Dihydrolipoic acid inhibits 15-lipoxygenase-dependent lipid peroxidation

Free Radical Biology & Medicine, Vol. 35, No. 10, pp. 1203–1209, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter

doi:10.1016/S0891-5849(03)00508-2

Original Contribution DIHYDROLIPOIC ACID INHIBITS 15-LIPOXYGENASE-DEPENDENT LIPID PEROXIDATION DOMENICO LAPENNA, GIULIANO CIOFANI, SANTE DONATO PIERDOMENICO, MARIA ADELE GIAMBERARDINO, FRANCO CUCCURULLO

and

Dipartimento di Medicina e Scienze dell’Invecchiamento and Centro di Scienze dell’Invecchiamento-Fondazione Universita’ G. d’Annunzio, Chieti, Italy (Received 11 November 2002; Revised 17 June 2003; Accepted 17 July 2003)

Abstract—The potential antioxidant effects of the hydrophobic therapeutic agent lipoic acid (LA) and of its reduced form dihydrolipoic acid (DHLA) on the peroxidation of either linoleic acid or human non-HDL fraction catalyzed by soybean 15-lipoxygenase (SLO) and rabbit reticulocyte 15-lipoxygenase (RR15-LOX) were investigated. DHLA, but not LA, did inhibit SLO-dependent lipid peroxidation, showing an IC50 of 15 ␮M with linoleic acid and 5 ␮M with the non-HDL fraction. In specific experiments performed with linoleic acid, inhibition of SLO activity by DHLA was irreversible and of a complete, noncompetitive type. In comparison with DHLA, the well-known lipoxygenase inhibitor nordihydroguaiaretic acid and the nonspecific iron reductant sodium dithionite inhibited SLO-dependent linoleic acid peroxidation with an IC50 of 4 and 100 ␮M, respectively, while the hydrophilic thiol N-acetylcysteine, albeit possessing iron-reducing and radical-scavenging properties, was ineffective. Remarkably, DHLA, but not LA, was also able to inhibit the peroxidation of linoleic acid and of the non-HDL fraction catalyzed by RR15-LOX with an IC50 of, respectively, 10 and 5 ␮M. Finally, DHLA, but once again not LA, could readily reduce simple ferric ions and scavenge efficiently the stable free radical 1,1-diphenyl-2-pycrylhydrazyl in ethanol; DHLA was considerably less effective against 2,2⬘-azobis(2-amidinopropane) dihydrochloride-mediated, peroxyl radical-induced non-HDL peroxidation, showing an IC50 of 850 ␮M. Thus, DHLA, at therapeutically relevant concentrations, can counteract 15-lipoxygenasedependent lipid peroxidation; this antioxidant effect may stem primarily from reduction of the active ferric 15lipoxygenase form to the inactive ferrous state after DHLA-enzyme hydrophobic interaction and, possibly, from scavenging of fatty acid peroxyl radicals formed during lipoperoxidative processes. Inhibition of 15-lipoxygenase oxidative activity by DHLA could occur in the clinical setting, eventually resulting in specific antioxidant and antiatherogenic effects. © 2003 Elsevier Inc. Keywords—Lipoic acid, Dihydrolipoic acid, 15-Lipoxygenase, Lipid peroxidation, Iron, Free radicals, Antioxidant, Atherosclerosis, Free radicals

INTRODUCTION

LOXs have been identified, namely 5-, 12-, and 15-LOX, which insert dioxygen, respectively, at C5, C12, and C15 positions of arachidonic acid [1–5]. Of particular interest is 15-LOX, since it can also oxidize esterified fatty acids in biological membranes and lipoproteins, forming 15hydroperoxy-eicosatetraenoic acid (15-HPETE) from arachidonic acid and 13-hydroperoxy-octadecadienoic acid (13-HPODE) from linoleic acid [1,2,4,5]. Remarkably, 15-LOX has been implicated for its specific oxidative effects in the pathogenesis of atherosclerosis [1,4 – 6]. Soybean lipoxygenase-1 (SLO) is a plant-derived 15LOX that efficiently catalyzes the oxidation of linoleic acid to 13-HPODE. Because of structural and functional similarities with mammalian LOXs, SLO is commonly

Lipoxygenases (LOXs) are a family of nonheme ironcontaining dioxygenases able to induce enzymatic peroxidation of polyunsaturated fatty acids [1,2]. In general, LOXs contain an essential iron atom, which is present as Fe2⫹ in the inactive enzyme form; enzymatic activation occurs through hydroperoxide-driven oxidation of Fe2⫹ to Fe3⫹ [1–3]. LOXs are widely distributed among plants and animals [1,2]. In mammals, three major types of Address correspondence to: Prof. Domenico Lapenna, Patologia Medica, Policlinico di Colle dell’Ara, Via dei Vestini, 66013 Chieti Scalo, Italy; Tel: ⫹39 871 358098; Fax: ⫹39 871 551615; E-Mail: [email protected]. 1203

1204

D. LAPENNA et al.

used for both mechanistic and inhibitory studies and is widely accepted as a model for LOXs from other sources [1,2,7–10]. In such a context, reduction of the enzyme Fe3⫹ to Fe2⫹ and scavenging of peroxyl radicals generated from LOX-polyunsaturated fatty acid interaction may be involved in the pharmacological inhibition of enzymatic lipid peroxidation [1–3,9,10]. Lipoic (or thioctic) acid (LA), the naturally occurring coenzyme of pyruvate and ␣-ketoglutarate dehydrogenase, is a lipophilic therapeutic agent used in a variety of diseases including neurological and liver disorders [11,12]. Patients with diabetic neuropathy and alcoholic liver disease may benefit from LA therapy, which is thought to act through antioxidant mechanisms [11,12]. LA undergoes substantial conversion in vivo to dihydrolipoic acid (DHLA), which is an effective reducing compound with radical-scavenging and metal-chelating antioxidant properties [11–13]. LA also has an intrinsic antioxidant activity, partly related to chelation of catalytic transition metals [11,12]. Despite seemingly extensive characterization of LA and DHLA antioxidant effects, it is still unkown whether these compounds could inhibit 15-LOX-dependent lipid peroxidation. We have, therefore, addressed this issue here, showing that only DHLA has specific antilipoperoxidative properties. MATERIALS AND METHODS

Reagents and lipoprotein preparation Reagents were generally obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA) including Fluka-produced SLO (EC 1.13.11.12), which was used as purchased. The rabbit reticulocyte 15-LOX (RR15-LOX) was a product of Calbiochem (San Diego, CA, USA) and also used as purchased; and, 2,2⬘-azobis(2-amidinopropane) dihydrochloride was from Polysciences Inc. (Warrington, PA, USA). The non-high-density lipoprotein (non-HDL) fraction, namely LDL plus VLDL, was obtained from EDTA plasma of healthy adults, as reported previously [14], using dextrane sulfate (mol. wt. 500,000) plus MgCl2 to precipitate the fraction itself and remove EDTA. LA and DHLA, as well as nordihydroguaiaretic acid (NDGA), were predissolved in ethanol, using the same alcohol aliquots in control experiments. 15-LOX-dependent lipid peroxidation The effects of LA and DHLA were at first tested on SLO-dependent linoleic acid peroxidation. Reactions were carried out in quartz cuvettes containing, in a final volume of 1.0 ml, 0.3 ␮M SLO, the agents tested (predissolved or not in ethanol as appropriate), and 35 ␮M

linoleic acid in 10 mM KH2PO4-KOH buffer (KB), pH 7.4, plus 1% Tween-20; incubation was for 120 min at 37°C. According to previous reports and considering that specific enzyme activity in the inhibitor absence is independent of the sequence of reagent addition [9,10], lipoperoxidative reactions were started by substrate addition. Enzymatic lipid peroxidation was assessed through continuous spectrophotometric monitoring of absorbance increase at 234 nm due to formation of conjugated diene hydroperoxides (CD) during specific oxidative processes [9,10,14]. Reference cuvettes contained linoleic acid (or the non-HDL fraction when it was used as the lipid oxidizable substrate) with or without the agents tested, as appropriate. Molar extinction coefficient of CD was considered to be 29,500 at 234 nm [14]. Minimal drug concentrations significantly inhibiting lipid peroxidation (ICmin) and drug concentrations inhibiting by 50% (IC50) and 100%, i.e., totally (IC100) lipid peroxidation, were determined [14]. For comparative purposes, we also evaluated the LOX inhibitor NDGA, the nonspecific iron reductant sodium dithionite (SDT), and the hydrophilic thiol N-acetylcysteine (NAC) on the peroxidation of 35 ␮M linoleic acid catalyzed by 0.3 ␮M SLO. The effects of LA and DHLA were further tested on SLO-dependent human lipoprotein peroxidation. Optimal operative conditions for continuous spectrophotometric monitoring at 234 nm of CD formation during lipid peroxidation were found using 0.1 ␮M SLO and 0.1 mg non-HDL protein/ml, in 10 mM KB, pH 7.4, plus 5 mM sodium deoxycholate; incubation was for 60 min at 37°C with and without LA or DHLA. In additional experiments, we assessed a possible inhibitory activity of LA and DHLA on mammalian 15-LOX-catalyzed lipid peroxidation. As for SLO, reactions were carried out in quartz cuvettes containing, in a final volume of 1.0 ml, 0.15 ␮M RR15-LOX, the agents tested, and 200 ␮M linoleic acid in 10 mM KB, pH 7.4, plus 1% Tween-20; incubation was for 60 min at 37°C. Moreover, the effects of LA and DHLA were assessed on the peroxidation of the non-HDL fraction (0.1 mg nonHDL protein/ml) induced by 0.1 ␮M RR15-LOX in 10 mM KB, pH 7.4, plus 5 mM sodium deoxycholate; CD formation was monitored spectrophotometrically for 120 min at 37°C as reported above. Iron reduction To avoid possible interference of buffers and phosphates with simple iron ions, drug capability to reduce Fe3⫹ to Fe2⫹ was evaluated in 0.15 M NaCl plus 1% Tween-20; reaction mixtures contained 5 ␮M FeCl3, LA, or DHLA, and the specific iron(II) colorimetric detector bathophenanthroline disulfonic acid disodium salt (BPD)

Dihydrolipoic acid as a 15-lipoxygenase inhibitor

1205

at 100 ␮M final concentration. Formation of the BPDFe2⫹ complex was assessed spectrophotometrically at 535 nm after 120 min incubation at 37°C, using for calculation a molar extinction coefficient of 22,000. Iron(III)-reducing effects of NDGA, SDT, and NAC were also tested. Radical-scavenging assessment Radical-scavenging activity of LA and DHLA, as well as of NDGA, SDT, and NAC, was evaluated by assessing their capacity to interact with and scavenge the stable free radical 1,1-diphenyl-2-pycrylhydrazyl (DPPH), resulting in 1,1-diphenyl-2-picrylhydrazine formation with DPPH bleaching [14,15]. The reaction system contained 35 ␮M DPPH in ethanol, with or without the agents tested; after 5 min incubation at 37°C, DPPHrelated absorbance values at 517 (A517) were recorded spectrophotometrically against appropriate drug-containing blanks. Furthermore, we studied whether LA and DHLA could be able to counteract lipid peroxidation induced by peroxyl radicals generated thermally by the azo-initiator AAPH. Experimental tubes contained 0.1 mg non-HDL protein/ml and 2 mM AAPH, in 10 mM KB, pH 7.4, plus 5 mM sodium deoxycholate and 0.1 mM diethylenetriaminepentaacetic acid, with and without LA or DHLA; incubation was for 60 min at 37°C. CD formation was monitored spectrophotometrically at 234 nm as reported above for enzymatic lipid peroxidation.

Fig. 1. The inhibitory effect of DHLA on SLO-dependent linoleic acid peroxidation evaluated by continuous spectrophotometric monitoring of absorbance increase at 234 nm due to CD formation. Trace 1: control; traces 2– 4: 4, 15, and 30 ␮M DHLA, respectively. The results are representative of seven similar experiments carried out with 0.3 ␮M SLO and 35 ␮M linoleic acid.

From experiments performed with various linoleic acid and DHLA concentrations and represented graphically as Yoshino’s fractional velocity plot [17], inhibition of SLO activity by DHLA appeared to be of a complete, noncompetitive type (Fig. 2). In fact, as shown in Fig. 2, the straight line obtained in this plot goes through the origin [17]; according to Yoshino [17], noncompetitive inhibition is confirmed by the constant slope

Statistics Data were calculated as means ⫾ SD and analyzed by one-way analysis of variance (ANOVA) plus StudentNewman-Keuls test [16]; p ⬍ .05 was considered statistically significant [16]. RESULTS

The drug effect on 15-LOX-dependent lipid peroxidation DHLA, but not LA, could significantly counteract enzymatic peroxidation of linoleic acid and of the nonHDL fraction. As depicted in Fig. 1, DHLA inhibited, in a dose-related manner, SLO-dependent linoleic acid peroxidation with ICmin, IC50, and IC100 values of 4, 15, and 30 ␮M, respectively. Indeed, 0.17 ⫾ 0.015 nmol CD/min were produced by SLO in control experiments as compared to 0.14 ⫾ 0.012 and 0.085 ⫾ 0.007 nmol CD/min with 4 and 15 ␮M DHLA, respectively (both p ⬍ .05 vs. control; 4 vs. 15 ␮M DHLA, p ⬍ .05; n ⫽ 7). No significant CD formation was detectable with 30 ␮M DHLA (Fig. 1), which totally inhibited SLO-catalyzed linoleic acid peroxidation.

Fig. 2. Yoshino’s fractional velocity plot vs. reciprocal DHLA concentration, namely 1/[DHLA]. Concentrations of DHLA were those corresponding to its values of ICmin (4 ␮M), IC50 (15 ␮M), and IC100 (30 ␮M), detected with 0.3 ␮M SLO and 35 ␮M linoleic acid, which we used at the various concentrations of 17.5, 35, and 70 ␮M. The yield of linoleic acid peroxidation catalyzed by 0.3 ␮M SLO in the absence (V0) and presence (V) of DHLA was assessed spectrophotometrically at 234 nm as CD formation and calculated as nmol CD/min. See Results for further explanations.

1206

D. LAPENNA et al.

Fig. 3. The inhibitory effect of DHLA on SLO-dependent human non-HDL peroxidation evaluated by continuous spectrophotometric monitoring of absorbance increase at 234 nm due to CD formation. Trace 1: control; traces 2– 4: 1.8, 5, and 20 ␮M DHLA, respectively. The results are representative of six similar experiments carried out with 0.1 ␮M SLO and 0.1 mg non-HDL protein/ml.

of the plot (single straight line) irrespective of the change in linoleic acid concentration (Fig. 2). SLO continued to be inhibited by about 50% after 30 min dialysis against 100 ml KB, pH 7.4, of 1.0 ml mixtures containing 0.3 ␮M SLO and a concentration of DHLA corresponding to its IC50 value (15 ␮M). Accordingly, after such dialytic procedure, SLO oxidized 35 ␮M linoleic acid at a rate of 0.083 ⫾ 0.008 nmol CD/min in comparison with 0.16 ⫾ 0.014 nmol CD/min of specific control experiments (p ⬍ .05; n ⫽ 6). Hence, DHLA acted as an irreversible enzymic inhibitor. As expected, NDGA showed strong antioxidant effects on the peroxidation of 35 ␮M linoleic acid induced by 0.3 ␮M SLO, inhibiting CD formation with an IC50 of 4 ␮M (0.089 ⫾ 0.006 vs. 0.18 ⫾ 0.016 nmol CD/min of control experiments, p ⬍ .05; n ⫽ 6). SDT counteracted SLO-dependent CD formation with an IC50 of 100 ␮M, which resulted in 0.090 ⫾ 0.0085 nmol CD/min in comparison with 0.18 ⫾ 0.016 nmol CD/min of control experiments (p ⬍ .05; n ⫽ 6), while NAC was ineffective even at 1 mM concentration (0.17 ⫾ 0.015 vs. 0.18 ⫾ 0.016 nmol CD/min of control experiments, p ⫽ NS; n ⫽ 6). In the experiments performed with the human nonHDL fraction, DHLA also inhibited SLO-catalyzed lipid peroxidation in a dose-dependent manner (Fig. 3) with an ICmin, IC50, and IC100 of 1.8, 5, and 20 ␮M, respectively. Indeed, enzymic lipoprotein peroxidation resulted in 74 ⫾ 8.7 nmol CD/mg non-HDL protein in control experiments, and it was significantly lowered to 61 ⫾ 7 and 37 ⫾ 4.3 nmol CD/mg non-HDL protein by, respectively, 1.8 and 5 ␮M DHLA (both p ⬍ .05 vs. control; 1.8 vs. 5 ␮M DHLA, p ⬍ .05; n ⫽ 6). Total inhibition of

Fig. 4. The inhibitory effect of DHLA on RR15-LOX-dependent human non-HDL peroxidation evaluated by continuous spectrophotometric monitoring of absorbance increase at 234 nm due to CD formation. Trace 1: control; traces 2– 4: 2, 5, and 22 ␮M DHLA, respectively. The results are representative of six similar experiments carried out with 0.1 ␮M RR15-LOX and 0.1 mg non-HDL protein/ml.

SLO-catalyzed lipoprotein peroxidation was evident with 20 ␮M DHLA (Fig. 3). Under the experimental conditions used, RR15-LOX oxidized linoleic acid yielding 0.061 ⫾ 0.0053 nmol CD/min in control experiments (n ⫽ 6). The ICmin of DHLA was 5.5 ␮M, which resulted in 0.05 ⫾ 0.0045 nmol CD/min (p ⬍ .05 vs. controls; n ⫽ 6). DHLA at 10 ␮M halved RR15-LOX-dependent linoleic acid peroxidation (0.03 ⫾ 0.0027 nmol CD/min, p ⬍ .05 vs. both controls and 5.5 ␮M DHLA; n ⫽ 6), while at 50 ␮M DHLA had a totally antilipoperoxidative effect. Moreover, DHLA inhibited RR15-LOX-dependent peroxidation of the non-HDL fraction (Fig. 4). In this experimental setting, ICmin and IC50 of DHLA were 2 and 5 ␮M, which resulted in 26.3 ⫾ 3 and 16 ⫾ 1.8 nmol CD/mg non-HDL protein, respectively, as compared to 32 ⫾ 3.8 nmol CD/mg non-HDL protein of control experiments (both p ⬍ .05 vs. control; 2 vs. 5 ␮M DHLA, p ⬍ .05; n ⫽ 6). Finally, 22 ␮M DHLA totally inhibited RR15LOX-catalyzed non-HDL peroxidation (Fig. 4). The drug effect on iron reduction Absorbance at 535 nm of the BPD-Fe2⫹ complex after 120 min incubation of 5 ␮M FeCl3 with 5 and 10 ␮M DHLA was 0.054 ⫾ 0.003 and 0.108 ⫾ 0.005, respectively (n ⫽ 6). Similar values of absorbance at 535 nm, corresponding to mean Fe2⫹ concentrations of 2.5 and 5 ␮M and, hence, to 50% and 100% Fe3⫹ reduction, were detected in the presence of 0.4 and 1 ␮M NDGA (0.053 ⫾ 0.002 and 0.107 ⫾ 0.004, n ⫽ 6), 20 and 40 ␮M SDT (0.055 ⫾ 0.003 and 0.109 ⫾ 0.005, n ⫽ 6), or 5 and 20 ␮M NAC (0.054 ⫾ 0.003 and 0.110 ⫾ 0.006, n ⫽ 6). Thus, the order of potency of iron reduction by the agents tested was: NDGA ⬎ DHLA ⬎ NAC ⬎ SDT.

Dihydrolipoic acid as a 15-lipoxygenase inhibitor

Regarding LA, it was incapable of reducing Fe3⫹ under the experimental conditions used. Drug radical-scavenging activity DHLA was the most efficient radical scavenger, inducing the bleaching of 50% of DPPH at a drug concentration of 15 ␮M (0.130 ⫾ 0.005 vs. 0.261 ⫾ 0.009 A517 of control experiments, p ⬍ .05; n ⫽ 6). NDGA and NAC induced the bleaching of 50% of DPPH at concentrations of 30 and 220 ␮M, respectively, while both LA and SDT had no specific radical-scavenging activity (not shown). DHLA, but not LA, significantly counteracted AAPHmediated, peroxyl radical-induced non-HDL peroxidation with an IC50 of 850 ␮M, which gave 27 ⫾ 3.3 nmol CD/mg non-HDL protein in comparison with 54 ⫾ 6 nmol CD/mg non-HDL protein of control experiments (p ⬍ .05; n ⫽ 6).

DISCUSSION

The present study shows that DHLA can inhibit 15LOX-catalyzed linoleic acid and human lipoprotein peroxidation while LA is ineffective. Since DHLA has reducing properties, it is conceivable that drug-dependent reduction of Fe3⫹ to Fe2⫹ at the enzyme-active site is responsible for the inhibition of enzymatic lipid peroxidation. Such peroxidation could be favored by peroxyl radicals generated from 15-LOX-unsaturated lipid interaction [1–3,18]; thus, scavengers of peroxyl radicals, such as DHLA [11–13], might exert specific antilipoperoxidative effects. Yet, our results show that DHLA is considerably less effective as a peroxyl radical scavenger than as a 15-LOX inhibitor. While the IC50 of DHLA against AAPH-mediated, peroxyl radical-induced lipoprotein peroxidation is as high as 850 ␮M, that against SLO- or RR15-LOX-catalyzed lipoprotein peroxidation is only 5 ␮M. So, given also the capability of DHLA to readily reduce simple ferric ions, it may be suggested that the inhibitory activity of DHLA on 15LOX-dependent lipid peroxidation is primarily due to reduction of the enzyme iron resulting in 15-LOX inactivation. However, it should be considered that the azoinitiator AAPH generates peroxyl radicals essentially in the aqueous phase; it cannot be, therefore, totally excluded that the lipophilic compound DHLA could scavenge some fatty acid peroxyl radicals formed from 15LOX-polyunsaturated fatty acid interaction directly within the enzyme and/or lipid hydrophobic environment, thereby decreasing lipid peroxidation. In this regard, it is of note that DHLA does efficiently scavenge the stable free radical DPPH in the organic solvent ethanol.

1207

The importance of enzyme iron reduction in 15-LOX inhibition is further pointed out by the finding that NDGA, which has been reported to reduce the active ferric form of SLO to the inactive ferrous state [2], is the most effective iron reductant and SLO inhibitor. Moreover, SDT, which reduces iron without acting as a radical scavenger, inhibits SLO activity, though less efficiently than NDGA and DHLA, which are the best iron reductants and also have radical-scavenging properties. On the other hand, the hydrophilic thiol NAC, although able to reduce simple iron ions and to act as a radical scavenger, does not result in SLO inhibition, suggesting that other factors like drug hydrophobicity may be relevant to such inhibition. Accordingly, it has been reported that enhancing the hydrophobicity of SLO inhibitors makes them more effective [10] and that n-alcohols, as well as synthetic hydrophobic thiols, inhibit SLO by virtue of their hydrophobic interactions with the catalytic enzyme site [19,20]. Thus, especially Fe3⫹ reduction and, possibly, radical scavenging by DHLA after hydrophobic drugenzyme interaction may result in total inhibition of 15LOX-catalyzed lipid peroxidation. Similar to a previous study performed with 1-octanethiol and other n-alkylthiols [19], the inhibition of SLO activity by DHLA is of a noncompetitive type, indicating that the unsaturated lipid substrate and DHLA bind to the enzyme at independent sites [17]. In this regard, it is known that the coordinated iron of SLO faces two large internal cavities, namely cavities I and II, connecting the metal to the exterior of the molecule [7]. While cavity II is implicated in the interaction of SLO with polyunsaturated fatty acids [7], cavity I could be involved in that with DHLA, resulting in noncompetitive enzymatic inhibition. Also similar to another study performed with synthetic hydrophobic thiols including 1-octanethiol [20], the inhibition of SLO activity by DHLA is not reversed by dialysis, indicating that the substance irreversibly inactivates the enzymes. This phenomenon is of potential therapeutic relevance, since DHLA formed in vivo from LA may induce a persistent inhibition of 15-LOX-dependent lipid peroxidation even after acute or during intermittent LA administration. The aforementioned considerations are based also on experiments performed with SLO. As in other investigations, SLO has been here used for its large availability and relative inexpensiveness; more importantly, SLO may represent a suitable model for mammalian and human 15-LOX activity. Given also the polyunsaturated fatty acid oxygenation sites, SLO has been reported to be most like the mammalian 15-LOX [1]. Furthermore, although SLO is not totally identical to human 15-LOX, there is a 39% identity between these enzymes based on amino acid sequences, with maximal alignment in two conserved histidine regions thought to be the substrate

1208

D. LAPENNA et al.

binding at the catalytic site [1,2,21,22]. Thus, inhibitors of SLO may be expected to also inhibit human 15-LOX. The capacity of several enzymatic inhibitors to inactivate both SLO and human 15-LOX, sometimes acting even more efficiently on the latter than on the former enzyme, has been shown [21]. Therefore, as already noted, SLO may be accepted as a model for mammalian and human 15-LOX [1,2,7–10,21]. In such a context, it is noticeable that DHLA can also inhibit linoleic acid and lipoprotein peroxidation catalyzed by RR15-LOX; this enzyme shares an 81% overall sequence identity with the corresponding human form [22], further suggesting possible inhibitory effects of DHLA against lipid peroxidation induced by human 15-LOX. LA reaches mean plasma concentrations of about 15 ␮M after a single dose of 600 mg administered orally to humans [23]. Considerably higher drug concentrations may be reached, however, at tissue level, where LA accumulates undergoing a substantial conversion to DHLA [11,12]. As such, it is remarkable that DHLA counteracts 15-LOX-dependent lipid peroxidation at therapeutically achievable concentrations. Moreover, DHLA has been shown to be present both intra- and extracellularly after incubation with LA, leading to augmentation of cell antioxidant defenses and function [11,12,24]. Since LOXs, including 15-LOX, are intracellular enzymes, DHLA could inhibit enzymatic lipid peroxidation within the cell, preventing the transferring across plasma membranes of lipoperoxides able to “seed” extracellular lipoproteins [25]. The inhibition of enzymatic lipid peroxidation by DHLA could, however, also occur in the extracellular space, where 15-LOX may be released from disrupted cells in inflammatory sites or in growing atherosclerotic lesions. Collectively, inhibition of 15-LOX by DHLA appears feasible in vivo, eventually resulting in antioxidant and antiatherogenic effects. It is indeed worth noting that pharmacological inhibition of 15-LOX or disruption of the 12-/15-LOX gene can prevent the development of experimental atherosclerosis [25,26]. When extrapolated to the clinical setting, our results suggest LA administration for therapeutic and/or preventive purposes in pathological conditions characterized by 12-/15-LOX activation, such as hypercholesterolemia, arterial hypertension, cancer, and asthma [5,27–33]. Interestingly, 15LOX has also been demonstrated in coronary graft atherosclerotic lesions of patients with transplant coronary artery disease [34], which may be a specific clinical target of LA treatment. Thus, the therapeutic spectrum of LA-DHLA may be broader and more specific than that previously recognized involving diabetes mellitus, especially diabetic neuropathy, and alcoholic liver disease [11,12].

In conclusion, we have herein shown that DHLA is an efficient 15-LOX inhibitor, counteracting at therapeutically relevant concentrations enzyme-dependent peroxidation of linoleic acid and human non-HDL fraction. DHLA is considered a universal antioxidant both in the membrane and in the aqueous phase and interacts with endogenous antioxidants, regenerating, for example, ascorbic acid and increasing cell glutathione levels [11– 13,24]. For its inhibitory activity against 15-LOX-catalyzed lipid peroxidation, DHLA now appears an even more universal antioxidant, which is already available for human therapy.

REFERENCES [1] Yamamoto, S. Mammalian lipoxygenases: molecular structures and functions. Biochim. Biophys. Acta 1128:117–131; 1992. [2] Gardner, H. W. Recent investigations into the lipoxygenase pathways of plants. Biochim. Biophys. Acta 1084:221–239; 1991. [3] Jones, G. D.; Russell, L.; Darley-Usmar, V. M.; Stone, D.; Wilson, M. T. Role of lipid hydroperoxides in the activation of 15-lipoxygenase. Biochemistry 35:7197–7203; 1996. [4] Sigal, E.; Laughton, C. W.; Mulkins, M. A. Oxidation, lipoxygenase, and atherogenesis. Ann. NY Acad. Sci. 714:211–224; 1994. [5] Cornicelli, J. A.; Trivedi, B. K. 15-lipoxygenase and its inhibition: a novel therapeutic target for vascular disease. Curr. Pharm. Des. 5:11–20; 1999. [6] Khu¨ n, H.; Heydek, D.; Hugou, I.; Gniwotta, C. In vivo action of 15-lipoxygenase in early stages of human atherogenesis. J. Clin. Invest. 99:888 – 893; 1997. [7] Boyington, J. C.; Gaffney, B. J.; Amzel, L. A. The three-dimensional structure of an arachidonic acid 15-lipoxygenase. Science 260:1482–1486; 1993. [8] Clemens, F.; Drutkowski, G.; Wiese, M.; Frohberg, P. The inactivation of lipoxygenase-1 from soybeans by amidrazones. Biochim. Biophys. Acta 1549:88 –98; 2001. [9] Sud’ina, G. F.; Mirzoeva, O. K.; Pushkareva, M. A.; Korshunova, G. A.; Sumbatyan, N. V.; Varfolomeev, S. D. Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett. 329:21–24; 1993. [10] Maccarrone, M.; Veldink, G. A.; Vliegenthart, F. G.; Finazzi Agro’, A. Inhibition of soybean lipoxygenase-1 by chain-breaking antioxidants. Lipids 30:51–54; 1995. [11] Packer, L.; Witt, E. H.; Tritschler, H. J. Alpha-lipoic acid as a biological antioxidant. Free Radic. Biol. Med. 19:227–250; 1995. [12] Bustamante, J.; Lodge, J. K.; Marcocci, L.; Tritschler, H. J.; Packer, L.; Rihn, B. H. Alpha-lipoic acid in liver metabolism and disease. Free Radic. Biol. Med. 24:1023–1039; 1998. [13] Kagan, V.; Shvedova, A.; Serbinova, E.; Khan, S.; Swanson, C.; Powell, R.; Packer, L. Dihydrolipoic acid: a universal antioxidant both in the membrane and in the aqueous phase. Biochem. Pharmacol. 44:1637–1649; 1992. [14] Lapenna, D.; Ciofani, G.; Bruno, C.; Pierdomenico, S. D.; Cuccurullo, F. Antioxidant activity of amiodarone on human lipoprotein oxidation. Br. J. Pharmacol. 133:739 –745; 2001. [15] Blois, M. S. Antioxidant determinations by the use of a stable free radical. Nature 158:1199 –1200; 1958. [16] Glantz, S. A. Primer of biostatistics. New York: McGraw-Hill; 1987. [17] Yoshino, M. A graphical method for determining inhibition parameters for partial and complete inhibitors. Biochem. J. 248: 815– 820; 1987. [18] Chamulitrat, W.; Mason, R. P. Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. J. Biol. Chem. 264:20968 –20973; 1989.

Dihydrolipoic acid as a 15-lipoxygenase inhibitor [19] Kuninori, T.; Nishiyama, J.; Shirakawa, M.; Shimoyama, A. Inhibition of soybean lipoxygenase-1 by n-alcohols and n-alkylthiols. Biochim. Biophys. Acta 1125:49 –55; 1992. [20] Clapp, C. H.; Grandizio, A. M.; Yang, Y.; Kagey, M.; Turner, D.; Bicker, A.; Muskardin, D. Irreversible inactivation of soybean lipoxygenase-1 by hydrophobic thiols. Biochemistry 41:11504 – 11511; 2002. [21] Gleason, M. M.; Rojas, C. J.; Learn, K. S.; Perrone, M. H.; Bilder, G. E. Characterization and inhibition of 15-lipoxygenase in human monocytes: comparison with soybean lipoxygenase. Am. J. Physiol. 268:C1301–C1307; 1995. [22] Sigal, E. The molecular biology of mammalian arachidonic acid metabolism. Am. J. Physiol. 260:L13–L28; 1991. [23] Breithaupt-Gro¨ gler, K.; Niebch, G.; Schneider, E.; Erb, K.; Hermann, R.; Blume, H. H.; Schug, B. S.; Belz, G. G. Dose-proportionality of oral thioctic acid— coincidence of assessments via pooled plasma and individual data. Eur. J. Pharm. Sci. 8:57– 65; 1999. [24] Jones, W.; Li, X.; Qu, Z.-C.; Perriot, L.; Whitesell, R. R.; May, J. M. Uptake, recycling, and antioxidant actions of ␣-lipoic acid in endothelial cells. Free Radic. Biol. Med. 33:83–93; 2002. [25] Cyrus, T.; Witzum, J. L.; Rader, D. J.; Tangirala, R.; Fazio, S.; Linton, M. F.; Funk, C. D. Disruption of the 12/15 lipoxygenase gene diminishes atherosclerosis in apoE-deficient mice. J. Clin. Invest. 103:1597–1604; 1999. [26] Sendobry, S. M.; Cornicelli, J. A.; Welch, K.; Bocan, T.; Tait, B.; Trivedi, B. K.; Colbry, N.; Dyer, R. D.; Feinmark, S. J.; Daugherty, A. Attenuation of diet-induced atherosclerosis in rabbits with

[27]

[28]

[29] [30] [31] [32] [33]

[34]

1209

a highly selective lipoxygenase inhibitor lacking significant antioxidant properties. Br. J. Pharmacol. 120:1199 –1206; 1997. Balley, J. M.; Makheja, A. N.; Lee, R.; Simon, T. H. Systemic activation of 15-lipoxygenase in heart, lung, and vascular tissues by hypercholestreolemia: relationship to lipoprotein oxidation and atherogenesis. Atherosclerosis 113:247–258; 1995. Stern, N.; Nozawa, K.; Golub, M.; Eggena, P.; Knoll, E.; Tuck, M. L. The lipoxygenase inhibitor phenidone is a potent hypotensive agent in the spontaneously hypertensive rat. Am. J. Hypertens. 6:52–58; 1993. Sasaki, M.; Hori, M. T.; Hino, T.; Golub, M. S.; Tuck, M. L. Elevated 12-lipoxygenase activity in the spontaneously hypertensive rat. Am. J. Hypertens. 10:371–378; 1997. DelliPizzi, A.; Guan, H.; Tong, X.; Takizawa, H.; Nasjletti, A. Lipoxygenase-dependent mechanisms in hypertension. Clin. Exp. Hypertens. 22:181–192; 2000. Ikawa, H.; Kamitani, H.; Calvo, B. F.; Foley, J. F.; Eling, T. E. Expression of 15-lipoxygenase-1 in human colorectal cancer. Cancer Res. 59:360 –366; 1999. Natarajan, R.; Nadler, J. Role of lipoxygenases in breast cancer. Front. Biosci. 3:E81–E88; 1998. Bradding, P.; Redington, A. E.; Djukanovic, R.; Conrad, D. J.; Holgate, S. T. 15-lipoxygenase immunoreactivity in normal and in asthmatic airways. Am. J. Respir. Crit. Care Med. 151:1201– 1204; 1995. Ravalli, S.; Marboe, C. C.; D’Agati, V. D.; Michler, R. E.; Sigal, E.; Cannon, P. Immunohistochemical demonstration of 15-lipoxygenase in transplant coronary artery disease. Arterioscler. Thromb. Vasc. Biol. 15:340 –348; 1995.