Diet is a major life style factor affecting human health, thus emphasizing the need for evidence-based dietary guidelines for primary disease prevention. While current recommendations promote intake of fruit and vegetables, we have limited understanding of plant-derived bioactive food constituents other than those representing the small number of essential nutrients and minerals. This limited understanding can be attributed to some extent to a lack of fundamental data describing the absorption, distribution, metabolism and excretion (ADME) of bioactive compounds. Consequently, we selected the flavanol (−)-epicatechin (EC) as an example of a widely studied bioactive food constituent and investigated the ADME of [2-14C](−)-epicatechin (300 μCi, 60 mg) in humans (n = 8). We demonstrated that 82 ± 5% of ingested EC was absorbed. We also established pharmacokinetic profiles and identified and quantified >20 different metabolites. The gut microbiome proved to be a key driver of EC metabolism. Furthermore, we noted striking species-dependent differences in the metabolism of EC, an insight with significant consequences for investigating the mechanisms of action underlying the beneficial effects of EC. These differences need to be considered when assessing the safety of EC intake in humans. We also identified a potential biomarker for the objective assessment of EC intake that could help to strengthen epidemiological investigations.

Accumulating data suggest that diets rich in flavanols and procyanidins are beneficial for human health. In this context, there has been a great interest in elucidating the systemic levels and metabolic profiles at which these compounds occur in humans. Although recent progress has been made, there still exist considerable differences and various disagreements with regard to the mammalian metabolites of these compounds, which in turn are largely a consequence of the lack of availability of authentic standards that would allow for the directed development and validation of expedient analytical methodologies. In this study, we developed a method for the analysis of structurally related flavanol metabolites using a wide range of authentic standards. Applying this method in the context of a human dietary intervention study using comprehensively characterized and standardized flavanol- and procyanidin-containing cocoa, we were able to identify the structurally related (-)-epicatechin metabolites (SREM) postprandially extant in the systemic circulation of humans. Our results demonstrate that (-)-epicatechin-3'-β-D-glucuronide, (-)-epicatechin-3'-sulfate, and a 3'-O-methyl-(-)-epicatechin-5/7-sulfate are the predominant SREM in humans and further confirm the relevance of the stereochemical configuration in the context of flavanol metabolism. In addition, we also identified plausible causes for the previously reported discrepancies regarding flavanol metabolism, consisting, to a significant extent, of interlaboratory differences in sample preparation (enzymatic treatment and sample conditioning for HPLC analysis) and detection systems. Thus, these findings may also aid in the establishment of consensus on this topic.

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SummaryZinc deficiency is characterized by an attenuation of growth factor signaling pathways and an amplification of p53 pathways. This outcome is facilitated by hypo-phosphorylation of AKT and ERK secondary to zinc deficiency, which are permissive events to the activation of the intrinsic cell death pathway. Low zinc concentrations provide an environment that is also conducive to the production of reactive oxygen/reactive nitrogen species (ROS/ RNS) and caspase activation. Additionally, during zinc deficiency endogenous survival pathways such as NF-k B are inhibited in their transactivation potential. The above factors contribute to the irreversible commitment of the zinc deficient cell to death. IUBMB Life, 57: 661-670, 2005

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T he vascular endothelium is crucially involved in the fundamental regulation of blood flow matching demand and supply of tissue. After transient ischemia, arterial inflow increases. As a response to increased shear forces during reactive hyperemia, healthy arteries dilate via release of NO or other endothelium-derived vasoactive substances. This endothelium-dependent flow-mediated vasodilation (FMD) is impaired in atherosclerosis.1 As the presence of endothelial dysfunction is closely associated with cardiovascular risk and outcome, the measurement of FMD in the brachial artery has become a standard method for the assessment of endothelial function in patients and to evaluate therapeutic interventions targeting atherosclerosis. To date, this has been impossible in mice, although highly desirable to study mechanisms affecting endothelial function and in particular femoral artery dilation in transgenic mouse models.2 Therefore, we here characterize the physiology of FMD in living mice using a methodology analogous to humans and present important examples of vascular pathologies. Materials and MethodsMaterials and Methods are available in the online-only Data Supplement. ResultsIn healthy humans, the relative increase in brachial artery diameter at 45 to 60 sec after resolution of forearm ischemia is typically in the 5% to 10% range. To be able to detect such small diameter changes expected in mice (10-30 μm change at a baseline diameter of ≈200-300 μm) with a high heart rate, we used a high-resolution, high-frequency digital imaging platform and a 30 to 70 MHz linear array microscan transducer © 2014 American Heart Association, Inc. Objective-Endothelium-dependent, flow-mediated vasodilation after an increase in shear stress at the endothelial lining of conduit arteries during reactive hyperemia after ischemia is a fundamental principle of vascular physiology adapting blood flow to demand of supplied tissue. Flow-mediated vasodilation measurements have been performed in human studies and are of diagnostic and prognostic importance, but have been impossible because of technical limitations in transgenic mice to date, although these represent the most frequently used animal model in cardiovascular research. Approach and Results-Using high-frequency ultrasound, we visualized, quantified, and characterized for the first time endothelium-dependent dilation of the femoral artery after temporal ischemia of the lower part of the hindlimb and demonstrated that the signaling was almost exclusively dependent on stimulation of endothelial nitric oxide synthase, similar to acetylcholine, completely abolished after pharmacological or genetic inhibition of endothelial nitric oxide synthase and endothelial denudation, substantially impaired in mice of increasing age and cholesterol-fed ApoE knock outs and increased by the dietary polyphenol (−)-epicatechin. Intra-and interindividual variability were similar to the human methodology. Conclusions-The

In humans, endothelial vasodilator function serves as a surrogate marker for cardiovascular health and is measured as changes in conduit artery diameter after temporary ischemia [flow-mediated dilation (FMD)]. Here we present an FMD-related approach to study femoral artery (FA) vasodilation in anesthetized rats. Diameter and Doppler flow were monitored in the FA. Using high-resolution ultrasound (35 MHz) and automated analysis software, we detected dose-dependent vasodilation using established endothelium-independent [intravenous nitroglycerin EC(50) = 3.3 x 10(-6) mol/l, peak 21Delta% (SD 4)] and endothelium-dependent [intra-arterial acetylcholine EC(50) = 1.3 x 10(-6) mol/l, peak 27Delta% (SD 4)] pharmacological vasodilators. Wall shear stress induced by intra-aortic injection of adenosine and infusion of saline at increasing rates (1.5-4.5 ml/min) led to vasodilation at 1 to 2 min. Transient hindlimb ischemia by common iliac occlusion (5 min) led to reactive hyperemia with flow velocity and wall shear stress increase and was followed by FA dilation [16Delta% (SD 2)], the latter of which was completely abolished by nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-L-arginine [1Delta% (SD 2)]. FMD was significantly reduced in adult 20-24-wk-old animals compared with 9- to 10-wk-old animals, consistent with age-dependent endothelial dysfunction [16Delta% (SD 3) vs. 10Delta% (SD 3), P < 0.05]. Whereas FMD was completely NOS dependent in 9- to 10-wk-old animals, NOS-dependent mechanisms accounted for only half of the FMD in 20-24-wk-old animals, with the remainder being blocked by charybdotoxin and apamin, suggesting a contribution of endothelium-derived hyperpolarizing factor. To our knowledge, this is the first integrative physiological model to reproducibly study FMD of conduit arteries in living rats.

Epidemiology studies suggest that the consumption of diets rich in flavonoids is associated with reduced risk of cardiovascular disease. Plant-derived foods and beverages, such as red wine, tea, grape and grape juice, cocoa and chocolate, can be rich in 1 particular class of flavonoid, the flavan-3-ols. There is now an increasing body of research that suggests that consuming flavanol-rich foods can positively affect hemostasis, through mechanisms that either directly affect platelet function or increase certain endothelium-derived factors that maintain platelet acquiescence or increase fibrinolysis. In this paper, we will review a series of in vivo studies on the effects of flavanol-rich cocoa and chocolate on platelet activation and platelet-dependent hemostasis. In addition, we will briefly review the body of literature with regard to other flavanol-rich foods and beverages, and possible mechanisms of action.