On-line EPR study of free radicals induced by peroxidase/H 2 O 2 in human low-density lipoprotein Donatella Pietraforte a, * , Laura Turco a , Elena Azzini b , Maurizio Minetti a a Laboratorio di Biologia Cellulare, Istituto Superiore di Sanita `, Viale Regina Elena 299, 00161, Rome, Italy b Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, Via Ardeatina 546, 00178, Rome, Italy Received 5 February 2002; received in revised form 15 April 2002; accepted 19 April 2002 Abstract The aim of this study was to use direct electron paramagnetic resonance (EPR) spectroscopy at 37 jC and spin trapping techniques to study radical species formed during horseradish peroxidase/H 2 O 2 -initiated low-density lipoprotein (LDL) oxidation. Using direct EPR, we obtained evidence for the formation not only of the a-tocopheroxyl radical but also of a protein radical(s), assigned to a tyrosyl radical(s) of apolipoprotein B-100 (apo B-100). Spin trapping with 2-methyl-2-nitrosopropane revealed (i) the formation of a mobile adduct with h-hydrogen coupling assigned to a lipid radical and (ii) a partially immobilised adduct detected in LDL as well as in apo B-100, assigned after proteolytic digestion to the trapping of a radical centred on a tertiary carbon atom of an aromatic residue, probably tyrosine. Our results support the hypothesis that radicals are initiators of the oxidative process, and show that their formation is an early event in peroxidase- mediated oxidation. We also tested the effects of resveratrol (RSV), a polyphenolic antioxidant present in red wine. Our data indicate that 1–10 AM RSV is able to accelerate a-tocopherol consumption, conjugated dienes formation and the decay kinetics of LDL-centred radicals. Since phenols are substrates for peroxidases, this result may be ascribed to a RSV-mediated catalysis of peroxidase activity. D 2002 Elsevier Science B.V. All rights reserved. Keywords: LDL; Horseradish peroxidase; Free radical; EPR; Resveratrol 1. Introduction The pathogenesis of atherosclerosis is a multifaceted process that has been firmly correlated with the oxidation of lipoproteins [1–3]. A key role in the onset of athero- sclerotic disease has been attributed to low-density lip- oprotein (LDL) and to the oxidative processes leading to its modification into an atherogenic agent [1–5]. The prominent physiologically important modification found in LDL is the derivatisation of its constituents with the formation of lipid – protein crosslinkages. This mechanism is consistent with a cross-reaction of lipid oxidation prod- ucts, such as the radical-mediated formation of hydroper- oxides and aldehydes, with free amino groups of LDL [6,7]. These lipid–protein crosslinkings, recently demonstrated also in vivo by immunohistochemical studies [8], convert native LDL into a more negatively charged molecule. Oxidised LDL is a ligand for the scavenger receptor of macrophages, a key event in the formation of foam cells and atheromatous plaque [1,3]. Notwithstanding evidence of the presence of oxidatively modified LDL in human and animal atherosclerotic lesions [9], the physiologically relevant mechanism for LDL oxi- dation in vivo has not been completely identified. Recent studies have suggested a possible inflammatory aetiology of atherosclerosis sustained by oxidising agents present in the vascular system or derived from leukocytes. Inflammatory cells present in the wall of an atherosclerotic artery are activated to produce reactive oxygen and nitrogen species, as well as chlorinating compounds [3,5]. In this regard, there is increasing interest in peroxidase-mediated metal-inde- pendent oxidation of LDL, since an increase in peroxidase activity has been demonstrated in human atherosclerotic tissues [10]. Moreover, myeloperoxidase, a potent macro- 1388-1981/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S1388-1981(02)00211-1 Abbreviations: Apo B-100, apolipoprotein B-100; apo B-100 S , apolipoprotein B-100-centred radical(s); DTPA, diethylenetriaminepenta- acetic acid; EPR, electron paramagnetic resonance spectroscopy; HRP, horseradish peroxidase; LDL, low-density lipoprotein; MNP, 2-methyl-2- nitrosopropane; RSV, resveratrol; a-Toc S , a-tocopheroxyl radical * Corresponding author. Tel.: +39-6-49902506; fax: +39-6-49387143. E-mail address: pietraf@iss.it (D. Pietraforte). www.bba-direct.com Biochimica et Biophysica Acta 1583 (2002) 176 – 184