Atherosclerosis 211 (2010) 283–286
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Decreased endothelin-1 levels after acute consumption of red wine and de-alcoholized red wine Tuomas O. Kiviniemi a,∗ , Antti Saraste a,b , Terho Lehtimäki c , Jyri O. Toikka a,d , Markku Saraste a , Olli T. Raitakari a , Jaakko J. Hartiala a , Jorma Viikari b , Juha W. Koskenvuo a a
Department of Clinical Physiology and Nuclear Medicine, Turku University Hospital, Turku, Finland Department of Medicine, Turku University Hospital, Turku, Finland Laboratory of Atherosclerosis Genetics, Department of Clinical Chemistry, Tampere University Hospital and the Medical School, University of Tampere, Finland d Department of Clinical Physiology, Tampere University Hospital, Tampere, Finland b c
a r t i c l e
i n f o
Article history: Received 12 October 2009 Received in revised form 12 January 2010 Accepted 13 January 2010 Available online 25 January 2010 Keywords: Coronary artery diameter Endothelin-1 Red wine De-alcoholized red wine
a b s t r a c t Background: Red wine consumption may inﬂuence on vasoconstrictive peptide endothelin-1 levels, and this may be one mechanism leading to improved vasodilation after red wine consumption. Endothelin-1 levels and their association with coronary epicardial diameter and ﬂow rate, however, have not been studied in vivo after consumption of red wine and de-alcoholized red wine. The purpose of this randomized trial was to determine the acute effects of these beverages on endothelin-1 levels and compare them to coronary artery epicardial diameter and ﬂow rate. Methods: Twenty-two healthy men consumed a high dose (8.1 ± 0.9 dL) of alcohol-containing red wine and de-alcoholized red wine in a cross-over design at one sitting with a two-week washout period. Endothelin-1 levels were determined and coronary artery diameter and ﬂow rate assessed using transthoracic echocardiography before and acutely after intervention. Results: Red wine and de-alcoholized red wine signiﬁcantly decreased endothelin-1 levels (0.75 ± 0.26 pg/mL to 0.61 ± 0.20 pg/mL, p = 0.002; 0.74 ± 0.32 pg/mL to 0.63 ± 0.24 pg/mL, p = 0.04, respectively), but did not have a signiﬁcant effect on epicardial diameter (1.1 ± 0.3 mm vs. 1.1 ± 0.3 mm, p = 0.58; and 1.1 ± 0.3 mm vs. 1.1 ± 0.2 mm, p = 0.10, respectively) or ﬂow rate (7.8 ± 4.0 mL/min to 6.4 ± 3.6 mL/min, p = 0.07; and 7.8 ± 4.0 mL/min to 7.4 ± 3.2 mL/min, p = 0.53, respectively). Conclusions: Red wine and de-alcoholized red wine decreased plasma endothelin-1 levels after acute consumption, but this change was not reﬂected in coronary epicardial diameters or ﬂow rate. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The early stages of atherosclerosis are characterized by endothelial dysfunction. Endothelium-derived relaxing and contracting factors maintain arterial homeostasis, but this balance may be disrupted by the presence of cardiovascular risk factors and inﬂammation, thus leading the vasculature to become susceptible to atheroma formation. Both the alcohol and the phenolic compounds found in red wine appear to favorably maintain endothelial function and limit atherosclerosis . Red wine contains several phenolic compounds, of which resveratrol and oligomeric procyanidins are considered the most potent in terms of beneﬁcial cardiovascular effects. Resveratrol augments endothelial NO synthase expression and activity and subsequent NO release from endothelial cells . Procyanidins in particular are linked to inhibi-
tion of potent vasoconstrictive peptide endothelin-1 synthesis . It is, however, not known if these experimental ﬁndings have clinical relevance in humans. Red wine has been shown to enhance coronary ﬂow reserve (CFR) in moderate  and high doses , whereas de-alcoholized red wine and pure ethanol have been found ineffective in this context. Furthermore, whether there is association between endothelin-1 levels and coronary epicardial diameters or ﬂow rate remains unknown. The purpose of this randomized study was to determine the acute effects of these beverages on endothelin-1 levels and compare them to coronary epicardial diameter as assessed using transthoracic echocardiography. 2. Methods 2.1. Subjects and study protocol
∗ Corresponding author. Tel.: +358 2 3131 935; fax: +358 2 3131 666. E-mail address: [email protected]
ﬁ (T.O. Kiviniemi). 0021-9150/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2010.01.017
In total, 22 healthy, non-smoking, Caucasian men (mean age 23 ± 1.8 years, body mass index 24 ± 2.3 kg/m2 ) with normal
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cholesterol and plasma glucose levels were included in the study. None of the subjects were taking any medication. The study was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association and was approved by the Ethics Committee of the Southwest Finland Health Care District. All subjects gave their written, informed consent. The subjects fasted overnight. They were instructed to avoid caffeine and tobacco for 12 h and alcohol for 36 h before the studies. The subjects were randomly assigned in a cross-over design to take doses of red wine or de-alcoholized red wine at one sitting on separate days with a washout period of at least two weeks. The dose of red wine contained a high amount of ethanol: 1.0 g/kg . De-alcoholized red wine was ingested in an equal volume as red wine. The full doses of red wine and de-alcoholized red wine were 8.1 ± 0.9 dL, which corresponds with approximately 6–7 glasses of wine (á 12 cl). Coronary ﬂow velocity and coronary epicardial diameter were measured using transthoracic echocardiography at the distal segment of the left anterior descending coronary artery before drinking and again 30 min after each dose. Flow rate was calculated as velocity time integral of ﬂow velocity × heart rate × epicardial coronary artery radius2 × , as previously described . Flow velocity and diameter were measured as an average of three measurements at baseline and again 30 min after the full dose of each beverage. The intra- and interobserver variabilities (coefﬁcient of variation CV [%]) for diameter measurements were 6.5 ± 7.5 and 5.7 ± 5.6, respectively; and for ﬂow velocities 4.9 ± 4.7 and 11.0 ± 10.7, respectively. The CVs of ﬂow rate measurements were larger (24.5 ± 9.2 and 22.2 ± 21.1, respectively). 2.2. Phenolic content of the beverages Perlage Cabernet del Veneto 2003 (Italy) and Ebony Vale Cabernet Sauvignon 2003 (grapes from Italy; de-alcoholization in Germany) were chosen as the red wine and de-alcoholized red wine, respectively. A total of four wines were prescreened for their total phenolic content, and those chosen had the most similar content. The de-alcoholization was performed via the vacuum distillation method, which consists of separating the wine into different fractions at very low pressure and normal room temperature, in a high cylinder made out of glass and standing in a hermetically closed room with walls of glass. Under vacuum, the boiling point of the alcohol is reduced so that it actually evaporates at room temperature. The phenolic proﬁles of the beverages were determined using an analytical HPLC method as previously described [7,8]. Flavonoids (anthosyans, cathecins, oligomeric procyanidines, ﬂavonols) and other phenolic substances (resveratrol) were analyzed in their natural form. Hydroxybenzoic acid and ellagic acid were quantiﬁed with the use of gallic acid; hydroxycinnamic acid with chlorogenic acid; cathecin and oligomeric procyanidines with procyanidine B2; ﬂavonols with rutins; and anthosyans with cyanidine-3-glucocide. Samples were analyzed in three parallel determinations. The phenolic content of the beverages and the total amounts of phenolic substances given to the subjects are presented in Table 1. Procyanidins were detected in both beverages. Resveratrol was abundant only in red wine. The phenolic substances anthosyans, ﬂavonols and cathekins were detected in both red wine and dealcoholized red wine. 2.3. Blood samples Venous blood samples were obtained and EDTA plasma was separated by low-speed centrifugation for the measurement of endothelin-1 levels before and 2 h after drinking. Endothelin-1 levels were measured in duplicate according to the manufacturer’s
Fig. 1. Endothelin-1 levels before and after acute consumption of red wine (n = 20) and de-alcoholized red wine (n = 21). There was no signiﬁcant difference between endothelin-1 levels after consumption of red wine vs. de-alcoholized red wine (p = 0.69) nor between the changes in the levels (p = 0.78).
instructions before and after the interventions by using Human Endothelin-1 Immunoassay (Human Endothelin cat. no: BBE-5, manufacturer: R&D Systems, Minneapolis, USA). 2.4. Statistical analysis The data are presented as means ± SD. A paired T-test with Bonferroni correction (three addressed hypotheses) was used to compare serum levels, epicardial diameters, heart rate and ﬂow rate values before and after beverage ingestion. p-values lower than 0.05 (or 0.017 with Bonferroni correction) were considered signiﬁcant. The normality of the variables was tested using a Kolmogorov–Smirnov test. Statistical analyses were done using SPSS 15.0 (SPSS Inc). 3. Results Endothelin-1 levels decreased signiﬁcantly after consumption of red wine and de-alcoholized red wine (0.75 ± 0.26 pg/mL to 0.61 ± 0.20 pg/mL, p = 0.002; 0.74 ± 0.32 pg/mL to 0.63 ± 0.24 pg/mL, p = 0.04; respectively) (Fig. 1). There was no signiﬁcant difference between endothelin-1 levels after consumption of red wine as compared to after consumption of de-alcoholized red wine (p = 0.69), nor between the changes in the levels before and after consumption (p = 0.78). No signiﬁcant changes in epicardial diameters were detected after consumption of red wine and de-alcoholized red wine, (1.1 ± 0.3 mm vs. 1.1 ± 0.3 mm, p = 0.58; and 1.1 ± 0.3 mm vs. 1.1 ± 0.2 mm, p = 0.10, respectively). Heart rate increased signiﬁcantly after red wine consumption (62 ± 10 to 67 ± 11 p = 0.018), whereas the opposite trend was observed with de-alcoholized red wine (62 ± 10 to 59 ± 10, p = 0.16). There were no signiﬁcant changes in ﬂow rate after consumption of red wine and de-alcoholized red wine (7.8 ± 4.0 mL/min to 6.4 ± 3.6 mL/min, p = 0.07; and 7.8 ± 4.0 mL/min to 7.4 ± 3.2 mL/min, p = 0.53, respectively).
Table 1 Amounts (mg) of phenolic substances ingested by the subjects. Phenolic compounds (mg)
De-alcoholized red wine
Hydroxybenzoic acid Hydroxycinnamic acids Ellagic acid Cathecins (mono- and dimers) Procyanidines–oligomers Flavonols Antosyans Resveratrol
0.24 ± 0.03 36.6 ± 4.0 2.51 ± 0.28 15.15 ± 1.68 22.84 ± 2.54 11.50 ± 1.28 2.35 ± 0.26 2.59 ± 0.29
7.37 ± 0.8 37.1 ± 4.1 0.73 ± 0.08 8.26 ± 0.92 33.21 ± 3.69 8.26 ± 0.92 0.57 ± 0.06 0
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4. Discussion This randomized cross-over trial shows for the ﬁrst time a signiﬁcant decrease in endothelin-1 levels in healthy men after consumption of red wine and de-alcoholized red wine. This decrease, however, was not accompanied by changes in coronary epicardial diameter or ﬂow rate. One explanation for the negligible effect of the decreased endothelin-1 levels on epicardial diameter and ﬂow rate is that the concentration of endothelin-1 was too low and its change too minor. While receptor sensitivities are in the nanomolar range , endothelin-1 levels in the plasma samples yielded concentrations in the picomolar range. Although not precisely representing actual interstitial levels of endothelin-1, plasma levels are likely to reﬂect directional changes in interstitial levels . Another explanation is that both NO and prostanoids act together to blunt the coronary vasoconstrictor inﬂuence of endothelin-1 on maintaining vasomotor homeostasis . In fact, no single mediator accounts for the regulation of the whole coronary ﬂow during hyperemia; instead, many mediators act together. For example, if NO or prostanoid receptors are blocked, other factors maintain hyperemic ﬂow . Levels of other coronary ﬂow mediators were not measured in this study, and therefore, this issue needs to be clariﬁed through future studies. Endothelin-1 is a highly potent vasoconstrictor peptide  produced by the vascular endothelium. The biosynthesis of endothelin-1 occurs through several proteolytic steps from inactive precursors. The 203-amino acid translation product preproendothelin-1 is cleaved by furin-like proteases to form the inactive intermediate big endothelin-1. Proteolytic cleavage of big endothelin-1 by endothelin converting enzyme-1 (ECE-1), a membrane-bound zinc metalloproteinase, results in active endothelin-1. The endothelin-A receptor, expressed predominantly on vascular smooth muscle cells, mediates potent and sustained vasoconstriction through a complex signal transduction system resulting in release of intracellular calcium . Numerous stimuli increase endothelin-1 production in endothelial cells, including epinephrine, angiotensin II, thrombin, and cytokines  whereas key vasodilators inhibit endothelin-1 production. Moreover, agents that increase cGMP, such as atrial natriuretic peptide, prevent endothelin-1 release . Interestingly, endothelial cells release approximately 80% of endothelin-1 to their basolateral side, and circulating endothelin-1 is removed rapidly, mostly by lungs and kidneys . The binding of endothelin-1 to the endothelin-A and endothelin-B receptors on vascular smooth muscle leads to vasoconstriction . On the contrary, the binding of endothelin-1 to endothelin-B receptors at low doses on the endothelium leads to production of NO and prostacyclin [15,16], which induce vasodilation. Moreover, in a previous study, administration of exogenous endothelin caused endothelin-B-receptor-mediated vasodilation at low doses but endothelin-A-receptor-mediated constriction at high doses, indicating that the endothelin-B receptor on the endothelium is more sensitive to endothelin-1 than the receptors on vascular smooth muscle at low doses . The net effect of endothelin-1 on vascular tone is likely to depend on the complex interplay of endothelin-1-induced vasoconstriction and vasodilation which, on the other hand, may be modiﬁed by vasomotor tone as well as by receptor density . Previously, endothelin-1 synthesis decreased in an experimental study where cultured bovine aortic endothelial cells were treated with non-alcoholic red wine extract . Corder et al. demonstrated that mainly procyanidins accounted for the inhibition . We extend these ﬁndings, showing that the decrease in endothelin-1 levels can also be seen in vivo. However, this change in endothelin-1 levels may be due to factors other than protein synthesis. Namely, as early as 1990, seven-fold increases of ET-1
levels within 2–4 min and full recovery to baseline levels within 10–15 min was described using an experimental set-up in humans . Using immunoelectron microscopy, Russell et al. convincingly described in 1998 that storage for both endothelin-1 and endothelin converting enzyme-1 (ECE-1) is localized in the Weibel-Palade bodies of endothelial cells . Therefore, plasma ET-1 levels can be changed abruptly via two mechanisms: (1) release of active ET-1 from Weibel-Palade bodies, and (2) changes in endothelial converting enzyme activity, which can happen within seconds. However, our study only demonstrated a decreased level of ET-1 following administration of standard and de-alcoholized red wine but left open the question of whether the decrease was due to decreased secretion or increased elimination of ET-1 or decreased activation of ECE-1. Investigating the in vivo effects of endothelin-1 is challenging, and the biological effects of endothelin-1 are poorly understood in humans. This lack of understanding was one driving force of the current study. The increased heart rate after consumption of alcoholcontaining red wine was most likely due to the high ethanol dose . The increased heart rate probably affected ﬂow rate values after consumption of alcohol-containing red wine; however, this change did not reach statistically signiﬁcant levels. Improved coronary vasodilation has been reported previously after moderate  and heavy  red wine consumption. In this study, where epicardial diameters and ﬂow rate were assessed at baseline without hyperemic stimulus, we could not detect signiﬁcant changes after either study beverage when consumed in high doses. This probably reﬂects that slight changes in vascular tone mediators do not inﬂuence epicardial diameter or ﬂow rate. TTE has been used to measure coronary artery diameters with relatively good correlations (r = 0.83–0.93) when compared to quantitative coronary angiography [21,22], intracoronary ultrasound , and epicardial echocardiography . Moreover, the variation of repeated ofﬂine diameter measurements by TTE has been low (CV 5.4–7.5%) . The intra- and interobserver variabilities for diameter and ﬂow velocities measurements were low. The CV of ﬂow rate measurements was much larger, however, due to the fact that ﬂow rate is calculated using a formula multiplying ﬂow velocity, heart rate and squared diameter values together, meaning a small change in any of the values yields great variability. Some limitations of the present study should be pointed out. First, we studied healthy young men only. This homogenous population was chosen to diminish the role of confounding factors. Moreover, atherosclerotic processes begin early in life, and changes in these soluble markers may be one of their earliest signs. Therefore, it makes sense to study subjects in this age group. Second, despite the double blind setting, the subjects may have been able to distinguish the red wine from the de-alcoholized version by the taste. Third, the amount of ethanol was relatively high (1 g/kg), corresponding to binge drinking amounts. Taken together, although endothelin-1 levels were shown to decrease after consumption of red wine and de-alcoholized red wine, there was not a detectable change in coronary epicardial diameter or ﬂow rate. Conﬂict of interest None. Acknowledgments We would like to thank Ms. Nina Peltonen for her skillful technical assistance. Funding: The study is part of the project “Molecular Imaging in Cardiovascular and Metabolic Research”, which belongs to the
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Centre of Excellence programme of the Academy of Finland. The study was also supported by the Turku University Hospital Research Foundation, the Tampere University Hospital Medical Fund, the Turku University Foundation, the Emil Aaltonen Foundation, the Orionpharma Research Foundation, the Instrumentarium Research Foundation, the Finnish Foundation for Cardiovascular Research and the Paulo Foundation. References  Szmitko PE, Verma S. Antiatherogenic potential of red wine: clinician update. Am J Physiol Heart Circ Physiol 2005;288:H2023–30.  Leikert JF, Rathel TR, Wohlfart P, et al. Red wine polyphenols enhance endothelial nitric oxide synthase expression and subsequent nitric oxide release from endothelial cells. Circulation 2002;106:1614–7.  Corder R, Douthwaite JA, Lees DM, et al. Endothelin-1 synthesis reduced by red wine – Red wines confer extra beneﬁt when it comes to preventing coronary heart disease. Nature 2001;414:863–4.  Kiviniemi TO, Saraste A, Toikka JO, et al. A moderate dose of red wine, but not de-alcoholized red wine increases coronary ﬂow reserve. Atherosclerosis 2007;195:e176–81.  Shimada K, Watanabe H, Hosoda K, Takeuchi K, Yoshikawa J. Effect of red wine on coronary ﬂow velocity reserve. Lancet 1999;354, 1002-.  Kiviniemi TO, Toikka JO, Koskenvuo JW, et al. Vasodilation of epicardial coronary artery can be measured with transthoracic echocardiography. Ultrasound Med Biol 2007;33:362–70.  Kähkönen MP, Hopia AI, Heinonen M. Berry phenolics and their antioxidant activity. J Agric Food Chem 2001;49:4076–82.  Lamuela-Raventos RM, Waterhouse AL. A direct HPLC separation of wine phenolics. Am J Enol Vitic 1994;45:1–5.  Duncker DJ, Bache RJ. Regulation of coronary blood ﬂow during exercise. Physiol Rev 2008;88:1009–86.  Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332: 411–5.
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