Age-related decrease in high-density lipoproteins antioxidant activity is due to an alteration in the PON1's free sulfhydyl groups


      The aim of this study was to evaluate the antioxidant activity of HDL with aging and to investigate the implication of PON1 in this process. The study involved 54 healthy subjects distributed in two age groups, young (20–25 years) and elderly (65–85 years). Lipid peroxidation was induced by OH and O2 oxygen free radicals produced by γ-radiolysis of water. LDL oxidation was followed by the measurement of conjugated diene (CD), lipid peroxide (LP) and malondialdehyde (MDA) formation. PON1 was purified separately from young (Y-PON1) and elderly subjects (E-PON1). PON1 activity and structure was followed by measurement of PON1 paraoxonase (p.ase) activity, titration of the SH groups, and electrophoretic mobility by SDS-PAGE. Our results show a significant decrease in the HDL antioxidant activity: percentage of protection against CD formation = 27.70% (p < 0.01) for E-HDL versus 73.08% (p < 0.001) for Y-HDL. Moreover, E-PON1 showed a lower antioxidant activity when compared to Y-PON1 47.08% versus 78.14%, respectively (p < 0.0001). Exposition of PON1 to OH and O2 oxygen free radicals induced a significant decrease in PON1 p.ase activity as well as a reduction in the number of PON1's free sulfhydryl groups. Moreover, our results show a close association between PON1's free sulfhydryl groups and its capacity to protect LDL against lipid peroxidation. There was a significant decrease in the number of free sulfhydryls between Y-PON1 and E-PON1 with respect to cysteine-284 amino acid residues (p < 0.0092).


      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'


      Subscribe to Atherosclerosis
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Durrington P.N.
        • Mackness B.
        • Mackness M.I.
        Paraoxonase and atherosclerosis.
        Arteriosclerosis. 2001; 21: 473-480
        • Watson A.D.
        • Berliner J.A.
        • Hama S.Y.
        • et al.
        Protective effect of high density lipoprotein associated paraoxonase. Inhibition of the biological activity of minimally oxidized low density lipoprotein.
        J Clin Invest. 1995; 96: 2882-2891
        • Aviram M.
        • Billecke S.
        • Sorenson R.
        • et al.
        Paraoxonase active site required for protection against LDL oxidation involves its free sulfhydryl group and is different from that required for its arylesterase/paraoxonase activities: selective action of human paraoxonase allozymes Q and R.
        Arterioscler Thromb Vasc Biol. 1998; 18: 1617-1624
        • Aviram M.
        • Rosenblat M.
        • Billecke S.
        • et al.
        Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants.
        Free Radic Biol Med. 1999; 26: 892-904
        • Sorenson R.C.
        • Primo-Parmo S.L.
        • Kuo C.L.
        • et al.
        Reconsideration of the catalytic center and mechanism of mammalian paraoxonase/arylesterase.
        Proc Natl Acad Sci USA. 1995; 92: 7187-7191
        • Jaouad L.
        • Milochevitch C.
        • Khalil A.
        PON1 paraoxonase activity is reduced during HDL oxidation and is an indicator of HDL antioxidant capacity.
        Free Radic Res. 2003; 37: 77-83
        • Sanguinetti S.M.
        • Brites F.D.
        • Fasulo V.
        • et al.
        HDL oxidability and its protective effect against LDL oxidation in Type 2 diabetic patients.
        Diabetes Nutr Metab. 2001; 14: 27-36
        • Gowri M.S.
        • Van der Westhuyzen D.R.
        • Bridges S.R.
        • Anderson J.W.
        Decreased protection by HDL from poorly controlled type 2 diabetic subjects against LDL oxidation may be due to the abnormal composition of HDL.
        Arterioscler Thromb Vasc Biol. 1999; 19: 2226-2233
        • Seres I.
        • Paragh G.
        • Deschene E.
        • Fulop Jr., T.
        • Khalil A.
        Study of factors influencing the decreased HDL associated PON1 activity with aging.
        Exp Gerontol. 2004; 39: 59-66
        • Gussekloo J.
        • Schaap M.C.
        • Frolich M.
        • Blauw G.J.
        • Westendorp R.G.
        Creactive protein is a strong but nonspecific risk factor of fatal stroke in elderly persons.
        Arterioscler Thromb Vasc Biol. 2000; 20: 1047-1051
        • Ershler W.B.
        • Keller E.T.
        Age-associated increased interleukin-6 gene expression, late-life diseases, and frailty.
        Annu Rev Med. 2000; 51: 245-270
        • Milochevitch C.
        • Khalil A.
        Study of the paraoxonase and platelet-activating factor acetylhydrolase activities with aging.
        Prostaglandins Leukot Essent Fatty Acids. 2001; 65: 241-246
        • Sattler W.
        • Mohr D.
        • Stocker R.
        Rapid isolation of lipoproteins and assessment of their peroxidation by high-performance liquid chromatography postcolumn chemiluminescence.
        Meth Enzymol. 1994; 233: 469-489
        • Eckerson H.W.
        • Romson J.
        • Wyte C.
        • La Du B.N.
        The human serum paraoxonase polymorphism: identification of phenotypes by their response to salts.
        Am J Hum Genet. 1983; 35: 214-227
        • Smolen A.
        • Eckerson H.W.
        • Gan K.N.
        • Hailat N.
        • La Du B.N.
        Characteristics of the genetically determined allozymic forms of human serum paraoxonase/arylesterase.
        Drug Metab Dispos. 1991; 19: 107-112
        • Gan K.N.
        • Smolen A.
        • Eckerson H.W.
        • La Du B.N.
        Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities.
        Drug Metab Dispos. 1991; 19: 100-106
        • Khalil A.
        • Fortin J.P.
        • LeHoux J.G.
        • Fulop Jr., T.
        Age-related decrease of dehydroepiandrosterone concentrations in low density lipoproteins and its role in the susceptibility of low density lipoproteins to lipid peroxidation.
        J Lipid Res. 2000; 41: 1552-1561
        • Fricke H.
        The chemical action of Röntgen rays on dilute ferrosulfate solutions as measure of dose.
        Am J Roentgenol Radium Ther Nucl Med. 1927; 18: 429-432
        • Spink J.W.T.
        • Wood R.J.
        An introduction to radiation chemistry.
        Wiley/Interscience, New York1990
        • Bielski B.H.J.
        Reevalution of the spectral and kinetic of hydroperoxide and supereroxide anion free radicals.
        Photochem Photobiol. 1978; 28: 645-649
        • Bonnefont-Rousselot D.
        • Motta C.
        • Khalil A.
        • et al.
        Physicochemical changes in human high-density lipoproteins (HDL) oxidized by gamma radiolysis-generated oxyradicals. Effect on their cholesterol effluxing capacity.
        Biochim Biophys Acta. 1995; 1255: 23-30
        • el-Saadani M.
        • Esterbauer H.
        • el-Sayed M.
        • et al.
        spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent.
        J Lipid Res. 1989; 30: 627-630
        • Agarwal R.
        • Chase S.D.
        Rapid, fluorimetric-liquid chromatographic determination of malondialdehyde in biological samples.
        J Chromatogr B: Analyt Technol Biomed Life Sci. 2002; 775: 121-126
        • Zago V.
        • Sanguinetti S.
        • Brites F.
        • et al.
        Impaired high-density lipoprotein antioxidant activity in healthy postmenopausal women.
        Atherosclerosis. 2004; 177: 203-210
        • Hu M.L.
        Measurement of protein thiol groups and glutathione in plasma.
        Meth Enzymol. 1994; 233: 380-385
        • Laemmli U.K.
        Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
        Nature. 1970; 182: 499-520
        • Khalil A.
        • Wagner J.R.
        • Lacombe G.
        • Dangoisse V.
        • Fulop Jr., T.
        Increased susceptibility of low-density lipoprotein (LDL) to oxidation by gamma-radiolysis with age.
        FEBS Lett. 1996; 392: 45-48
        • Khalil A.
        • Jay-Gerin J.P.
        • Fulop Jr., T.
        Age-related increased susceptibility of high-density lipoproteins (HDL) to in vitro oxidation induced by gamma-radiolysis of water.
        FEBS Lett. 1998; 435: 153-158
        • Ferretti G.
        • Bacchetti T.
        • Busni D.
        • Rabini R.A.
        • Curatola G.
        Protective effect of paraoxonase activity in high-density lipoproteins against erythrocyte membranes peroxidation: a comparison between healthy subjects and type 1 diabetic patients.
        J Clin Endocrinol Metab. 2004; 89: 2957-2962
        • Valabhji J.
        • McColl A.J.
        • Schachter M.
        • et al.
        High-density lipoprotein composition and paraoxonase activity in Type I diabetes.
        Clin Sci. 2001; 101: 659-670
        • Mertens A.
        • Verhamme P.
        • Bielicki J.K.
        • et al.
        Increased low-density lipoprotein oxidation and impaired high-density lipoprotein antioxidant defense are associated with increased macrophage homing and atherosclerosis in dyslipidemic obese mice: LCAT gene transfer decreases atherosclerosis.
        Circulation. 2003; 107: 1640-1646
        • Abbott C.A.
        • Mackness M.I.
        • Kumar S.
        • Boulton A.J.
        • Durrington P.N.
        Serum paraoxonase activity, concentration, and phenotype distribution in diabetes mellitus and its relationship to serum lipids and lipoproteins.
        Arterioscler Thromb Vasc Biol. 1995; 15: 1812-1818
        • Tomas M.
        • Senti M.
        • Garcia-Faria F.
        • et al.
        Effect of simvastatin therapy on paraoxonase activity and related lipoproteins in familial hypercholesterolemic patients.
        Arterioscler Thromb Vasc Biol. 2000; 20: 2113-2119
        • McElveen J.
        • Mackness M.I.
        • Colley C.
        • et al.
        Distribution of paraoxon hydrolytic activity in the serum of patients after myocardial infarction.
        Clin Chem. 1986; 32: 671-673
        • Ikeda Y.
        • Suehiro T.
        • Inoue M.
        • et al.
        Serum paraoxonase activity and its relationship to diabetic complications in patients with non-insulin-dependent diabetes mellitus.
        Metabolism. 1998; 47: 598-602
        • Eisenberg S.
        High density lipoprotein metabolism.
        J Lipid Res. 1984; 25: 1017-1058
        • Mackness M.I.
        • Abbott C.
        • Arrol S.
        • Durrington P.N.
        The role of high-density lipoprotein and lipid-soluble antioxidant vitamins in inhibiting low-density lipoprotein oxidation.
        Biochem J. 1993; 294: 829-834
        • Romanchik J.E.
        • Morel D.W.
        • Harrison E.H.
        Distributions of carotenoids and alpha-tocopherol among lipoproteins do not change when human plasma is incubated in vitro.
        J Nutr. 1995; 125: 2610-2617
        • Bonnefont-Rousselot D.
        • Khalil A.
        • Gardes-Albert M.
        • Delattre J.
        Reciprocal protection of LDL and HDL oxidised by OH free radicals in the presence of oxygen.
        FEBS Lett. 1997; 403: 70-74
        • Shih D.M.
        • Gu L.
        • Xia Y.R.
        • et al.
        Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis.
        Nature. 1998; 394: 284-287
        • Teiber J.F.
        • Draganov D.I.
        • La Du B.N.
        Purified human serum PON1 does not protect LDL against oxidation in the in vitro assays initiated with copper or AAPH.
        J Lipid Res. 2004; 45: 2260-2268
        • Senti M.
        • Aubo C.
        • Tomas M.
        Differential effects of smoking on myocardial infarction risk according to the Gln/Arg 192 variants of the human paraoxonase gene.
        Metabolism. 2000; 49: 557-559