Original ContributionThe potent vasodilator ethyl nitrite is formed upon reaction of nitrite and ethanol under gastric conditions
Introduction
During the last decade, a lot of attention has been given to the biochemistry of nitrite in the human organism [1], [2], [3], [4], [5]. It is now clear that in the body, the nonenzymatic reduction of nitrite to •NO can occur under acidic conditions [6] such as in the stomach lumen [7], [8], [9], in acidified urine [10], on the skin surface [11], [12] and in the oral cavity [13]. The equilibrium of nitrite with nitrous acid and other reactive nitrogen oxides at low pH is described by reactions 1 to 3:NO2- + H+ ⇆ HNO22HNO2 ⇆ •NO + •NO2 + H2O•NO + •NO2 ⇆ N2O3
Nitrite, the precursor of HNO2, derives from nutritional sources, from the reduction of dietary nitrate by oral commensal bacteria, and from oxidation of endogenous •NO [2], [5]. The stomach is continuously exposed to nitrite through the swallowing of saliva, which is particularly rich in nitrite. The general view is that nitrite is only harmful to the GI tract because it can promote the generation of carcinogenic nitrosamines [14], [15], [16], [17], [18]. However, with the recent discovery of in vivo generation of •NO from nitrite, a number of potentially beneficial effects are being revealed. These studies suggest a role for nitrite, •NO and its reaction products in host defense against gut pathogens [19], in regulation of gastric mucosal blood flow and mucous production [20], [21], and in protection against gastric ulcers [22].
Recently, we have shown that the consumption of red wine greatly increases the in vivo formation of •NO in the stomachs of healthy volunteers, via mechanisms that include the reduction of nitrite to •NO by wine polyphenols [23]. Ethanol represents another major constituent of wine that could potentially interact with nitrite under gastric conditions. From a chemical viewpoint, nitrite-derived reactive nitrogen species can react with aliphatic alcohols to yield alkyl nitrites by O-nitrosation of the corresponding alcohol [24]. Alkyl nitrites belong to the family of organic nitrites which are known for their potent vasodilatory activity. Considering that both, HNO2 [25] and N2O3 [26], [27] may undergo such a reaction (reaction 4 and 5) they are likely candidates for a nitrosation reaction of ethanol in the acidic stomach generating ethyl nitrite in vivo.H3O+ROH + HNO2 ⇆ RONO + H20ROH + N2O3 ⇆ RONO + HNO2
There have been a few earlier reports on the generation of ethyl nitrite in vivo. Macdonald and Jonsson detected ethyl nitrite in the breath of subjects who had been smoking and drinking concurrently, and the reaction of nitrogen oxides in the smoke with ethanol was suggested as its source [28]. More recently, Deng and colleagues showed that ethyl nitrite can be form in vivo after intraperitoneal administration of ethanol in rats and they proposed that its production proceeded via reaction of ethanol with peroxynitrite [29].
We aimed to study if ethyl nitrite is formed from alcoholic drinks and nitrite under simulated gastric conditions, and how such generation would be influenced by the concentration of nitrite and ethanol, pH, and the type of alcoholic drink (red wine or brandy). Moreover, we attempted to confer physiological relevance of such interaction by studying the vasoactivity of a nitrite:ethanol mixture.
Section snippets
Chemicals and buffer solutions
Ethanol p.a. (Ph.Eur) was obtained from Merck; ethyl nitrite (10%-20% in ethanolic solution) was obtained from Aldrich and quantified as described below; [1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) was obtained from Tocris Cookson (Bristol, UK), acetylcholine (ACh), Nω-nitro-l-arginine methyl ester (L-NAME), phenylephrine, sodium nitrite, sodium nitroprusside (SNP), glutathione (GSH) and diethylenetriaminepentaacetic acid (DTPA) were obtained from Sigma Chemicals Co. Unless otherwise
Ethyl nitrite detection and quantification
The electron impact ionization mass spectrum for ethyl nitrite in the mixture of 250 µM nitrite in 10% ethanol at pH 2.0 (Fig. 1) is consistent with those reported in the literature [29], [31]. Mass spectrum was acquired at retention time (RT) of 1.15 min and it is also consistent with that obtained from ethyl nitrite standard at pH 2.0 (data not shown), clearly identifying the production of ethyl nitrite in the nitrite:ethanol mixture.
Quantification of ethyl nitrite in a range of
Discussion
Nitrite is now emerging as an important alternative substrate for •NO generation in processes that are independent on •NO synthases. [1], [2], [3], [4], [5], [6]. •NO formation from nitrite proceeds via enzymatic as well as non-enzymatic pathways. Nonenzymatic acid-catalyzed reduction of nitrite to •NO is the predominate pathway in the stomach [6], [8], [9].
In a previous study we have shown that consumption of red wine increases intragastric •NO formation following the intake of nitrate and
Acknowledgments
This work was supported by FCT (Portugal) and FEDER, grant PTDCI/AGR-ALI/71262/2006, Vinnova (Chronic inflammation, KIDaT), EU’s 6th Framework Program (Eicosanox, LSMH-CT-2004-005003), Torsten and Ragnar Söderbergs Foundation, The Swedish research Council and Heart-Lung Foundation. Bruno Gago is a PDBEB student from Center for Neurosciences and Cell Biology (CNC) supported by fellowship SFRH/BD/15228/2004.
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