Coenzyme Q improves LDL resistance to ex vivo oxidation but does not enhance endothelial function in hypercholesterolemic young adults

Free Radical Biology & Medicine, Vol. 28, No. 7, pp. 1100 –1105, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

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Original Contribution COENZYME Q IMPROVES LDL RESISTANCE TO EX VIVO OXIDATION BUT DOES NOT ENHANCE ENDOTHELIAL FUNCTION IN HYPERCHOLESTEROLEMIC YOUNG ADULTS OLLI T. RAITAKARI,*,§ ROBYN J. MCCREDIE,* PAUL WITTING,† KAYE A. GRIFFITHS,* JACINTA LETTERS,† DAVID SULLIVAN,‡ ROLAND STOCKER,† and DAVID S. CELERMAJER*㛳 *Department of Cardiology, Royal Prince Alfred Hospital, Sydney, Australia; †Biochemistry Group, The Heart Research Institute, Sydney, Australia; ‡Department of Biochemistry, Royal Prince Alfred Hospital, Sydney, Australia; §Department of Clinical Physiology, University of Turku, Turku, Finland; and 㛳Department of Medicine, University of Sydney, Sydney, Australia (Received 30 August 1999; Revised 9 February 2000; Accepted 9 February 2000)

Abstract—Oxidative modification of low-density lipoprotein (LDL) may cause arterial endothelial dysfunction in hyperlipidemic subjects. Antioxidants can protect LDL from oxidation and therefore improve endothelial function. Dietary supplementation with coenzyme Q (CoQ10) raises its level within LDL, which may subsequently become more resistant to oxidation. Therefore, the aim of this study was to assess whether oral supplementation of CoQ10 (50 mg three times daily) is effective in reducing ex vivo LDL oxidizability and in improving vascular endothelial function. Twelve nonsmoking healthy adults with hypercholesterolemia (age 34 ⫾ 10 years, nine women and three men, total cholesterol 7.4 ⫾ 1.1 mmol/l) and endothelial dysfunction (below population mean) at baseline were randomized to receive CoQ10 or matching placebo in a double-blind crossover study (active/placebo phase 4 weeks, washout 4 weeks). Flow-mediated (FMD, endothelium-dependent) and nitrate-mediated (NMD, smooth muscle-dependent) arterial dilatation were measured by high-resolution ultrasound. CoQ10 treatment increased plasma CoQ10 levels from 1.1 ⫾ 0.5 to 5.0 ⫾ 2.8 ␮mol/l (p ⫽ .009) but had no significant effect on FMD (4.3 ⫾ 2.4 to 5.1 ⫾ 3.6 %, p ⫽ .99), NMD (21.6 ⫾ 6.1 to 20.7 ⫾ 7.8 %, p ⫽ .38) or serum LDL-cholesterol levels (p ⫽ .51). Four subjects were selected randomly for detailed analysis of LDL oxidizability using aqueous peroxyl radicals as the oxidant. In this subgroup, CoQ10 supplementation significantly increased the time for CoQ10H2 depletion upon oxidant exposure of LDL by 41 ⫾ 19 min (p ⫽ .04) and decreased the extent of lipid hydroperoxide accumulation after 2 hours by 50 ⫾ 37 ␮mol/l (p ⫽ .04). We conclude that dietary supplementation with CoQ10 decreases ex-vivo LDL oxidizability but has no significant effect on arterial endothelial function in patients with moderate hypercholesterolemia. © 2000 Elsevier Science Inc. Key words—Antioxidants, Vascular reactivity, Ultrasound, Dyslipidemia, Free radicals

INTRODUCTION

tion during heart surgery [6]. The antioxidant properties of CoQ10 raise the possibility of clinically relevant antioxidant function in terms of decreasing the oxidation of low-density lipoprotein (LDL) and/or improving arterial endothelial function. Oxidative modification of LDL is thought to contribute significantly to the development of vascular endothelial dysfunction, a key early event in atherosclerosis [7,8]. LDL is protected against oxidative modification by a variety of antioxidant defenses [9]. Among these, the lipophilic antioxidants ␣-tocopherol (␣-TOH) and CoQ10H2 are associated with LDL. Observational studies have suggested that the concentration of ␣-TOH (the most abundant form of vitamin E) in the blood [10] and

Coenzyme Q (CoQ10, also known as ubiquinone-10) has an important role in cellular respiration and ATP production [1]. Ubiquinol-10 (CoQ10H2, the reduced form of coenzyme Q) is an endogenous product of the mevalonate pathway, and a lipid-soluble antioxidant with potentially cell protective effects [2]. Previous clinical studies have focused on the use of CoQ10 in heart failure [3,4], ischemic heart disease [5], and myocardial protecAddress correspondence to: David S. Celermajer, Department of Cardiology, Royal Prince Alfred Hospital, Missenden Road, Camperdown, New South Wales 2050, Sydney, Australia; Tel: ⫹612 95156111; Fax: ⫹612 9550-6262; E-Mail: [email protected]. 1100

Coenzyme Q and endothelial function

dietary intake of vitamin E [11] are inversely correlated with the risk of cardiovascular disease. Recent data, however, have shown that vitamin E alone may not be an effective antioxidant for LDL, because the antioxidant function of vitamin E in LDL relies on the presence of other suitable reducing agents, such as vitamin C [12] or CoQ10H2 [13]. The concentration of CoQ10H2 in plasma and plasma-derived lipoproteins is much lower than that of ␣-TOH [14]. Therefore, we sought to determine whether oral supplementation of CoQ10 is effective in reducing LDL oxidizability, and if so, how this relates to improving vascular endothelial function in patients with moderate hypercholesterolemia. METHODS

Subjects We studied 12 consecutively recruited adults who met the prospectively defined criteria of age 16 – 45 years, life-long nonsmokers, no diabetes mellitus or hypertension, LDL-cholesterol ⬎ 3.8 mmol/l, no regular use of antioxidant vitamins and who had endothelial dysfunction at their baseline study, defined here as flow-mediated dilatation (FMD) less than the population mean for vessel size [15]. On average, the baseline FMD levels were 1.9 standard deviations below the population mean (range 0 –3 standard deviations). None of the subjects were taking any lipid lowering medication or other antioxidants during the study period. All studies were approved by the local committee on ethical practice, and all subjects gave their informed consent. Study design The subjects were randomized to receive CoQ10 (50 mg three times daily) or matching placebo in a doubleblind crossover study (active/placebo phase 4 weeks, washout 4 weeks). Thus, every subject was examined on four occasions (baseline, after active and placebo treatments, after washout phase). CoQ10 was dispersed in palm oil (Blackmores, Sydney, Australia), and the mixture was supplemented in the form of dark capsules. Capsules designated as placebo were identical, but lacking in CoQ10. The compliances were determined by return tablet count, and were 91 ⫾ 13% during CoQ10 treatment and 92 ⫾ 8% during placebo treatment. EXPERIMENTAL PROCEDURES

Phosphate buffer (pH 7.4, 50 mM) was prepared from nanopure water. All reagents employed were of the highest purity available. Buffers were stored over Chelex-100 (BioRad, Richmond, CA, USA) at 4°C for 24 h to remove

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contaminating transition metals. 2,2⬘-Azobis(2-amidinopropane) (AAPH) was obtained from Polysciences (Warrington, PA). ␣-TOH (purity 96%) was obtained as a gift (Henkel Corporation, La Grange, IL, USA). A standard of cholesteryl linoleate hydroperoxide (used for CE-OOH) was prepared [16] and stored in ethanol at ⫺20°C; standards were quantified by spectroscopy using ⑀234nm ⬃ 29,500 M⫺1cm⫺1. Serum lipoproteins Fasting serum total cholesterol and triglyceride concentrations were measured using standard enzymatic methods (Boehringer Mannheim GmbH) with a fully automated analyzer (Hitachi 917 or 747; Hitachi Ltd, Tokyo, Japan). High-density lipoprotein cholesterol (HDL-cholesterol) was measured after precipitation with dextran sulphate-magnesium. The LDL-cholesterol concentration was calculated as described [17] using the Friedewald formula. Oxidation of LDL Four subjects (two men, two women, age: 36 ⫾ 7 years, LDL-cholesterol 5.7 ⫾1.5 mmol/l) were randomly selected for detailed analysis of LDL oxidizability using aqueous peroxyl radicals (ROO•) as the oxidant. AAPH thermally decomposes to yield ROO• at a constant rate [18]. LDL was isolated by the rapid ultra-centrifugation method [19] and used on the same day for ex vivo oxidation experiments. LDL was chilled to 4°C and combined with AAPH and the reaction mixture then incubated at 37°C. For oxidation, LDL was used at 1.8 ⫾ 0.8 and 2.1 ⫾ 0.8 ␮mol/l for baseline and active, respectively. AAPH was used at 1 mM final concentration. Aliquots (50 ␮l) of the reaction mixture were extracted (at times indicated) into 5 ml hexane and 1 ml methanol/ acetic acid (0.2% v/v) for lipid analyses by high-performance liquid chromatography (HPLC). Analyses of lipids, lipid hydroperoxides and hydroxides, and ␣-TOH Analyses of LDL lipids and antioxidants were performed using reversed-phase HPLC as described [19,20], except that cholesteryl linoleate hydroperoxides and hydroxides (together referred to as CE-O(O)H), which show similar retention times under the HPLC conditions used, were estimated using UV234nm rather than postcolumn chemiluminescence detection. Cholesterol remained unoxidized throughout the experiments and was employed as the internal standard for all lipid-soluble components analyzed. Ubiquinone and ubiquinol quan-

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tities were determined using HPLC with electrochemical detection as described previously [19]. The majority of coenzyme Q in human plasma is present as CoQ10H2 [14]. Except for the subgroup of samples used in oxidation experiments, we measured the oxidized, antioxidant-inactive form of coenzyme Q (i.e., CoQ10) in the plasma samples, because this form is more stable than CoQ10H2 and no precaution was taken to preserve CoQ10H2 in plasma. Indeed, plasma extracts did not contain significant amounts of CoQ10H2, as verified by HPLC. However, because CoQ10H2 autoxidizes stoichiometrically to CoQ10, the CoQ10 measured in these samples reflects total coenzyme Q. Ultrasound studies All studies were performed using an Acuson 128XP/10 mainframe (Acuson, Mountain View, CA, USA) with a 7.0 MHz linear array transducer. The ultrasound method for measuring endothelium-dependent and smooth muscle-dependent arterial dilatation has been described previously [21,22]. In brief, brachial artery diameter was measured from B-mode ultrasound images. In all studies, scans were obtained at rest, during reactive hyperemia, again at rest, and after sublingual administration of nitrate. The subjects lay quietly for 10 min before the first scan. The brachial artery was scanned in longitudinal section 2 to 15 cm above the elbow. Depth and gain settings were set to optimize images of the lumen/arterial wall interface, images were magnified using a resolution box function, and machine operating parameters were not changed during any study. When a satisfactory transducer position was found, the skin was marked and the arm remained in the same position throughout the study. A resting scan was recorded, and arterial flow velocity was measured using a Doppler signal [21,22]. Increased flow was then induced by inflation of a pneumatic tourniquet placed around the forearm (distal to the scanned part of the artery) to a pressure of 250 mmHg for 4.5 min, followed by release. A second scan was taken continuously for 30 s before and 90 s after cuff deflation, including a repeat flow velocity recording for the first 15 s after the cuff was released. Thereafter, 10 to 15 min was allowed for vessel recovery, after which a further resting scan was taken. Sublingual nitroglycerin spray (400 ␮g) was then administered, and 3 to 4 min later the last scan was acquired. All subjects tolerated the sublingual nitroglycerin well. Vessel diameter was measured in every case by two independent observers who were blinded to the subject’s clinical details and stage of the experiment. The arterial diameter was measured at a fixed distance from an anatomic marker (such as a fascial plane or a vein seen in cross section) using ultrasonic calipers. Measurements

were taken from the anterior to the posterior “m” line at end-diastole, incident with the R-wave on a continuously recorded electrocardiograph. The m line represents the edge of the intima-media interface in the ultrasound image of the arterial wall. For the reactive hyperemia scan, diameter measurements were taken 45 to 60 s after cuff deflation. Four cardiac cycles were analyzed for each scan, and the measurements for each observer were averaged. The vessel diameter in scans after reactive hyperemia and nitroglycerine administration was expressed as the percentage relative to the average diameter of the artery in the two resting (control) scans (100%). Volume flow at baseline and after cuff deflation was calculated from measurements of arterial flow velocity, heart rate, and vessel diameter, as previously described [21]. This method has been shown to be accurate and reproducible for measurement of small changes in arterial diameter [23], with low interobserver error for measurement of FMD and nitrateinduced arterial dilatation [21,23]. Endothelial function tested by the currently described method in the brachial artery correlates well with coronary endothelial function [24] and with the angiographically determined extent of coronary atherosclerosis [25].

Statistical methods The results of the crossover trial were analyzed as described by Armitage and Berry [26] taking treatment period and carryover effects into account. Differences were evaluated by means of paired and unpaired t-tests. With 12 subjects enrolled in a crossover trial, this study had 80% power to exclude a CoQ10-related improvement of 3% in FMD, compared to placebo, at the p ⬍ .05 level. Descriptive data are presented as mean ⫾ SD, and significance was inferred at two-tailed p ⬍ .05. RESULTS

The mean age of the study subjects was 34 ⫾ 10 years (mean ⫾ SD; range: 16 to 45 years), with body mass index 23 ⫾ 4 kg/m2 (mean ⫾ SD; range: 19 to 33 kg/m2). At baseline, the mean levels of total cholesterol, HDL-cholesterol, and serum triglycerides were 7.4 ⫾ 1.1, 1.4 ⫾ 0.4, and 1.3 ⫾ 0.9 mmol/l, respectively. The mean LDL-cholesterol was 5.5 mmol/l (range 3.9 to 7.6 mmol/l). The baseline level of plasma CoQ10 was 1.1 ⫾ 0.5 ␮mol/l. The mean FMD was 4.3 ⫾ 2.4% and mean nitrate-mediated dilatation (NMD) was 21.6 ⫾ 6.1% (Table 1). CoQ10 treatment increased plasma CoQ10 levels by 3.9 ⫾ 2.8 ␮mol/l (p ⫽ .009), but had no significant effect on FMD, NMD, or serum LDL-cholesterol levels (Table 1). In the subgroup of four subjects who underwent

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Table 1. Results of the Cross-over Trial

All subjects (N ⫽ 12) CoQ10 (␮mol/l) FMD (%) NMD (%) LDL-cholesterol (mmol/l) Subgroup (N ⫽ 4) LDL oxidation (␮mol/l)b CoQ consumption (min)c CoQ redox statusd LDL ␣-TOH (% remaining)e

Baseline

Active

Placebo

p valuea

1.1 ⫾ 0.5 4.3 ⫾ 2.4 21.6 ⫾ 6.1 5.5 ⫾ 1.7

5.0 ⫾ 2.8 5.1 ⫾ 3.6 20.7 ⫾ 7.8 5.5 ⫾ 1.9

1.3 ⫾ 0.6 5.6 ⫾ 3.4 19.5 ⫾ 10.3 5.7 ⫾ 1.8

.0009 .99 .38 .51

83 ⫾ 34 38 ⫾ 9 60 ⫾ 23 66.3 ⫾ 12.5

33 ⫾ 24 79 ⫾ 23 52 ⫾ 5 78.3 ⫾ 11.6

86 ⫾ 35 34 ⫾ 8 70 ⫾ 13 74.5 ⫾ 15.0

.038 .035 .81 .10

a

Paired t test (change during active treatment vs. change during placebo). Content of CEO(O)H after exposure of LDL to AAPH for 2 h. Time required for consumption of CoQ10H2 upon exposure of LDL to AAPH. The 100% values (molecules per LDL particle) were 0.18 ⫾ 0.08, 0.58 ⫾ 0.24, and 0.05 ⫾ 0.04 for ubiquinol-10, and 0.23 ⫾ 0.20, 0.80 ⫾ 0.66, and 0.35 ⫾ 0.30 for ubiquinone-10, for baseline, active, and placebo, respectively. d Defined as 100 ⫻ [CoQ10/(CoQ10 ⫹ CoQ10H2)]. e The 100% ␣-TOH values (molecules of ␣-TOH per LDL particle) were 10.7 ⫾ 6.1, 12.2 ⫾ 7.1, and 8.8 ⫾ 2.6 for baseline, active, and placebo, respectively. b c

detailed oxidizability assessment, CoQ10 supplementation increased the content of LDL CoQ10H2 and enhanced the resistance of lipoprotein to lipid peroxidation induced by ROO•. CoQ10 supplementation increased the time required for consumption of CoQ10H2 by 41 ⫾ 19 min (p ⫽ .04) and reduced the extent of CE-O(O)H accumulation. Thus, after 2 h of oxidation CE-O(O)H concentration was significantly lower (33 ⫾ 24 ␮mol/l) in the treated versus placebo (86 ⫾ 35 ␮mol/l) (p ⫽ .04). Placebo had no significant effect on any of the parameters measured, and no significant period or carryover effects were noted. Using median values for FMD and LDL-cholesterol as cut-points, the effects of CoQ10 on FMD were examined in two subgroups of subjects who might have been expected to have the best response to CoQ10; those with the worst baseline FMD (⬍4.2%) or those with the highest LDL-cholesterol (⬎5.2 mmol/l). In neither subgroup (n ⫽ 6) did treatment with CoQ10 improve FMD significantly. DISCUSSION

Oxidatively modified LDL may have an important role in the pathogenesis of early atherosclerosis, in part by decreasing the biological activity of nitric oxide and consequent impairment of endothelium-dependent dilatation [27]. Furthermore, recent clinical studies have demonstrated inverse relationships between vascular function and markers of LDL oxidation [7,8]. In the present study, we found that supplementation with oral CoQ10 for 4 weeks decreased the ex vivo oxidizability of LDL, in agreement with our previous studies [14,28] and those of others [29] where CoQ10 was given more

acutely. This treatment, however, was not associated with improvement in arterial endothelial dysfunction in patients with hypercholesterolemia. Antioxidants may enhance the bioavailability of endothelium-derived nitric oxide by scavenging free radicals within the extracellular space, but the regulation of the intracellular redox state may also be an important mechanism [30,31]. There may be several potential reasons for the lack of effect of CoQ10 supplementation on endothelial function in the present study. Several recent studies have shown that the treatment with another antioxidant agent, vitamin C, has an acute beneficial effect on endothelial function [32–35]. In contrast to lipid-soluble CoQ10, vitamin C is water-soluble and highly accessible to cells. Therefore, it might be that the administered CoQ10 was not taken up by endothelial cells in sufficient quantities to scavenge free radicals at the site of nitric oxide production. Indeed, oral CoQ10 may not be readily accessible to all tissues [36]. Beneficial effects may require higher doses of CoQ10or longer duration of administration to permit LDL with greater antioxidant content to penetrate the vessel wall and accumulate in sufficient quantity to influence endothelial nitric oxide production or bioavailability. Lack of improvement in endothelial function after antioxidant therapy is also consistent with oxidized LDL not being entirely responsible for endothelial dysfunction in subjects with hypercholesterolemia [37]. The arterial endothelium plays an important role in cardiovascular homeostasis, and, in particular, the role of endothelium-derived nitric oxide seems pivotal in promoting vasodilation and inhibiting platelet aggregation, monocyte adhesion, and smooth muscle proliferation [38]. In the present study, we have used a recently described and validated test of arterial endothelial func-

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tion that reflects mainly the endothelium-dependent release of nitric oxide in response to a physical stimulus (shear stress) [39,40]. Previous in vitro and in vivo data have implicated arterial endothelial dysfunction as a key early event in atherosclerosis [38,41], preceding plaque formation and clinical events. Therefore, the treatment of endothelial dysfunction may be an important strategy in the prevention of atherogenesis in its early stages. Both parenteral and oral administration of vitamin C has been shown to improve endothelial dysfunction acutely [32,35,42]. The long-term effects of antioxidants on endothelial function have been previously studied by Gilligan et al. [43]. These investigators gave a combination of antioxidant vitamin supplements (daily: 30 mg ␤-carotene, 1000 mg vitamin C, 800 IU vitamin E) for 1 month to hypercholesterolemic subjects and assessed endothelial function by using strain gauge plethysmography and acetylcholine infusions. They found, similarly to our study, that treatment with these antioxidants significantly reduced the oxidizability of LDL but did not have any measurable beneficial effect on endothelial function. We have recently documented a lack of benefit of long-term oral vitamin E therapy on arterial endothelial function in the healthy elderly [44]. In healthy smokers, we found that vitamin C administration acutely improved endothelial function, but there was no sustained beneficial effect after 2 months of therapy [45]. Contrary to these findings, some long-term studies have suggested beneficial effects of antioxidants on vascular endothelial function in certain conditions, such as in combination with lipid lowering therapy [46,47], in subjects with coronary artery disease [48,49] or high levels of risk factors [50], and in post-menopausal women [51]. The present study included a relatively small number of subjects. The subjects were, however, selected according to prespecified criteria and included young adults with moderate hypercholesterolemia and impaired endothelial function but no other cardiovascular risk factors. Prospective calculations suggested reasonable power to exclude a significant beneficial vascular effect in this study. Vascular physiology was measured in a peripheral artery; however, endothelial dysfunction is a diffuse process and endothelial function of the brachial artery has been shown to be closely associated with endothelial function in the coronary arteries [24]. A preliminary report of a longitudinal study in humans has examined the link between endothelial dysfunction and subsequent cardiovascular event rates, demonstrating a significant relationship [52]. In conclusion, we found that medium-term dietary supplementation with CoQ10 decreases ex vivo LDL oxidizability but has no measurable beneficial effect on arterial endothelial function, in healthy young patients with moderate hypercholesterolemia.

Acknowledgements — This study was financially supported by the Academy of Finland (O.T.R), the Medical Foundation of Sydney University, Australia (D.S.C.), the Australian National Health & Medical Research Council (R.S.), and Blackmores Ltd., Sydney, Australia.

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