid oxidation not only of low-density lipoprotein, but also of HDL itself. We investigated in vitro effects of some medical drugs on PON1 activity from human serum. K_i constants for oxytetracycline hydrochloride, netilmycin sulfate, lincomycin hydrochloride, clindamycin phosphate, and streptomycin sulfate were found as 0.2, 3.73, 18.30, 35.80, and 56.30 mM, respectively. Our results indicate that these commonly used drugs inhibit the activity of the enzyme at very low doses with different inhibition mechanisms.     

The importance of high-density lipoproteins for paraoxonase-1 secretion, stability, and activity

The association of paraoxonase-1 (PON1) with high-density lipoproteins (HDL) is a prerequisite for maintaining normal serum activity of the enzyme. The lipoprotein furnishes an amphipathic environment to shield the hydrophobic, N-terminal region of the enzyme, and such an environment may also be necessary for interaction of PON1 with its substrates. HDL provides the optimal physiological acceptor complex, in terms of both stimulating PON1 secretion and stabilizing the secreted peptide. Lipid and peptide components of HDL contribute to these effects, such that modulating HDL composition influences PON1 activity and function. In this context, understanding how PON1 associates with HDL, what governs the association, and the mechanism by which the PON1–HDL complex exerts its antioxidant function is of particular physiological relevance. Moreover, HDL is subject to substantial compositional variations under both normal and pathological metabolic conditions. It has implications for the influence of the enzyme on cardiovascular risk, as normal enzyme activity may not correlate with optimal functional (antioxidant) efficiency. We review evidence that HDL lipid and protein components interact to promote PON1 secretion and maintain serum enzyme activity. Emerging data on how the enzyme associates with HDL are discussed, and the consequences for PON1 function of modifications to HDL are outlined. Finally, we highlight questions concerning the HDL–PON1 association that remain unanswered but are of particular importance in defining PON1 efficiency.     

Cholesterol transfer from mitochondrial membranes and cells to human and rat serum lipoprotein fractions

We have investigated the transfer of [14C]cholesterol from labeled bovine heart mitochondria and Friend erythroleukemic cells to high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) fractions from human and rat plasma. The lipoprotein fractions were obtained by molecular sieve chromatography of plasma on agarose A-5m columns. For either membrane system, the highest rate of [14C]cholesterol transfer was observed with the human and the rat HDL fraction. Since the mitochondria lack the receptors for HDL, one may conclude that the observed preferential transfer is not governed by a receptor-controlled interaction of HDL with the membrane. Under conditions where the pool of free cholesterol in the lipoprotein fractions was the same, HDL was a much more efficient acceptor of [14C]cholesterol from mitochondria than LDL or VLDL. Similarly, transfer of [14C]cholesterol proceeded at a higher rate to HDL than to sonicated egg phosphatidylcholine (PC) vesicles, even under conditions where there was a tenfold excess of the vesicle-PC pool over the HDL phospholipid pool. This preferred transfer of [14C]cholesterol to HDL cannot be explained by a random diffusion of monomer cholesterol molecules. Rather, it shows that HDL has a specific effect on this process in the sense that it most likely enhances the efflux of cholesterol from the membrane. Treatment of HDL with trypsin reduced the rate of [14C]cholesterol transfer by 40-50%, indicating that protein component(s) are involved. One of these components appears to be apoA-I, as this protein was shown to enhance the transfer of [14C]cholesterol from mitochondria to lipid vesicles.