Abstract
In vitro studies have suggested that flavonoids may have specific vascular effects, but their mechanism of action has not been clarified. A subclass of flavonoids—flavan-3-ols and their oligomers (procyanidins)—are constituents of cocoa beans, which can be detected in human plasma after ingestion of cocoa. In turn, plant extracts rich in flavan-3-ols can increase the activity of nitric oxide synthase (NOS) in endothelial cells. Nitric oxide is an essential signaling molecule in vascular physiology. Nitric oxide bioactivity can be preserved in human plasma in a circulating pool via increases in a number of nitrosated compounds. Thus, it is possible that cocoa rich in flavan-3-ols may lead to improved endothelium-dependent dilation via an increase of nitric oxide bioactivity. However, commercially available cocoa drinks contain only small amounts of flavan-3-ols due to roasting and alkalization of cocoa beans, which are known to degrade flavan-3-ols. We tested the hypothesis that ingestion of flavan-3-ol rich cocoa can increase the circulating pool of nitric oxide in human plasma, thus increasing endothelium-dependent dilation. Participants were 26 outpatients with at least 1 cardiovascular risk factor, including history of coronary artery disease, hypertension, hyperlipidemia, diabetes, or current tobacco use. Individuals were excluded if they had C-reactive protein levels greater than 0.5 mg/dL, atrial fibrillation, acute coronary syndrome, or New York Heart Association class III or IV heart failure. Individuals were studied in the morning after a 12-hour fasting period. In an initial study involving the first 6 participants, we assessed the time course of flavan-3-ol effects on flow-mediated dilation (FMD). This was measured at 0, 2, 4, and 6 hours after ingestion of 100 mL of cocoa drink containing 176 mg of flavan-3-ols (70 mg of epicatechin plus catechin, 106 mg of procyanidins [The Positive Food Co, Wokingham, England]) (n = 6) or control (100 mL cocoa drink with <10 mg of flavan-3-ols [Dovedrink, Mars Inc, Hackettstown, NJ] or water) (n = 3). We then used these results to guide the timing of a double-blind crossover study. Twenty participants received 100 mL of cocoa drinks with high or low levels of flavan-3-ols, in random order, on 2 consecutive days. The sum of nitrosylated and nitrosated species (collectively referred to as RNO) was measured by reductive chemiluminescence assay 2 hours after ingestion on both days. Nitrate and nitrite levels were measured as previously described. Endothelium-dependent dilation was assessed by measuring FMD of the brachial artery. In addition, we measured a number of other vascular parameters that would not be expected to change as a result of flavan-3-ol, including blood pressure, heart rate, and plasma levels of nitrite and nitrate. Similarly, we measured endothelium-independent dilation of the brachial artery following sublingual application of 400 µg of glyceroltrinitrate, diameter of the brachial artery, and forearm blood-flow at rest and during reactive hyperemia, as assessed by venous occlusion plethysmography. (Technical details are available from the authors.) All variables except endothelium-independent dilation were measured both before and after ingestion of the cocoa. Endothelium-independent dilation was measured only after ingestion of each drink, as nitroglycerine could have interfered with measurement of the other variables. Differences were assessed by paired t tests, with P values for multiple comparisons adjusted by the Bonferroni criterion. Our study was approved by the ethics board of the Medical Faculty of the Heinrich Heine-University, and all participants gave written informed consent. We found that a single dose of a cocoa drink rich in flavan-3-ols transiently increased nitric oxide bioactivity in human plasma and significantly reversed endothelial dysfunction. The correlation between FMD and levels of RNO suggests that flavan-3-ols induce arterial dilation via their effects on nitric oxide availability, a conclusion that is supported by the negative results for the other vascular variables. The long-term clinical effect of flavan-3-ols, however, remains to be established.
