Bruno Gago

Dietary polyphenols generate nitric oxide from nitrite in the stomach and induce smooth muscle relaxation

Nitrite, considered a biological waste and toxic product, is being regarded as an important physiological molecule in nitric oxide (NO) biochemistry. Because the interaction of dietary phenolic compounds and nitrite would be kinetically (due to the high concentrations achieved) and thermodynamically (on basis of the redox potentials) feasible in the stomach, we have studied the potential reduction of nitrite by polyphenols present in several dietary sources. By measuring the time courses of *NO production in simulated gastric juice (pH 2), the efficiency of the compounds studied is as follows: Epicatechin-3-O-gallate>quercetin>procyanidin B8 dimer>oleuropein>procyanidin B2 dimer>chlorogenic acid>epicatechin>catechin>procyanidin B5 dimer. The initial rates of *NO production fall in a narrow range (ca. 1-5 microMs(-1)) but the distinct kinetics of the decay of *NO signals suggest that competition reactions for *NO are operative. The proof of concept that, in the presence of nitrite, phenol-containing dietary products induce a strong increase of *NO in the stomach was established in an in vivo experiment with healthy volunteers consuming lettuce, onions, apples, wine, tea, berries and cherries. Moreover, selected mixtures of oleuropein and catechin with low nitrite (1 microM) were shown to induce muscle relaxation of stomach strips in a structure-dependent way. Data presented here brings strong support to the concept that polyphenols consumed in a variety of dietary products, under gastric conditions, reduce nitrite to *NO that, in turn, may exert a biological impact as a local relaxant.

Dietary flavonoids with a catechol structure increase α-tocopherol in rats and protect the vitamin from oxidation in vitro

To identify dietary phenolic compounds capable of improving vitamin E status, male Sprague-Dawley rats were fed for 4 weeks either a basal diet (control) with 2 g/kg cholesterol and an adequate content of vitamin E or the basal diet fortified with quercetin (Q), (−)-epicatechin (EC), or (+)-catechin (C) at concentrations of 2 g/kg. All three catechol derivatives substantially increased concentrations of α-tocopherol (α-T) in blood plasma and liver. To study potential mechanisms underlying the observed increase of α-T, the capacities of the flavonoids to i) protect α-T from oxidation in LDL exposed to peroxyl radicals, ii) reduce α-tocopheroxyl radicals (α-T · ) in SDS micelles, and iii) inhibit the metabolism of tocopherols in HepG2 cells were determined. All flavonoids protected α-T from oxidation in human LDL ex vivo and dose-dependently reduced the concentrations of α-T · . None of the test compounds affected vitamin E metabolism in the hepatocyte cultures. In conclusion, fortification of the diet of Sprague-Dawley rats with Q, EC, or C considerably improved their vitamin E status. The underlying mechanism does not appear to involve vitamin E metabolism but may involve direct quenching of free radicals or reduction of the α-T · by the flavonoids.

Intragastric nitration by dietary nitrite: implications for modulation of protein and lipid signaling.

Inorganic nitrite, derived from the reduction of nitrate in saliva, has recently emerged as a protagonist in nitric oxide ((•)NO) biology as it can be univalently reduced to (•)NO, in the healthy human stomach. Important physiological implications have been attributed to nitrite-derived (•)NO in the gastrointestinal tract, namely modulation of host defense, blood flow, mucus formation and motility. At acidic pH, nitrite generates different nitrogen oxides depending on the local microenvironment (redox status, gastric content, pH, inflammatory conditions), including (•)NO, nitrogen dioxide ((•)NO(2)), dinitrogen trioxide (N(2)O(3)), and peroxynitrite. Thus, the gastric environment is a significant source of nitrating and nitrosating agents, especially in individuals consuming a nitrate/nitrite-rich diet on a daily basis. Both, the gastric lumen and mucosa contain putative targets for nitration, not only proteins and lipids from ingested aliments but also endogenous proteins secreted by the oxyntic glands. The physiological and functional consequences of nitration of gastric mediators will impact on local processes including food digestion and ulcerogenesis. Additionally, gastric nitration products (such as nitrated lipids) may be absorbed and affect systemic pathways. Thus, dietary ingestion of nitrate will have direct consequences for endogenous protein nitration, as indicated by our preliminary data.

Dietary Nitrite in Nitric Oxide Biology: A Redox Interplay with Implications for Pathophysiology and Therapeutics

Until recently, nitrite has been considered a stable oxidation inert metabolite of nitric oxide ((∙)NO) metabolism. This view is now changing as it has been shown that nitrite can be reduced back to (∙)NO and thus one may consider a reversible interaction regarding (∙)NO:nitrite couple. Not only physiological regulatory actions have been assigned to nitrite but also may represent, in addition to nitrate, the largest (∙)NO reservoir in the body. This notion has obvious importance when considering that (∙)NO is a ubiquitous regulator of cell functions, ranging from neuromodulation to the regulation of vascular tone. Particularly in the stomach, following ingestion of nitrate and food or beverages-containing polyphenols, a rich chemistry occurs in which (∙)NO, (∙)NO-derived species and nitroso or nitrated derivatives may be formed. Most of these molecules may play an important role in vivo. For instance, it has been shown that polyphenol-catalyzed nitrite reduction to (∙)NO may induce local vasodilation and that ethanol (from wine) reacts with (∙)NO-derived species yielding nitroso derivatives endowed with (∙)NO-donating properties. Thus, this review reveals new pathways for the biological effects of dietary nitrite encompassing its interaction with dietary components (polyphenols, red wine, lipids), yielding products with impact on human physiology and pathology, namely cardiovascular, urinary and gastrointestinal systems. Novel therapeutic strategies are therefore expected to follow the elucidation of the mechanisms of nitrite biology.

The potent vasodilator ethyl nitrite is formed upon reaction of nitrite and ethanol under gastric conditions

By acting as a bioreactor, affording chemical and mechanical conditions for the reaction between dietary components, the stomach may be a source of new bioactive molecules. Using gas chromatography-mass spectrometry we here demonstrate that, under acidic gastric conditions, ethyl nitrite is formed in microM concentrations from the reaction of red wine or distilled alcoholic drinks with physiological amounts of nitrite. Rat femoral artery rings and gastric fundus strips dose-dependently relaxed upon exposure to nitrite:ethanol mixtures. In contrast, when administered separately in the same dose ranges, nitrite evoked only minor vasorelaxation while ethanol actually caused a slight vasoconstriction. Mechanistically, the relaxation effect was assigned to generation of nitric oxide (*NO) as supported by direct demonstration of *NO release from ethyl nitrite and the absence of relaxation in the presence of the soluble guanylyl cyclase inhibitor, ODQ. In conclusion, these results suggest that ethanol in alcoholic drinks interacts with salivary-derived nitrite in the acidic stomach leading to the production of the potent smooth muscle relaxant ethyl nitrite. These findings reveal an alternative chemical reaction pathway for dietary nitrate and nitrite with possible impact on gastric physiology and pathophysiology.

LDL Isolated from Plasma-Loaded Red Wine Procyanidins Resist Lipid Oxidation and Tocopherol Depletion

Dietary phenolic compounds may act as antioxidants in vitro, but because of structural modifications during absorption, its role based on concentrations high enough to afford an antioxidant protection needs to be re-evaluated. We have explored the hypothesis that red wine procyanidins interact with low density lipoproteins (LDL) and that, at this location, the phenolic compounds efficiently protect LDL from oxidation and maintain LDL alpha-tocopherol at a high steady state concentration by recycling it back from the alpha-tocopheroxyl radical. To this end, human plasma was supplemented with wine procyanidins and isolated LDL were challenged with a constant flux of peroxyl radicals. As compared with LDL from plasma-free procyanidins, those LDL better resisted lipid oxidation and exhibited longer lag-phases of alpha-tocopherol consumption. The procyanidins, depending on their structure, were able to reduce the UV-induced alpha-tocopherol radical in a micellar system, as evidenced by electron paramagnetic ressonance. Mechanistically, the protection of LDL was interpreted in terms of quenching of peroxyl radicals and the recycling of alpha-tocopherol by the procyanidins bound to the lipoproteins. These results support the notion that, in human plasma, the procyanidins, via binding to LDL, may act as efficient local antioxidants.

Diffusion of nitric oxide through the gastric wall upon reduction of nitrite by red wine: physiological impact.

In this work we showed that nitric oxide produced via red wine- and ascorbate-dependent reduction of nitrite diffuses through the rat stomach, inducing smooth muscle relaxation. The studies encompassed ex vivo and in vivo models of diffusion. Regarding the former, luminal *NO generated from a mixture of physiologic nitrite and ascorbate or wine diffuses across the stomach wall, being 8-20% of that produced in the mucosal side detected at high microM range (>100 microM) in the serosal side. In order to evaluate whether cellular dysfunction was associated with *NO diffusion at the microM range, the gastric tissue exposed to *NO was evaluated in terms of carbachol-induced muscle contraction in fundal strips and mitochondrial respiration and showed to remain functional and metabolically active. Moreover, pre-contracted gastric strips were shown to relax 86.5+/-5.5% (control) and 75.0+/-4.0% (nitrite/ascorbate-exposed tissue) when challenged with acidified nitrite. The studies in the living animal support the diffusion of luminal *NO to the gastric vasculature as, following addition of nitrite/ascorbate to rat stomach in vivo, *NO was not detected in the serosal environment but concentrations as high as 31 microM of *NO were detected outside the stomach after cardiac arrest. Collectively, the results establish a link between the consumption of nitrite and dietary reductants (e.g., wine polyphenols) and stomach muscle relaxation via the local chemical generation of *NO.

Pepsin is nitrated in the rat stomach, acquiring antiulcerogenic activity: A novel interaction between dietary nitrate and gut proteins

Dietary nitrate is reduced to nitrite and nitric oxide ((•)NO) in the gut, producing reactive species able to nitrate proteins and lipids. We investigated intragastric production of (•)NO and nitrating agents in vivo by examining selective nitration of pepsinogen and pepsin. We further addressed the functional impact of nitration on peptic activity by evaluating the progression of secretagogue-induced ulcers. Pepsinogen nitration was assessed in healthy and diclofenac-induced ulcerated rat stomachs. Both groups were fed nitrite or water by oral gavage. Protein nitration was studied by immunofluorescence and immunoprecipitation. In parallel experiments, pentagastrin was administered to rats and nitrite was then instilled intragastrically. (•)NO levels were measured before and after nitrite administration by chemiluminescence. Macroscopic damage was assessed and nitrated pepsin was examined in the margin of ulcers. Protein nitration was detected physiologically in the stomach of healthy animals. Nitrite had a dual effect on intragastric nitration: overall nitration was decreased under physiological conditions but enhanced by acute inflammation. Pepsin and pepsinogen were also nitrated via a nitrite-dependent pathway. Nitration of both pepsin and its zymogen led to decreased peptic activity in response to classical substrates (e.g., collagen). Under conditions of acute ulceration, nitrite-dependent pepsin nitration prevented the development of gastric ulcers. Dietary nitrite generates nitrating agents in the stomach in vivo, markedly decreasing peptic activity. Under inflammatory and ulcerogenic conditions pepsin nitration attenuates the progression of gastric ulceration. These results suggest that dietary nitrite-dependent nitration of pepsin may have a novel antiulcerogenic effect in vivo.

Methicillin-resistant Staphylococcus aureus carrying the new mecC gene--a meta-analysis.

In 2011, a new mecA gene homolog, named mecC gene, was found in isolates from both humans and animals. The discovery of methicillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene has caused speculations about the origin, epidemiology, and impact of these isolates. The objective of this work is to perform a meta-analysis on the prevalence of mecC MRSA, based on previously published results. Meta-analysis showed that the overall pooled prevalence is 0.009% (95% confidence interval=0.05-0.013) and that there was evidence of heterogeneity (P<0.01, I(2)=97%). In conclusion, the very low reported prevalence provides an important baseline to monitor the epidemiology of this emerging form of MRSA.

Ethyl nitrite is produced in the human stomach from dietary nitrate and ethanol, releasing nitric oxide at physiological pH: potential impact on gastric motility

BACKGROUND Nitric oxide ((∙)NO), a ubiquitous molecule involved in a plethora of signaling pathways, is produced from dietary nitrate in the gut through the so-called nitrate-nitrite-NO pathway. In the stomach, nitrite derived from dietary nitrate triggers a network of chemical reactions targeting endogenous and exogenous biomolecules, thereby producing new compounds with physiological activity. OBJECTIVE The aim of this study was to ascertain whether compounds with physiological relevance are produced in the stomach upon consumption of nitrate- and ethanol-rich foods. DESIGN Human volunteers consumed a serving of lettuce (source of nitrate) and alcoholic beverages (source of ethanol). After 15 min, samples of the gastric headspace were collected and ethyl nitrite was identified by GC-MS. Wistar rats were used to study the impact of ethyl nitrite on gastric smooth muscle relaxation at physiological pH. RESULT Nitrogen oxides, produced from nitrite in the stomach, induce nitrosation of ethanol from alcoholic beverages in the human stomach yielding ethyl nitrite. Ethyl nitrite, a potent vasodilator, is produced in vivo upon the consumption of lettuce with either red wine or whisky. Moreover, at physiological pH, ethyl nitrite induces gastric smooth muscle relaxation through a cGMP-dependent pathway. Overall, these results suggest that ethyl nitrite is produced in the gastric lumen and releases (∙)NO at physiological pH, which ultimately may have an impact on gastric motility. Systemic effects may also be expected if ethyl nitrite diffuses through the gastric mucosa reaching blood vessels, therefore operating as a (∙)NO carrier throughout the body. CONCLUSION These data pinpoint posttranslational modifications as an underappreciated mechanism for the production of novel molecules with physiological impact locally in the gut and highlight the notion that diet may fuel compounds with the potential to modulate gastrointestinal welfare.