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Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine (M.E.W., N.A.H., E.A., M.H., D.F.K., J.F.K., J.A.V.) Boston, Massachusetts
DSM Nutritional Products, Inc. (J.G.E.) Parsippany, New Jersey
Address reprint requests to: Joseph A. Vita, MD, Section of Cardiology, Boston Medical Center, 88 East Newton Street, Boston, MA, 02118. E-mail: jvita{at}bu.edu
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ABSTRACT |
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 TOPFOOTNOTES ABSTRACT INTRODUCTIONMETHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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Objective: We hypothesized that dietary supplementation with epigallocatechin gallate (EGCG), a major catechin in tea, would improve endothelial function in humans.
Design: We examined the effects of EGCG on endothelial function in a double blind, placebo-controlled, crossover design study. We measured brachial artery flow-mediated dilation by vascular ultrasound at six time points: prior to treatment with EGCG or placebo, two hours after an initial dose of EGCG (300 mg) or placebo, and after two weeks of treatment with EGCG (150 mg twice daily) or placebo. The order of treatments (EGCG or placebo) was randomized and there was a one-week washout period between treatments.
Results: A total of 42 subjects completed the study, and brachial artery flow-mediated dilation improved from 7.1 ± 4.1 to 8.6 ± 4.7% two hours after the first dose of 300 mg of EGCG (P = 0.01), but was similar to baseline (7.8 ± 4.2%, P = 0.12) after two weeks of treatment with the final measurements made approximately 14 hours after the last dose. Placebo treatment had no significant effect, and there were no changes in reactive hyperemia or the response to sublingual nitroglycerin. The changes in vascular function paralleled plasma EGCG concentrations, which increased from 2.6 ± 10.9 to 92.8 ± 78.7 ng/ml after acute EGCG (P < 0.001), but were unchanged from baseline after two weeks of treatment (3.4 ± 13.1 ng/ml).
Conclusion: EGCG acutely improves endothelial function in humans with coronary artery disease, and may account for a portion of the beneficial effects of flavonoid-rich food on endothelial function.
Key words: endothelial function, tea, epigallocatechin gallate, coronary artery disease, catechins, flavonoids
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INTRODUCTION |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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Studies have also examined specific classes of flavonoids, and have demonstrated inverse relations between cardiovascular risk and intake of catechins [9]. The catechins are monomeric flavanols that include epicatechin, epigallocatechin, epigallocatechin gallate, and epicatechin gallate. They are found in a variety of foods including green and black tea, onions, grapes, apples, and chocolate. Many of these foods also contain polymeric flavanols such as thearubingins and theaflavins, which are found in fermented teas, and procyanidins, which are found in red grapes, red wine, apples, and chocolate. The mechanisms accounting for the benefits of flavanols remain incompletely defined, however, recent studies suggest that these nutrients may act, in part, by improving endothelial function [6,10–14].
The endothelium plays a central role in the regulation of vascular homeostasis, and maintenance of the normal vasodilator, anti-inflammatory, anti-thrombotic, and anti-proliferative properties of the endothelium may reduce cardiovascular risk [15,16]. We previously observed that acute and chronic black tea consumption reverses impaired flow-mediated dilation in patients with coronary artery disease [10], a response that reflects, in part, endothelial production of nitric oxide and other vasodilators [17]. Experimental studies have shown that flavanols, including epigallocatechin gallate (EGCG), enhance production and/or bioavailability of endothelium-derived nitric oxide in cultured endothelial cells and isolated arterial tissue [18–21]. In an effort to better understand the specific components of tea that improve endothelial function, the present study examined the effects of EGCG supplementation on brachial artery endothelial function.
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METHODS |
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Study Design
We examined the effect of EGCG on vascular function in a five-week, double blind, placebo-controlled, crossover study. Subjects received EGCG 150 mg twice daily (TEAVIGO®, DSM Nutritional Products, Inc., Parsippany, NJ) and placebo (gelatin capsules visually identical to EGCG capsules, supplied by DSM Nutritional Products, Inc., Parsippany, NJ) for two weeks for each treatment with a one-week washout period between treatments. The order of treatments was determined using computer-generated random numbers. Subjects visited our clinical research unit at the beginning and end of each of the two-week treatment periods. Prior to each visit, they discontinued all vasoactive medications for at least 24 hours and current smokers were asked to refrain from smoking for at least 12 hours. We also asked subjects to completely refrain from tea and red wine consumption during the 5-week study period.
The study design is outlined in Fig. 1. We tested vascular function at six time points: 1) before EGCG; 2) two hours after an initial 300 mg dose of EGCG; 3) after two weeks of EGCG 150 mg twice daily; 4) before placebo; 5) two hours after placebo; and 6) after two weeks of placebo. The two-hour time point doses of EGCG and placebo were administered with a light snack consisting of a bagel and standardized portions of margarine and jelly. We chose this time point for measurement of the acute effect of EGCG given the known pharmacokinetics in humans (1.5–1.6 hours to peak plasma concentration) [22] and our prior study showing a benefit of tea consumption at two hours [10]. During the two-week treatment portions of the study, subjects were instructed to take EGCG or placebo capsules with their morning and evening meals to limit the risk of gastrointestinal discomfort, based on the manufacturer's recommendations. We confirmed compliance by pill count. Subjects took their last dose of study medication the evening before the follow-up time points (3 and 6) or approximately 14 hours prior to measurement of vascular function. Thus, the studies performed at the end of two weeks of treatment were completed at the time of "trough" EGCG plasma concentration. At each of the six time points, we collected a blood sample and then measured blood pressure and heart rate using an automatic hemodynamic monitor after the subject rested supine for at least 10 minutes (Johnson and Johnson, Inc., New Brunswick, NJ). The blood pressure and heart rate measurements were made three times five minutes apart and averaged.
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Biochemical Analysis
We measured serum markers of cardiovascular disease risk at each visit (prior to the standard meal) and we measured plasma EGCG concentrations at the same six time points used for evaluation of vascular function. We also evaluated the safety of EGCG treatment by measuring renal, hepatic, muscle, and thyroid function at baseline and each of the two-week treatment periods. Serum glucose, blood urea nitrogen (BUN), creatinine, sodium, potassium, creatine kinase, calcium, total cholesterol, high density lipoprotein, triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase and total bilirubin were determined in freshly collected serum samples by automated analyzer (Bayer Advia 1640) and automated complete blood count was performed in the Boston Medical Center Clinical Laboratories. Thyroid Stimulating Hormone (TSH) and Free Thyroxine (FT4) were determined by chemiluminescence measured by automated analyzer (Bayer-Centaur). LDL cholesterol was calculated using the Friedewald formula [25]. Samples for EGCG concentration were prepared by mixing one ml of heparinized plasma with 1 ml of stabilizing buffer (1% ascorbic acid and 0.01% EDTA). Plasma EGCG concentration was measured by high-performance liquid chromatography with electrochemical detection [26]. Serum C-reactive protein was measured using a high-sensitivity nephelometric method on a commercial basis by the Brigham and Women's Hospital Department of Laboratory Medicine as previously described with a limit of detection of 0.17 mg/L [27]. Serum intracellular adhesion molecule-1 (ICAM-1) concentrations were measured with a commercially available enzyme linked immunoassay kit (R&D Systems, Minneapolis, MN) [28]. Samples for C-reactive protein, ICAM-1, and EGCG were frozen at –80 degrees Celsius until analyzed.
Statistical Analysis
The primary endpoint of the study was the effect of treatment (EGCG and placebo) on brachial artery flow-mediated dilation, expressed as percent change in diameter and calculated as the difference between peak brachial artery diameter at one minute following cuff release and baseline brachial diameter, divided by the baseline brachial artery diameter as previously described [23,24]. Secondary endpoints included the effect of treatment on flow-mediated dilation expressed as absolute change in diameter (millimeters), nitroglycerin-mediated dilation, baseline brachial artery diameter, hyperemic flow, blood pressure, heart rate, and the biochemical analyses. These analyses were completed using two-way mixed repeated measures analysis of variance (ANOVA) with terms for treatment and treatment order. When the overall model was statistically significant (P < 0.05), we completed post hoc pairwise comparisons using the Student-Newman Keuls test. The study was prospectively designed to have 90% power (
= 0.05) to detect a 1.5 percentage point improvement in flow-mediated dilation with a sample size of 40 subjects. We examined the comparability of the EGCG-First and Placebo-First groups using the unpaired t or 
2 tests for continuous and categorical variables, respectively. Since, the risk factor profile differed between groups, we also completed an analysis of each group separately. We examined the relation between change in flow-mediated dilation and change in EGCG concentration using the Pearson correlation coefficient. Variables are presented as mean ± SD, except as otherwise indicated.
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RESULTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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Table 1 describes the baseline characteristics of this 42-subject cohort and the characteristics of each treatment order subgroup. The two groups were similar in terms of age, gender, and race. Despite the randomized group assignment, however, the EGCG-First subjects had significantly higher systolic and diastolic blood pressures, and total cholesterol concentrations. There also was a non-significant trend for higher fasting glucose concentrations in the EGCG-First group.
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As noted above, subjects in the EGCG-First group had significantly higher blood pressures and total cholesterol concentrations than the Placebo-First group. At the beginning of the study, flow-mediated dilation was 6.2 ± 3.6% in the EGCG-First group and 8.0 ± 4.9% in Placebo-First group, but this difference was not statistically significant (P = 0.19). To explore the possibility that the differences in baseline risk factors might have confounded results, we completed an analysis of each of the two groups separately, recognizing that the smaller sample size for the individual groups would limit statistical power. As for the group as a whole, there was significant improvement in flow-mediated dilation two hours following EGCG treatment in the Placebo-First group (P = 0.02, n = 21), while flow-mediated dilation after two weeks of placebo treatment was similar to before treatment (P = 0.80). No significant differences were found with respect to %FMD in the EGCG-First group (n = 21), although the directionally of the change in %FMD between baseline and two hours was similar to the group as a whole.
Vascular Function and Plasma EGCG Concentrations
Overall, treatment affected EGCG concentration (P < 0.001 by ANOVA). Plasma concentrations of EGCG were undetectable before placebo and were 2.8 ± 13.7, 1.9 ± 12.2 ng/ml after short-term and long-term Placebo treatment, respectively (P = NS). EGCG concentrations were 2.6 ± 10.9 before EGCG and 92.8 ± 78.7 and 3.4 ± 13.1 ng/ml at the two-hour and two-week time points, respectively. As shown in Fig. 2, only short-term EGCG produced a significant change in EGCG concentration compared to before treatment (P < 0.001), and this change paralleled the change in vascular function. There was no correlation between change in flow-mediated dilation and change in EGCG concentration at the two-hour time point (P = 0.56, n = 39).
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DISCUSSION |
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The findings of the present study are consistent with several prior studies demonstrating improved endothelial function following acute consumption of a flavonoid-containing beverage [10,13,29]. We previously reported that brachial artery flow-mediated dilation improves two hours after consumption of black tea using the same methodology and patient population that we used in the present study [10]. It is notable that the relative improvement in flow-mediated dilation in our prior tea study (57%) was greater than the present study (21% following 300 mg of EGCG) [10]. Although the total catechin content of the tea consumed in our prior study was only 60 mg (17.5 mg of which was EGCG), the total polyphenol content of the tea was higher (733 mg), possibly accounting for the proportionally greater effect. The importance of noncatechin components of tea is further emphasized by our prior observation that changes in flow-mediated dilation did not correlate with changes in plasma catechin concentrations following tea consumption [30]. Agewall and colleagues reported a similar relative improvement in flow-mediated dilation (44%) one hour after consumption of de-alcoholized wine with total polyphenol content of 1110 mg in healthy volunteers [29]. Heiss and colleagues observed a doubling of brachial artery flow-mediated dilation two hours after consumption of flavanol-rich cocoa with a catechin plus procyanidins content of only 176 mg, however total polyphenols were not reported [13]. Thus, EGCG has the ability to acutely improve endothelial function, but other components of these beverages may have a similar effect, including other catechins and thearubigins. It is possible that vascular function may relate most strongly to total polyphenols content of these beverages.
In addition to an acute effect, consumption of flavonoid-containing beverages, including black tea, also has a cumulative effect on endothelial function following chronic consumption that persists for at least 14 hours after the last beverage consumption [10–12,31,32]. The lack of improvement in endothelial function after two weeks of EGCG treatment in the present study could reflect the relatively low dose in comparison to the total flavonoid content in flavonoid rich beverages. Moreover, the findings are consistent with the relatively short plasma half-life of EGCG (approximately 4 hours) [22,33] as reflected by the return of plasma concentrations to baseline the morning after the last dose of EGCG. Other tea catechins have similarly short half-lives [22,33]. Thus, the effects of flavonoid-containing beverages on the vascular endothelium are unlikely to be attributable to EGCG or other catechins and are more likely attributable to other tea components or metabolites that persist in plasma and/or accumulate in vascular tissue.
Recent experimental studies provide information about how catechins and other polyphenols might improve vasodilator function. Endothelium-dependent relaxation and cyclic guanosine-3',5'-monophosphate (cGMP) accumulation were greater in arterial tissue isolated from rats consuming de-alcoholized red wine or a catechin-rich diet compared to a control diet, and these effects were attributable to an increase in the activity, but not the expression of endothelial nitric oxide synthase (eNOS) [19]. Epicatechin had comparable effects on eNOS activity in cultured endothelial cells [34]. Interestingly, polymeric procyanidins increased eNOS activity to a greater extent than monomers in aortic endothelial cells [18], potentially providing insight into the greater effects of whole beverages compared to pure monomeric catechin. Recently, Lorenz and colleagues specifically examined the effects of EGCG on endothelial function [21]. They observed that EGCG produces eNOS-dependent relaxation of isolated rat aorta and increases eNOS activity in cultured endothelial cells. Under these conditions, activation of eNOS is associated with activation of phosphatidylinositol-3-hydroxy kinase and Akt and phosphorlyation of eNOS at serine 1179. Vasodilator effects began to occur with EGCG concentrations as low as 1 µM, and although the tissue concentrations achieved in the present study are unknown, the plasma concentrations are within an order of magnitude (0.2 µM). Thus, these experimental studies are consistent with our findings in human subjects. Further studies will be needed to elucidate the upstream signals that account for these effects.
Flavonoids have a number of other effects that might reduce cardiovascular disease risk. Of recent interest is the contribution of systemic inflammation to atherosclerosis, and several studies suggest that catechins and other flavonoids have anti-inflammatory effects. We investigated the possibility that EGCG might have such an effect in human subjects by examining changes in the soluble form of the endothelial adhesion molecule ICAM-1, which is shed from the endothelial surface and correlates with cardiovascular risk factors and prevalent cardiovascular disease [28]. We also measured changes in C-reactive protein, a general marker of systemic inflammation and increasingly accepted marker of cardiovascular risk [33]. We observed no effect of EGCG on ICAM-1 or C-reactive protein in the present study, but these findings may be limited by the relatively short period of treatment. The results are consistent with previous data in smokers showing no significant change in C-reactive protein with four weeks of tea consumption [34]. In addition, the lack of effect on these markers likely relates to the relatively low dose of catechin and inability to achieve sustained plasma concentrations with 2 weeks of treatment.
Our study had a number of limitations. Endothelial function was examined in a peripheral artery and extension of the findings to more clinically relevant circulations must be made with caution. However, prior studies suggest a correlation between endothelial function of the brachial artery and that of coronary circulation [35]. We observed an imbalance in risk factor profile in the two treatment groups, which likely is chance finding that relates to the relatively small sample size. However, all subjects received both treatments and the results were directionally similar in both groups, and thus, there is no indication that this imbalance affected the results. Although not statistically significant, flow-mediated dilation was numerically lower before EGCG than before placebo, making it harder to interpret the results. Based on manufacturer experience, EGCG was administered after a standardized meal to avoid gastro-intestinal side effects, and it is possible that the food interfered with EGCG absorption and blunted the improvement in endothelial function, however, plasma levels of EGCG were markedly increased two hours after EGCG administration, confirming bioavailability. Finally, the study was completed in subjects with established cardiovascular disease. The effects of EGCG supplementation in lower risk populations and in patients not taking medications that known to influence endothelial function, including angiotensin-II converting enzyme inhibitors [36] and HMG-CoA reductase inhibitors [37–39] will require further study.
The present study may have clinical relevance. Endothelial dysfunction in the coronary and peripheral circulations is increasingly recognized as a useful barometer of cardiovascular risk [15], and many interventions that improve endothelial function have been proven to lower cardiovascular disease risk [16]. The results of the present study provide further evidence that the inverse association between flavonoid consumption and cardiovascular disease may relate, in part, to improved endothelial function. Many of the prior studies examining flavonoid consumption and endothelial function involved ingestion of large amounts of beverage that would be difficult for most individuals to sustain over the long term. Administration of appropriate amounts of the active components of these beverages in supplement form clearly has therapeutic potential, although which components or combination of components will yield the greatest effect remains unknown. The present study demonstrates a benefit of acute EGCG treatment on endothelial function. Further studies will be needed to determine the relative merits of flavonoid consumption as supplements or whether consumption of these compounds in whole foods and beverages is preferable, as is currently recommended by the American Heart Association [40].
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ACKNOWLEDGMENTS |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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TOPFOOTNOTESABSTRACTINTRODUCTIONMETHODSRESULTSACKNOWLEDGMENTSREFERENCES |
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This study was funded by a grant from DSM Nutritional Products, Inc., Parsippany, NJ.
Received October 28, 2004. Accepted February 15, 2006.
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