5-(3',4'-Dihydroxyphenyl)-γ-Valerolactone Is a Substrate for Human Paraoxonase: A Novel Pathway in Flavan-3-ol Metabolism.

SCOPE: Dietary flavan-3-ols are known to mediate cardiovascular benefits. Currently, it is assumed that the levels of flavan-3-ol catabolites detected in humans, 5-(3',4'-dihydroxyphenyl)-γ-valerolactone (γVL) and 5-(3',4'-dihydroxyphenyl)-γ-valeric acid (γVA), and their corresponding phase II metabolites, are determined exclusively by the action of the gut microbiome. However, a family of human proteins, paraoxonase (PON), can theoretically hydrolyze γVL metabolites into the corresponding γVAs. This study aims to determine if PON is involved in γVL and γVA metabolism in humans. METHODS AND RESULTS: A rapid conversion of γVL into γVA is detected in serum ex vivo (half-life = 9.8 ± 0.3 min) that is catalyzed by PON1 and PON3 isoforms. Phase II metabolites of γVL are also reacted with PON in serum. Following an intake of flavan-3-ol in healthy males (n = 13), the profile of γVA metabolites detected is consistent with that predicted from the reactivity of γVL metabolites with PON in serum. Furthermore, common PON polymorphisms are evaluated to assess the use of γVL metabolites as biomarkers of flavan-3-ol intake. CONCLUSION: PONs are involved in flavan-3-ol metabolic pathway in humans. PON polymorphisms have a minor contribution to inter-individual differences in the levels of γVL metabolites, without affecting their use as a nutritional biomarker.

Impact of polyphenol oxidase on the bioavailability of flavan-3-ols in fruit smoothies: a controlled, single blinded, cross-over study.

Flavan-3-ols are bioactive compounds found in a variety of fruits and vegetables (F&V) that have been linked to positive health benefits. Increasing habitual flavan-3-ol intake is challenged by the generally low consumption of F&V. While smoothies are a commonly endorsed, consumer-accepted means to increase the daily intake of these important foods, fruits used for smoothie preparation can have a high polyphenol oxidase (PPO) activity and thus potentially affect the content and bioavailability of flavan-3-ols. To assess whether or not consuming freshly prepared smoothies made with different PPO-containing fruit impacts the bioavailability of the flavan-3-ols, a controlled, single blinded and cross-over study was conducted in healthy men (n = 8) who consumed a flavan-3-ol-containing banana-based smoothie (high-PPO drink), a flavan-3-ol-containing mixed berry smoothie (low-PPO drink) and flavan-3-ols in a capsule format (control). The peak plasma concentration (Cmax) of flavan-3-ol metabolites after capsule intake was 680 ± 78 nmol L-1, which was similar to the levels detected after the intake of the low PPO drink. In contrast, the intake of the high PPO drink resulted in a Cmax of 96 ± 47 nmol L-1, 84% lower than that obtained after capsule intake. In a subsequent study (n = 11), flavan-3-ols were co-ingested with a high-PPO banana drink but contact prior to intake was prevented. In this context, plasma flavan-3-ol levels were still reduced, suggesting an effect possibly related to post-ingestion PPO activity degrading flavan-3-ols in the stomach. There was a substantial range in the PPO activity detected in 18 different fruits, vegetables and plant-derived dietary products. In conclusion, bioavailability of flavan-3-ols, and most likely other dietary polyphenol bioactives, can be reduced substantially by the co-ingestion of high PPO-containing products, the implications of which are of importance for dietary advice and food preparation both at home and in industrial settings.

Flavan-3-ol-methylxanthine interactions: Modulation of flavan-3-ol bioavailability in volunteers with a functional colon and an ileostomy.

Flavan-3-ols, including the flavan-3-ol monomer (-)-epicatechin, are dietary bioactives known to mediate beneficial cardiovascular effects in humans. Recent studies showed that flavan-3-ols could interact with methylxanthines, evidenced by an increase in flavan-3-ol bioavailability with a concomitant increase in flavan-3-ol intake-mediated vascular effects. This study aimed at elucidating flavan-3-ol-methylxanthine interactions in humans in vivo by evaluating the specific contributions of theobromine and caffeine on flavan-3-ol bioavailability. In ileostomists, the effect of methylxanthines on the efflux of flavan-3-ol metabolites in the small intestine was assessed, a parameter important to an understanding of the pharmacokinetics of flavan-3-ols in humans. In a randomized, controlled, triple cross-over study in volunteers with a functional colon (n = 10), co-ingestion of flavan-3-ols and cocoa methylxanthines, mainly represented by theobromine, increased peak circulatory levels (Cmax) of flavan-3-ols metabolites (+21 ± 8%; p < 0.05). Conversely, caffeine did not mediate a statistically significant effect on flavan-3-ol bioavailability (Cmax = +10 ± 8%, p = n.s.). In a subsequent randomized, controlled, double cross-over study in ileostomists (n = 10), cocoa methylxanthines did not affect circulatory levels of flavan-3-ol metabolites, suggesting potential differences in flavan-3-ol bioavailability compared to volunteers with a functional colon. The main metabolite in ileal fluid was (-)-epicatechin-3'-sulfate, however, no differences in flavan-3-ol metabolites in ileal fluid were observed after flavan-3-ol intake with and without cocoa methylxanthines. Taken together, these results demonstrate a differential effect of caffeine and theobromine in modulating flavan-3-ol bioavailability when these bioactives are co-ingested. These findings should be considered when comparing the effects mediated by the intake of flavan-3-ol-containing foods and beverages and the amount and type of methylxanthines present in the ingested matrixes. Ultimately, these insights will be of value to further optimize current dietary recommendations for flavan-3-ol intake. CLINICAL TRIAL REGISTRATION NUMBER: This work was registered at clinicaltrials.gov as NCT03526107 (study part 1, volunteers with functional colon) and NCT03765606 (study part 2, volunteers with an ileostomy).

Absorption, distribution, metabolism and excretion of apigenin and its glycosides in healthy male adults.

The bioavailability of apigenin and its O-glycosides in humans was investigated with apigenin-4'-glucuronide (Ap-4'-GlcUA), apigenin-7-glucuronide and apigenin-7-sulfate being identified as in vivo metabolites. Apigenin per se was poorly absorbed with metabolites equivalent to 0.5% of intake excreted in urine 0-24 h post-intake. Consumption of a parsley drink containing apigenin-7-O-(2″-O-apiosyl)glucoside resulted in the peak plasma concentration (Cmax) of Ap-4'-GlcUA occurring after 4 h, indicative of absorption in the lower gastrointestinal tract (GIT). Urinary excretion of the three metabolites corresponded to 11.2% of intake. Ingestion of dried powdered parsley leaves with yogurt extended the Cmax of Ap-4'-GlcUA to 6 h. Consumption of chamomile tea containing apigenin-7'-O-glucoside resulted in a 2 h Cmax of Ap-4'-GlcUA, in keeping with absorption in the upper GIT. Urinary excretion was equivalent to 34% of intake. Intake of the parsley drink provided information on intra- and inter-individual variations in the level of excretion of the apigenin metabolites. CLINICAL TRAIL REGISTRATION NUMBER: This trail was registered at clinicaltrials.gov as NCT03526081.

Evaluation of (-)-epicatechin metabolites as recovery biomarker of dietary flavan-3-ol intake.

Data from dietary intervention studies suggest that intake of (-)-epicatechin mediates beneficial vascular effects in humans. However, population-based investigations are required to evaluate associations between habitual intake and health and these studies rely on accurate estimates of intake, which nutritional biomarkers can provide. Here, we evaluate a series of structurally related (-)-epicatechin metabolites (SREM), particularly (-)-epicatechin-3'-glucuronide, (-)-epicatechin-3'-sulfate and 3'-O-methyl-(-)-epicatechin-5-sulfate (SREMB), as flavan-3-ol and (-)-epicatechin intake. SREMB in urine proved to be a specific indicator of (-)-epicatechin intake, showing also a strong correlation with the amount of (-)-epicatechin ingested (R2: 0.86 (95% CI 0.8l; 0.92). The median recovery of (-)-epicatechin as SREMB in 24 h urine was 10% (IQR 7-13%) and we found SREMB in the majority of participants of EPIC Norfolk (83% of 24,341) with a mean concentration of 2.4 ± 3.2 µmol/L. Our results show that SREMB are suitable as biomarker of (-)-epicatechin intake. According to evaluation criteria from IARC and the Institute of Medicine, the results obtained support use of SREMB as a recovery biomarker to estimate actual intake of (-)-epicatechin.

Evaluation at scale of microbiome-derived metabolites as biomarker of flavan-3-ol intake in epidemiological studies.

The accurate assessment of dietary intake is crucial to investigate the effect of diet on health. Currently used methods, relying on self-reporting and food composition data, are known to have limitations and might not be suitable to estimate the intake of many bioactive food components. An alternative are nutritional biomarkers, which can allow an unbiased assessment of intake. They require a careful evaluation of their suitability, including: (a) the availability of a precise, accurate and robust analytical method, (b) their specificity (c) a consistent relationship with actual intake. We have evaluated human metabolites of a microbiome-derived flavan-3-ol catabolite, 5-(3',4'-dihydroxyphenyl)-[gamma]-valerolactone (gVL), as biomarker of flavan-3-ol intake in large epidemiological studies. Flavan-3-ols are widely consumed plant bioactives, which have received considerable interest due to their potential ability to reduce CVD risk. The availability of authentic standards allowed the development of a validated high-throughput method suitable for large-scale studies. In dietary intervention studies, we could show that gVL metabolites are specific for flavan-3-ols present in tea, fruits, wine and cocoa-derived products, with a strong correlation between intake and biomarker (Spearman's r = 0.90). This biomarker will allow for the first time to estimate flavan-3-ol intake and further investigation of associations between intake and disease risk.

Hyperhomocysteinemia evoked by folate depletion: effects on coronary and carotid arterial function.

High circulating concentrations of homocysteine (ie, hyperhomocysteinemia [Hhcy]) impair the vascular function of peripheral conduit arteries and arterioles perfusing splanchnic and skeletal muscle regions. The effects of HHcy on coronary resistance vessel function and other indexes of vascular function, ie, arterial permeability and stiffening, are unclear. We tested the hypotheses that HHcy impairs coronary resistance vessel reactivity; increases carotid arterial permeability; and initiates arterial stiffening. Male rats that consumed folate-replete (CON, n=44) or folate-deplete (HHcy, n=48) chow for 4 to 5 weeks had total plasma homocysteine concentrations of 7+/-2 or 58+/-4 micromol/L, respectively. Maximal acetylcholine-evoked relaxation (approximately 40% vs approximately 60%) and tension development from baseline in response to nitric oxide synthase inhibition (approximately 20% vs approximately 40%) were lower (both P<0.05) in coronary resistance vessels (approximately 120 microm, internal diameter) isolated from HHcy versus CON animals, respectively, whereas sodium nitroprusside-evoked relaxation and contractile responses to serotonin and potassium chloride were similar between groups. Permeability to 4400 MW and 65 000 MW fluorescently labeled (TRITC) dextran reference macromolecules (quantitative fluorescence microscopy) was approximately 44% and approximately 24% greater (P<0.05), respectively, in carotid arteries from HHcy versus CON rats. Maximal strain, evaluated by using a vessel elastigraph, was less ( approximately 32% vs 42%, P<0.05) in carotid arterial segments from HHcy versus CON animals, respectively. Finally, estimates of oxidative (copper-zinc+manganese superoxide dismutase activity) and glycoxidative (pentosidine) stress were elevated (P<0.05) in arterial tissue from HHcy versus CON rats. These findings suggest that moderately severe HHcy evoked by folate-depletion impairs endothelium-dependent relaxation of coronary resistance vessels, increases carotid arterial permeability, and initiates arterial stiffening. HHcy may produce these effects by a mechanism associated with increased oxidative and glycoxidative stress.

Safety and efficacy of cocoa flavanol intake in healthy adults: a randomized, controlled, double-masked trial.

BACKGROUND: Evidence from dietary intervention studies shows that the intake of flavanols and procyanidins can be beneficial for cardiovascular health. Nevertheless, there is a clear need for advancing our understanding with regard to safe amounts of intake for these bioactives. OBJECTIVE: The aim was to investigate in healthy adults the effects of cocoa flavanol (CF) intake amount and intake duration on blood pressure, platelet function, metabolic variables, and potential adverse events (AEs). DESIGN: This investigation consisted of 2 parts. Part 1 was an open-label, intake-amount escalation study, in which 34 healthy adults (aged 35-55 y) consumed escalating amounts of CFs, ranging from 1000 to 2000 mg/d over 6 wk. Primary outcomes were blood pressure and platelet function, select metabolic variables, and the occurrence and severity of AEs. Secondary outcomes included plasma concentrations of CF-derived metabolites and methylxanthines. On the basis of the outcomes of study part 1, and assessing the same outcome measures, part 2 of this investigation was a controlled, randomized, double-masked, 2-parallel-arm dietary intervention study in which healthy participants (aged 35-55 y) were asked to consume for 12 consecutive weeks up to 2000 mg CFs/d (n = 46) or a CF-free control (n = 28). RESULTS: Daily intake of up to 2000 mg CFs/d for 12 wk was not associated with significant changes in blood pressure or platelet function compared with CF-free controls in normotensive, healthy individuals who exhibited a very low risk of cardiovascular disease. There were no clinically relevant changes in the metabolic variables assessed in either of the groups. AEs reported were classified as mild in severity and did not significantly differ between study arms. CONCLUSION: The consumption of CFs in amounts up to 2000 mg/d for 12 wk was well tolerated in healthy men and women. This trial was registered at clinicaltrials.gov as NCT02447770 (part 1) and NCT02447783 (part 2).

Long-term high copper intake: effects on indexes of copper status, antioxidant status, and immune function in young men.

BACKGROUND: Short-term high copper intake does not appear to affect indexes of copper status or functions related to copper status, but the effects of long-term high copper intake are unknown. OBJECTIVE: A study was conducted in men to determine the effect of long-term high copper intake on indexes of copper status, oxidant damage, and immune function. DESIGN: Nine men were confined to a metabolic research unit (MRU) for 18 d and were fed a 3-d rotating menu providing an average of 1.6 mg Cu/d. The men continued the study under free-living conditions for 129 d and supplemented their usual diets with 7 mg Cu/d. The men then returned to the MRU for 18 d of the same diet as during the first period, except that copper intake was 7.8 mg/d. Plasma copper, ceruloplasmin activity, ceruloplasmin protein, plasma malondialdehyde, benzylamine oxidase activity, erythrocyte superoxide dismutase, hair copper, urinary copper, and urinary thiobarbituric acid-reactive substances were measured during each MRU period. RESULTS: Ceruloplasmin activity, benzylamine oxidase, and superoxide dismutase were significantly higher at the end of the second MRU period than at the end of the first. Urinary copper excretion, hair copper concentrations, and urinary thiobarbituric acid-reactive substances were significantly higher during the second MRU period than during the first. Polymorphonuclear cell count, the percentage of white blood cells, lymphocyte count, and interleukin 2R were affected by copper supplementation. Antibody titer for the Beijing strain of influenza virus was significantly lower in supplemented subjects after immunization than in unsupplemented control subjects. CONCLUSIONS: Under highly controlled conditions, long-term high copper intake results in increases in some indexes of copper status, alters an index of oxidant stress, and affects several indexes of immune function. The physiologic implications of these changes are unknown.

Improved coronary vascular function evoked by high-intensity treadmill training is maintained in arteries exposed to ischemia and reperfusion.

We hypothesized that myocardial contractile function and coronary arterial function are greater after ischemia and reperfusion in high-intensity treadmill-trained vs. sedentary rats. Rats performed 10 x 4-min bouts of treadmill running consisting of 2 min at 13 m/min + 2 min at 45-60 m/min (Etr) or were sedentary (Sed) for 12 wk. Animals then were instrumented to measure left ventricular (LV) contractility in response to three 15-min coronary occlusion (O) and 5-min reperfusion (R) cycles (Isc) or a sham operation (Sham). After the Isc and Sham protocols, hearts were excised and coronary arterial ( approximately 105 microm ID) function was evaluated by using isometric techniques. LV developed pressure, the first derivative of LV pressure at a developed pressure of 40 mmHg, and systolic blood pressure were not different between Etr (n = 14) and Sed (n = 7) rats before or after the Sham protocol. Furthermore, hemodynamic variables were similar in Etr (n = 14) and Sed (n = 13) animals before the Isc protocol and were depressed to the same degree by the three O-R cycles. Therefore, Etr did not alter myocardial contractile function in rats that were (i.e., Isc) or were not (i.e., Sham) exposed to ischemia and reperfusion. Acetylcholine-evoked relaxation (10-8 to 3 x 10-5 M) was greater (P < 0.05) in coronary arteries from Sham-Etr vs. Sham-Sed animals (5 of 8 doses tested) and Isc-Etr vs. Isc-Sed rats (3 of 8 doses tested). Maximal relaxation produced by sodium nitroprusside (10-4 M) was similar among groups. Vasocontractile responses produced by KCl (10-100 mM) and endothelin-1 (10-11-10-4 M) were greater (P < 0.05) in the presence vs. the absence of nitric oxide synthase inhibition (10-6 M NG-monomethyl-l-arginine) in vessels from Sham-Etr but not Sham-Sed rats and from Isc-Etr but not Isc-Sed rats. These findings suggest that Etr-evoked improvements in coronary function are maintained in small arteries even when exposed to ischemia and reperfusion.

Reducing arginase activity via dietary manganese deficiency enhances endothelium-dependent vasorelaxation of rat aorta.

L-Arginine is a common substrate for the enzymes arginase and nitric oxide synthase (NOS). Acute inhibition of arginase enzyme activity improves endothelium-dependent vasorelaxation, presumably by increasing availability of substrate for NOS. Arginase is activated by manganese (Mn), and the consumption of a Mn-deficient (Mn-) diet can result in low arginase activity. We hypothesize that endothelium-dependent vasorelaxation is greater in rats fed Mn- versus Mn sufficient (Mn+) diets. Newly weaned rats fed Mn+ diets (0.5 microg Mn/g; n = 12) versus Mn+ diets (45 microg Mn/g; n = 12) for 44 +/- 3 days had (i) lower liver and kidney Mn and arginase activity (P < or = 0.05), (ii) higher plasma L-arginine (P < or = 0.05), (iii) similar plasma and urine nitrate + nitrite, and (iv) similar staining for endothelial nitric oxide synthase in thoracic aorta. Vascular reactivity of thoracic aorta (approximately 720 microm i.d.) and small coronary arteries (approximately 110 microm i.d.) was evaluated using wire myographs. Acetylcholine (ACh; 10(-8)-10(-4) M) produced greater (P < or = 0.05) vasorelaxation in thoracic aorta from Mn- rats (e.g., maximal percent relaxation, 79 +/- 7%) versus Mn + rats (e.g., maximal percent relaxation, 54 +/- 9%) at 5 of 7 evaluated doses. Tension produced by NOS inhibition using N(G) monomethyl-L-arginine (L-NMMA; 10(-3) M) and vasorelaxation evoked by (i) arginase inhibition using difluoromethylornithine (DFMO; 10(-7) M), (ii) ACh (10(-8)-10(-4) M) in the presence of DFMO, and (iii) sodium nitroprusside (10(-9)-10(-4) M) were unaffected by diet. No differences existed between groups concerning these responses in small coronary arteries. These findings support our hypothesis that endothelium-dependent vasorelaxation is greater in aortic segments from rats that consume Mn- versus Mn+ diets; however, responses from small coronary arteries were unaffected.

Food effects on the absorption and pharmacokinetics of cocoa flavanols.

Macronutrients in food and gastric acid are known to have a pronounced effect on the metabolism of many xenobiotics, an effect that impacts their efficacy as bioactive agents. In this investigation we assessed the impact of select food treatments and the histamine H(2)-receptor antagonist Famotidine (Pepcid-AC) on flavanol absorption and metabolism. Four crossover intervention studies were conducted with 6 subjects each. Volunteers consumed sugar-free, flavanol-rich cocoa (0.125 g/kg body wt) alone, with macronutrient-rich foods (8.75 or 17.5 kJ/kg subject body wt) or Famotidine (Pepcid-AC). Blood samples were drawn at 5 time points including baseline. Plasma samples were analyzed for epicatechin and catechin flavanols by HPLC. Pharmacokinetic parameters were assessed using non-compartmental methodology. When provided at 17.5 kJ/kg subject body weight (approximately 4 kcal/kg), sugar and bread test meals increased flavanol area under the curve (AUC) values to 140% of control values (P < 0.05). A corresponding tendency for plasma antioxidant capacity to increase was observed for the cocoa treatment at 1.5 and 2.5 h (P < 0.17, P < 0.06, respectively). The ability of treatment meals to affect AUC values was positively correlated with treatment carbohydrate content (r = 0.83; P< 0.02). In contrast to carbohydrate rich meals, lipid and protein rich meals and Famotidine treatment had minimal effects on flavanol absorption. Based on C(max) and AUC values, this data suggests that the uptake of flavanols can be increased significantly by concurrent carbohydrate consumption.

An 18-month follow-up study on the influence of smoking on blood antioxidant status of teenage girls in comparison with adult male smokers in Korea.

OBJECTIVES: The influence of cigarette smoking on blood antioxidant status in teenage girls with a history of short-term smoking was followed over 18 mo. METHODS: Data obtained from female senior high school students (ages 14 to 18 y) in Korea were compared with data obtained from adult male smokers (ages 36 to 51 y) with a long history of smoking and living in the same geographic areas as the teenage subjects. A smoker was a person who had smoked at least three cigarettes a day for at least 1 y for teenagers (n = 35) or at least 10 cigarettes a day for at least 13 y for adults (n = 20). Serum, urine, and anthropometric data were obtained from teenagers every 6 mo over an 18-mo period. Samples were collected once from adults. Data were analyzed by Student's t test and Fisher's protected least significant difference test for comparing smokers and non-smokers and for analyzing period effects in each group. RESULTS: Serum nicotine and cotinine concentrations were higher in smokers than in non-smokers. Blood pressures were higher in teenage (at 0 and 12 mo) and adult smokers than in non-smokers. Extracellular superoxide dismutase activities and concentrations of serum vitamin C and folate were lower in smokers in the teenage (at 0, 12, or 18 mo) and adult groups. Serum ceruloplasmin activities and thiobarbituric acid-reactive substance production were not influenced by smoking. In adults, serum copper concentrations were higher in smokers than in non-smokers. This parameter for teenagers did not change consistently throughout the study. CONCLUSIONS: Similar to adults, cigarette smoking by teenagers has a negative effect on oxidant defense systems.

Antioxidative activities of oolong tea.

While the antioxidative properties of green and black tea have been extensively studied, less attention has been given to these properties in oolong tea. The reducing powers, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities, the amount of total phenolic compounds, the inhibitory effect on FeCl(2)/H(2)O(2) (Fenton reaction system)-induced DNA damage, and the inhibitory effect on erythrocyte hemolysis of an oolong tea water extract (OTE) were evaluated in the present study. The OTE was found to have strong antioxidative activities in all of the model systems tested. When the OTE was separated into different fractions according to molecular weight, it was found that the fractions with higher amounts of phenolic compounds (lower molecular weight) have stronger antioxidative activities. The present results support the concept that oolong tea contains several low molecular weight antioxidants that may have health promotion activities.

Stability of the flavan-3-ols epicatechin and catechin and related dimeric procyanidins derived from cocoa.

Cocoa flavanols and procyanidins possess wide-ranging biological activities. The present study investigated the stability of the cocoa monomers, (-)-epicatechin and (+)-catechin, and the dimers, epicatechin-(4beta-8)-epicatechin (Dimer B2) and epicatechin-(4beta- 6)-epicatechin (Dimer B5), in simulated gastric and intestinal juice and at different pH values. The dimers were less stable than the monomers at both acidic and alkaline pH. Incubation of Dimer B2 and Dimer B5 in simulated gastric juice (pH 1.8) or acidic pH resulted in degradation to epicatechin and isomerization to Dimer B5 and Dimer B2, respectively. When incubated in simulated intestinal juice or at alkaline pH, all four compounds degraded almost completely within several hours. These results suggest that the amount, and type, of flavanols and procyanidins in the gastrointestinal tract following the consumption of cocoa can be influenced by the stability of these compounds in both acidic and alkaline environments.

Chronic consumption of flavanol-rich cocoa improves endothelial function and decreases vascular cell adhesion molecule in hypercholesterolemic postmenopausal women.

Endothelial dysfunction characterizes many disease states including subclinical atherosclerosis. The consumption of flavanol-rich cocoa and cocoa-based products has been shown to improve endothelial function in both compromised and otherwise normal, healthy individuals when administered either acutely or over a period of several days, or weeks. Women experience increased risk for cardiovascular disease after menopause, which can be associated with endothelial dysfunction. Whether a flavanol-rich cocoa-based product can improve endothelial function in hypercholesterolemic postmenopausal women is not known. The purpose of the present study was to determine whether chronic dietary administration of flavanol-rich cocoa improves endothelial function and markers of cardiovascular health in hypercholesterolemic postmenopausal women. Thirty-two postmenopausal hypercholesterolemic women were randomly assigned to consume a high-flavanol cocoa beverage (high cocoa flavanols (CF)--446 mg of total flavanols), or a low-flavanol cocoa beverage (low CF--43 mg of total flavanols) for 6 weeks in a double-blind study (n=16 per group). Endothelial function was determined by brachial artery-reactive hyperemia. Plasma was analyzed for lipids (total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol), hormones (follicle-stimulating hormone), total nitrate/nitrite, activation of cellular adhesion markers (vascular cell adhesion molecule 1, intercellular adhesion molecule 1, E-Selectin, P-Selectin), and platelet function and reactivity. Changes in these plasma markers were then correlated to brachial reactivity. Brachial artery hyperemic blood flow increased significantly by 76% (P<0.05 vs. baseline) after the 6-week cocoa intervention in the high CF group, compared with 32% in the low CF cocoa group (P=ns vs. baseline). The 2.4-fold increase in hyperemic blood flow with high CF cocoa closely correlated (r2=0.8) with a significant decrease (11%) in plasma levels of soluble vascular cell adhesion molecule-1. Similar responses were not observed after chronic use of low CF. There were no significant differences between high and low CF in other biochemical markers and parameters measured. This study is the first to identify beneficial vascular effects of flavanol-rich cocoa consumption in hypercholesterolemic postmenopausal women. In addition, our results suggest that reductions in plasma soluble vascular cell adhesion molecule-1 after chronic consumption of a flavanol-rich cocoa may be mechanistically linked to improved vascular reactivity.