Dietrich Rein

Epicatechin in Human Plasma: In Vivo Determination and Effect of Chocolate Consumption on Plasma Oxidation Status

Diets that are rich in plant foods have been associated with a decreased risk for specific disease processes and certain chronic diseases. In addition to essential macronutrients and micronutrients, the flavonoids in a variety of plant foods may have health-enhancing properties. Chocolate is a food that is known to be rich in the flavan-3-ol epicatechin and procyanidin oligomers. However, the bioavailability and the biological effects of the chocolate flavonoids are poorly understood. To begin to address these issues, we developed a method based on HPLC coupled with electrochemical (coulometric) detection to determine the physiological levels of epicatechin, catechin and epicatechin dimers. This method allows for the determination of 20 pg (69 fmol) of epicatechin, which translates to plasma concentrations as low as 1 nmol/L. We next evaluated the absorption of epicatechin, from an 80-g semisweet chocolate (procyanidin-rich chocolate) bolus. By 2 h after ingestion, there was a 12-fold increase in plasma epicatechin, from 22 to 257 nmol/L (P < 0.01). Consistent with the antioxidant properties of epicatechin, within the same 2-h period, there was a significant increase of 31% in plasma total antioxidant capacity (P < 0.04) and a decrease of 40% in plasma 2-thiobarbituric acid reactive substances (P < 0.01). Plasma epicatechin and plasma antioxidant capacity approached baseline values by 6 h after ingestion. These results show that it is possible to determine basal levels of epicatechin in plasma. The data support the concept that the consumption of chocolate can result in significant increases in plasma epicatechin concentrations and decreases in plasma baseline oxidation products.

The effects of flavanol-rich cocoa and aspirin on ex vivo platelet function

BACKGROUND Flavanols modulate platelet function in vitro, but less is known of their in vivo effects and how they compare to pharmacological platelet inhibitors. We investigated the effect of a flavanol-rich cocoa beverage (897 mg/ml) in combination with and in comparison to aspirin on platelet function and activation in healthy subjects. METHODS AND RESULTS On separate test days in a crossover design, 16 healthy adults consumed aspirin (81 mg), cocoa (as a beverage), or aspirin plus cocoa. Platelet activation was measured by surface expression of P-selectin and PAC-1 binding to the activated conformation of the GPIIb/IIIa receptor (GPIIb/IIIa-act). Platelet function was measured on an analyzer (the PFA-100) that measures shear stress-induced platelet plug formation in response to collagen-epinephrine or collagen-ADP. Plasma epicatechin concentrations peaked approximately 2 h after subjects were given either the cocoa or aspirin plus cocoa. After 6 h, cocoa inhibited epinephrine-induced platelet function. Epinephrine-induced platelet function was inhibited 2 and 6 h after aspirin, and after aspirin plus cocoa. Epinephrine-stimulated P-selectin expression was inhibited by aspirin at 6 h, and after 2 and 6 h by aspirin plus cocoa. ADP-stimulated P-selectin expression was not affected by the treatments. Cocoa and aspirin, given separately, reduced epinephrine-stimulated GPIIb/IIIa-act expression at 2 and 6 h, respectively, and at 2 and 6 h when given together, suggesting an additive effective. ASA plus cocoa inhibited ADP-stimulated GPIIb/IIIa-act expression at 6 h. CONCLUSIONS Flavanol-rich cocoa inhibited epinephrine-stimulated platelet activation and function. These effects were qualitatively similar to aspirin, but less profound. These results emphasize the need to further examine the effects of food flavonoids for platelet modulating effects.

Cocoa and Wine Polyphenols Modulate Platelet Activation and Function

There is speculation that dietary polyphenols can provide cardioprotective effects due to direct antioxidant or antithrombotic mechanisms. We report in vitro and postingestion ex vivo effects of cocoa procyanidins, a procyanidin-rich cocoa beverage and dealcoholized red wine (DRW) on human platelet activation. In a series of in vitro studies, cocoa procyanidin trimers, pentamers or DRW (3 and 10 micromol/L) were incubated with citrated peripheral whole blood in the presence and absence of platelet agonists. Platelet activation was detected using fluorescent-labeled monoclonal antibodies recognizing the fibrinogen binding conformation of GPIIb-IIIa (referred to herein as PAC-1 binding) and the activation-dependent platelet epitope CD62P (P-selectin). The percentage of CD42a-positive platelets coexpressing PAC-1 binding and/or CD62P was determined by multiparameter flow cytometry. Procyanidin trimers, pentamers and DRW added to whole blood in vitro increased PAC-1 binding and P-selectin expression. In contrast, procyanidin trimers, pentamers and DRW inhibited the platelet activation in response to epinephrine. The effects on platelet activation of cocoa beverage and DRW consumption were also studied in healthy subjects. Citrated blood was obtained before and 2 and 6 h after the ingestion of a cocoa beverage, a caffeine-containing beverage, DRW or water. Platelet activation was measured by flow cytometry. The consumption of DRW did not affect the expression of activation-dependent platelet antigens, either unstimulated or after ex vivo activation with epinephrine. However, the consumption of DRW increased PAC-1 binding in response to 100 micromol/L ADP ex vivo. Cocoa consumption reduced platelet response to agonists ex vivo. The ingestion of water had no effect on platelet activation, whereas a caffeine-containing beverage augmented the response of platelets to epinephrine. In summary, select cocoa procyanidins and DRW added to whole blood in vitro increased expression of platelet activation markers in unstimulated platelets but suppressed the platelet activation response to epinephrine. In contrast, cocoa consumption suppressed unstimulated and stimulated platelet activation in whole blood. This suppressive effect observed on platelet reactivity may explain in part the reported cardioprotective effects of dietary polyphenols.

Effect of dietary catechin and vitamin E on aortic fatty streak accumulation in hypercholesterolemic hamsters

Male golden Syrian hamsters were fed for 16 weeks on a hypercholesterolemic diet containing, per kg, 150 g of lipids (90 g butterfat, 35 g vitamin E-stripped corn oil and 25 g fish oil), 2 g cholesterol and either 3 IU vitamin E (3 IU E), 3 IU vitamin E and 200 mg catechin hydrate (3 IU E-200 Cat) or 30 IU vitamin E (30 IU E). More fatty streaks, measured by Oil Red O staining, were deposited in aortas of hamsters fed 3 IU E than in those fed either 3 IU E-200 Cat or 30 IU E. Lipid staining increased with plasma low-density lipoprotein cholesterol (LDL-C) in all animals. At the same concentration of LDL-C, animals fed either 3 IU E-200 Cat or 30 IU E developed less fatty streaks than those fed 3 IU E. Plasma LDL-C and total cholesterol were highest in hamsters fed 3 IU E and LDL-C and total cholesterol in animals fed 3 IU-200 Cat were not different from those fed either 3 IU E or 30 IU E. This study showed the importance of circulating plasma LDL-C on atherogenesis and the inhibitory effect on this process of both dietary vitamin E and catechin.

Flavanols and platelet reactivity.

Platelet activity and platelet-endothelial cell interactions are important in the acute development of thrombosis, as well as in the pathogenesis of cardiovascular disease. An increasing number of foods have been reported to have platelet-inhibitory actions, and research with a number of flavanol-rich foods, including, grape juice, cocoa and chocolate, suggests that these foods may provide some protection against thrombosis. In the present report, we review a series of in vivo studies on the effects of flavanol-rich cocoa and chocolate on platelet activation and platelet-dependent primary hemostasis. Consumption of flavanol-rich cocoa inhibited several measures of platelet activity including, epinephrine- and ADP-induced glycoprotein (GP) IIb/IIIa and P-Selectin expression, platelet microparticle formation, and epinephrine-collagen and ADP-collagen induced primary hemostasis. The epinephrine-induced inhibitory effects on GP IIb/IIIa and primary hemostasis were similar to, though less robust than those associated with the use of low dose (81 mg) aspirin. These data, coupled with information from other studies, support the concept that flavanols present in cocoa and chocolate can modulate platelet function through a multitude of pathways.

Fluorescent Lipid Oxidation Products and Heme Spectra Index Antioxidant Efficacy in Kidney Tissue of Hamsters

Studies ranging from epidemiological to molecular suggest that dietary antioxidants protect against chronic diseases. The hypothesis was investigated that hamster kidney homogenate are better protected against induced oxidative damage after animals were fed a purified vitamin E deficient diet supplemented with 30 international units (IU) vitamin E/kg (30 IU diet) than fed the minimum required 3 IU vitamin E/kg (3 IU diet). The addition of 200 mg (+)-catechin to the 3 IU diet may offer protection. The effects of dietary (+)-catechin and alpha-tocopherol on tissue oxidizability were investigated by measuring (i) fluorescent products in lipid extracts, (ii) heme protein oxidation and (iii) heme destruction. A rapid initial increase of oxidation markers was measured over the 4 h incubation period for iron + ascorbate induced oxidation and a constant increase for lipoxygenase catalyzed products. Analysis of covariance over time and comparison at specific incubation times showed that iron + ascorbate induced homogenates from hamsters fed the 30 IU diet generated less fluorescent products and oxidized heme proteins than homogenates from the 3 IU or the 3 IU plus (+)-catechin fed animals. Incubation with lipoxygenase produced more lipid fluorescent products and heme protein oxidation in the 3 IU than in the 30 IU vitamin E group. In conclusion, supplementary dietary vitamin E but not supplementary (+)-catechin in a diet containing the minimum requirement of vitamin E for the species enhances oxidative resistance of kidney tissue.

Dietary vitamin e in an atherogenic hamster model

Regular consumption of fruits, vegetables and cereals moderates development of coronary heart disease and atherosclerosis in humans, possibly as a result of antioxidants present in these foods. The aim of this study was to develop a hamster model sufficiently sensitive to test the effects of nutritional rather than pharmacological amounts of food components on the development of atherosclerosis. The effect of vitamin E on aortic lipid deposition was investigated in hamsters fed a hypercholesterolemic diet containing per kilogram 2 g cholesterol, 90 g butterfat, 35 g vitamin E-stripped corn oil, 25 g fish oil and either the minimum requirement of 3 international units (IU) vitamin E or 30 IU. After 30 weeks, lipoprotein cholesterol fractions did not differ between groups, and the 6:1 LDL-cholesterol : HDL-cholesterol ratio was atherogenic. Early atherosclerosis was measured by lipid staining of aortic arch sections with oil red O and quantified by a photomicroscopy color-scanning technique. The area of lipid deposits in the 30 IU vitamin E group (7.9 ± 1.3%, mean ± SEM) was 58% less than in the 3 IU vitamin E group (18.7 ± 4.4%, p < 0.03). Hamsters fed a mixture of saturated and n-6 and n-3 polyunsaturated fatty acids plus cholesterol became hypercholesterolemic and were sensitive to vitamin E with respect to development of atherosclerosis. This model provides a tool to test atheroprotective effects of individual food components in vivo.