We aimed to investigate whether dark chocolate consumption also affects other markers of endothelial health, and whether chocolate enrichment with flavanols has additional health benefits. We investigated the effect of acute and prolonged consumption of flavanol‐enriched dark chocolate and of regular dark chocolate on markers important for a more complete assessment of endothelial health, such as FMD, blood pressure (BP), augmentation index (AIX), leukocyte count, leukocyte cell surface activation markers, and plasma cytokines involved in cellular adherence, coagulation and inflammation. We also investigated whether prolonged daily intake of high flavanol chocolate will alter the vascular resilience capacity to a high‐fat challenge test and whether an increased flavanol content affects the sensory profile of chocolate and thereby the appreciation and the motivation to consume these chocolates for a longer period.

The type of flavanols considered to be responsible for both the beneficial health effect and bitter taste are the (–)‐epicatechins ( 8 ). Several in vitro experiments in endothelial cells demonstrated that (–)‐epicatechins can increase NO bioavailability ( 9 , 10 ). NO is known to maintain endothelial health not only by influencing vasodilation, but also by preventing leukocyte adhesion and by lowering coagulatory and inflammatory factors. The assessment of endothelial health based on a single outcome measure, such as FMD, would provide a simplified picture of endothelial health status. Most research so far has focused on FMD, and the effects of cocoa flavanols on inflammation, leukocyte adhesion, and leukocyte activation capacity are less well determined ( 1 ). Recent findings also demonstrated that intake of flavanol‐rich dark chocolate for 3 d reduced the postprandial impairment of vascular function after a glucose tolerance test ( 11 ). This raises the question of whether cocoa flavanol consumption makes subjects also more resistant to atherogenic nutritional challenges, such as a high‐fat meal, known to reduce FMD postprandially ( 12 – 14 ).

Several human intervention studies demonstrated that flavanol‐enriched chocolate and cocoa intake can improve flow mediated dilatation (FMD; refs. 1 , 2 ). The European Food and Safety Authority (EFSA) therefore approved the health claim: “cocoa flavanols help maintain endothelium‐dependent vasodilation, which contributes to normal blood flow” ( 3 ). The scientific evidence for this EFSA health claim is based on intervention studies in which healthy subjects consumed chocolate or cocoa powder beverages with an increased flavanol content. As the predominantly consumed cocoa‐derived product is chocolate, increasing chocolate flavanol content might be of importance to improve vascular health and thereby to decrease cardiovascular disease (CVD) risk at the population level. However, flavanols have been described as astringent and bitter. Increasing the flavanol content of chocolate might therefore affect taste perception ( 4 ), hence affecting the motivation to consume such chocolates ( 5 – 7 ).

Statistical analysis was performed by linear mixed models for repeated measures (PASW Statistics 18.0.3; IBM SPSS, Chicago, IL, USA). Study outcomes of study part I were analyzed by using treatment (HFC or NFC), time (T 0 or T 2h ), and treatment * time as fixed effects. Study outcomes of study part II were analyzed using treatment (HFC or NFC), time (before or after 4 wk intervention), and treatment * time as fixed effects. Study outcomes of the high‐fat challenge test were analyzed by using treatment (HFC or NFC background), time (T 0 , T 1.5h , or T 3h ), and treatment * time as fixed effects. Baseline was included in the model as covariate for all study parts.

In a sensory panel test separate from the main study, 31 untrained subjects (aged 25±7 yr; BMI 22.1±2.8 kg/m 2 ) evaluated the analytical sensory properties of the HFC and NFC on a 9‐point scale. Samples were offered in pieces of 5–7 g at room temperature (~20°C) in a randomized order. Between samples, subjects cleaned their palate and tongue with crackers and water. The 20 attributes in the sensory test were generated by means of a panel of 6 independent subjects that had experience with sensory testing. Next to the intervention chocolates, two commercially available chocolates with a comparable cocoa content, Verkade Puur (Koninklijke Verkade, Zaandam, The Netherlands) and Delicata Puur (Albert Heijn, Zaandam, The Netherlands), were used for attribute generation and descriptive analyses.

During the intervention, subjects filled out questionnaires about the motivation to consume chocolate on d 1,7,14, 21, and 27. Questions were asked about sensory boredom; desire to eat something sweet, desire to eat something savory, and desire to eat chocolate, and questions on the liking and wanting of the chocolate; i.e., pleasantness and desire finish. Questions on sensory boredom were answered before consumption, questions on wanting and liking of the chocolate after consumption of one bite. All questions were answered on a 9‐point scale.

Expression of leukocyte cell surface markers was determined by flow cytometry (FACSCanto II, Becton‐Dickinson, Breda, The Netherlands). Markers were selected based on the involvement in activation and adherence to the endothelium. In short, whole blood was stained with fluorescent‐labeled monoclonal antibodies (mAbs; Becton‐Dickinson): FITC‐conjugated CD66b, APC‐cy7‐conjugated CD11b, APC‐conjugated CD62l, PE‐conjugated CD11c, and PerCP‐cy5.5‐conjugated CD45. Cell populations were identified by scatter properties and CD45 expression (FACSDiva 6.1.2 software; Becton‐Dickinson). Nonspecific binding was ruled out by using isotype‐matched mAbs. Expression of cell surface markers is expressed as mean florescence intensity (MFI) in arbitrary units (AU).

All vascular measurements were performed after 10 min of rest. Brachial SBP, DBP, and heart rate (HR) were assessed automatically (Dinamap Pro 100; GE Healthcare, Little Chal‐font, UK) for 10 min with a 3 min interval. Central SBP (CSBP) and the HR‐corrected AIX, a measure of wave reflection and arterial stiffness ( 21 ), were assessed by pulse wave analysis of the radial artery (SphygmoCor CP System; ATcor Medical, Sydney, NSW, Australia) as described previously ( 20 ). FMD was assessed as described previously ( 22 ). In short, after baseline recordings, a pressure cuff on the forearm was inflated and kept constant at a pressure of 200 mmHg for 5 min. Thereafter, the cuff was released, and short movies of the artery were made every 20 s for 4 min (Picus, Art.Lab 2.1; Esaote Benelux BV, Maastricht, The Netherlands). FMD was computed as maximum vessel diameter after cuff release divided by baseline and expressed as a percentage. A nitro‐glycerin dose was administrated sublingually by spray at the end of each day to assess endothelium‐independent dilation. FMD vessel recordings were analyzed and judged by a technician in a blinded procedure. One subject in study part I and 4 subjects in part II were removed from the analysis due to bad recordings.

The high‐fat challenge test consisted of a milkshake containing 95 g of fat, high in monounsaturated fatty acids (MUFAs). Previous work demonstrated that vascular responses after a high‐fat MUFA challenge were more pronounced if compared to challenges high in saturated fatty acids (SFAs) or n‐3 polyunsaturated fatty acids (PUFAs) ( 20 ). The shake contained low‐fat yogurt, low‐fat milk, strawberry flavor, 7.5 g sugar and 95 g high‐oleic acid sunflower oil (Aldoc BV, Schiedam, The Netherlands). This reflected a macronutrient composition of 10 g protein, 19 g carbohydrates and 95 g fat (of which 83% of total fat energy was from MUFAs) and contained 990 kcal [Nederlands Voedingsstoffenbestand (NEVO) 2007 database, available from: http://www.rivm.nl ].

HFC and NFC were provided by Barry Callebaut (Lebbeke, Belgium). The NFC was commonly produced chocolate with a cocoa content of 58%. The HFC had similar cocoa content and caffeine and theobromine concentrations as the NFC. The NFC was chosen in the original study design because of the seemingly relative low dose of flavanols. Reanalyzed flavanol concentrations substantially deviated from the original product sheet. We therefore decided to analyze flavanol content independently from the manufacturer based on a method previously described ( 18 , 19 ). This analysis showed that 70 g HFC contained 1078 mg flavanols, of which 349 mg was (–)‐epicatechins, and 70 g NFC contained 259 mg flavanols, of which 97 mg was (–)‐epicatechins. Chocolate flavanol concentrations and macronutrient composition are summarized in Supplemental Table S1.

A personal dietary consultation was planned before the intervention to prevent weight gain. During this consult, participants were advised to refrain from certain energy‐dense food products from their normal diet. If weight gain or loss >1 kg was recorded during their weekly visit, an additional consultation was scheduled to correct. Participants were not allowed to consume other chocolates or to consume more than 2 cups of tea, 2 glasses of red wine, or 1 apple per day, which are important sources of dietary flavanols. On the day prior to each testing day, subjects consumed a standardized low‐fat evening meal, were directed to refrain from alcohol or strenuous exercise, and were not allowed to eat or drink anything except water after 8:00 P.M.

Fasting vascular measurements, differentiated blood count, plasma cytokines, leukocyte activation markers, plasma metabolic markers and plasma liver function markers were determined before and after both intervention periods. Flavanol concentrations in 24 h urine samples were determined at the end of each intervention period as a marker of compliance. After the fasting measurements at the end of each intervention period, participants received a high‐fat challenge test containing 95 g of fat. At 3 h after consumption of this high‐fat shake, measures of vascular function, differentiated blood count, leukocyte activation markers, plasma cytokines, and plasma metabolic markers were determined. Plasma metabolic markers were also determined 1.5 h after high‐fat shake consumption.

An additional 15 volunteers were recruited to start up, with a total of 44 participants for study part II. Part II consisted of a double‐blind randomized crossover 4 wk intervention study in which participants consumed 70 g of chocolate daily in two 4 wk periods, randomly assigned to either HFC or NFC, with a 4 wk washout between periods. Chocolates were dispensed weekly in bars of 35 g; one bar was consumed in the afternoon and one in the evening. Extra bars were distributed, and participants were asked to bring back empty packages and leftover chocolates and to keep a diary to record at what time the bars were eaten.

Part I consisted of a double‐blind randomized crossover acute intervention study, in which 29 participants visited the university 2 times. On each study day, subjects consumed 70 g HFC or NFC. Vascular measurements, differentiated blood count, plasma cytokines, plasma (–)‐epicatechins, plasma metabolic markers, and plasma liver function markers were determined at baseline and 2 h after chocolate consumption. This timeframe was chosen because effects of flavanol consumption on vascular function are most pronounced 2 h after consumption ( 17 ). A minimum of 1 wk washout period was established between the two study days. All participants of part I also entered part II.

Study design. Part I: crossover acute intervention study. Samples were collected at baseline and 2 h postprandially after chocolate intake. Part II: crossover 4 wk intervention study. Fasting samples were collected before and after the intervention. A high‐fat postprandial challenge test was implemented at the end of both intervention periods. Vascular measurements were performed 3 h after the high‐fat challenge, and blood was collected 1.5 and 3 h after the high‐fat challenge. Determined study outcomes: vascular measures ( a ), plasma metabolic markers ( b ), plasma cytokines ( c ), differentiated blood cell count ( d ), leukocyte cell surface markers ( e ), plasma flavanols ( f ), urinary flavanols ( g ), plasma ALAT/ASAT ( h ).

The study consisted of two study parts ( Fig. 1 ). Part I comprised an acute intervention study in which the postprandial effects of either high‐flavanol chocolate (HFC) or normal‐flavanol chocolate (NFC) consumption on vascular health was investigated. In part II, the effects of daily intake of HFC or NFC for 4 wk on vascular health were investigated. In addition, the responsiveness to a postprandial high‐fat challenge test after a 4 wk chocolate background of either HFC or NFC was investigated. Randomization was performed for both study parts by an independent research assistant using a computer‐generated table. For study part II, we constructed 25 blocks with a size of 2. Researchers as well as participants were blinded to randomization until after data analysis. Sample size was calculated with assumption that within‐patient sd of the FMD measurement was 2.13. Effect size between treatments were estimated 2.1 U FMD% for study part I and 1.6 U FMD% for study part II ( 15 , 16 ). This study was performed at the Wageningen University from January until July 2011, according to the principles of the Declaration of Helsinki and in accordance with the Medical Research Involving Human Subjects Act (WMO).

Overweight (predefined BMI 25–32 kg/m 2 ) middle‐aged male subjects between 45–70 yr old were recruited. All subjects were nonsmoking, normoglycemic (World Health Organization criteria) and not diagnosed with any long‐term medical condition or high BP [systolic BP (SBP) >160 mmHg and/or diastolic BP (DBP) >100 mmHg]. Furthermore, subjects were not allowed to use medication or to take food supplements known to interfere with glucose homeostasis, BP, coagulation, or inflammation. All subjects gave written informed consent, and the study was approved by the Medical Ethics Committee of Wageningen University and registered at ClinicalTrials.gov (ID: NCT01308892; U.S. National Institutes of Health, Bethesda, MD, USA; http://clinicaltrials.gov ).

The sensory profile of the HFC and NFC are visualized in radar charts ( Fig. 3 ). The HFC chocolate was less grainy, less hard, more soft, less creamy, less sweet, more astringent, and more bitter and had a higher off‐flavor compared to the NFC. The aftertaste of the HFC was described as more bitter, less sweet, higher aftertaste, and higher dry mouth. These differences in taste did affect motivation scores of the participants during the intervention ( Table 3 ). The desire for chocolate score was, on average, 1.7 points lower if subjects received HFC compared to NFC. The chance that they would choose the chocolates themselves was on average 2.8 points lower for HFC compared to NFC. The wanting to finish the chocolate score was 2.5 points lower for the HFC group compared to the NFC group.

Table 2 lists the effects of a 4 wk HFC or NFC background on the postprandial response to a high‐fat shake. High‐fat shake consumption decreased FMD by 1.8% point, AIX by 4% point and DBP by 2 mmHg on average, with no difference between an HFC and NFC background. High‐fat shake consumption increased hematocrit and number of thrombocytes, lymphocytes, monocytes and neutrophils. Plasma concentrations of sICAM1, sVCAM1, sICAM3, P‐selectin, IL‐8 and TNF‐a concentrations were increased, whereas plasma concentrations of IL‐6 were decreased 3 h after shake consumption. In addition, shake consumption increased lymphocyte CD11c and CD11b expression, monocyte CD11c and CD11b expression, and neutrophil CD62l and CD11b expression, while monocyte CD62l expression was decreased. No differences between an HFC and NFC background were observed on leukocyte count, plasma cytokines, or leukocyte activation markers. No differences in response in plasma glucose, TG, insulin, and FFA between a HFC or NFC background were observed (Supplemental Table S3).

Table 2 lists the effects of 4 wk daily consumption of HFC and NFC on vascular measurements, differentiated leukocyte count, plasma cytokines and leukocyte activation markers in the fasting state, including the postprandial outcomes after the high‐fat challenge test. Chocolate consumption for 4 wk increased fasting FMD by 1% point and decreased fasting AIX by 1% point on average, with no difference in response between HFC and NFC. Both FMD and AIX values returned to baseline after the washout period ( Fig. 2 ). Chocolate consumption for 4 wk additionally decreased the number of leukocytes and decreased plasma sICAM1 and sICAM3 concentrations in the fasting state, with no difference between HFC and NFC. Plasma TNF‐α concentrations were decreased after HFC and increased after NFC intervention. Chocolate consumption for 4 wk also lowered expression of several cell surface molecules on leukocytes; on lymphocytes, CD62L and CD11b expression decreased by 4.8 and 5.0% on average, respectively. On monocytes, CD62l expression decreased by 17.9% on average. On neutrophils, CD66b and CD11c expression decreased by 3.9 and 10.2% on average, respectively. No significant differences in leukocyte cell surface molecule expression were observed between the HFC and NFC intervention. Chocolate consumption did not affect fasting ALAT or ASAT (Supplemental Table S3). A minor but significant increase of 0.1 mL in fasting plasma glucose and 0.3 mL in plasma FFA was observed after 4 wk of chocolate intervention, with no difference in response between HFC and NFC (Supplemental Table S3).

Participants did not gain or lose weight during the intervention period ( P =0.565). Compliance with the study protocol was good, as reflected by the consumed bars and urinary flavanol concentrations. In total, 4930 of 4952 bars were consumed during the intervention, 99.3% of the HFC bars and 99.7% of the NFC bars. At the end of the intervention, urinary flavanol concentrations were 1.34 ± 0.99 ug/ml after the NFC period and 8.75 ± 7.79 ug/ml after the HFC period ( P <0.001).

Chocolate ingestion decreased AIX, with no difference in postprandial response between HFC and NFC. Other measures of vascular function, such as FMD, were not affected 2 h after chocolate ingestion. Hematocrit and number of thrombocytes, lymphocytes, monocytes and neutrophils were increased 2 h after chocolate ingestion. Plasma sICAM1 concentrations were increased after HFC ingestion only. Plasma sICAM3, IL‐1β, and vWF concentrations were increased, and plasma IL‐6 concentration was decreased, 2 h after chocolate intake, with no difference between HFC and NFC. Chocolate ingestion increased plasma glucose and TAG and decreased plasma FFA. Insulin was increased 2 h after ingestion of both chocolates, with a more pronounced increase after HFC compared to NFC (Supplemental Table S2).

Three participants dropped out or were excluded during study part II, one due to medical reasons not related to the study, one due to disliking of the chocolate and one due to failure to adhere to the treatment. A total of 29 participants finished part I (age, 64±4 yr; BMI, 27.8±2.6 kg/m 2 ), and 41 participants finished part II (age, 63±5 yr; BMI, 27.6±2.3 kg/m 2 ).

DISCUSSION

We characterized the effect of chocolate consumption on endothelial health and investigated whether high‐flavanol dark chocolate in this respect is preferred above common dark chocolate. A 4 wk daily consumption of dark chocolate improved FMD and decreased AIX, leukocyte cell count, plasma concentrations of soluble adhesion molecules, and protein expression of adhesion markers on leukocytes. Increased flavanol content did not have an additional beneficial effect on markers of endothelial health, but did affect taste and had a negative effect on the motivation to eat chocolate.

The 1% point increase in FMD after 4 wk daily consumption of dark chocolate is in line with the health claim by EFSA that cocoa flavanols have a positive effect on endothelium‐dependent vasodilation (3). Each 1% increase in FMD is associated with a relative risk of cardiovascular events of 0.87 (95% CI: 0.83, 0.91; ref. 25). Our observed changes in FMD after chocolate intervention may therefore contribute to a cardiovascular disease risk reduction. Next to the improvement in FMD, we are the first demonstrating that 4 wk daily intake of dark chocolate additionally lowers the AIX. The AIX is a measure of wave reflection and arterial stiffness, and a decrease in AIX is associated with a decrease in relative risk of cardiovascular events (26). Based on the observed improvements in FMD and AIX, our results support the hypothesis that chocolate consumption improves vascular function.

We also aimed to elucidate how chocolate can affect other markers of endothelial health. The changes in leukocyte cell counts, plasma cytokines, and leukocyte adherence markers after chocolate consumption point toward a less‐activated state of cellular adherence and, hence, a less atherogenic milieu. First, 4 wk daily consumption of dark chocolate lowered leukocyte cell counts in the circulation. Leukocytes can transmigrate through the endothelium and therefore play a crucial role in the formation of atherosclerosis. In addition, elevated leukocyte cell count is a marker for systemic inflammation and associated with an increased CVD risk (27, 28). Second, dark chocolate consumption decreased protein expression of lymphocyte CD62L and CD11b, monocyte CD62L, and neutrophil CD66b and CD11c. These cell surface molecules are involved in leukocyte recruitment and adherence to the endothelium during the initial steps of atherosclerosis. CD11b is part of MAC‐1 integrin, and, together with CD11c and CD62L, they can bind to ICAMs and CD34 on endothelial cells, thereby activating the endothelium (29). CD66b is a degranulation marker of neutrophils and upregulated after activation (30). Third, chocolate consumption decreased plasma concentrations of soluble adhesion molecules sICAM1 and sICAM3. Both sICAMs can be secreted by the endothelium and leukocytes. Lower levels of these soluble adhesion molecules have been postulated to be associated with lower risk of cardiovascular events (29). The combination of lower numbers of leukocytes, decreased leukocyte adhesion molecule expression, and decreased plasma soluble adhesion molecules after 4 wk dark chocolate consumption point toward reduced leukocyte adherence and subsequently reduced activation of the endothelium, the initial state of atherosclerosis. Interestingly, this decrease in endothelial activation is coherent with our observed improvement in vascular function.

Previous reports investigating the effect of chocolate consumption on factors of cellular adherence are limited or evaluated by only a few measures. The effect of chocolate consumption on leukocyte cell count has not been reported before. One previous study investigated the effect of cocoa powder intake on leukocyte adherence markers and observed that 4 wk daily intake of cocoa did not affect CD11b (31). The difference with our decrease in CD11b may be because the researchers included patients at high risk of CVD and used cocoa powder instead of chocolate. In addition to CD11b, we also studied CD66b, CD11c, and CD62L. Chocolate and cocoa intervention studies investigating soluble adherence molecules in plasma are more abundant but inconclusive. Most are performed with cocoa powder (31–33) or in patients at risk of CVD (31–34), whereas our observations are in overweight, but apparently healthy men.

In the same study population, we investigated how chocolate ingestion affects vascular health postprandially and whether a background of 4 wk NFC or HFC consumption can improve the capacity to respond to a high‐fat MUFA challenge test. We did not observe an effect on FMD after acute intake of chocolate. The increase in soluble adhesion molecules sICAM1 and sICAM3 and the lack of a positive effect on FMD after acute intake of chocolate are most likely an effect of the high energy, sugar, or fat content of chocolate, which may have abolished the positive effect of the flavanols. We therefore can neither verify nor rebut that chocolate flavanol intake acutely improves vascular function (2). Our observed postprandial high‐fat shake responses are in line with our previous findings that a high‐fat shake affects vascular function and leukocyte adherence capacity (20). Since this high‐fat challenge test was implemented at the end of both interventions only, we could not determine whether 4 wk chocolate consumption, independent of the type, affected the capacity to respond to a high‐fat challenge test.

No differences were observed between NFC and HFC consumption on measures of endothelial health. This observation is supported by a meta‐analysis, in which the researchers found that chronic chocolate or cocoa powder intake improved FMD regardless of the dose consumed (2). Our results indicate that flavanol‐enriched chocolate was not healthier than regular dark chocolate with respect to vascular health markers. The absence of a difference in effect between the NFC and HFC cannot be explained by the maximal flavanol absorption capacity in the gut, considering the 5‐fold increase in plasma (–)‐epicatechins 2 h after intake and the 6.5‐fold increase in 24 h urine flavanol concentrations after 4 wk intake of HFC if compared to NFC intake. But the maximal effect on vascular health might have been reached by the flavanol dose in the NFC. If the vascular health effects are due to flavanols, it might still be relevant from a public health perspective to increase chocolate flavanol content in order to reduce saturated fat and sugar intake per portion. However, this strategy would work only if extra flavanols would not negatively affect the high standards of chocolate flavor, which was the case in our study.

An important aspect of this study was the independent analyses of chocolate flavanol content. Flavanol concentrations strongly deviated from the original product sheets. Apparently flavanol concentrations are not constant and vary between batches. Previous chocolate intervention studies barely report self‐analyzed flavanol content of the intervention chocolates, and deviating concentrations in previously published studies can, therefore, be expected.

As no differences in endothelial health effects were observed between both chocolates, we cannot rule out an intervention effect. We have several reasons to conclude that both the NFC and HFC are inducing the described beneficial effects on endothelial health. First, vascular measures including FMD, returned to baseline levels in the washout period. Second, several markers related to vascular health were changed in the same beneficial direction. Third, subjects remained weight stable during the intervention. Fourth, the infringement in the participants' habits and lifestyle during intervention were minimal. Still, chocolate contains many potentially bioactive constituents in addition to flavanols (35). These components or the synergy of these components may also be accountable for the observed beneficial effects.

This study provide new insights on how chocolate affects the endothelium by demonstrating that chocolate consumption besides improving vascular function also lowers the adherence capacity of leukocytes in the circulation and that extra flavanols did not augment these effects. Extra flavanols did affect flavor and thereby the motivation to eat chocolate. Chocolate‐producing companies that want to increase the amount of flavanols in chocolate, for vascular benefit product claims, will have to deal with the preservation of taste.