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.     

Arylesterase activity of paraoxonase 1 (PON1) on HDL3 and HDL2: Relationship with Q192R,C-108T,and L55M polymorphisms

BackgroundControversy exists regarding the role of the subfractions of high-density lipoproteins (HDL₂ and HDL₃) in cardiovascular disease. The functionality of these particles, and their protective role, is due in part to the paraoxonase 1 (PON1) presence in them. The polymorphisms rs662 (Q192R, A/G), rs854560 (L55 M, T/A), and rs705379 (C-108T) of the PON1 gene have been related to enzyme activity and, with the anti-oxidative capacity of the HDL. The objective was to determine the arylesterase PON1 activity in HDL₃ and HDL₂ and its relationship with the polymorphisms mentioned, in a young population.MethodsThe polymorphisms were determined through mini-sequencing (SnaPshot). The HDL subpopulations were separated via ionic precipitation, cholesterol was measured with enzymatic methods, and PON1 activity was measured through spectrophotometry.ResultsThe results show that the PON1 polymorphisms do not influence the cholesterol in the HDL. A variation between 40.02 and 43.9 mg/dL was in all the polymorphisms without significant differences. Additionally, PON1 activity in the HDL₃ subfractions was greater (62.83 ± 20 kU/L) than with HDL₂ (35.8 ± 20.8 kU/L) in the whole population and in all the polymorphisms (p < 0.001), and it was independent of the polymorphism and differential arylesterase activity in the Q192R polymorphism (QQ > QR > RR). Thus, 115.90 ± 30.7, 88.78 ± 21.3, 65.29 ± 10.2, respectively, for total HDL, with identical behavior for HDL₃ and HDL₂.ConclusionsPON1 polymorphisms do not influence the HDL_-c, and the PON activity is greater in the HDL₃ than in the HDL₂, independent of the polymorphism, but it is necessary to delve into the functionality of these findings in different populations.     

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.     

Effect of 3,4-dihydroxyphenylalanine on Cu(2+)-induced inactivation of HDL-associated paraoxonasel and oxidation of HDL; inactivation of paraoxonasel activity independent of HDL lipid oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

Effect of 3,4-dihydroxyphenylalanine on Cu2+-induced Inactivation of HDL-associated Paraoxonase1 and Oxidation of HDL; Inactivation of Paraoxonase1 Activity Independent of HDL Lipid Oxidation

Paraoxonase1 (PON1), one of HDL-asssociated antioxidant proteins, is known to be sensitive to oxidative stress. Here, the effect of endogenous reducing compounds on Cu²⁺-mediated inactivation of PON1 was examined. Cu²⁺-mediated inactivation of PON1 was enhanced remarkably by catecholamines, but not by uric acid or homocysteine. Furthermore, catecholamines such as 3,4-dihydroxyphenylalanine (DOPA), dopamine or norepinephrine were more effective than caffeic acid or pyrocatechol in promoting Cu²⁺-mediated inactivation of PON1, suggesting the importance of dihydroxybenzene group as well as amino group. DOPA at relatively low concentrations showed a concentration-dependent inactivation of PON1 in a concert with Cu²⁺, but not Fe²⁺. The DOPA/Cu²⁺-induced inactivation of PON1 was prevented by catalase, but not hydroxyl radical scavengers, consistent with Cu²⁺-catalyzed oxidation. A similar result was also observed when HDL-associated PON1 (HDL-PON1) was exposed to DOPA/Cu²⁺. Separately, it was found that DOPA at low concentrations (1-6 μM) acted as a pro-oxidant by enhancing Cu²⁺-induced oxidation of HDL, while it exhibited an antioxidant action at ≥10 μM. In addition, Cu²⁺-oxidized HDL lost the antioxidant action against LDL oxidation. Meanwhile, the role of DOPA/Cu²⁺-oxidized HDL differed according to DOPA concentration; HDL oxidized with Cu²⁺ in the presence of DOPA (60 or 120 μM) maintained antioxidant activity of native HDL, in contrast to an adverse effect of DOPA at 3 or 6 μM. These data indicate that DOPA at micromolar level may act as a pro-oxidant in Cu²⁺-induced inactivation of PON1 as well as oxidation of HDL. Also, it is proposed that the oxidative inactivation of HDL-PON1 is independent of HDL oxidation.     

A complete backbone spectral assignment of human apolipoprotein AI on a 38 kDa preβHDL (Lp1-AI) particle

ApoAI is the major protein component of the high-density lipoprotein (HDL) that has been a hot subject of interests because of its anti-atherogenic properties. ApoAI/preβ-HDL is the most effective acceptors specifically for free cholesterol in human plasma and serves as the precursor of HDL particles. Here we report a complete backbone assignment of human apoAI on a 38 kDa preβHDL (Lp1-AI) particle.     

Effects of ABCG1 knockout on proteomic composition of HDL in mice on a chow diet and a high-fat diet

ATP-binding cassette transporter G1 (ABCG1) is a cellular transmembrane protein that transports oxysterol efflux from cells to high-density lipoprotein (HDL) particles in the plasma. Previous studies have demonstrated that an ABCG1 deficiency exerts an antiatherosclerotic function through the effects of oxysterol accumulation in cells to enhance apoptosis and regulate inflammatory processes. However, whether the deficiency of ABCG1 and the corresponding changes in the efflux of oxysterols could take a series of impacts on the proteomic composition of HDL remains unclear. Here, plasma HDL of ABCG1(−/−) mice and their wild-type controls on a normal chow diet (NCD) or a high-fat diet (HFD) were isolated by ultracentrifugation. The proportion of 7-ketocholesterol and the proteomic composition of samples were comparatively analyzed by LC-MS/MS. In NCD-fed mice, lipid metabolism-related protein (arachidonate 12-lipoxygenase) and antioxidative protein (pantetheinase) exhibited increased accumulation, and inflammatory response protein (alpha-1-antitrypsin) was decreased in accumulation in ABCG1(−/−) mice HDL. In HFD-fed mice, fewer proteins were detected than that of NCD-fed mice. The ABCG1(−/−) mice HDL exhibited increased accumulation of lipid metabolism-related proteins (e.g., carboxylesterase 1C, apolipoprotein (apo)C-4) and decreased accumulation of alpha-1-antitrypsin, as well as significantly reduced proportion of 7-ketocholesterol. Additionally, positive correlations were found between 7-ketocholesterol and some essential proteins on HDL, such as alpha-1-antitrypsin, apoA-4, apoB-100, and serum amyloid A (SAA). These results suggest a detrimental impact of oxysterols on HDL composition, which might affect the antiatherosclerotic properties of HDL.     

Logic regression analysis of association of gene polymorphisms with low HDL: Tehran Lipid and Glucose Study

Introduction

Logic regression is a generalized regression method that can recognize complex Boolean interactions of binary variables. It has been successfully applied to single-nucleotide polymorphism (SNP) data because of the importance of interactions in SNP association studies. The objective of this study is to assess the association between high-density lipoprotein (HDL) function and some related gene polymorphisms after adjusting for potential confounders using logic regression.

Methods

Subjects in this cross-sectional study were randomly selected from among participants of the Tehran Lipid and Glucose Study (TLGS). A total of 436 subjects (172 men and 264 women), aged ≥ 20 years, were selected to be included in the current study. Logic regression analysis was used in order to recognize the combination of genetic main effects and possible interactions associated with HDL-C level. Cross validation and randomization test were carried out to avoid over fitting of the models.

Results

The cross validation test suggested three Boolean combinations with four predictors for a fully-adjusted logic model. The fully adjusted model showed that those who carry an Apo E gene E3 allele or have high TG level have an odds ratio of 2.35 (95% CI:1.3–4.25) for having low HDL compared to other subjects. In addition, subjects with high TG level have an odds ratio of 2.73 (95% CI: 1.65, 4.53) for having low HDL.

Conclusion

The results showed that logic regression is a powerful method to find the interaction between high TG level and Apo E polymorphism associated with low HDL.     

Human carotid plaque phosphatidylcholine specifically interacts with paraoxonase 1, increases its activity,and enhances its uptake by macrophage at the expense of its binding to HDL

Human carotid atherosclerotic plaque is in direct contact with circulatory blood components. Thus, plaque and blood components may affect each other. The current study presents the effects of plaque chloroform:methanol (C:M) extract on the HDL-associated enzyme paraoxnase 1 (PON1). This study is part of our investigation on the mutual effects of the interactions between atherosclerotic lesions and blood components. Recombinant PON1 (rePON1) was incubated with the human carotid plaques C:M extract and PON1 activities were analyzed. Lactonase and paraoxonase activities were elevated due to C:M treatment, by 140 and by 69%, respectively. Analytical chemistry analyses revealed specific phosphatidylcholines (PCs) as the plaque active components. Tryptophan fluorescence quenching assay, together with molecular docking, shows that PON1 activity is enhanced in correlation with the level of PC affinity to PON1. Molecular docking revealed that PCs interact specifically with H2-PON1 α-helix, which together with H1 enzyme α-helix links the protein to the HDL surface. These findings are supported by additional results from the PON1 ∆20 mutant that lack its H1-α-helix. Incubation of this mutant with the plaque C:M extract increased PON1 activity by only 20%, much less than the wild-type PON1 that elevated PON1 activity at the same concentration by as much as 95%. Furthermore, as much as the affinity of the enzyme to the PC was augmented, the ability of PON1 to bind to the HDL particle decreased. Finally, PON1 interaction with PC enhance its uptake into the macrophage cytoplasm. In conclusions, Specific lesion phosphatidylcholines (PCs) present in the human carotid plaque significantly enhance PON1 catalytic activities due to their interaction with the enzyme. Such a lesion׳s PC–PON1 interaction, in turn, competes with HDL PCs and enhances PON1 uptake by macrophage at the expense of PON1 binding to the HDL.     

北京鸭血清HDL主要载脂蛋白的高效液相层析分离及其理化性质

用高效液相层析(HPLC)对北京鸭血清高密度脂蛋白(HDL)的所有载脂蛋白(apo)分离纯化,得五个主要apo峰,经SDS-PAGE鉴定,均为单一带;并做了IEF、糖基含量分析;对其中含量高的四个apo峰样进行了氨基酸组成测定和N-端部分氨基酸序列分析;从所做理化性质的研究,发现北京鸭血清HDL中的主要apo为:A-Ⅰ、C-Ⅲ_o、apoC-Ⅲ_s(s=1,2)、apoC's及可能的A-Ⅲ,几乎不含E和A-Ⅱ_o北京鸭HDL中apo的上述组成特点明显不同于易患动脉粥样硬化(As)的人及兔等动物,在一定程度上决定了北京鸭HDL的形成与代谢具有其特点,该特点与北京鸭不易形成As密切相关。     

Cellular localization and interaction of ABCA1 and caveolin-1 in aortic endothelial cells after HDL incubation

The goal of this study was to investigate the cellular localization and the interaction between caveolin-1 and ABCA1 in cholesterol-loaded aortic endothelial cells after HDL incubation. Immunofluorescence confocal microscopy showed that ABCA1 was found primarily on the cell surface, whereas caveolin-1 was revealed on the cell surface and in the cytoplasm. The HDL appeared to colocalize with ABCA1 and caveolin-1 on the cell surface. No free HDL was revealed in the cytoplasm. The HDL was colocalized neither with early endosome marker (CD71) nor with late endosome marker (LAMP2). The chemical cross-linking and immunoprecipitation analysis revealed that ABCA1 binds directly to both HDL and caveolin-1, whereas HDL does not bind directly to caveolin-1. The studies provide evidence for a direct interaction between ABCA1 and HDL, ABCA1 and caveolin-1, but not HDL and caveolin-1, indicating that ABCA1 may act as a structural platform between HDL and caveolin-1 on the cell surface during cellular cholesterol efflux.     

Oxidative inactivation of lactonase activity of purified human paraoxonase 1 (PON1)

Paraoxonase1 (PON1), one of HDL-associated antioxidant proteins, is known to lose its activity in vivo systems under oxidative stress. Here, we examined the effect of various oxidants on lactonase activity of PON1, and tried to protect the lactonase activity from oxidative inactivation. Among the oxidative systems tested, the ascorbate/Cu²⁺ system was the most potent in inactivating the lactonase activity of purified PON1; in contrast to a limited role of Fe²⁺, Cu²⁺ (0.05–1.0 µM) remarkably enhanced the inactivation of PON1 in the presence of ascorbate (0.02–0.1 mM). Moreover, Cu²⁺ alone inhibited the lactonase activity at concentrations as low as 1 µM. The ascorbate/Cu²⁺-mediated inactivation of PON1 lactonase activity was prevented by catalase, but not general hydroxyl radical scavengers, suggesting the implication of Cu²⁺-bound hydroxyl radicals in the oxidative inactivation. Compared to arylesterase activity, lactonase activity appears to be more sensitive to Cu²⁺-catalyzed oxidation. Separately, ascorbate/Cu²⁺-mediated inactivation of lactonase activity was prevented by oleic acid as well as phoshatidylcholine. Taken together, our data demonstrate that Cu²⁺-catalyzed oxidation may be a primary factor to cause the decrease of PON1 lactonase activity under oxidative stress and that lactonase activity of PON1 is most susceptible to ascorbate/Cu²⁺ among PON1 activities. In addition, we have showed that radical-induced inactivation of lactonase activity is prevented by some lipids.     

Effect of HDL1 infusion on biliary secretion in perfused rat liver

The effects of HDL1 lipoprotein infusion on biliary lipid secretion were studied in thein vitro model of rat perfused liver. A strong increase in bile flow was observed during and after lipoprotein infusion. This caused a significant rise in cholesterol, phospholipid and bile salt secretions. However, only the percentage of cholesterol increased with respect to the other bile lipids. The changes observed in the cholesterol/phospholipid molar ratio values of liver membrane subfractions (i.e., liver plasma membrane, mitochondria plus lysosomes and microsomes) isolated from the perfused rat liver after HDL1 administration were not significant.     

Activity of the antioxidant enzyme paraoxonase in Argentinean children living at high altitude

Background: Children living at high altitude in San Antonio de los Cobres (SAC), Argentina, were shown to have lower high-density lipoprotein cholesterol (HDL-C) levels than Buenos Aires (BA) children. HDL antioxidant capacity is mainly attributed to paraoxonase1 (PON1).

Objective: To compare PON1 activity in indigenous SAC vs. BA children.

Methods: A cross-sectional study compared 158 SAC vs. 97 BA children (6–16 years). Anthropometric data and lipoprotein profile were measured. PON1 was evaluated employing paraoxon (PON) and phenylacetate (ARE) activity.

Results: The prevalence of overweight/obesity was lower in SAC than in BA children (18.3 vs. 30.9%). Triglycerides (1.34 vs. 0.90?mmol/l), apo B (0.84 vs.0.72?g/l), apo A-I (1.33 vs. 1.27?g/l), and ARE activity (100 vs. 90?µmol/ml/min) were higher, while HDL-C (1.16 vs. 1.32?mmol/l) and PON activity (170 vs. 203?nmol/ml/min) were lower in SAC than in BA. Separate multiple linear regression analyses showed that SAC children had significantly higher triglyceride (Beta ?0.38), apo B (Beta ?0.34), and ARE (Beta ?0.36) plus lower HDL-C (Beta 0.33) and PON (Beta 0.25) compared with BA; adjusted for age, gender, and BMI.

Conclusion: SAC showed an unfavorable lipoprotein profile, lower PON and higher ARE activities compared with BA children, suggesting the presence of altered HDL metabolism and antioxidant capacity.     

Stimulation of lipolysis enhances the rate of cholesterol efflux to HDL in adipocytes

Adipose tissue constitutes a major location for cholesterol storage and, as such, it may play a role in the regulation of circulating cholesterol levels. A possible metabolic link between the lipolytic activity of adipocytes and their ability to release cholesterol to reconstituted human high density lipoprotein, HDL, was investigated in 3T3-L1 adipocytes. In the presence of HDL, composed of human apoA-I and phosphatidylcholine, adipocytes release cholesterol in a lipoprotein-dose and time dependent fashion. β-adrenergic activation of the lipolysis promotes a 22% increase in the extent of cholesterol efflux to reconstituted discoidal HDL particles. Activation of lipolysis promotes a rapid decrease in the cholesterol content of the plasma membrane and a concomitant increase in lipid droplet cholesterol. This change is independent of the presence of HDL. Activation of the lipolysis does not affect the levels of ABCA1 and SR-BI. Therefore, the enhancement of cholesterol efflux is not due to the level of plasma membrane cholesterol, or to the levels of the cholesterol transporters ABCA1 and scavenger receptor SR-BI. Brefeldin A did not affect the rate of cholesterol efflux under basal lipolytic conditions, but it abolished the lipolysis-dependent enhancement of cholesterol efflux to HDL. This study suggests that activation of lipolysis is accompanied by an increase in BFA-sensitive vesicular transport that in turn enhances cholesterol efflux to HDL. The study supports a metabolic link between the lipolytic activity of adipocytes and the rate of cellular cholesterol efflux to HDL.     

Developmental change in the position of the fetal human larynx

The position of the mammalian larynx has been shown to be an important determinant in breathing, swallowing, and vocalizing patterns. While the growth of the adult human larynx has been studied extensively, detailed examination of fetal development has not been undertaken. Thus, crucial developmental change in the fetal period and the effects of this change on normal maturation are still unclear. This study has examined the development of the larynx and its topography during the fetal period. Thirty specimens were preserved in 10% buffered formaldehyde solution for a period of 6 weeks, after which mid-sagittal sections were performed. Fetal ages were calculated from femur diaphyseal lengths and ranged from 15 to 29 weeks. Direct measurements were taken to determine the growth and position of the larynx and trachea relative to the vertebral column and soft palate. Results show that the upper and lower levels of the larynx correspond to the basiocciput and the lower border of the third to upper border of the fourth cervical vertebrae, respectively. The epiglottic cartilage was present at 15 weeks. By 21 weeks, the epiglottis was well developed and in close palatal apposition. At 23 to 25 weeks, the epiglottis and soft palate were found to be in full contact. The acquisition of this contact may be related to fetal respiratory viability.     

Monoclonal antibodies to plasma high density lipoprotein (HDL) of eel (Anguilla japonica)

Six week-old female mice (Balb/c) injected intraperitonealy with 50 μg of eel high density lipoprotein (HDL) emulsified with equal volume of adjuvant three times every two weeks. Three weeks after the third injection, hyperimmunized mice were boosted by injection of 100 μg of HDL. After 5 days, the best responding mouse to injected HDL was sacrificed, and spleen cells were fused with mouse myeloma cells (Sp2/O–Ag14), and hybridomas were cultured in a selection medium. Monoclonal antibodies specific to apolipoprotein A-I or A-II (apoA-I or apoA-II) of HDL were obtained by cloning and recloning the hybridomas. Eighteen monoclonal antibodies specific to apoA-I and/or apoApII were isolated. Antibodies in the culture medium were purified by a HiTrap Protein G or an eel-HDL column. These purified antibodies belong to the subclass IgG1. The monoclonal antibodies specific to eel apoA-I and apoA-II secreted by clone 10D12 and 2G3, respectively, interact with serum proteins of some fish species such as red-sea bream and carp. The anti-eel apoA-I antibody of 10D12 did not bind to serum proteins of rat, rabbit, and chicken, while the anti-eel apoA-II of 2G3 antibody did.