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Contents lists available at ScienceDirect
Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
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Short communication
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In vitro oxidation of LDL by ozone
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Gerd Hörl a , Gerhard Ledinski a , Gerd Kager a , Seth Hallström a , Erwin Tafeit a , Martin Koestenberger b , Günther Jürgens a , Gerhard Cvirn a,∗ a b
Institute of Physiological Chemistry, Medical University of Graz, Austria Department of Pediatrics, Medical University of Graz, Austria
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Article history: Received 3 February 2014 Received in revised form 5 May 2014 Accepted 6 May 2014 Available online xxx
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Keywords: Atherosclerosis Lipid hydroperoxides Low-density lipoprotein Oxidation-specific immune epitopes Ozone
Recent studies suggest that ozone is present in atherosclerotic lesions. Since these lesions are characterized by a dramatic accumulation of low-density lipoprotein (LDL), we aimed to investigate whether ozone is capable of oxidizing LDL, thereby rendering this lipoprotein atherogenic. Lipid hydroperoxide (LPO) concentrations and thiobarbituric acid reactive substances (TBARS) were measured to assess the oxidative status of the lipid part of LDL. Relative electrophoretic mobility (REM) and oxidation-specific immune epitopes were measured to assess the oxidative status of the protein part (apoB) of the LDL particle. Ozone turned out to be a potent oxidant of LDL. LPO concentrations, TBARS, REM, and oxidationspecific immune epitopes significantly increased upon ozonization. Our results suggest that ozonization of LDL may be a novel pathway which supports atherogenesis. Ozone is capable of oxidizing the lipid part of LDL, followed by immediate oxidation of the protein part of LDL, rendering the lipoprotein atherogenic. © 2014 Published by Elsevier Ireland Ltd.
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1. Introduction Previously, 5,6-secosterols (i.e., atheronal A and B) have been discovered within human atherosclerotic plaque material in vivo (Wentworth et al., 2003). Particularly the formation of atheronal A is most likely due to a reaction unique to the ozonolysis of cholesterol (Loscalzo, 2004). It therefore has been concluded that ozone or an ozone-like oxidant is being endogenously generated during atherosclerosis progression (Tomono et al., 2011). In various studies utilizing biologically relevant systems the oxidation of cholesterol by ozone has been investigated thereupon in detail (Wentworth et al., 2009).
Abbreviations: ApoB, apolipoprotein B; BHT, butylated hydroxytoluene; DELFIA, dissociation-enhanced lanthanide fluorescence immunoassay; DTPA, diethylenetriamine-pentaacetic acid; EDTA, ethylene-diamine-tetraacetic acid; Eu, europium; IgG, immunoglobulin G; IRAK-1, interleukin-1 receptor-associated kinase-1; LDL, low-density lipoprotein; LPO, lipid hydroperoxides; MPO, myeloperoxidase; NF-B, nuclear factor kappa-light-chain-enhancer of activated B cells; OB 04, monoclonal antibody raised against copper-oxidized LDL; oxLDL, oxidatively modified LDL; REM, relative electrophoretic mobility; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances. ∗ Corresponding author at: Institute of Physiological Chemistry, Medical University of Graz, Harrachgasse 21/II, A-8010 Graz, Austria. Tel.: +43 316 380 4174; fax: +43 316 380 9610. E-mail address: [email protected] (G. Cvirn).
The present study was undertaken to investigate whether ozone is capable of modifying not only cholesterol, but the entire lipid part as well as the apoB part of the LDL particle, the major carrier of cholesterol in human blood. LDL in its native form is not atherogenic. However, oxidatively modified LDL (oxLDL) has been shown to play an eminent role in the development of atherosclerosis (Jürgens et al., 1987; Hutter et al., 2013). Thus, oxidation of LDL by ozone generated endogenously in inflammatory arteries might be a physiological pathway contributing to pathogenesis of atherosclerosis. In the present study, assumed ozone-provoked peroxidation of the lipid part of the LDL particle was assessed by measuring LPO concentrations and the amount of TBARS. Presumably, oxidative modification of the lipid part of the LDL particle is followed by a modification of apoB (Esterbauer et al., 1987). We assessed the degree of oxidation of the apoB by measuring relative electrophoretic mobilities (REMs). The oxidation of apoB involves derivatization of lysine residues by adducts that neutralize the positively charged epsilon-amino groups (Jürgens et al., 1995). Thus, the net charge of the LDL particle negatively increases depending on the oxidation rate. This change in the LDL surface charge was assessed in our study by determining the respective relative electrophoretic mobility (REM). Moreover, the extent of oxidation of apoB was also assessed by quantifying oxidationspecific immune epitopes by means of a fluorescence immunoassay using specific antibodies against oxLDL (Hammer et al., 1995).
http://dx.doi.org/10.1016/j.chemphyslip.2014.05.002 0009-3084/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Hörl, G., et al., In vitro oxidation of LDL by ozone. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.05.002
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Human LDL was obtained from the plasma of normolipemic, 62Q2 fasting young male donors by ultracentrifugation (Jürgens et al., 1990). Ozone was prepared in a Humazon ozone-generator (Tech63 nomed GmbH, Karlsruhe, Germany). The reaction vessel contained 64 2.0 g/mL ozone. During the entire incubation time the samples 65 were rotated (10 rpm, 75◦ angle of rotation) at 23 ◦ C to provide 66 optimal contact of the ozone with the LDL solution (250 L at 67 a concentration of 1 mg/mL). Each experiment was performed in 68 triplicate. Student’s paired t-test was used for statistical evalua69 tion with significance defined as P < 0.05. Details are stated in the 70 Appendix A. 71
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Fig. 2. Ozone-induced oxidation of the protein part (apoB) of the LDL particle.
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3. Results Fig. 1A shows that the LPO content of LDL time-dependently increased during the first 6 min of ozonization. After this almost linear increase a plateau of approximately 370 nmol LPO/mg LDL was reached. Despite continued ozonization no further increase of the LPO content of the LDL particle was observed. Similar results were obtained for TBARS formation, which also time-dependently increased during the first 6 min of ozonization. The measured TBARS values reached a maximum of approximately 40 nmol/L with no further increase after 6 min (Fig. 1B). As shown in Fig. 2A, the REM of LDL samples time-dependently increased during the first 6 min of ozonization before reaching a constant level of approximately 1.63. Similar results were obtained for oxidation-specific epitopes which were formed within the first 8 min of ozonization, shown in Fig. 2B. Maximum level of oxidation-specific immune epitopes (140,000 counts) was reached and continued ozonization did not result in further formation of oxidation-specific immune epitopes.
LPO concentrations (panel A) and TBARS (panel B) significantly increased during ozonization. Data represent mean ± SD from three separate experiments. The reaction vessel contained 2.0 g/mL ozone. Experiments were performed at 37 ◦ C in 0.01 M phosphate buffer (pH = 7.4) containing 0.154 M NaCl. The LDL concentration was 1 mg/mL. ANOVA provided a statistical significance of P < 0.001 for both LPO and TBARS measurements. Panel A: Four homogenous sub-groups were identified, Grp. I: 0 h; Grp. II: 2 h; Grp. III: 4 h; Grp. IV: 6, 8, and 10 h. Panel B: Three homogenous groups were identified: Grp. I: 0 h; Grp. II: 2 and 4 h; Grp. III: 6, 8, and 10 h. Relative electrophoretic mobility (REM, panel A) and oxidationspecific immune epitopes (panel B) significantly increased during ozonization. Data represent mean ± SD from three separate experiments. The reaction vessel contained 2.0 g/mL ozone. Experiments were performed at 37 ◦ C in 0.01 M phosphate buffer (pH = 7.4) containing 0.154 M NaCl. The LDL concentration was 1 mg/mL. ANOVA provided a statistical significance of P < 0.001 for both REM and OB/04 measurements. Panel A: three homogenous sub-groups were identified, Grp. I: 0 h; Grp. II: 2 and 4 h; Grp. III: 6, 8, and 10 h. Panel B: Four homogenous groups were identified: Grp. I: 0 h; Grp. II: 2 h; Grp. III: 4 and 6 h; Grp. IV: 8 and 10 h. 4. Discussion Elevated levels of LDL, the main carrier of plasma cholesterol in humans, are a major risk factor for atherosclerosis (Imazu et al., 2008). LDL in its native form is not atherogenic, but oxidatively modified LDL has been shown to promote the development of atherosclerosis (Steinberg and Lewis, 1997). Several lines of evidence demonstrated that oxidation of LDL does indeed occur in vivo (Steinberg, 1997). Endothelial cells, macrophages, and muscle cells have been shown to be capable of oxidizing LDL by means
Please cite this article in press as: Hörl, G., et al., In vitro oxidation of LDL by ozone. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.05.002
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of, amongst others, 15-lipoxygenase and myeloperoxidase (MPO, Delporte et al., 2014). Moreover, transition metal ions have been shown to be crucial for oxidative modification of LDL (Esterbauer et al., 1992), and ferryl hemoglobin is also a possible physiological mediator for LDL oxidation (Potor et al., 2013). However, the mechanisms by which LDL is oxidized in vivo are at present not completely understood. We show herein that ozone is capable of oxidizing the lipid part of LDL followed by an immediate oxidation of apoB. Thus, oxidation of LDL by ozone might be such a pathway leading to the formation of atherogenic oxLDL. Regarding the lipid part, ozone induces the formation of lipid hydroperoxides as well as the formation of TBARS, the end products of the decomposition of lipid peroxidation products. Particularly the rapid formation of TBARS indicates that ozone is apparently an efficient initiator of lipid peroxidation. Oxidation of the lipid part of LDL was followed by an immediate and efficient oxidation of apoB. We found increasing REM and increasing formation of oxidation-specific immune epitopes in LDL exposed to ozone. Increasing REM indicates derivatization of the lysine residues of apoB. We assume that this derivatization is caused by the peroxidation-derived aldehydes and not by ozone itself. Mudd et al. have shown that lysine is not susceptible to oxidation by ozone in aqueous solutions (Mudd et al., 1969). The rapid formation of oxidation-specific immune epitopes in LDL upon ozonization further characterizes ozone as an efficient oxidant of LDL. It has to be stated that the assumption that ozone is produced in atherosclerosic lesions is still in debate and that the physiological concentrations of ozone are unknown to date. On one hand it has been shown that the formation of secosterols, indicating preceding generation of ozone, is mediated by a MPO-dependent system in vivo at least partly through an oxidant with the chemical structure of ozone (Tomono et al., 2011). On the other hand, it has been reported that secosterols were generated in an ozoneindependent manner via the Hock-cleavage of 5␣-hydroperoxy cholesterol, which can arise from the singlet oxygen ene reaction with cholesterol (Brinkhorst et al., 2008). Moreover, not only endogenously generated ozone but also ozone derived from environmental pollution is of physiological relevance (Robertson et al., 2013). It has been shown that exposure of rats to ozone causes increased aortic LOX-1 (lectine-like oxidized low-density lipoprotein receptor-1) mRNA and protein (Kodavanti et al., 2011). LOX-1 binds oxidatively modified lipids (e.g., oxLDL) and stimulates downstream signaling involving nuclear factor kappa B (Navarra et al., 2009), a mechanism that leads to vascular complications.
5. Conclusions In conclusion, ozone-induced oxidation of LDL might be a physiological relevant pathway which promotes atherogenesis. In an on-going study, the lipid composition and the poly-unsaturated fatty acids in LDL sensitive for oxidation by ozone have to be investigated. Presumably, the physiological role of ozonated LDL is not restricted to atherogenesis. Ozonated LDL generally might play a role in inflammatory signaling. It has recently been shown that ozonated LDL is capable of inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells and interleukin1 receptor-associated kinase-1-associated signaling which may impair immune function and promote apoptosis (Cappello et al., 2007). Thus, ozonated LDL might be relevant to various inflammatory or malignant diseases.
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Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgement We thank Prof. Otto Schmut for intellectual input and Barbara Scherz for technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphyslip. 2014.05.002. References Brinkhorst, J., Nara, S.J., Pratt, D.A., 2008. Hock cleavage of cholesterol 5alpha-hydroperoxide: an ozone-free pathway to the cholesterol ozonolysis products identified in arterial plaque and brain tissue. J. Am. Chem. Soc. 130, 12224–12225. Cappello, C., Saugel, B., Huth, K.C., Zwergal, A., Krautkrämer, M., Furman, C., Rouis, M., Wieser, B., Schneider, H.W., Neumeier, D., Brand, K., 2007. Ozonized low density lipoprotein (ozLDL) inhibits NF-B and IRAK-1-associated signalling. Arterioscler. Thromb. Vasc. Biol. 27, 226–332. Delporte, C., Boudjeltia, K.Z., Noyon, C., Furtmüller, P.G., Nuyens, V., Slomianny, M.C., Madhoun, P., Desmet, J.M., Raynal, P., Dufour, D., Koyani, C.N., Reye, F., Rousseau, A., Vanhaeverbeek, M., Ducobu, J., Michalski, J.C., Neve, J., Vanhamme, L., Obinger, C., Malle, E., Van Antwerpen, P., 2014. Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100. J. Lipid Res. 55, 747–757. Esterbauer, H., Gebicki, J., Puhl, H., Jürgens, G., 1992. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic. Biol. Med. 13, 341–390. Esterbauer, H., Jürgens, G., Quehenberger, O., Koller, E., 1987. Autooxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J. Lipid Res. 28, 495–509. Hammer, A., Kager, G., Dohr, G., Rabl, H., Ghassempur, I., Jürgens, G., 1995. Generation characterization, and histochemical application of monoclonal antibodies selectively recognizing oxidatively modified apoB-containing lipoproteins. Arterioscler. Thromb. Vasc. Biol. 15, 704–713. Hutter, R., Speidl, W.S., Valdiviezo, C., Sauter, B., Corti, R., Fuster, V., Badimon,.J.J., 2013. Macrophages transmit potent proangiogenic effects of oxLDL in vitro and in vivo involving HIF-1˛ activation: a novel aspect of angiogenesis in atherosclerosis. J. Cardiovasc. Transl. Res. 6, 558–569. Imazu, M., Ono, K., Tadehara, F., Kajiwara, K., Yamamoto, H., Sumii, K., Tasaki, N., Oiwa, J., Shimohara, Y., Gomyo, Y., Itabe, H., 2008. Plasma levels of oxidized low density lipoprotein are associated with stable angina pectoris and modalities of acute coronary syndrome. Int. Heart J. 49, 515–524. Jürgens, G., Fell, A., Ledinski, G., Chen, Q., Paltauf, F., 1995. Delay of copper-catalyzed oxidation of low density lipoprotein by in vitro enrichment with choline or ethanolamine plasmalogens. Chem. Phys. Lipids 77, 25–31. Jürgens, G., Hoff, H.F., Chisolm, G.M., Esterbauer, H., 1987. Modification of human serum low low-density lipoprotein by oxidation-characterization and pathophysiological implications. Chem. Phys. Lipids 45, 315–336. Kodavanti, U.P., Thomas, R., Ledbetter, A.D., Schladweiler, M.C., Shannahan, J.H., Wallenborn, J.G., Lund, A.K., Campen, M.J., Butler, E.O., Gottipolu, R.R., Nyska, A., Richards, J.E., Andrews, D., Jaskot, R.H., McKee, J., Kotha, S.R., Patel, R.B., Parinandi, N.L., 2011. Vascular and cardiac impairments in rats inhaling ozone and diesel exhaust particles. Environ. Health Perspect. 119, 312–318. Loscalzo, J., 2004. Ozone-from environmental pollutant to atherogenic determinant. N. Engl. J. Med. 350, 834–835. Mudd, J.B., Leavitt, R., Ongun, A., McManus, T.T., 1969. Reaction of ozone with amino acids and proteins. Atmos. Environ. 3, 669–682. Navarra, T., Del Turco, S., Berti, S., Basta, G., 2009. The lectine-like oxidized low-density lipoprotein receptor-1 and its soluble form: cardiovascular implications. J. Atheroscler. Thromb. 17, 317–331. Potor, L., Banyai, E., Becs, G., Soares, M.P., Balla, G., Balla, J., Jeney, V., 2013. Atherogenesis may involve the prooxidant and proinflammatory effects of ferryl hemoglobin. Oxid. Med. Cell. Longev. 2013, 676425, http://dx.doi.org/10.1155/2013/676425. Robertson, S., Colombo, E.S., Lucas, S.N., Hall, P.R., Febbraio, M., Paffett, M.L., Campen, M.J., 2013. CD36 mediates endothelial dysfunction downstream of circulating factors induced by O3 exposure. Toxicol. Sci. 134, 304–311.
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Steinberg, D., 1997. Low density lipoprotein oxidation and its pathological significance. J. Biol. Chem. 272, 20963–20966. Steinberg, D., Lewis, A., 1997. Conner memorial lecture. Oxidatative modification of LDL and atherogenesis. Circulation 95, 1062–1071. Tomono, S., Miyoshi, N., Shiokawa, H., Iwabuchi, T., Aratani, Y., Higashi, T., Nukaya, H., Ohshima, H., 2011. Formation of cholesterol ozonolysis products in vitro and in vivo through a myeloperoxidase-dependent pathway. J. Lipid Res. 52, 87–97.
Wentworth, A.D., Song, B.D., Nieva, J., Shafton, A., Tripurenani, S., Wentworth Jr., P., 2009. The ration of cholesterol 5,6-secosterols formed from ozone and singlet oxygen offers insights into the oxidation of cholesterol in vivo. Chem. Commun. (Camb.) 21, 3098–3100. Wentworth Jr., P., Nieva, J., Takeuchi, C., Galve, R., Wentworth, A.D., Dilley, R.B., DeLaria, G.A., Saven, A., Babior, B.M., Jand a, K.D., Eschenmoser, A., Lerner, R.A., 2003. Evidence for ozone formation in human atherosclerotic arteries. Science 302, 1053–1056.
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Contents lists available at ScienceDirect
Chemistry and Physics of Lipids journal homepage: www.elsevier.com/locate/chemphyslip
1
Short communication
2
In vitro oxidation of LDL by ozone
3 4 5 6
Q1
Gerd Hörl a , Gerhard Ledinski a , Gerd Kager a , Seth Hallström a , Erwin Tafeit a , Martin Koestenberger b , Günther Jürgens a , Gerhard Cvirn a,∗ a b
Institute of Physiological Chemistry, Medical University of Graz, Austria Department of Pediatrics, Medical University of Graz, Austria
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a r t i c l e
i n f o
a b s t r a c t
9 10 11 12 13 14
Article history: Received 3 February 2014 Received in revised form 5 May 2014 Accepted 6 May 2014 Available online xxx
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Keywords: Atherosclerosis Lipid hydroperoxides Low-density lipoprotein Oxidation-specific immune epitopes Ozone
Recent studies suggest that ozone is present in atherosclerotic lesions. Since these lesions are characterized by a dramatic accumulation of low-density lipoprotein (LDL), we aimed to investigate whether ozone is capable of oxidizing LDL, thereby rendering this lipoprotein atherogenic. Lipid hydroperoxide (LPO) concentrations and thiobarbituric acid reactive substances (TBARS) were measured to assess the oxidative status of the lipid part of LDL. Relative electrophoretic mobility (REM) and oxidation-specific immune epitopes were measured to assess the oxidative status of the protein part (apoB) of the LDL particle. Ozone turned out to be a potent oxidant of LDL. LPO concentrations, TBARS, REM, and oxidationspecific immune epitopes significantly increased upon ozonization. Our results suggest that ozonization of LDL may be a novel pathway which supports atherogenesis. Ozone is capable of oxidizing the lipid part of LDL, followed by immediate oxidation of the protein part of LDL, rendering the lipoprotein atherogenic. © 2014 Published by Elsevier Ireland Ltd.
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1. Introduction Previously, 5,6-secosterols (i.e., atheronal A and B) have been discovered within human atherosclerotic plaque material in vivo (Wentworth et al., 2003). Particularly the formation of atheronal A is most likely due to a reaction unique to the ozonolysis of cholesterol (Loscalzo, 2004). It therefore has been concluded that ozone or an ozone-like oxidant is being endogenously generated during atherosclerosis progression (Tomono et al., 2011). In various studies utilizing biologically relevant systems the oxidation of cholesterol by ozone has been investigated thereupon in detail (Wentworth et al., 2009).
Abbreviations: ApoB, apolipoprotein B; BHT, butylated hydroxytoluene; DELFIA, dissociation-enhanced lanthanide fluorescence immunoassay; DTPA, diethylenetriamine-pentaacetic acid; EDTA, ethylene-diamine-tetraacetic acid; Eu, europium; IgG, immunoglobulin G; IRAK-1, interleukin-1 receptor-associated kinase-1; LDL, low-density lipoprotein; LPO, lipid hydroperoxides; MPO, myeloperoxidase; NF-B, nuclear factor kappa-light-chain-enhancer of activated B cells; OB 04, monoclonal antibody raised against copper-oxidized LDL; oxLDL, oxidatively modified LDL; REM, relative electrophoretic mobility; TBA, thiobarbituric acid; TBARS, thiobarbituric acid reactive substances. ∗ Corresponding author at: Institute of Physiological Chemistry, Medical University of Graz, Harrachgasse 21/II, A-8010 Graz, Austria. Tel.: +43 316 380 4174; fax: +43 316 380 9610. E-mail address: [email protected] (G. Cvirn).
The present study was undertaken to investigate whether ozone is capable of modifying not only cholesterol, but the entire lipid part as well as the apoB part of the LDL particle, the major carrier of cholesterol in human blood. LDL in its native form is not atherogenic. However, oxidatively modified LDL (oxLDL) has been shown to play an eminent role in the development of atherosclerosis (Jürgens et al., 1987; Hutter et al., 2013). Thus, oxidation of LDL by ozone generated endogenously in inflammatory arteries might be a physiological pathway contributing to pathogenesis of atherosclerosis. In the present study, assumed ozone-provoked peroxidation of the lipid part of the LDL particle was assessed by measuring LPO concentrations and the amount of TBARS. Presumably, oxidative modification of the lipid part of the LDL particle is followed by a modification of apoB (Esterbauer et al., 1987). We assessed the degree of oxidation of the apoB by measuring relative electrophoretic mobilities (REMs). The oxidation of apoB involves derivatization of lysine residues by adducts that neutralize the positively charged epsilon-amino groups (Jürgens et al., 1995). Thus, the net charge of the LDL particle negatively increases depending on the oxidation rate. This change in the LDL surface charge was assessed in our study by determining the respective relative electrophoretic mobility (REM). Moreover, the extent of oxidation of apoB was also assessed by quantifying oxidationspecific immune epitopes by means of a fluorescence immunoassay using specific antibodies against oxLDL (Hammer et al., 1995).
http://dx.doi.org/10.1016/j.chemphyslip.2014.05.002 0009-3084/© 2014 Published by Elsevier Ireland Ltd.
Please cite this article in press as: Hörl, G., et al., In vitro oxidation of LDL by ozone. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.05.002
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Human LDL was obtained from the plasma of normolipemic, 62Q2 fasting young male donors by ultracentrifugation (Jürgens et al., 1990). Ozone was prepared in a Humazon ozone-generator (Tech63 nomed GmbH, Karlsruhe, Germany). The reaction vessel contained 64 2.0 g/mL ozone. During the entire incubation time the samples 65 were rotated (10 rpm, 75◦ angle of rotation) at 23 ◦ C to provide 66 optimal contact of the ozone with the LDL solution (250 L at 67 a concentration of 1 mg/mL). Each experiment was performed in 68 triplicate. Student’s paired t-test was used for statistical evalua69 tion with significance defined as P < 0.05. Details are stated in the 70 Appendix A. 71
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3. Results Fig. 1A shows that the LPO content of LDL time-dependently increased during the first 6 min of ozonization. After this almost linear increase a plateau of approximately 370 nmol LPO/mg LDL was reached. Despite continued ozonization no further increase of the LPO content of the LDL particle was observed. Similar results were obtained for TBARS formation, which also time-dependently increased during the first 6 min of ozonization. The measured TBARS values reached a maximum of approximately 40 nmol/L with no further increase after 6 min (Fig. 1B). As shown in Fig. 2A, the REM of LDL samples time-dependently increased during the first 6 min of ozonization before reaching a constant level of approximately 1.63. Similar results were obtained for oxidation-specific epitopes which were formed within the first 8 min of ozonization, shown in Fig. 2B. Maximum level of oxidation-specific immune epitopes (140,000 counts) was reached and continued ozonization did not result in further formation of oxidation-specific immune epitopes.
LPO concentrations (panel A) and TBARS (panel B) significantly increased during ozonization. Data represent mean ± SD from three separate experiments. The reaction vessel contained 2.0 g/mL ozone. Experiments were performed at 37 ◦ C in 0.01 M phosphate buffer (pH = 7.4) containing 0.154 M NaCl. The LDL concentration was 1 mg/mL. ANOVA provided a statistical significance of P < 0.001 for both LPO and TBARS measurements. Panel A: Four homogenous sub-groups were identified, Grp. I: 0 h; Grp. II: 2 h; Grp. III: 4 h; Grp. IV: 6, 8, and 10 h. Panel B: Three homogenous groups were identified: Grp. I: 0 h; Grp. II: 2 and 4 h; Grp. III: 6, 8, and 10 h. Relative electrophoretic mobility (REM, panel A) and oxidationspecific immune epitopes (panel B) significantly increased during ozonization. Data represent mean ± SD from three separate experiments. The reaction vessel contained 2.0 g/mL ozone. Experiments were performed at 37 ◦ C in 0.01 M phosphate buffer (pH = 7.4) containing 0.154 M NaCl. The LDL concentration was 1 mg/mL. ANOVA provided a statistical significance of P < 0.001 for both REM and OB/04 measurements. Panel A: three homogenous sub-groups were identified, Grp. I: 0 h; Grp. II: 2 and 4 h; Grp. III: 6, 8, and 10 h. Panel B: Four homogenous groups were identified: Grp. I: 0 h; Grp. II: 2 h; Grp. III: 4 and 6 h; Grp. IV: 8 and 10 h. 4. Discussion Elevated levels of LDL, the main carrier of plasma cholesterol in humans, are a major risk factor for atherosclerosis (Imazu et al., 2008). LDL in its native form is not atherogenic, but oxidatively modified LDL has been shown to promote the development of atherosclerosis (Steinberg and Lewis, 1997). Several lines of evidence demonstrated that oxidation of LDL does indeed occur in vivo (Steinberg, 1997). Endothelial cells, macrophages, and muscle cells have been shown to be capable of oxidizing LDL by means
Please cite this article in press as: Hörl, G., et al., In vitro oxidation of LDL by ozone. Chem. Phys. Lipids (2014), http://dx.doi.org/10.1016/j.chemphyslip.2014.05.002
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of, amongst others, 15-lipoxygenase and myeloperoxidase (MPO, Delporte et al., 2014). Moreover, transition metal ions have been shown to be crucial for oxidative modification of LDL (Esterbauer et al., 1992), and ferryl hemoglobin is also a possible physiological mediator for LDL oxidation (Potor et al., 2013). However, the mechanisms by which LDL is oxidized in vivo are at present not completely understood. We show herein that ozone is capable of oxidizing the lipid part of LDL followed by an immediate oxidation of apoB. Thus, oxidation of LDL by ozone might be such a pathway leading to the formation of atherogenic oxLDL. Regarding the lipid part, ozone induces the formation of lipid hydroperoxides as well as the formation of TBARS, the end products of the decomposition of lipid peroxidation products. Particularly the rapid formation of TBARS indicates that ozone is apparently an efficient initiator of lipid peroxidation. Oxidation of the lipid part of LDL was followed by an immediate and efficient oxidation of apoB. We found increasing REM and increasing formation of oxidation-specific immune epitopes in LDL exposed to ozone. Increasing REM indicates derivatization of the lysine residues of apoB. We assume that this derivatization is caused by the peroxidation-derived aldehydes and not by ozone itself. Mudd et al. have shown that lysine is not susceptible to oxidation by ozone in aqueous solutions (Mudd et al., 1969). The rapid formation of oxidation-specific immune epitopes in LDL upon ozonization further characterizes ozone as an efficient oxidant of LDL. It has to be stated that the assumption that ozone is produced in atherosclerosic lesions is still in debate and that the physiological concentrations of ozone are unknown to date. On one hand it has been shown that the formation of secosterols, indicating preceding generation of ozone, is mediated by a MPO-dependent system in vivo at least partly through an oxidant with the chemical structure of ozone (Tomono et al., 2011). On the other hand, it has been reported that secosterols were generated in an ozoneindependent manner via the Hock-cleavage of 5␣-hydroperoxy cholesterol, which can arise from the singlet oxygen ene reaction with cholesterol (Brinkhorst et al., 2008). Moreover, not only endogenously generated ozone but also ozone derived from environmental pollution is of physiological relevance (Robertson et al., 2013). It has been shown that exposure of rats to ozone causes increased aortic LOX-1 (lectine-like oxidized low-density lipoprotein receptor-1) mRNA and protein (Kodavanti et al., 2011). LOX-1 binds oxidatively modified lipids (e.g., oxLDL) and stimulates downstream signaling involving nuclear factor kappa B (Navarra et al., 2009), a mechanism that leads to vascular complications.
5. Conclusions In conclusion, ozone-induced oxidation of LDL might be a physiological relevant pathway which promotes atherogenesis. In an on-going study, the lipid composition and the poly-unsaturated fatty acids in LDL sensitive for oxidation by ozone have to be investigated. Presumably, the physiological role of ozonated LDL is not restricted to atherogenesis. Ozonated LDL generally might play a role in inflammatory signaling. It has recently been shown that ozonated LDL is capable of inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells and interleukin1 receptor-associated kinase-1-associated signaling which may impair immune function and promote apoptosis (Cappello et al., 2007). Thus, ozonated LDL might be relevant to various inflammatory or malignant diseases.
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Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgement We thank Prof. Otto Schmut for intellectual input and Barbara Scherz for technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemphyslip. 2014.05.002. References Brinkhorst, J., Nara, S.J., Pratt, D.A., 2008. Hock cleavage of cholesterol 5alpha-hydroperoxide: an ozone-free pathway to the cholesterol ozonolysis products identified in arterial plaque and brain tissue. J. Am. Chem. Soc. 130, 12224–12225. Cappello, C., Saugel, B., Huth, K.C., Zwergal, A., Krautkrämer, M., Furman, C., Rouis, M., Wieser, B., Schneider, H.W., Neumeier, D., Brand, K., 2007. Ozonized low density lipoprotein (ozLDL) inhibits NF-B and IRAK-1-associated signalling. Arterioscler. Thromb. Vasc. Biol. 27, 226–332. Delporte, C., Boudjeltia, K.Z., Noyon, C., Furtmüller, P.G., Nuyens, V., Slomianny, M.C., Madhoun, P., Desmet, J.M., Raynal, P., Dufour, D., Koyani, C.N., Reye, F., Rousseau, A., Vanhaeverbeek, M., Ducobu, J., Michalski, J.C., Neve, J., Vanhamme, L., Obinger, C., Malle, E., Van Antwerpen, P., 2014. Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100. J. Lipid Res. 55, 747–757. Esterbauer, H., Gebicki, J., Puhl, H., Jürgens, G., 1992. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic. Biol. Med. 13, 341–390. Esterbauer, H., Jürgens, G., Quehenberger, O., Koller, E., 1987. Autooxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J. Lipid Res. 28, 495–509. Hammer, A., Kager, G., Dohr, G., Rabl, H., Ghassempur, I., Jürgens, G., 1995. Generation characterization, and histochemical application of monoclonal antibodies selectively recognizing oxidatively modified apoB-containing lipoproteins. Arterioscler. Thromb. Vasc. Biol. 15, 704–713. Hutter, R., Speidl, W.S., Valdiviezo, C., Sauter, B., Corti, R., Fuster, V., Badimon,.J.J., 2013. Macrophages transmit potent proangiogenic effects of oxLDL in vitro and in vivo involving HIF-1˛ activation: a novel aspect of angiogenesis in atherosclerosis. J. Cardiovasc. Transl. Res. 6, 558–569. Imazu, M., Ono, K., Tadehara, F., Kajiwara, K., Yamamoto, H., Sumii, K., Tasaki, N., Oiwa, J., Shimohara, Y., Gomyo, Y., Itabe, H., 2008. Plasma levels of oxidized low density lipoprotein are associated with stable angina pectoris and modalities of acute coronary syndrome. Int. Heart J. 49, 515–524. Jürgens, G., Fell, A., Ledinski, G., Chen, Q., Paltauf, F., 1995. Delay of copper-catalyzed oxidation of low density lipoprotein by in vitro enrichment with choline or ethanolamine plasmalogens. Chem. Phys. Lipids 77, 25–31. Jürgens, G., Hoff, H.F., Chisolm, G.M., Esterbauer, H., 1987. Modification of human serum low low-density lipoprotein by oxidation-characterization and pathophysiological implications. Chem. Phys. Lipids 45, 315–336. Kodavanti, U.P., Thomas, R., Ledbetter, A.D., Schladweiler, M.C., Shannahan, J.H., Wallenborn, J.G., Lund, A.K., Campen, M.J., Butler, E.O., Gottipolu, R.R., Nyska, A., Richards, J.E., Andrews, D., Jaskot, R.H., McKee, J., Kotha, S.R., Patel, R.B., Parinandi, N.L., 2011. Vascular and cardiac impairments in rats inhaling ozone and diesel exhaust particles. Environ. Health Perspect. 119, 312–318. Loscalzo, J., 2004. Ozone-from environmental pollutant to atherogenic determinant. N. Engl. J. Med. 350, 834–835. Mudd, J.B., Leavitt, R., Ongun, A., McManus, T.T., 1969. Reaction of ozone with amino acids and proteins. Atmos. Environ. 3, 669–682. Navarra, T., Del Turco, S., Berti, S., Basta, G., 2009. The lectine-like oxidized low-density lipoprotein receptor-1 and its soluble form: cardiovascular implications. J. Atheroscler. Thromb. 17, 317–331. Potor, L., Banyai, E., Becs, G., Soares, M.P., Balla, G., Balla, J., Jeney, V., 2013. Atherogenesis may involve the prooxidant and proinflammatory effects of ferryl hemoglobin. Oxid. Med. Cell. Longev. 2013, 676425, http://dx.doi.org/10.1155/2013/676425. Robertson, S., Colombo, E.S., Lucas, S.N., Hall, P.R., Febbraio, M., Paffett, M.L., Campen, M.J., 2013. CD36 mediates endothelial dysfunction downstream of circulating factors induced by O3 exposure. Toxicol. Sci. 134, 304–311.
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Steinberg, D., 1997. Low density lipoprotein oxidation and its pathological significance. J. Biol. Chem. 272, 20963–20966. Steinberg, D., Lewis, A., 1997. Conner memorial lecture. Oxidatative modification of LDL and atherogenesis. Circulation 95, 1062–1071. Tomono, S., Miyoshi, N., Shiokawa, H., Iwabuchi, T., Aratani, Y., Higashi, T., Nukaya, H., Ohshima, H., 2011. Formation of cholesterol ozonolysis products in vitro and in vivo through a myeloperoxidase-dependent pathway. J. Lipid Res. 52, 87–97.
Wentworth, A.D., Song, B.D., Nieva, J., Shafton, A., Tripurenani, S., Wentworth Jr., P., 2009. The ration of cholesterol 5,6-secosterols formed from ozone and singlet oxygen offers insights into the oxidation of cholesterol in vivo. Chem. Commun. (Camb.) 21, 3098–3100. Wentworth Jr., P., Nieva, J., Takeuchi, C., Galve, R., Wentworth, A.D., Dilley, R.B., DeLaria, G.A., Saven, A., Babior, B.M., Jand a, K.D., Eschenmoser, A., Lerner, R.A., 2003. Evidence for ozone formation in human atherosclerotic arteries. Science 302, 1053–1056.
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