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.

There is no direct competition between arginase and nitric oxide synthase for the common substrate l-arginine.

AIMS: Extrahepatic arginases are postulated to be involved in cardiovascular-related pathologies by competing with nitric oxide synthase (NOS) for the common substrate l-arginine, subsequently decreasing nitric oxide production. However, previous models used to study arginase and NOS competition did not account for steady state level of l-arginine pool, which is dependent on conditions of l-arginine supply and utilization pathways. This work aimed at revisiting the concept of NOS and arginase competition while considering different conditions of l-arginine supply and l-arginine utilization pathways. METHODS AND RESULTS: Mouse macrophage-like RAW cells and human vascular endothelial cells co-expressing NOS and arginase were used to reevaluate the concept of substrate competition between arginase and NOS under conditions of l-arginine supply that mimicked either a continuous (similar to in vivo conditions) or a limited supply (similar to previous in vitro models). Enzyme kinetics simulation models were used to gain mechanistic insight and to evaluate the tenability of a substrate competition between the two enzymes. In addition to arginase and NOS, other l-arginine pathways such as transporters and utilization towards protein synthesis were considered to understand the intricacies of l-arginine metabolism. Our results indicate that when there is a continuous supply of l-arginine, as is the case for most cells in vivo, arginase does not affect NOS activity by a substrate competition. Furthermore, we demonstrate that l-arginine pathways such as transporters and protein synthesis are more likely to affect NOS activity than arginase. CONCLUSIONS: Arginase does not outcompete NOS for the common substrate l-arginine. Findings from this study should be considered to better understand the role of arginase in certain pathologies and for the interpretation of in vivo studies with arginase inhibitors.

Arginase inhibitor, Nω-hydroxy-L-norarginine, spontaneously releases biologically active NO-like molecule: Limitations for research applications.

There has been a renewed interest in the enzyme arginase for its role in various physiological and pathological processes that go beyond the urea cycle. One such role ascribed to arginase has been that of regulating nitric oxide (NO) production by a substrate (l-arginine) competition between arginase and nitric oxide synthase (NOS). Several arginase inhibitors have been developed to investigate the biological roles of arginase, of which Nω-hydroxy-l-norarginine (nor-NOHA) is commercially available and is used widely from cell culture models to clinical investigations in humans. Despite the prevalence of nor-NOHA to investigate the substrate competition between arginase and NOS, little is known regarding interferences that nor-NOHA could have on common methods to assess NO production. Therefore, we investigated if nor-NOHA has unintended consequences on common NO assessment methods. We show that nor-NOHA spontaneously releases biologically active NO-like molecule in cell culture media by reacting with riboflavin. This NO-like molecule is indistinguishable from an NO donor (NOR-3) using common methods to assess NO. Besides riboflavin, nor-NOHA spontaneously reacts with H2O2 to diminish H2O2 content and produce NO-like molecule in the process. Our investigation provides detailed evidence on unintended artefacts related to nor-NOHA that can limit its use in cell culture, as well as some ex vivo and in vivo models. Future studies on arginase should take into consideration the limitations presented here when using nor-NOHA as a research tool, not only in investigations related to arginase and NOS competition, but also for investigating other biological roles of arginase.

The metabolome of [2-(14)C](-)-epicatechin in humans: implications for the assessment of efficacy, safety, and mechanisms of action of polyphenolic bioactives.

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-(14)C](-)-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.

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).

Measurement of endothelium-dependent vasodilation in mice--brief report.

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 physiology of flow-mediated vasodilation in mice resembles that in humans underscoring the significance of this novel technology to noninvasively, serially, and reliably quantify flow-mediated vasodilation in transgenic mice.

Structurally related (-)-epicatechin metabolites in humans: assessment using de novo chemically synthesized authentic standards.

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'-ß-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.

The stereochemical configuration of flavanols influences the level and metabolism of flavanols in humans and their biological activity in vivo.

Extensive epidemiological and clinical evidence associates diets high in flavanol-containing foods with cardiovascular health benefits in humans. Catechin and epicatechin, the most common flavanols in foods, are present in the diet in different enantiomeric forms. This study investigated the influence of the stereochemical configuration of flavanols on their absorption, metabolism, and biological activity. Healthy adult males were asked to consume equal amounts of the stereochemically pure flavanols (-)-epicatechin, (-)-catechin, (+)-catechin, and (+)-epicatechin (1.5mg/kg bw) in a well-defined cocoa-based, dairy-containing drink matrix, and flavanol levels were subsequently determined in plasma and 24-h urine. The results obtained show that the stereochemical configuration of flavanols has a profound influence on their uptake and metabolism in humans. In addition, we assessed the vasodilatory activity of each flavanol stereoisomer in vivo and found (-)-epicatechin to be the single stereoisomer capable of mediating a significant arterial dilation response. Importantly, this effect was independent of the classic antioxidant properties of flavanols. Overall, these results indicate that the proposed beneficial health effects associated with the consumption of flavanol-containing foods will significantly depend on the stereochemical configuration of the flavanols ingested.

Functional foods for health promotion: state-of-the-science on dietary flavonoids. Extended abstracts from the 12th Annual Conference on Functional Foods for Health Promotion, April 2009.

The extended abstracts in this report are based on presentations from the 12(th) Special Conference on Functional Foods for Health Promotion, cosponsored by the North American branch of the International Life Sciences Institute (ILSI North America) Project Committee on Flavonoids and the American Society for Nutrition at the Experimental Biology meeting in April 2009. The theme of this year's special conference was "State-of-the-Science on Dietary Flavonoids." The conference began with a general introduction and overview of flavonoids and their presence in the diet as well as the estimated intake levels in the US population. Subsequent presentations addressed issues pertaining to study design and interpretation, mechanisms of action, and the potential health impacts related to inflammation, the vasculature, and the brain. The present summary of the current science indicates that dietary flavonoids, particularly flavanols, show promising potential for reducing cardiovascular disease risk via reduction of inflammation and improvement in vascular function. However, the existing data must be interpreted cautiously, with consideration given to the compound tested (i.e., parent or metabolite), the use of controls, and the practicality of the concentrations used. While more data are needed on the long-term health impacts of dietary flavonoids in humans, including the efficacious dose, current data indicate it may soon be possible to develop public health messages about flavonoid-rich foods.

Cocoa flavanols lower vascular arginase activity in human endothelial cells in vitro and in erythrocytes in vivo.

The availability of l-arginine can be a rate-limiting factor for cellular NO production by nitric oxide synthases (NOS). Arginase competes with NOS for l-arginine as the common substrate. Increased arginase activity has been linked to low NO levels, and an inhibition of arginase activity has been reported to improve endothelium-dependent vasorelaxation. Based on the above, we hypothesized that an increase in the circulating NO pool following flavanol consumption could be correlated with decreased arginase activity. To test this hypothesis we (a) investigated the effects of (-)-epicatechin and its structurally related metabolites on endothelial arginase expression and activity in vitro; (b) evaluated the effects of dietary flavanol-rich cocoa on kidney arginase activity in vivo; and (c) assessed human erythrocyte arginase activity following flavanol-rich cocoa beverage consumption in a double-blind intervention study with cross-over design. The results demonstrate that cocoa flavanols lower arginase-2 mRNA expression and activity in HUVEC. Dietary intervention with flavanol-rich cocoa caused diminished arginase activity in rat kidney and, erythrocyte arginase activity was lowered in healthy humans following consumption of a high flavanol beverage in vivo.

Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins.

One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.

In vivo measurement of flow-mediated vasodilation in living rats using high-resolution ultrasound.

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.

Dietary flavanols and platelet reactivity.

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.

Zinc deficiency-induced cell death.

Zinc 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-kappaB are inhibited in their transactivation potential. The above factors contribute to the irreversible commitment of the zinc deficient cell to death.

Alterations in protein kinase C activity and processing during zinc-deficiency-induced cell death.

Protein kinases C (PKCs) are a family of serine/threonine kinases that are critical for signal transduction pathways involved in growth, differentiation and cell death. All PKC isoforms have four conserved domains, C1-C4. The C1 domain contains cysteine-rich finger-like motifs, which bind two zinc atoms. The zinc-finger motifs modulate diacylglycerol binding; thus, intracellular zinc concentrations could influence the activity and localization of PKC family members. 3T3 cells were cultured in zinc-deficient or zinc-supplemented medium for up to 32 h. Cells cultured in zinc-deficient medium had decreased zinc content, lowered cytosolic classical PKC activity, increased caspase-3 processing and activity, and reduced cell number. Zinc-deficient cytosols had decreased activity and expression levels of PKC-alpha, whereas PKC-alpha phosphorylation was not altered. Inhibition of PKC-alpha with Gö6976 had no effect on cell number in the zinc-deficient group. Proteolysis of the novel PKC family member, PKC-delta, to its 40-kDa catalytic fragment occurred in cells cultured in the zinc-deficient medium. Occurrence of the PKC-delta fragment in mitochondria was co-incident with caspase-3 activation. Addition of the PKC-delta inhibitor, rottlerin, or zinc to deficient medium reduced or eliminated proteolysis of PKC-delta, activated caspase-3 and restored cell number. Inhibition of caspase-3 processing by Z-DQMD-FMK (Z-Asp-Gln-Met-Asp-fluoromethylketone) did not restore cell number in the zinc-deficient group, but resulted in processing of full-length PKC-delta to a 56-kDa fragment. These results support the concept that intracellular zinc concentrations influence PKC activity and processing, and that zinc-deficiency-induced apoptosis occurs in part through PKC-dependent pathways.

Zinc influences the in vitro development of peri-implantation mouse embryos.

BACKGROUND: For humans, it is estimated that over 70% of concepti are lost during early development. In culture, mouse peri-implantation embryos can mimic development from the blastocyst to the egg cylinder stage of development, a period during which implantation occurs in vivo. We describe a novel application of this model to investigate nutritional factors that may influence this stage of development. We investigated the influence of zinc (Zn) deficiency on embryonic development at the time of embryo implantation. METHODS: Mouse blastocysts were cultured for 144 hr in low Zn, Zn-replete or control medium. RESULTS: Embryos developed normally when they were cultured in the control and Zn-replete media. Embryos cultured in the low Zn medium were significantly impaired in forming egg cylinder morphology. This was associated with a reduction in extraembryonic endoderm as determined by immunohistochemistry for markers of visceral and parietal endoderm and correlated with an increase in TUNEL positive cells in the low Zn group. There was no change in the frequency of cells positive for phosphorylated Histone H3, a marker for S-phase, indicating that an increase in apoptosis was primarily responsible for the smaller size and reduction in extraembryonic endoderm. The increased cell death was not associated with an increase in reactive oxygen species (ROS) detected by dichlorodihydrofluorescein staining. CONCLUSIONS: These data support an important role for Zn in promoting differentiation and cell survival in the early embryo and suggest that sub-optimal nutrition is an important factor that contributes to defects in primary germ layers and early embryonic loss.