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Specificity and inhibition by antioxidant of lipid peroxidation by lipoxygenase: effects of substrate, lipoxygenase and milieu
International Congress Series 1233 (2002) 355 – 356
Specificity and inhibition by antioxidant of lipid peroxidation by lipoxygenase: effects of substrate, lipoxygenase and milieu Noriko Noguchi, Hiromasa Yamashita, Hartmut Kuhn, Etsuo Niki * Research Center for Advanced Science and Technology, University of Tokyo, Komaba, Tokyo 153-8904, Japan University Clinicum Charite, Humboldt University, 10115 Berlin, Germany Human Stress Signal Research Center, Ikeda 563-8577, Japan
Abstract Although lipid peroxidation by lipoxygenases usually results in a site-, stereo- and enantiospecific mechanism, it was found that the oxidation of phospholipids incorporated into the liposomal membrane by soybean lipoxygenase, but not by rabbit reticulocyte 15-lipoxygenase, was proceeded entirely by a nonspecific mechanism. The radical-scavenging antioxidants such as a-tocopherol did not inhibit specific oxidation; however, they did suppress the nonspecific oxidation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Lipoxygenase; Lipid peroxidation; Antioxidant; Free radical
Lipid peroxidation induces membrane damage and lipoprotein modification [1]. It proceeds in (1) a free radical-mediated chain mechanism, (2) nonradical stoichiometric oxidation, and (3) enzymatic oxidation. The characteristic of enzymatic oxidation is its site, stereo and enantio specificity [2]. For example, 15-lipoxygenase oxidizes linoleic acid moieties to give 13(S)-hydroperoxy-9Z,11E-octadecadienoate (HPODE), specifically. Such specific oxidation products have been accepted as a marker for enzymatic oxidation. In the present study, it was found that under certain circumstances, lipid peroxidation by lipoxygenase might proceed entirely in a nonspecific, random mechanism, and that the radical-scavenging antioxidants were only capable of inhibiting random oxidation.
* Corresponding author. Human Stress Signal Research Center, 1-8-31 Midorigaoka, Ikeda 563-8577, Japan. Tel: +81-727-51-9991; fax: +81-727-51-9964. E-mail address: [email protected] (E. Niki). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 1 6 3 - 2
356
N. Noguchi et al. / International Congress Series 1233 (2002) 355–356
It was confirmed that, as previously reported by many investigators [2], the oxidation of linoleic acid is proceeded entirely by a specific mechanism to give 13(S)-9Z,11E-HPODE independent of the reaction milieu, that is aqueous solution, micelles, phosphatidylcholine liposomal membranes, and low-density lipoprotein (LDL) by either 15-lipoxygenase isolated from rabbit reticulocytes (15-LOX) or soybean lipoxygenase (SLO). The rabbit 15-LOX oxidized phosphatidylcholine in LDL and in liposomal membranes, predominantly, by specific mechanism to give 13(S)-9Z,11E-HPODE. On the other hand, when palmitoyl-linoleoyl-PC (PLPC) was oxidized in liposomal membranes by SLO, four isomeric oxidation products were formed, that is 13-9Z,11E-, 13-9E,11E-, 9-10E,12Z-, and 9-10E,12E-HPODE were observed. 13-9Z,11E- and 9-10E,12Z-HPODE were formed in equal amounts, while 13-9E,11E- and 9-10E,12E-HPODE were formed equally. Furthermore, 13-9Z,11E-HPODE was found to be racemic by a chiral-phase HPLC analysis. These results clearly show that the oxidation of PLPC by SLO in liposomal membranes is preceded by a random mechanism like free radical-mediated oxidation. To confirm the contribution of free radicals, the action of radical-scavenging antioxidants was studied against the oxidation of PLPC in liposomal membranes induced by 15LOX and SLO. a-Tocopherol was incorporated into PLPC liposomal membranes and oxidized by either 15-LOX or SLO. a-Tocopherol did not exert an appreciable antioxidant effect against 15-LOX-induced oxidation; however, it suppressed the oxidation by SLO in a dose-dependent manner. The formation of radicals during the lipoxygenase-induced lipid peroxidation has been suggested. The 15-lipoxygenase binds to linoleate and abstracts 13-S-hydrogen, selectively, to yield the 13-linoleate carbon-centered radical, to which oxygen adds to give the 13-S-peroxyl radical followed by hydrogen atom transfer to give 13(S)-9Z,11E-HPODE. Under these circumstances, the linoleate radical is not literally free but bound to the enzyme, and the reaction proceeds in a concerted mechanism and the antioxidant may not be able to trap such a bound radical. On the other hand, if the life of linoleate carboncentered radicals and/or peroxyl radicals is long enough to give free linoleate and/or peroxyl radicals, they must undergo isomerization to give random products just like free radical-mediated peroxidation. The radical-scavenging antioxidants may well have a chance to trap such free radicals. In conclusion, it was found that the lipid peroxidation in solution, liposomal membranes and LDL by rabbit reticulocyte 15-LOX were proceeded selectively by a specific mechanism; however, the peroxidation of PC in liposomal membranes by SLO was proceeded exclusively by a nonspecific mechanism to give site-, stereo-, and enantiorandom hydroperoxides. Peroxidation in solution by SLO gave specific products. aTocopherol did not exert an antioxidant capacity against specific oxidation; however, it suppressed the random oxidation.
References [1] E. Niki, Lipid peroxidation and its inhibition: overview and perspectives, J. Oleo Sci. 50 (2001) 313 – 320. [2] H. Kuhn, B.J. Thiele, The diversity of the lipoxygenase family: many sequence data but little information on biological significance, FEBS Lett. 449 (1999) 7 – 11.
Specificity and inhibition by antioxidant of lipid peroxidation by lipoxygenase: effects of substrate, lipoxygenase and milieu Noriko Noguchi, Hiromasa Yamashita, Hartmut Kuhn, Etsuo Niki * Research Center for Advanced Science and Technology, University of Tokyo, Komaba, Tokyo 153-8904, Japan University Clinicum Charite, Humboldt University, 10115 Berlin, Germany Human Stress Signal Research Center, Ikeda 563-8577, Japan
Abstract Although lipid peroxidation by lipoxygenases usually results in a site-, stereo- and enantiospecific mechanism, it was found that the oxidation of phospholipids incorporated into the liposomal membrane by soybean lipoxygenase, but not by rabbit reticulocyte 15-lipoxygenase, was proceeded entirely by a nonspecific mechanism. The radical-scavenging antioxidants such as a-tocopherol did not inhibit specific oxidation; however, they did suppress the nonspecific oxidation. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Lipoxygenase; Lipid peroxidation; Antioxidant; Free radical
Lipid peroxidation induces membrane damage and lipoprotein modification [1]. It proceeds in (1) a free radical-mediated chain mechanism, (2) nonradical stoichiometric oxidation, and (3) enzymatic oxidation. The characteristic of enzymatic oxidation is its site, stereo and enantio specificity [2]. For example, 15-lipoxygenase oxidizes linoleic acid moieties to give 13(S)-hydroperoxy-9Z,11E-octadecadienoate (HPODE), specifically. Such specific oxidation products have been accepted as a marker for enzymatic oxidation. In the present study, it was found that under certain circumstances, lipid peroxidation by lipoxygenase might proceed entirely in a nonspecific, random mechanism, and that the radical-scavenging antioxidants were only capable of inhibiting random oxidation.
* Corresponding author. Human Stress Signal Research Center, 1-8-31 Midorigaoka, Ikeda 563-8577, Japan. Tel: +81-727-51-9991; fax: +81-727-51-9964. E-mail address: [email protected] (E. Niki). 0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 1 6 3 - 2
356
N. Noguchi et al. / International Congress Series 1233 (2002) 355–356
It was confirmed that, as previously reported by many investigators [2], the oxidation of linoleic acid is proceeded entirely by a specific mechanism to give 13(S)-9Z,11E-HPODE independent of the reaction milieu, that is aqueous solution, micelles, phosphatidylcholine liposomal membranes, and low-density lipoprotein (LDL) by either 15-lipoxygenase isolated from rabbit reticulocytes (15-LOX) or soybean lipoxygenase (SLO). The rabbit 15-LOX oxidized phosphatidylcholine in LDL and in liposomal membranes, predominantly, by specific mechanism to give 13(S)-9Z,11E-HPODE. On the other hand, when palmitoyl-linoleoyl-PC (PLPC) was oxidized in liposomal membranes by SLO, four isomeric oxidation products were formed, that is 13-9Z,11E-, 13-9E,11E-, 9-10E,12Z-, and 9-10E,12E-HPODE were observed. 13-9Z,11E- and 9-10E,12Z-HPODE were formed in equal amounts, while 13-9E,11E- and 9-10E,12E-HPODE were formed equally. Furthermore, 13-9Z,11E-HPODE was found to be racemic by a chiral-phase HPLC analysis. These results clearly show that the oxidation of PLPC by SLO in liposomal membranes is preceded by a random mechanism like free radical-mediated oxidation. To confirm the contribution of free radicals, the action of radical-scavenging antioxidants was studied against the oxidation of PLPC in liposomal membranes induced by 15LOX and SLO. a-Tocopherol was incorporated into PLPC liposomal membranes and oxidized by either 15-LOX or SLO. a-Tocopherol did not exert an appreciable antioxidant effect against 15-LOX-induced oxidation; however, it suppressed the oxidation by SLO in a dose-dependent manner. The formation of radicals during the lipoxygenase-induced lipid peroxidation has been suggested. The 15-lipoxygenase binds to linoleate and abstracts 13-S-hydrogen, selectively, to yield the 13-linoleate carbon-centered radical, to which oxygen adds to give the 13-S-peroxyl radical followed by hydrogen atom transfer to give 13(S)-9Z,11E-HPODE. Under these circumstances, the linoleate radical is not literally free but bound to the enzyme, and the reaction proceeds in a concerted mechanism and the antioxidant may not be able to trap such a bound radical. On the other hand, if the life of linoleate carboncentered radicals and/or peroxyl radicals is long enough to give free linoleate and/or peroxyl radicals, they must undergo isomerization to give random products just like free radical-mediated peroxidation. The radical-scavenging antioxidants may well have a chance to trap such free radicals. In conclusion, it was found that the lipid peroxidation in solution, liposomal membranes and LDL by rabbit reticulocyte 15-LOX were proceeded selectively by a specific mechanism; however, the peroxidation of PC in liposomal membranes by SLO was proceeded exclusively by a nonspecific mechanism to give site-, stereo-, and enantiorandom hydroperoxides. Peroxidation in solution by SLO gave specific products. aTocopherol did not exert an antioxidant capacity against specific oxidation; however, it suppressed the random oxidation.
References [1] E. Niki, Lipid peroxidation and its inhibition: overview and perspectives, J. Oleo Sci. 50 (2001) 313 – 320. [2] H. Kuhn, B.J. Thiele, The diversity of the lipoxygenase family: many sequence data but little information on biological significance, FEBS Lett. 449 (1999) 7 – 11.