Nitrates in the human diet - good or bad?
`Nigel Benjamin
`
`To cite this version:
`Nigel Benjamin. Nitrates in the human diet - good or bad?. Annales de zootechnie, INRA/EDP
`Sciences, 2000, 49 (3), pp.207-216. ￿10.1051/animres:2000118￿. ￿hal-00889892￿
`
`HAL Id: hal-00889892
`https://hal.archives-ouvertes.fr/hal-00889892
`Submitted on 1 Jan 2000
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`Ann. Zootech. 49 (2000) 207–216
`© INRA, EDP Sciences
`
`207
`
`Review article
`
`Nitrates in the human diet – good or bad?
`
`Nigel BENJAMIN*
`
`Department of Clinical Pharmacology, Charterhouse Square, St.Bartholomew’s and The Royal
`London School of Medicine and Dentistry, London EC1M 8BQ, UK
`
`(Received 15 November 1999; accepted 17 April 2000)
`
`Abstract — Although the nitrate and nitrite have been used for centuries, it has only recently been
`discovered that nitrate is manufactured in mammals by the oxidation of nitric oxide and that the
`nitrate formed also has the potential for disinfecting the food we eat. The mechanisms by which
`nitric oxide and other nitrogen oxides provide selective toxicity towards pathogens is not yet completely
`understood, and it is likely that the mechanisms will be different with different organisms. Whereas
`it is clear that acidified nitrite is produced on mucosal surfaces, and that this combination is effective
`in killing a variety of human gut and skin pathogens, there is no definite evidence as yet that this mech-
`anism is truly protective in humans exposed to a contaminated environment. Further understanding
`of the complex chemistry of nitrogen oxides may also help develop new antimicrobial therapies
`based on augmenting what seems to be a simple and effective host defence system.
`
`nitrates / nitrites / human diet / health
`
`Résumé — Les nitrates dans l’alimentation humaine : une bonne ou une mauvaise chose ?
`Bien que les nitrates soient connus depuis des siècles, c’est seulement depuis peu qu’il a été décou-
`vert que les nitrates sont synthétisés chez les mammifères à partir du monoxyde d’azote, et que les
`nitrates formés ont la capacité de tuer les bactéries présentes dans les aliments consommés. Les
`mécanismes par lesquels le monoxyde d’azote et d’autres oxydes d’azote manifestent une toxicité sélec-
`tive vis-à-vis de bactéries pathogènes ne sont pas encore totalement élucidés. Il est probable que
`ces mécanismes dépendent des organismes considérés. Alors qu’il est clair que les nitrites acidifiés
`sont produits à la surface des muqueuses, et que cela permet de tuer de nombreux agents pathogènes
`de la peau et de l’intestin de l’Homme, une incertitude demeure quant aux mécanismes de protection
`réels de l’Homme exposé à un environnement contaminé. Une meilleure compréhension de la chimie
`complexe des oxydes d’azote permettra d’aider au développement de nouvelles thérapies antibacté-
`riennes fondées sur l’accroissement de ce qui semble être un système de défense de l’hôte simple et
`efficace.
`
`nitrates / nitrites / alimentation humaine / santé humaine
`
`* Correspondence and reprints
`e-mail: n.benjamin@qmw.ac.uk
`
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`208
`
`N. Benjamin
`
`1. INTRODUCTION
`
`There has been considerable controversy
`concerning the possible harmful effects of
`nitrate in our diet and in the environment.
`Nitrate has, in general, been considered
`harmful, although the evidence for this has
`become less convincing with time. Recently,
`since the discovery of nitric oxide as an
`important biological molecule which regu-
`lates many bodily functions and provides
`host defence against numerous micro-organ-
`isms, the image of nitrate has, to some extent
`been rehabilitated. This is because of the
`discovery of a novel biochemical pathway
`which can lead to the formation of large,
`possibly protective, amounts of nitric oxide
`in mammals from the sequential reduction of
`inorganic nitrate. The purpose of this paper
`is to consider the possible harmful and ben-
`eficial properties of inorganic nitrates which
`are an essential component of biological
`systems and which are encountered in large
`amounts in our diet every day.
`–) have been
`Inorganic nitrates (NO3
`added as a food preservative, especially pig
`meat, to make ham and bacon for centuries.
`As well as its beneficial effect to limit the
`growth of serious pathogens such as
`Clostridium botulinum [30], nitrate, or more
`specifically its reduction product nitrite
`–) has also the dubious benefit of ren-
`(NO2
`dering muscle tissue a bright pink colour,
`by the formation of nitrosomyoglobin. It
`has subsequently become clear that nitrate is
`generally non-reactive with organic
`molecules and has to be chemically or enzy-
`matically reduced to nitrite to be effective as
`an antimicrobial agent [5]. Nitrate is also
`found in large quantities in green, leafy veg-
`etables such as lettuce and spinach, partic-
`ularly when grown under low light condi-
`tions (see below and Tab. I).
`Despite its long use, there have been con-
`siderable concerns about the use of nitrate
`and nitrite as a preservative in food and
`about the content of these ions in vegeta-
`bles and drinking water. There are two rea-
`sons for this.
`
`Table I. Contribution (%) of various foodstuffs
`to dietary intake of nitrate and nitrite (after Com-
`mittee on Nitrite and Alternative Curing Agents
`in Food [7]).
`
`Food [30]
`
`Nitrate
`
`Nitrite
`
`Cured meats
`Fresh meats
`Vegetables
`Fruit / juices
`Baked foods / cereals
`Milk / milk products
`Water
`
`1.6
`0.8
`87
`6
`1.6
`0.2
`2.6
`
`39
`7.7
`16
`1.3
`34
`1.3
`1.3
`
`Firstly, the theoretical possibility of form-
`ing carcinogenic N-nitroso compounds in
`food to which these ions are added and in
`humans in vivo, due to nitrosation of sec-
`ondary amines also present in the diet or
`ingested as drug therapy [37]. Nitrosation
`of amines and other chemicals will occur
`rapidly under acidic conditions (such as in
`the human stomach) when nitrite is present,
`due to the formation of nitrous acid [42]. It
`will also occur in the stomach under more
`neutral pH when there are bacteria present.
`The mechanism of this presumably enzy-
`matic nitrosation is not understood.
`
`Nitrous acid is an effective nitrosating
`agent due to its ability to donate an NO+
`group. These nitrosation reactions are catal-
`ysed by halide ions (such as chloride) and
`thiocyanate due to the formation of nitroso-
`halide (such as NOCl) and nitrosothio-
`cyanate (NOSCN) respectively, and these
`intermediates will more effectively donate
`NO+ groups. Both chloride and thiocyanate
`ions are present in gastric juice in high con-
`centrations, the latter is derived from the
`diet (particularly brassicas such as cabbage)
`and is concentrated in saliva. Nitrate in the
`diet is similarly concentrated in saliva fol-
`lowing absorption and reduced to nitrite in
`the mouth. It therefore seems that the stom-
`ach is an ideal reaction chamber for the
`nitrosation of susceptible swallowed chem-
`icals.
`
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`Nitrates in the human diet – good or bad?
`
`209
`
`This scheme of human metabolism of
`nitrate and nitrite seems to be counterpro-
`ductive for human health and has prompted
`a reconsideration of exactly how these ions
`are handled following ingestion. We have
`formulated a scheme whereby the forma-
`tion of nitrogen oxides in the stomach and on
`the skin surface, derived from dietary nitrate
`and nitrite, may protect against bacterial,
`and possibly viral, pathogens.
`A second potential mechanism for nitrite
`toxicity is the formation of methaemoglobin
`due to oxidation of the iron in haemoglobin.
`This is due to the reaction of nitrite with
`oxyhaemoglobin, where it is oxidised to
`form nitrate. It should also be noted that
`nitric oxide also reacts quickly with oxy-
`haemoglobin to produce methaemoglobin
`and nitrate. This may be more important
`than nitrite in causing this problem in infants
`fed on well water.
`Methaemoglobinaemia is generally only
`a problem in young infants, but has been
`the main reason for statutory limitations on
`the concentrations of nitrate in drinking
`water. For nitrate to cause methaemoglobi-
`naemia it has to be reduced to nitrite in food,
`water or in the body. The mechanisms by
`which this may occur will be considered in
`the next section.
`
`2. NITRATE METABOLISM
`BY BACTERIA AND PLANTS
`
`In nature, nitrogen is continually cycled
`between nitrogen gas, and the fully oxidised
`nitrogen molecule – nitrate and the fully
`reduced nitrogen molecule – ammonia.
`Plants and bacteria have somewhat different
`uses for nitrogen molecules. In general,
`plants need nitrogen as ammonia as a pre-
`cursor to protein synthesis. They have the
`appropriate enzymes to reduce nitrate
`through to ammonia, a process requiring
`energy provided by photosynthesis. This
`occurs in at least two distinct steps catal-
`ysed by separate enzymes – nitrate reduction
`
`to nitrite (nitrate reductase) and then nitrite
`reduction to ammonia (nitrite reductase).
`Nitrate is present in the soil due to organic
`decomposition or the fixing of atmospheric
`nitrogen by nitrogen-fixing bacteria which
`may be associated with roots of certain legu-
`minous plants. Nitrate is commonly stored in
`the leaves of vegetables such as lettuce and
`spinach, and accumulates to very high con-
`centrations under low light conditions. This
`is presumably because of a reduction in the
`flux of nitrate through to ammonia when
`insufficient energy is available from pho-
`tosynthesis. As a result, especially in cool
`climates, green vegetables contribute to our
`nitrate intake more than any other single
`source [6].
`Although some micro-organisms will
`also reduce nitrate to ammonia for protein
`synthesis, many also use nitrate reduction
`for the purpose of anaerobic respiration. The
`enterobacteriacae such as E. coli can switch
`from using oxygen to burn available fuel to
`using nitrate as an electron acceptor (or oxi-
`dant) which is then converted to nitrite. The
`facultative anaerobes which inhabit the
`human mouth allow little further reduction
`of nitrite, presumably because they lack the
`nitrite reductase enzyme.
`
`3. NITRATE AND NITRITE
`METABOLISM IN MAN
`
`It was first shown in 1916 that humans
`excrete more nitrate than they consume [27].
`This was confirmed in careful studies by
`Tannenbaum’s group in the 1970’s and
`1980’s [16, 17] and is now known to be due,
`at least in part, to the formation of nitric
`oxide by mammalian cells from the amino
`acid L-arginine.
`
`3.1. Nitric oxide synthesis
`via NO synthase
`
`Following the demonstration in 1980 by
`Furchgott and Zawadski [15] that an intact
`
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`210
`
`N. Benjamin
`
`vascular endothelium was necessary for
`blood vessels to relax when exposed to the
`neurotransmitter acetylcholine, it was clear
`that endothelial cells were able to synthe-
`sise a short-lived vasodilator substance. It
`took seven years to identify this substance
`which turned out to be the simple molecule
`nitric oxide (NO) [29]. It is now known that
`there are three distinct nitric oxide synthase
`enzymes.
`Two of these enzymes, the endothelial
`isoform and the neuronal isoform, contin-
`ually synthesise NO whereas the inducible
`isoform produces this molecule from argi-
`nine only when the appropriate cell is
`exposed to bacterial cell wall products or
`pro-inflammatory cytokines such as inter-
`feron gamma and tumour necrosis factor
`alpha.
`The function of the endothelial isoform is
`to provide continual vasodilatation. Inhibi-
`tion of NO synthesis with arginine analogues
`such as methylarginine causes high blood
`pressure in animals and man, and intrigu-
`ingly, it has recently been shown that nitric
`oxide synthesis is impaired in patients with
`hypertension [14]. Although many central
`(brain) neurones contain the neuronal form
`of NO synthase, the precise function of NO
`in central nervous function is yet unclear.
`It is likely that these two isoforms contribute
`to the majority of NO synthesis which is
`rapidly converted to nitrate when this
`molecule encounters oxidised haemoglobin
`or superoxide.
`Inducible nitric oxide synthase is easily
`demonstrated within a few hours in mouse
`macrophages exposed to bacterial lipopolysac-
`charide (LPS). Indeed if rodents are exposed
`to LPS or made septicaemic there is a large
`rise in plasma and urinary nitrate concen-
`tration. It has been much more difficult to
`demonstrate synthesis of NO in human cells
`exposed to LPS, although it is clear that
`overwhelming infection will increase nitrate
`synthesis in man [28]. Merely injecting
`killed bacteria, as in a vaccine, is not effec-
`tive in enhancing NO synthesis [23]. The
`one infection which will cause a large
`
`increase in NO synthesis is gastroenteritis.
`We do not yet know that this is due to induc-
`tion of NO synthase. The large rise in
`plasma nitrate in this condition may, how-
`ever, be effective in preventing the recircu-
`lation of pathogens through the stomach by
`mechanisms discussed below.
`
`4. NITRATE AND NITRITE
`IN THE DIET
`
`4.1. Nitrate
`
`Because of the potential for toxicity, there
`have been many studies in the last 20 years
`which have studied the effect of nitrate in
`the human diet and estimated the intake of
`this ion in different populations. For those
`people who eat a large amount of vegeta-
`bles, then the main source of nitrate will be
`the green leaves of plants such as lettuce
`and spinach. Significant amounts are also
`found in root vegetables such as beetroot
`and carrots. For those who eat few vegeta-
`bles then the nitrate concentration of tap
`water becomes an important factor deter-
`mining nitrate intake. The concentration of
`nitrate in drinking water has been limited
`to 50 mg.l–1 in Europe, mainly because of
`concern about methaemoglobinaemia in
`infants (although this is now extremely rare).
`As it is now known that nitrite is the effec-
`tive antimicrobial product of nitrate, it is
`less common that nitrate itself is used as a
`preservative for meat products such as
`sausage and ham, and these foods generally
`have little nitrate content, although nitrate is
`available from pharmacies to use as a meat
`preservative.
`
`4.2. Nitrite
`
`The average intake of nitrite is consid-
`erably less than that of nitrate. The main
`source is again vegetables which will con-
`vert nitrate to nitrite on storage and when
`contaminated by nitrate-reducing bacteria.
`Occasionally this can result in very high
`
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`

`Nitrates in the human diet – good or bad?
`
`211
`
`concentrations of nitrite in vegetable juices
`in particular. Nitrite is also a component of
`preserved meats such as sausage and ham.
`However much of this nitrite is chemically
`altered following addition to food (Tab. I).
`The amount of nitrite ingested from food,
`however, is only a fraction of that swal-
`lowed as a result of nitrate reduction in the
`mouth. The average concentration of nitrite
`in saliva is around 200 m M. Given that we
`swallow approximately 500 ml saliva each
`hour then we must ingest approximately
`2.4 mmol of nitrite each day from this mech-
`anism. For this reason, limitations on the
`nitrite content of food seem inappropriate.
`
`5. METABOLISM OF NITRATE
`IN HUMANS
`
`It was found in the mid 1970s that this
`anion was handled in a peculiar way in the
`human body [35, 38]. When swallowed it
`is rapidly absorbed and at least 25% is con-
`centrated in the salivary glands by an as yet
`uncharacterised mechanism, so that the
`nitrate concentration of saliva is at least
`10 times that found in plasma. The nitrate is
`–) in the
`then rapidly reduced to nitrite (NO2
`mouth by mechanisms which will be dis-
`cussed below. Saliva containing large
`amounts of nitrite will be acidified in the
`normal stomach to produce nitrous acid
`which could potentially nitrosate amines to
`form N-nitrosamines, which experimentally
`are powerful carcinogens [8]. From this the-
`oretical understanding of nitrate metabolism
`a number of studies have been performed
`which looked at the relationship between
`nitrate intake and cancer (particularly gastric
`cancer) in humans. In general it was found
`that there was either no relationship or an
`inverse relationship, so that those individu-
`als who had a high nitrate intake had a lower
`rate of cancer [1, 13, 20, 39]. Similarly, in
`animal studies, it has been generally impos-
`sible to demonstrate an increased risk of
`cancer (or any other adverse effect) when
`nitrate intake is increased [7].
`
`It is now thought that endogenous nitrate
`synthesis derives from constitutive NO syn-
`thetase (NOS) enzymes acting on L-argi-
`nine [18]. The NO formed is rapidly
`oxidised to nitrate when it encounters super-
`oxide or oxidised haemoglobin. It is still not
`clear whether all endogenous nitrate syn-
`thesis derives from this route as, following
`prolonged infusion of 15N-labelled arginine,
`the enrichment of urinary nitrate with this
`heavy isotope is only about one half of the
`steady state of 15N arginine enrichment [21].
`This may mean that nitrate also derives from
`another source, or that the intracellular
`enrichment of labelled arginine is less than
`that in the plasma due to transamination
`reactions. This means that even on a nitrate-
`free diet, there are considerable concentra-
`tions of nitrate in plasma (around 30 m M)
`and in the urine (around 800 m mol per
`24 h). Although it is not protein bound,
`nitrate has a long half-life of 5–8 hours [40],
`which seems to be because it is about 80%
`reabsorbed from the renal tubule by an
`active transport mechanism [19].
`This peculiar metabolism of nitrate –
`renal salvage, salivary concentration and
`conversion to nitrite in the mouth made us
`consider that this may be a purposeful mech-
`anism to provide oxides of nitrogen in the
`mouth and stomach to provide host defence
`against swallowed pathogens [3]. The first
`studies we performed were to investigate
`the mechanism of nitrate reduction to nitrite
`in the mouth.
`
`5.1. Oral nitrate reduction
`
`Although Tannenbaum et al. [38] had
`considered that salivary bacteria may be
`reducing nitrate to nitrite, Sasaki and Matano
`[31] showed that in humans this activity is
`present almost entirely on the surface of the
`tongue. They considered that the nitrate
`reductase enzyme was most likely to be a
`mammalian nitrate reductase. Using a rat
`tongue preparation, we also found that the
`dorsal surface of the tongue in this animal
`had very high nitrate reductase activity,
`
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`212
`
`N. Benjamin
`
`which was confined to the posterior two-
`thirds [11]. Microscopic analysis of the
`tongue surface revealed a dense population
`of gram negative and gram-positive bacteria,
`80% of which, in vitro, showed marked
`nitrate reducing activity.
`Our suspicion that the nitrate reduction
`was being accomplished by bacteria was
`strengthened by the observation that the
`tongues of rats bred in a germ-free envi-
`ronment, which had no colonisation of bac-
`teria, demonstrated no nitrate reducing activ-
`ity on the tongue. Furthermore, treatment
`of healthy volunteers with the broad spec-
`trum antibiotic amoxycillin results in
`reduced salivary nitrite concentrations [10].
`Although we have not been able to charac-
`terise the organisms in normal human
`tongues (this would require a deep biopsy as
`the majority of the bacteria are at the bot-
`tom of the papillary clefts of the tongue
`surface), the most commonly found nitrite-
`producing organisms in the rat were Staphy-
`lococcus sciuri, followed by Staphylococ-
`cus intermedius, Pasteurella spp. and finally
`Streptococcus spp. [21]. Both morphomet-
`ric quantification of bacteria on tongue sec-
`tions and enumeration of culturable bacteria
`showed an increase in the density of bacte-
`ria towards the posterior tongue.
`We now believe that these organisms are
`true symbionts, and that the mammalian
`host actively encourages the growth of
`nitrite-forming organisms on the surface of
`the tongue. The bacteria are facultative
`anaerobes which, under hypoxic conditions,
`use nitrate instead of oxygen as an electron
`acceptor for oxidation of carbon compounds
`to derive energy. For the bacteria, nitrite is
`an undesirable waste product of this pro-
`cess, but is, we believe, used by the mam-
`malian host for its antimicrobial potential
`elsewhere.
`
`5.2. Acidification of nitrite – production
`of NO in the mouth and stomach
`
`Nitrite formed on the tongue surface
`can be acidified in two ways. It can be
`
`swallowed into the acidic stomach, or it may
`encounter the acid environment around the
`teeth provided by organisms such as Lac-
`tobacillus or Streptococcus mutans which
`are thought to be important in caries pro-
`duction.
`
`Acidification of nitrite produces nitrous
`acid (HNO2) which has an acid dissociation
`constant of 3.2, so that in the normal fasting
`stomach (pH 1–2) complete conversion will
`occur. Nitrous acid is unstable and will spon-
`taneously decompose to NO and nitrogen
`dioxide (NO2). Under reducing conditions
`more NO will be formed than NO2.
`Lundberg et al. [22] were the first to show
`that there was a very high concentration of
`NO in gas expelled from the stomach in
`healthy volunteers, which increased when
`nitrate intake was increased and reduced
`when stomach acidification was impaired
`with the proton pump inhibitor omeprazole.
`We have conducted further studies on the
`amount of NO produced following inges-
`tion of inorganic nitrate, measured more
`directly using nasogastric intubation of
`healthy human volunteers. Following
`1 mmol of inorganic nitrate, the amount of
`nitrate found in a large helping of lettuce,
`there follows a pronounced increase in stom-
`ach headspace gas NO which peaks at about
`1 hour and continues to be elevated above
`the control for at least 6 hours [26]. The
`concentration of NO measured in the
`headspace gas of the stomach in these exper-
`iments would be lethal after about 20 min-
`utes if breathed continuously.
`
`The concentration of NO in the stomach
`is much higher than would be expected from
`the concentration of nitrite in saliva and the
`measured pH in the gastric lumen. In vitro
`studies suggest that these concentrations of
`nitrite and acid would generate about one
`tenth of the NO that is actually measured
`(McKnight, Smith and Benjamin, unpub-
`lished data). It is likely that a reducing
`substance such as ascorbic acid [32–34],
`which is actively secreted into the stomach,
`or reduced thiols (which are in high
`
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`Nitrates in the human diet – good or bad?
`
`213
`
`concentrations in the gastric mucosa) are
`responsible for the enhanced NO produc-
`tion. We were surprised to find that NO is
`also generated in the oral cavity from sali-
`vary nitrite [11] as saliva is generally neutral
`or slightly alkaline.
`The most likely mechanism for this pro-
`duction is acidification at the gingival mar-
`gins as noted above. It will be important to
`determine if this is the case as the nitric
`oxide formed in this way may be able to
`inhibit the growth of organisms which gen-
`erate acid. Such a mechanism for local NO
`synthesis from nitrite may in part explain
`the importance of saliva in protection from
`caries. As in the stomach, acidification of
`saliva results in larger amounts of nitric
`oxide production than would be expected
`from the concentration of nitrite present.
`
`5.3. Nitric oxide synthesis from the skin
`
`Generation of nitric oxide from normal
`human skin can also readily be detected
`using a simple apparatus such as a glass jar
`sealed around the hand, with nitric oxide-
`free gas passed through it to a chemilumi-
`nescence detector [41]. As nitric oxide has
`the ability to diffuse readily across mem-
`branes, we first considered it most likely
`that we were measuring nitric oxide which
`had escaped from vascular endothelium to
`the skin surface, manufactured by constitu-
`tive NOS. However, when the NOS antag-
`onist monomethyl arginine was infused into
`the brachial artery of healthy volunteers in
`amounts sufficient to maximally reduce fore-
`arm blood flow, we found that the release of
`NO from the hand was not affected. Fur-
`thermore, application of inorganic nitrite
`substantially elevated skin NO synthesis.
`This coupled with the observations that NO
`release was enhanced by acidity, and
`reduced by antibiotic therapy makes it likely
`that again NO is being formed by nitrite
`reduction. Normal human sweat contains
`nitrite at a concentration of about 5 m M, and
`this concentration is precisely in line with
`
`that which we would predict would be nec-
`essary to generate the amount of nitric oxide
`release we observed from the skin. The
`source of nitrite is not clear, but is likely to
`be from bacterial reduction of sweat nitrate
`by skin commensal organisms which are
`known to elaborate the nitrate reductase
`enzyme.
`This observation has led us to the hypoth-
`esis that skin nitric oxide synthesis may also
`be designed as a host defence mechanism
`to protect against pathogenic skin infections,
`especially against fungi. The release of nitric
`oxide is inevitably increased following lick-
`ing of the skin (due to the large amount of
`nitrite in saliva), which may explain why
`animals and humans have an instinctive urge
`to lick their wounds [4].
`
`6. IMPORTANCE OF NITROGEN
`OXIDES IN HOST DEFENCE
`
`Much of the evidence for the importance
`of the arginine-nitric oxide system in host
`defence comes from the observation that
`inhibition of nitric oxide synthesis using
`arginine analogues impairs the ability of
`inflammatory cells (such as macrophages) or
`whole animals to kill invading pathogens
`[9]. This is particularly the case for intra-
`cellular organisms such as Leishmania major
`and M. tuberculosis [24, 25, 36], where there
`is particular evidence that the ability of the
`host to synthesise nitric oxide may be impor-
`tant in containing latent infections. The abil-
`ity to synthesise adequate amounts of nitric
`oxide may also be important in reducing the
`severity of falciparum malaria infections in
`humans [2].
`
`6.1. Mechanisms of nitric oxide-mediated
`microbial killing
`
`This subject has been extensively
`reviewed recently by Fang [12]. It is clear
`that many organisms are not killed by nitric
`oxide alone, but require the synthesis of
`
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`214
`
`N. Benjamin
`
`other, more reactive nitrogen oxide species.
`The reaction of nitric oxide with superoxide
`anion (which is also produced by activated
`inflammatory cells) to produce peroxyni-
`trite has received most attention [12]:
`– = ONOO–.
`NO + O2
`This reaction is very rapid, indeed more
`rapid than the reaction of superoxide with
`the enzyme superoxide dismutase. Perox-
`ynitrite is a very reactive species, which can
`easily be protonated to form peroxynitrous
`acid (ONOOH) which may then cleave to
`produce nitrogen dioxide and hydroxyl.
`
`6.2. Antimicrobial activity
`of acidified nitrite
`
`Following the observation that nitrite is
`manufactured in the mouth and then acidi-
`fied in the stomach, we went on to deter-
`mine the susceptibility of common food
`pathogens to acidified nitrite solutions. We
`found that the susceptibility varied as fol-
`lows: Y. enterocolitica > S. enteriditis >
`S. typhymurium = Shig. sonnei (P < 0.05).
`E. coli 0157 and Shig. sonnei are most resis-
`tant to acid; they survive exposure at pH 2.1
`for 30 minutes which kills the other micro-
`organisms. However, E. coli 0157 shows
`inhibition of growth up to pH 4.2 when the
`other organisms apart from Y. enterocolitica,
`manage to maintain growth unless nitrite is
`present in the solution. It seems that E. coli
`0157 manages to survive a relatively acid
`environment by slowing down growth activ-
`ity. Its ability to survive this way is undone
`by the addition of nitrite to the medium.
`Helicobacter pylori clearly must be able
`to tolerate the nitrosative stress found in the
`stomach, and indeed, this organism is more
`resistant than some other organisms to the
`combination of nitrite and acid [24]. The
`reason for this is not evident. It may be that
`it can withstand the effect of nitrogen oxides
`as it is protected against acid stress by the
`generation of ammonia from urea via
`urease enzyme. Alternatively it may have
`
`developed specific biochemical mechanisms
`for protection. If this is the case, such mech-
`anisms would be an attractive target for erad-
`ication of this important pathogen.
`
`7. CONCLUSIONS
`
`Although nitrate and nitrite have been
`used for centuries, it has only recently been
`discovered that nitrate is manufactured in
`mammals by the oxidation of nitric oxide
`and that the nitrate formed also has the
`potential for disinfecting the food we eat.
`We do not yet completely understand the
`mechanisms by which nitric oxide and other
`nitrogen oxides provide selective toxicity
`towards pathogens, and it is likely that the
`mechanisms will be different with different
`organisms. Whereas it is clear that acidified
`nitrite is produced on mucosal surfaces, and
`that this combination is effective in killing a
`variety of human gut and skin pathogens,
`we have no definite evidence as yet that this
`mechanism is truly protective in humans
`exposed to a contaminated environment.
`Further understanding of the complex
`chemistry of nitrogen oxides may also help
`develop new antimicrobial therapies based
`on augmenting what seems to be a simple
`and effective host defence system.
`
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