The Nitrate-Nitrite-Nitric Oxide Pathway

Nathan S. Bryan

Institut of molecular medicine, Health Science Center University of Texas Houston USA

The short-lived, free radical molecule nitric oxide (NO) has emerged as one of the most versatile cell signaling transmitters produced by mammalian biological systems. The discovery of the formation of NO from the semi-essential amino acid L-arginine through one of three isoforms of nitric oxide synthase provided a key therapeutic target, which is still the focus of much research today. Recently, the oxidative ‘waste’ products of nitric oxide, nitrite and nitrate, have been evaluated in a new context, due to their own ability form NO independent of nitric oxide synthase enzymes, through reductive electron exchanges. Since nitrate (as well as nitrite) are primarily ingested in the form of fruits and vegetables, which have been known for some time to protect against diseases from atherosclerosis to cancer, a new paradigm has emerged regarding the role of these once feared nitrogen oxides. There is now a recognized human nitrogen cycle consisting of commensal bacteria in the oral cavity, which serve a reductive role in the conversion of approximately 20% of ingested nitrate to nitrite, now appears to provide a significant NOS-independent source of NO generation. This new paradigm may have revolutionary implications in terms of developing strategies to combat heart disease and many other
contemporary diseases associated with a NO deficiency. Perhaps now we should consider nitrite and nitrate as the bioactive food components that account for the protective phenotype of certain foods and diets. Recent work has shown various cardioprotective effects from modest supplementation of nitrite and nitrate. Nitrite, in particular, has been shown to prevent hypercholesterolemic microvascular inflammation and protect against injury from ischemic events. The broader context of research regarding nitrate, nitrite, and nitric oxide suggests these simple nitrogen oxides serve as a critical dietary component for protection against various chronic diseases. Currently, heart disease and cancer lead the nation in cause of deaths. Concurrently, the dietary patterns of the West have transitioned towards heavily processed foods and lack significant quantities of fruits and vegetables. The explanations have been varied but overlook simple molecules known to play critical roles in multiple organ systems through the chemical messenger NO. The dietary contributions to normal NO homeostasis would not only help explain significantly lower rates of cardiovascular disease in those who regularly consume fruits and vegetables, but also arm scientists and physicians with a relatively simple and inexpensive therapeutic intervention. The objective of this presentation is to review the important roles nitrite and nitrate play in biological systems and NO homeostasis. A risk benefit analysis will be discussed to show nitrite and nitrate present no danger when consumed in modest quantities and preferably with antioxidants. In fact, research appears to suggest nitrite acts as a redundant NO reservoir when NOS activity is insufficient or stress requires a secondary source. The future use of nitrite/nitrate in dietary considerations will likely have a significant impact on current public health policy.

Monoxyde d'azote, nitrate et nitrite dans le rein

Dussaule JC, Guerrot D, Placier S, Boffa JJ, Chatziantoniou C
Unité UMRS 702 Inserm et UPMC (Paris)

Il est largement démontré que le monoxyde d'azote (NO) est un facteur essentiel de la régulation de l'hémodynamique rénale. Le blocage pharmacologique de sa synthèse endothéliale entraîne une néphropathie vasculaire comportant une composante inflammatoire et une composante fibrotique qui concourent à la perte fonctionnelle du rein. Dans le laboratoire, nos travaux pharmacologiques ont porté sur le modèle de carence en NO par administration de L-NAME qui inhibe l'activité de la NO-synthase chez le rat et la souris. Dans ce modèle expérimental, nous avons caractérisé deux marqueurs précoces, d'origine vasculaire, de la néphropathie : l'E-sélectine et la périostine. L'action pro-inflammatoire et profibrosante du L-NAME est prévenue par les antagonistes des récepteurs de l'angiotensine II et de l'endothéline, ce qui témoigne de l'équilibre entre facteurs rénaux vasodilatateurs et vasoconstricteurs dans les mécanismes physiopathologiques rénaux. La production rénale de NO est donc un élément clé de la prévention des néphropathies vasculaires. Les agents qui bloquent sa production comme l'ADMA (asymmetric dimethylarginine) qui s'accumule au cours de l'insuffisance rénale chronique, ont un effet pathogène certain. A l'inverse, certains travaux expérimentaux suggèrent que la supplémentation en nitrites et nitrates, en favorisant la synthèse de NO, aurait un effet protecteur contre la progression des maladies rénales chroniques.

Interaction of mitochondrial Complex I with NO

Alexander Galkin

Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast, BT9 7BL, UK

Mitochondrial complex I plays a critical role in regulating cellular energy generation and the production of reactive oxygen species (ROS). Two catalytically and structurally distinct forms of mitochondrial complex I have been characterised in enzyme preparations: one is a fully catalytically competent, active (A)-form and the other is a dormant, silent or de-activated (D)-form. When deprived of substrate, at physiological temperatures the idle enzyme undergoes conversion into the D-form. This can gradually convert back to the A-form in the presence of substrate (NADH and ubiquinone) during slow turnover(s) of the enzyme. In the D-form of complex I a critical cysteine-39 of the ND3 subunit becomes exposed to the outside of the enzyme and is susceptible to covalent modification. Previously we have found that the conformational state (A or D) of complex I is an important factor for the interaction of the enzyme with NO-metabolites in vitro, since only the D-form was susceptible to modification by nitrosothiols and peroxynitrite resulting in inhibition of respiration. Recently, we showed that the A-to-D transition occurs in cells and tissues deprived of oxygen when the respiratory chain is reduced. We demonstrated that re-activation of the accumulated D-form could be prevented by treatment with NO-donors or endogenously-generated nitric oxide (NO). Therefore, in some circumstances in situ, presence of NO may lead to modification of complex I when it is in its D-form and so impede its return to the active state. Indeed, accumulation of the covalently modified D-form is likely to be responsible for the so-called persistent inhibition of cellular respiration that occurs in cells when NO is present. The detrimental effect of such irreversible locking of complex I in the D-form could be because of the decrease in overall respiration rate and due to the fact that the modified D-form of the enzyme generates ROS at a higher rate than the A-form. Thus, a combination of changes in mitochondrial ROS production, a change in NAD/NADH ratio and a decline in the rate of oxidative phosphorylation could lead to cellular death and might be responsible for ischaemic damage as well as for the early stages of neurodegeneration.

Nitrate and Host Defence

Nigel Benjamin
Honorary Professor of Medicine
Peninsula School of Medicine and Dentistry
University of Exeter, UK

Exposure of humans to inorganic nitrate is inevitable. Every day, on average we synthesise approximately 1 millimole of nitrate from the oxidation of nitric oxide, which is continually generated in the vascular endothelium and neuronal tissues from the amino acid L-arginine. Following infections, and particularly gastroenteritis, the synthesis of nitric oxide increases, up to ten fold due to increased nitric oxide manufacture by inflammatory cells.We now know that nitric oxide is important in protecting us from a range of microbial pathogens. We also are exposed to 1-3millimoles of inorganic nitrate in our diet. Green, leafy vegetables and some root vegetables (such as beetroot) contain large amounts. This nitrate, together with that synthesised from nitric oxide, undergoes a complex reprocessing to allow more nitric oxide to be made. This process, known as the enterosalivary circulation of nitrate is proving to be important in maintaining cardiovascular health as well as protecting us from infection. Nitrate is concentrated at least ten-fold in salivary glands and on the surface of the tongue is converted to nitrite by facultative anaerobic bacteria which use nitrate as an alternative electron acceptor. This nitrite is swallowed and then, when acidified in the stomach, generates a complex mix of nitrogen oxides. We have shown that this mix of nitrogen oxides, including nitric oxide, is effective in killing a wide range of potential pathogens which cause food-borne illness, including C.Difficile, Salmonella typhi, Shigella, Campylobacter and E.Coli O157.Understanding of this mechanism suggests that increasing nitrate intake may be important in preventing illness from swallowed pathogens, and also suggests that that the reason antibiotic therapy may predispose towards C.Difficile infections may be due to prevention of oral conversion of nitrate to nitrite by tongue symbionts. We have also shown that nitrate in sweat may be important in protecting the skin from bacterial and fungal infection, and others have suggested that nitric oxide synthesis via bacterial reduction of the large amount of nitrate in urinereduction may have a rôle in preventing urine infections in susceptible patients. Future research will determine whether enhancing dietary nitrate intake is effective in augmenting host
defence against pathogens by increasing nitrogen oxide synthesis.

Nitrate, endogenous nitrosation and colorectal cancer risk

Theo M. de Kok,
Department of Toxicogenomics, Maastricht University, The Netherlands.

Increased intake of nitrate in drinking water has been shown to raise endogenous formation on N-nitroso compounds (NOC). Epidemiological studies have shown that dietary factors linked to the stimulation of endogenous nitrosation, a process resulting in the formation of this class of compounds, are associated with increased risk of various cancers, including colorectal cancer. Although NOC are known rodent carcinogens, there is only very limited direct evidence for a carcinogenic potential of NOC in humans. In a series of human studies we established that at physiologically relevant exposure levels in vivo, NOCexposure is associated with validated markers of carcinogenicity, and induces gene expression profiles in colonic tissue that are relevant in the carcinogenic process. These findings indicate that NOC exposure may be relevant in the development of cancer, thereby mechanistically linking nitrate intake to human cancer risk.

Méthémoglobinémie du nourrisson. Un seuil bactériologique.

Dr Jean-Louis L’hirondel
Rhumatologie, CHU Caen, France

Les autorités sanitaires internationales, notamment la Commission de l'Environnement de la Santé publique et de la Sécurité alimentaire (ENVI) du Parlement européen et l’Organisation Mondiale de la Santé (OMS) considèrent que les nitrates de l’eau de boisson constituent un danger sanitaire ; selon ces instances, les nitrates de l’eau de boisson seraient transformés en nitrites dans l’organisme, et les nitrites transforment l’hémoglobine des globules rouges en méthémoglobine ; d’où un risque de méthémoglobinémie du nourrisson. L’auteur montre qu’en fait, ce raisonnement en viendrait à interdire toute préparation alimentaire à base de légumes, souvent très riche en nitrates. En réalité, les nitrates de l’eau ou de la préparation alimentaire du nourrisson ne pourraient éventuellement être réduits en nitrites, que si une condition impérative était respectée, à savoir, que la préparation alimentaire soit bactériologiquement contaminée et contienne plus de 106 ou 107 germes ml-1. Il convient donc avant tout de garantir strictement les conditions sanitaires, quelles que soient les teneurs en nitrates de la préparation alimentaire.

Dietary nitrate and exercise performance

Andrew M Jones
Sport and Health Sciences, College of Life and Environmental Sciences, St. Luke’s Campus, University of
Exeter, Exeter EX1 2LU, United Kingdom

Nitric oxide (NO) is an important physiological signaling molecule that may modulate skeletal muscle function through its role in the regulation of blood flow, muscle contractility, glucose and calcium homeostasis, and mitochondrial biogenesis and respiration. In recent studies, we have shown that enhancing NO bioavailability through dietary nitrate supplementation reduces the O2 cost of exercise and improves exercise performance. In our first study (1), we found that 4-6 days of dietary nitrate supplementation (0.5 L of beetroot juice per day containing ~ 6 mmol nitrate) reduced the ‘steady-state’ O2 cost of sub-maximal cycle exercise by 5% and extended the time-to-exhaustion during high-intensity cycling by 16%. In a follow-up study (2), we used 31P- magnetic resonance spectroscopy (MRS) to investigate the mechanistic bases of this phenomenon. We found that the same dietary nitrate supplementation regimen as used in our first study resulted in both a reduced pulmonary O2 uptake and a reduced muscle metabolic perturbation (i.e., blunted changes in muscle phosphocreatine, adenosine diphosphate and inorganic phosphate concentrations) enabling high-intensity knee-extension exercise to be tolerated for a greater period of time. The estimated total ATP turnover rate was reduced by ~ 30% during both low-intensity and high-intensity exercise. These data imply that the reduced O2 cost of
exercise following dietary nitrate supplementation is related to a reduced ATP cost of muscle force production, although concurrent changes in the efficiency of mitochondrial respiration are also possible. We have also demonstrated that the positive effects of nitrate supplementation on muscle efficiency can be manifest acutely (i.e. 2.5 hours following a 6 mmol nitrate ‘bolus’) and that this effect is maintained if supplementation at the same dose is maintained for 15 days (3). Because beetroot juice contains compounds other than nitrate that might also be bioactive, we have developed a nitrate-depleted beetroot juice as a placebo (4). We found that nitrate-depleted beetroot juice had no physiological effects relative to a control condition whereas nitrate-rich beetroot juice reduced the O2 cost of both walking and running
and extended the time-to-exhaustion by 15% (4). Most recently, we have investigated the influence of acute dietary nitrate supplementation on 4 km and 16.1 km time trial (TT) performance in competitive cyclists (5). We found that cyclists were able to produce a greater power output for the same rate of pulmonary O2 uptake, resulting in a 2.7% reduction in the time to complete both TT distances. Collectively, our studies indicate that dietary nitrate supplementation profoundly and consistently reduces the O2 cost of physical activity, and enhances exercise performance. While these findings are clearly of considerable interest to athletes, it is possible that clinical populations and the elderly may also benefit if dietary nitrate intake can be shown to reduce the O2 cost of the ‘activities of daily living’.

1. Bailey SJ et al. J Appl Physiol. 107:1144-55, 2009.
2. Bailey SJ et al. J Appl Physiol. 109:135-48, 2010.
3. Vanhatalo A et al. Am J Physiol Regul Integr Comp Physiol. 299:R1121-31, 2010.
4. Lansley KE et al. J Appl Physiol. 2010 [Epub ahead of print].
5. Lansley KE et al. Med Sci Sports Exerc. 2011 [Epub ahead of print].

A propos de quelques erreurs ou imprécisions sur les nitrates dans la
littérature scientifique

Dr. Jean-Louis L’hirondel
Rhumatologie, CHU Caen, France

L’auteur recense les erreurs ou imprécisions sur les nitrates qu’il a pu déceler dans les articles scientifiques parus au cours des dernières années. Il décrit des omissions de certains champs du savoir, des conceptions erronées du métabolisme des nitrates, une méconnaissance du constat de Donahoe (1949), c’est-à-dire de l’absence de lien statistique entre les taux en nitrates de l’eau de puits et le risque méthémoglobinémie du nourrisson, enfin des erreurs méthodologiques à l’occasion d’études épidémiologiques sur la responsabilité éventuelle que les apports en nitrates par l’intermédiaire de l’alimentation solide ou de l’eau de boisson sont censés avoir dans l’apparition d’effets défavorables au long cours.

Nitrate and Nitrite in the WHO Guidelines for Drinking Water Quality

John Fawell,
Member of the Expert Committee on the WHO Guidelines for Drinking-water Quality

Nitrate, and to a lesser extent nitrite, have long been known to be anthropogenic contaminants in drinking water sources. Nitrite can be easily converted to nitrate but nitrate is difficult and expensive to remove from drinking water, particularly groundwater. As a consequence a great deal of research of varying quality has been directed at the potential adverse health effects of nitrate. The Joint WHO/FAO Expert Committee on Food Additives and Contaminants (JECFA) primarily used data from experiments in laboratory animal in developing tolerable daily intakes (TDI) for nitrate and nitrite. However, it is very important to consider whether there are differences in metabolism that mean that the animal model is or is not appropriate for extrapolation to humans. Equally the epidemiology database that has considered a number of endpoints provides equivocal evidence for long-term health effects such as cancer, although there is credible evidence for a plausible mechanism through nitrosation. WHO has a long standing guideline value of 50 mg/litre of nitrate which is based on methaemoglobinaemia in bottle-fed infants. The guideline for nitrite is 3 mg/litre on the same basis but the guidelines require that both are taken into consideration. However, WHO recognises the uncertainty in the data on methaemoglobinaemia and the complicating factor of infantile diarrhoea, and recommends that this value should not be seen as an absolute cut-off; providing guidance on applying the guideline value flexibly. Currently WHO regards the guideline value as a sensible and valid compromise in the face of conflicting scientific evidence but continues to monitor new studies as they emerge.

 Compte Rendu Coordination Rurale Centre - Colloque Nitrate ~ Nitrite ~ Oxyde nitrique