Nitrate and Human Health (1)

PERSPECTIVES

flora has also been implicated in the patho-genesis of gastric cancer and other malignan-cies and, for gastric cancer, much research hasfocused on the nitrate-reducing bacteria6–11.Facultative anaerobic bacteria in the oral cav-ity reduce salivary nitrate to nitrite12 and thisnitrite enhances the gastric generation ofN-nitrosamines, a versatile class of carcino-gens in animals13. However, despite extensiveresearch over the past 50 years, the linkbetween commensal bacteria, nitrate andhuman gastric cancer is still unclear14–18. Infact, there is increasing evidence that thenitrite that is formed in the mouth can subse-quently be used by the host to form biologi-cally useful nitrogen oxides (including nitricoxide, NO) that are important for hostdefence and for the maintenance of normalphysiological homeostasis in the stomach andelsewhere17,19–26. This newly described path-way for the generation of reactive nitrogenintermediates (RNIs) in mammals comple-ments the production of RNIs by nitric oxidesynthase (NOS) in white blood cells27–29.

Here, we consider the possibility thatnitrate-reducing bacteria on mucosal surfacesare truly symbiotic and suggest that the previ-ous view that dietary nitrate has only harmfuleffects should be reconsidered. We will pre-sent evidence that there is an active nitrogencycle in mammals that depends on bacterialenzymes and which results in the generationof biologically active nitrogen oxides withpossible beneficial effects.

Sources of nitrate and its circulationExogenous sources of nitrate and nitrite. Themain dietary source of nitrate (NO

3–) is veg-

etables, which account for 60–80% of the daily

nitrate intake in people on a typical westerndiet30. Nitrite (NO

2–) is also found in some

foodstuffs — for example, it is used as a foodadditive in meat to prevent botulism and toenhance its appearance. Other environmentalsources of nitrate and nitrite include cigarettesmoke31 and car exhausts. These and otherenvironmental pollutants contain volatilenitrogen oxides, some of which are convertedto nitrate or nitrite in the body.

Endogenous sources of nitrate. The mainsource of endogenous nitrate in mammals isthe L-arginine-NO pathway32, which is consti-tutively active in numerous cell typesthroughout the body. NO is produced fromthe amino acid L-arginine and molecular oxy-gen by NO synthases (NOSs). The generalbiological implications of NO have beenreviewed extensively elsewhere33,34. Althoughin simple aqueous systems NO is oxidized tonitrite, in mammals NO predominantlyreacts with oxidized haemoglobin and othercompounds to form nitrate35 (BOX 1). Underbasal conditions, the metabolites of endoge-nous NO in plasma are mainly derived fromthe L-arginine-NO pathway in the endo-thelium of blood vessels36–38 and possiblyneuronal tissue. However, during systemicinflammatory reactions or infections, whiteblood cells and other cells express an indu-cible NOS (iNOS), which produces largeamounts of NO and ultimately leads to aconsiderable increase in the concentrations ofnitrate in plasma39–41.

The entero-salivary circulation of nitrate. Themetabolic fate of nitrate in humans is out-lined in FIG. 1. After ingestion, nitrate israpidly and effectively absorbed proximallyfrom the gastrointestinal tract into the blood-stream, where it mixes with endogenouslysynthesized nitrate. Peak plasma concentra-tions are seen within 60 minutes of nitrateingestion and the half-life of nitrate in plasmais about 5 hours42. Most nitrate is ultimatelyexcreted in the urine but some is excreted in the saliva, sweat and possibly also the

Nitrate is generally considered a waterpollutant and an undesirable fertilizer residuein the food chain. Research in the 1970sindicated that, by reducing nitrate to nitrite,commensal bacteria might be involved in thepathogenesis of gastric cancers and othermalignancies, as nitrite can enhance thegeneration of carcinogenic N-nitrosamines.More recent studies indicate that thebacterial metabolism of nitrate to nitrite andthe subsequent formation of biologicallyactive nitrogen oxides could be beneficial.Here, we will consider the evidence thatnitrate-reducing commensals have a truesymbiotic role in mammals and facilitate apreviously unrecognized but potentiallyimportant aspect of the nitrogen cycle.

In an adult human, prokaryotic cells easilyoutnumber the eukaryotic cells as ~90% of allcells in the body are bacteria1. Most of thesebacteria reside in the gastrointestinal tract,with the highest density found in the oral cavity and the large intestine1. The complexinterplay between the host and the micro-organisms that inhabit the mucosal andepithelial surfaces has been intensely studied.That this relationship can be symbiotic isclearly illustrated, for instance, by vitamin Ksynthesis, which requires colonic bacteria2.A proper balance between the different speciescompeting for space in the gut is also essential,and disturbances in the flora, as a result ofexcessive use of antibiotics for example, cancause severe infections with opportunisticpathogens such as Clostridium difficile3.

In the gastrointestinal tract a relationshiphas been observed between Helicobacter pyloriinfection and gastric cancer4,5. The commensal

NATURE REVIEWS | MICROBIOLOGY VOLUME 2 | JULY 2004 | 593

Nitrate, bacteria and human health

Jon O. Lundberg, Eddie Weitzberg, Jeff A. Cole and Nigel Benjamin

O P I N I O N

594 | JULY 2004 | VOLUME 2 www.nature.com/reviews/micro

P E R S P E C T I V E S

in plasma. If we were all ‘germ-free’ and livedin a sterile environment, almost all nitrate —ingested or produced endogenously — wouldeventually leave the body unaltered becausehuman cells cannot metabolize this anion.However, in reality, some of the sites wherenitrate is excreted are heavily colonized bybacteria and, in contrast to mammalian cells,many of these microorganisms expressenzymes that can effectively reduce nitrate.Later, we will consider the relevance of bacter-ial nitrate metabolism for human health butbefore this, the general handling of nitrogenby bacteria in nature will be described.

Bacterial nitrate metabolismThe biological nitrogen cycle. FIGURE 2 shows aclassical, but simplified, overview of the bio-logical nitrogen cycle in which bacteria have adominant role. In aerobic soils and sediments(and also in conventional wastewater treat-ment plants), ammonia that is released fromhuman and animal excrement or applied asfertilizer is oxidized by aerobic nitrifying bac-teria to nitrate. Nitrate is then reduced to dini-trogen gas by anaerobic denitrifying bacteria,in a process that also requires a source of elec-trons. Denitrification dominates poorly aer-ated soils and sediments and is exploited inwastewater treatment. Life would be impossi-ble if the combined processes of nitrificationand denitrification were not compensated bya third process, dinitrogen fixation, whichcompletes the nitrogen cycle44.

Nitrate metabolism in bacteria. Despite theimportance of nitrification and denitrifica-tion to agriculture and the regeneration ofpotable water, neither process has significantrelevance to human health. Almost withoutexception, commensal bacteria do not deni-trify nitrate but instead catalyse a short-circuitin the nitrogen cycle — the rapid, anaerobicreduction of nitrate via nitrite to ammonia(FIG. 2) — in which the abundant supply ofelectrons that is provided by anaerobic car-bon-rich environments is exploited when thenormally more-favoured oxygen is not avail-able45,46. Superficially, this process seems to besimilar to nitrate assimilation, which is theprocess by which plants, many eukaryoticmicroorganisms and some bacteria usenitrate as a source of organic nitrogen com-pounds, but both the enzymes involved andtheir transcriptional control are distinct forthe two processes. As a broad generalization,the respiratory reduction of nitrate to ammo-nia is repressed by the presence of oxygen but is insensitive to the availability of ammo-nia or organic nitrogen compounds. It isinduced during anaerobic growth and further

in saliva are exceptionally high; up to 25% ofplasma nitrate is actively taken up by the sali-vary glands and secreted with saliva12, and theresulting salivary nitrate concentrations are atleast 10 times higher than the concentrations

intestines. The exact fate of all of the nitrate inthe body is still unresolved as only 60% of iso-topically labelled administered nitrate isrecovered in the urine43. For as-yet-unknownreasons, the concentrations of nitrate excreted

Box 1 | The biological chemistry of some reactive nitrogen intermediates

Nitric oxide synthesis

Arginine + O2

→ NO + CitrullineNitric oxide

Five-electron reduction of the amino acid arginine catalysed by nitric oxide synthase (NOS).

Nitric oxide oxidation

NO + O2

– → ONOO–

Nitric oxide Superoxide Peroxynitrite

ONOO– → NO3

–

Peroxynitrite Nitrate

ONOO– + H+ → HONOO → OH• + NO2

Peroxynitrite Peroxynitrous Hydroxyl Nitrogenacid radical dioxide

Nitric oxide reacts very rapidly with the superoxide radical to form the reactive intermediateperoxynitrite, which can isomerize to nitrate or can be protonated to form peroxynitrous acid.Peroxynitrous acid in turn can split into hydroxyl and nitrogen dioxide radicals.

NO + Hb2+O2

→ NO3

– + Hb3+

Nitric oxide Oxyhaemoglobin Nitrate Methaemoglobin

Nitric oxide is also rapidly oxidized by haemoglobin in red blood cells to form methaemoglobin,which in turn is reduced to normal haemoglobin by the enzyme methaemoglobin reductase.

Nitrate reduction

NO3

– + e– + 2H+ → NO2

– + H2O

Nitrate Nitrite

Nitrate is reduced by a bacterial nitrate reductase. Facultative anaerobic bacteria use nitrate asan alternative electron acceptor to oxygen under hypoxic conditions.

Nitrite acidification

NO2

– + H+ → HNO2

(pKa = 3.2)Nitrite Nitrous acid

2HNO2

→ N2O

3+ H

2O

Nitrous acid Dinitrogen trioxide

N2O

3→ NO + NO

2

Dinitrogen trioxide Nitric oxide Nitrogen dioxide

Nitrite is protonated under acidic conditions (such as those in the stomach) to generate nitrousacid, which will spontaneously yield dinitrogen trioxide, nitric oxide and nitrogen dioxide.Dinitrogen trioxide is a powerful nitrosating agent.

Nitrosation

N2O

3→ NO+ + NO

2–

Dinitrogen trioxide Nitrosonium ion Nitrite

NO+ + RSH → RSNO + H+

Nitrosonium ion Reduced thiol Nitrosothiol

NO+ + RR′NH → RR′NNO + H+

Nitrosonium ion Secondary amine N-nitrosamine

Dinitrogen trioxide is a powerful NO+ donor which can transfer to a great variety of organicside-groups, especially thiols to form S-nitroso compounds and secondary amines to formpotentially toxic N-nitrosamines.


It is rare for any single species to be ableto catalyse both denitrification and nitratereduction to ammonia. Although there are also few reports of the occurrence ofboth NirK and NirS in the same species,some enteric bacteria, such as E. coli and S. typhimurium, encode both the cytoplasmicnitrite reductase NirBD and the periplasmicnitrite reductase Nrf, and therefore haveboth a cytoplasmic pathway and a periplas-mic pathway for the reduction of nitrate vianitrite to ammonia (FIG. 3). High concentra-tions of nitrate induce expression of thecytoplasmic pathway, which is encoded bythe narGHJI and nirBD operons, but repressthe periplasmic pathway, which is encodedby the nap and nrf operons. At very lowexternal concentrations of nitrate andnitrite, similar to the concentrations foundin body fluids, it is the Nap–Nrf periplasmicpathway that is induced rather than the cyto-plasmic pathway. However, it is likely thatthe cytoplasmic NarG enzyme is responsiblefor the accumulation of nitrite from nitratein the mouth.

Chemostat competition experiments haveestablished that the ability to reduce nitrateand nitrite in the periplasm confers a selec-tive advantage relative to a strain that is ableto express only the cytoplasmic pathway52.Consequently, nitrate reduction in the peri-plasm of enteric bacteria is believed to be thephysiologically significant pathway53. How the expression of the genes involved in these

induced by the presence of nitrate or nitrite.Conversely, expression of the genes for nitrateassimilation is insensitive to the availability ofoxygen, but is tightly regulated by the nitrogenstatus of the cell47,48.

Multiple enzymes catalyse parallel pathways.A striking feature of respiratory bacterialnitrate metabolism is that multiple enzymescan catalyse each reaction. At least three typesof nitrate reductase catalyse the reduction ofnitrate to nitrite49. First, a soluble, assimilatory

nitrate reductase (NAS) is found in the cyto-plasm. Second, energy-conserving nitratereductases (NAR, for example, NarG, which isencoded by the first gene of the narGHJIoperon) with catalytic sites located in thecytoplasm are associated with the cytoplasmicmembrane, from which they receive electronsfor nitrate reduction50. Third, soluble, peri-plasmic nitrate reductases (NAP) are found inmany Gram-negative bacteria. All three typesare molybdoproteins and some bacteria, forexample, Paracoccus pantotrophus, possess allthree51. Escherichia coli and Salmonella enter-ica serovar Typhimurium also synthesizethree different nitrate reductases: althoughthey lack an assimilatory nitrate reductase, thegenes that encode the membrane-associatednitrate reductase are duplicated (the narGand narZ operons) and differentially regu-lated50,52, and they also express a periplasmicnitrate reductase, Nap, which is encoded bythe napFDAGHBC operon.

Bacterial nitrite reduction. Nitrite reductionis the reaction that defines whether bacteriacatalyse denitrification or nitrate reduction toammonia, and in each case, two distinctclasses of nitrite reductase are involved49. Allnitrite reductases are synthesized preferen-tially during anaerobic growth. The denitrifi-cation of nitrite to nitric oxide is catalysed bythe copper-containing NirK or the cyto-chrome cd nitrite reductase NirS, both ofwhich are located in the periplasm50. Two bio-chemically distinct nitrite reductases catalysethe reduction of nitrite to ammonia. TheNADH-dependent NirBD nitrite reductasereduces nitrite directly to ammonia in thecytoplasm of some bacteria (for example,Gram-negative enteric bacteria and Gram-positive bacteria such as Staphylococcuscarnosus and Bacillus subtilis). The role ofNirBD is to detoxify nitrite that is generatedby NarG (the membrane-associated nitratereductase) during anaerobic growth in thepresence of nitrate concentrations that aremuch greater than those found in warm-blooded animals52. More widely distributed isthe cytochrome c nitrite reductase Nrf, whichcatalyses the reduction of nitrite to ammoniain the periplasm of Gram-negative bacteria.This enzyme is the terminal component of an electron-transfer pathway in whichelectrons are transferred from physiologicalsubstrates, especially formate (hence the des-ignation Nrf, for nitrite reduction by for-mate). Nap, the periplasmic nitrate reductase,and Nrf, the periplasmic nitrite reductase, arecoordinately regulated to provide a pathwayfor the reduction of nitrate to ammonia inthe periplasm.


Stomach

Kidney

Bladder

Salivary glands

Bacteria on tongue surfacereduce nitrate to nitrite

Dietary nitrate(2–3 mM daily)

Salivary glandsconcentrate plasmanitrate tenfold to 200–1,000 µM

Nitric oxide produced is oxidisedto nitrate in blood

Stomach acid reacts with salivary nitrite to generate nitric oxide

Nitrate excreted in urine (2–3 mM daily)

Figure 1 | The entero-salivary circulation ofnitrate in humans. Vegetables, the maindietary source of nitrate, account for 60–80% ofthe daily nitrate intake (2–3 mM total) in peopleon a typical western diet30. Once ingested,nitrate is rapidly absorbed from thegastrointestinal tract and mixes withendogenously synthesized nitrate, which mainlycomes from the L-arginine-NO pathway32. Mostnitrate is ultimately excreted in the urine. Up to25% of plasma nitrate is actively taken up by thesalivary glands and secreted with saliva12; theresulting salivary nitrate concentrations can beat least 10 times higher than the concentrationsin plasma. Salivary nitrate is reduced to nitrite bycommensal bacteria in the oral cavity. In theacidic stomach, salivary nitrite is reduced to NOand other RNIs.

Denitrification Nitrification

Shortcircuit

Organic N

NO3–

?

NO2–

NH4+

N2O

N2

NO

Anaerobic process

Aerobic process

Anaerobic and aerobic process

N2 fixation

Figure 2 | The biological nitrogen cycle.Bacteria have a dominant role in this cycle. Mostcommensal bacteria do not denitrify nitrate butcatalyse a short-circuit in the nitrogen cycle — therapid, anaerobic reduction of nitrate via nitrite toammonia. The question mark indicates that themechanism of NO generation from nitrite duringnitrate reduction to ammonia is uncertain.



products is still very high. However, despiteefforts to minimize environmental exposure tonitrate, we still have to live with the nitrate thatis produced endogenously from the L-arginine-NO pathway. In healthy people on awestern diet, this constitutes as much as 50%of the total daily nitrate load.Although the car-cinogenic properties of N-nitroso compoundshave been well established in cell cultures andanimal experiments13, there is still no clear evi-dence for a link between nitrate intake and gas-tric cancer in humans. In fact, many studiesshow either no relationship or even an inverserelationship between a high intake of nitrateand the occurrence of gastric cancer14–18. Themechanism for such a protective effect isunknown, but it could be related to the highconcentrations of vitamin C and other antioxi-dants that are found in nitrate-rich foods suchas vegetables. It can also be speculated that gas-tric nitrogen oxides other than N-nitrosaminesprotect the gastric mucosa (see below) andinhibit the growth of H. pylori, which has beenimplicated as a causative factor for the devel-opment of gastric cancer in humans5.

Bladder cancer. Most dietary and endogenousnitrate is excreted in the urine. As urine isnormally sterile, no further reduction tonitrite occurs. However, during urinary-tractinfections, considerable amounts of nitritecan be formed by the invading bacteria.

Infection with the parasite Schistosomahaematobium is an important risk factor forthe development of cancer in the urinarybladder, and N-nitrosamines have been pro-posed to have a role in the pathogenesis ofthis cancer62,63. The proposed mechanism isthat chronic infection with nitrate-reducingbacteria, which often occurs with the parasiticdisease, increases the concentrations of uri-nary nitrite, thereby enhancing the generationof N-nitrosamines. An increased local rate ofendogenous NO production during thechronic parasitic infection has also been sug-gested to promote nitrosamine formation inthe bladder64.

Generating RNIs from nitriteN-nitrosamines are not the only compoundsthat are formed in the body from bacterial-derived nitrite. In 1994, it was shown that largeamounts of NO are generated in vivo fromsalivary nitrite in the acidic stomach19,24.Whennitrite is acidified, several other RNIs are alsogenerated, many of which have biologicaleffects26. RNIs are also produced by iNOS, forexample, in activated white blood cells, wherethey have an important role in primary hostdefence28,33. So, there are two parallel pathwaysfor the generation of RNIs in mammals.

Gastric cancer. The metabolism of nitrate andnitrite can result in the formation ofN-nitrosamines (BOX 1), which are carcino-genic57,58. Individuals can also be exposed topreformed N-nitrosamines — for example,from the diet, in certain occupational settings,and through the use of tobacco products. Theendogenous formation of N-nitroso com-pounds from nitrite in the stomach occurs inat least two ways (FIG. 4; BOX 1). In an acidicstomach, nitrite forms nitrous acid (pKa 3.2),which is not only a nitrosating agent (NO+

donor), but also spontaneously decomposesto a variety of nitrogen oxides, including thepotent nitrosating agents N

2O

3and N

2O

4.

In a situation in which gastric acidity isreduced — for example, by medication ordisease — bacteria can colonize the stomach.Some of these bacteria can catalyse the for-mation of N-nitroso compounds from nitriteat neutral pH values. The exact mechanismfor this is unknown, but it is believed toinvolve a bacterial nitrite reductase59, withexcess production of NO or related products.NO itself is not a nitrosating agent9,60.However, in the presence of oxygen, NO isoxidized to NO

2that exists in equilibrium

with N2O

3and N

2O

4, which in turn react

with secondary amines at neutral pH to formN-nitroso compounds10,61.

Reducing human exposure to preformedN-nitrosamines and nitrate is one approachto the prevention of cancer, and in manyinstances this has been successful, althoughexposure to N-nitrosamines in tobacco

different pathways is regulated has recentlybeen reviewed49. Even bacteria that lack deni-trification pathways and are known to reducenitrite directly to ammonia generate relativelyhigh concentrations of NO (REF. 54). The ques-tion mark in FIG. 2 indicates the unprovenpossibility that this NO is produced by one ofthe enzymes that reduce nitrite to ammonia(NrfA or NirBD). Consistent with the pro-posal that NO is an enzyme-bound interme-diate in this mode of nitrite reduction, it isknown that NrfA can reduce NO rapidly toammonia55.

Bacteria, nitrate and human disease Over the past 50 years inorganic nitrate hasgained a bad reputation, primarily due to itsproposed association with the developmentof cancer11 and methaemoglobinaemia, whichis a condition that mostly affects infants upto 12 months old and is caused by the oxida-tion, by nitrite or nitric oxide, of haemoglo-bin in red blood cells to an abnormal formknown as methaemoglobin that cannot bindor transport oxygen56. As a consequence, reg-ulatory authorities have expended enormousefforts in trying to minimize environmentalexposure to this anion. The proposed dele-terious effects of nitrate on human healthare not primarily related to the nitrate ionitself. In fact, nitrate has a remarkably lowtoxicity. Instead, the concern about nitratearises from its conversion to nitrite, a reac-tion that is catalysed by the bacterial enzymesdescribed above.

Periplasm

Cytoplasm

NO3–

NO3–

NO2–

NO2–

NO2–

NO2– NO3

–

NO2– NO3

–

NO2–

Nrf

NirBD

NAP(napF

operon)

NAR(narG

operon)

NAR(narZ

operon)

NH4+

NH4+

NO3–

NO2–

NarKNarU

NO2– NO2

–

NO2– NO2

–

NirCMembrane

Figure 3 | Multiple pathways for nitrate and nitrite transport and reduction in Escherichia coli.E. coli lacks an assimilatory nitrate reductase, but contains two membrane-associated nitratereductases, which are encoded by the narG and narZ operons. A periplasmic nitrate reductase,encoded by the napF operon, is also present. In the cytoplasm, the NADH-dependent NirBD nitritereductase reduces nitrite directly to ammonia. In the periplasm, this reaction is carried out by thecytochrome c nitrite reductase, Nrf.


Susceptibility to the complex mixture ofRNIs that is generated from acidified nitritevaries between different species. H. pylori76

and lactobacilli77 seem to be the least sensi-tive of the bacteria that have been tested. Inaddition, Salmonella, Yersinia and Shigellaspecies are more susceptible than E. coli 22.Local environmental conditions, such as theoxygen concentrations, the growth mediumand the presence of reducing agents (forexample, ascorbic acid or thiocyanate), willalso affect the antimicrobial effects of RNIs ona particular microorganism26,68.

The chemistry of RNIs in biological sys-tems is very complex and not yet fully charac-terized27,65,66. RNIs include different oxidationstates of nitrogenous compounds includingNO•, NO

2•, N

2O

3, N

2O

4, NO

2–, S-nitrosothiols,

ONOO– and NO3

–. The chemical nature andreactivity of any RNI in a biological system isdetermined by ambient factors, including oxy-gen tension, pH, proximity to haem-contain-ing proteins, redox state and thiol concentra-tion. RNIs also interact with reactive oxygenintermediates (ROIs), which are produced inlarge quantities by activated mammalianphagocytes. The classical example of this is thegeneration of peroxynitrite (ONOO–) fromthe reaction of NO with superoxide (O

2–)67.

Some of the central reactions involving RNIsin biological systems are shown in BOX 1.

Antimicrobial effects of RNIs. At neutral pHvalues, nitrite has a limited effect on bacterialgrowth and can even function as an alterna-tive oxidant under hypoxic conditions (seeabove). However, with increasing acidity thisanion shows potent antimicrobial activityagainst a wide variety of bacteria and fungi. Ata low pH value, nitrite is protonated tonitrous acid (HNO

2), which further decom-

poses to a variety of RNIs, many of whichhave antibacterial activity26,68 (BOX 1).

The exact mechanism for the antibacterialeffects of these nitrogen oxides is still notknown, but multiple cellular targets arealmost certainly involved27,68,69 (TABLE 1). DNAis one important target, and DNA damagecan occur through oxidative damage, deami-nation, interaction with DNA-repair systemsand several other alterations. Both cell-surfaceand intracellular proteins are also targets ofRNI-induced bacterial damage. Interactions

with proteins involve reactive thiols, iron–sulphur clusters, haem groups, zinc-containingmoieties, tyrosine residues, tyrosyl radicalsand amines.

The sensitivity to any particular nitrogenoxide differs profoundly between differentmicrobial pathogens. For example, NO• ismicrobicidal for Mycobacterium tuberculosis70,but has low antimicrobial activity against E. coli 71–73. On the other hand, E. coli is sensitive to other nitrogen oxides, including S-nitrosothiols74 and peroxynitrite71, whereasM. tuberculosis is not75.


NO3–Diet

Detrimental

Endogenous source (NO synthesis)

NO2–

NO2–

N2O3

NO,

RNIs

Vitamin CSNC

S-NONO,

NO2– + H+

Saliva

Stomach

High pH with bacterialover-growth

Low pH

NaR

NaR

NOC

NiR

Mutagenesis Bacterial killing

Mucus generation

Beneficial

Vasodilation

Figure 4 | The putative effects of nitrite in the stomach. Bacterial nitrate reductases (NaR) are shownin blue and nitrite reductases (NiR) in red. NOC, N-nitroso compounds; RNIs, reactive nitrogenintermediates; SNC, thiocyanate; S-NO, S-nitrosothiols.

Table 1 | Proposed mechanisms and cellular targets for antimicrobial effects of reactive nitrogen intermediates (RNIs)

Targets Mechanism Proposed RNI

DNA function

Oxidative DNA damage (for example, strand breaks, crosslinking or deamination) N-nitrosylation N2O3, ONOO–, NO2•

Filamentation S-nitrosylation S-NO

Protein function

Thiol groups (for example, in glyceraldehyde-3-phosphate dehydrogenase, S-nitrosylation, disulphide formation S-NO, N2O3, NO•

γ-glutamylcysteinyl synthetase)

Haem groups (for example, in cytochromes or catalase) Nitrosyl-haem formation NO•, S-NO

Iron/zinc-sulphur clusters (for example, in aconitase or DNA-binding proteins) S-nitrosylation NO•, ONOO–

Tyrosine residues (disruption of tyrosine phosphorylation, modification of Nitration ONOO–, NO2– + H2O2

protein function)

Tyrosyl radicals (for example, in ribonucleotide reductase) NO• radical interaction NO•

Amines N-nitrosylation N2O3, N2O4

Cell wall integrity

Lipids Lipid peroxidation NO2•, ONOO–

Surface thiols S-nitrosylation, disulphide formation S-NO, N2O3, NO•



the acid is buffered to pH 3 (REF. 81). This indi-cates an important role for dietary nitrate inprotection against ingested pathogens.

Gastric mucosal integrity. Experimental ani-mal studies82 as well as clinical studies inhumans83,84 clearly indicate that NO has animportant protective role in the stomach,probably by improving gastric mucosal bloodflow. The concentrations of NO in the stom-ach lumen (20–400 ppm) are several orders ofmagnitude higher than those that are requiredfor vasodilation85. As NO is known to easilytravel across biological membranes and asNO-donating drugs are gastroprotective, it hasbeen proposed that nitrite-derived NO, actingfrom the luminal side, could be involved in theregulation of gastric mucosal blood flow (FIG. 4). Several recent studies from differentlaboratories support this idea. Bjorne and col-leagues studied gastric mucosal blood flowand mucus secretion in a rat in vivo modelafter local application of human saliva to thegastric mucosa20. Mucosal blood flow andmucus secretion were increased after luminalapplication of nitrite-rich saliva, whereas salivafrom a fasting individual had no effect. Theseeffects were associated with the generation of NO and S-nitrosothiols. In addition, pre-treatment with an inhibitor of guanylyl cyclasemarkedly inhibited nitrite-mediated effects onblood flow. This indicates that the observedeffects were mediated by NO.

reports of the identification of bacterial per-oxynitrite and hydroxylamine reductases79,80,but much less is known about the mecha-nisms by which bacteria detoxify other RNIs.It is also apparent that multiple physiologicalroles have been assigned to individual proteins.Although the roles of the various proteinsfrom E. coli are, with notable exceptions, mod-erately well defined, few experimental data areavailable for the corresponding proteins frommost other bacteria.

Beneficial effects of acidified nitriteKilling ingested pathogens. Each day we pro-duce approximately one litre of saliva, whichcontinually flows into the acidic stomach. Thelevels of NO and other RNIs in the stomachwill depend on the nitrite concentration in thesaliva. The baseline concentration of NO in thestomach headspace gas is approximately 20parts per million (ppm)42.When consuming anitrate-free diet, the concentration of nitrite insaliva, even when acidified, is probably not suf-ficient to kill known enteric pathogens such asE. coli, Salmonella enterica serovar Typhi andSalmonella enterica serovar Enteritidis19,22.Following a meal containing high nitrate con-centrations (for example, a portion of lettuce),the levels of nitrate and nitrite in saliva increasemarkedly, resulting in an increase in stomachNO concentrations to >400 ppm42. Mostenteric pathogens are killed within 1 hourunder these conditions in vitro (FIG. 5), even if

Bacterial protection against RNIs. Bacteria inthe human body — like bacteria in any othernatural environment — are exposed to chem-ical attack by reactive nitrogen (and oxygen)species. It is hardly surprising, therefore, thatthey have evolved a wide variety of mecha-nisms to respond to environmental stress.Protection against ROIs has been studiedextensively and hundreds of gene productsare involved27. Despite the obvious implica-tions for human health, it is only recently thatthe biochemical mechanisms of resistance toRNIs have been studied, and the current pic-ture is incomplete. That NO is toxic to bacte-ria — and also an obligate intermediate indenitrification — was conclusively establishedby demonstrating that a mutation in the NOreductase gene was lethal to Pseudomonasstutzeri during anaerobic growth in the presence of nitrate or nitrite78.

Some of the defence systems seem tooverlap between ROIs and RNIs, but thereare also specific pathways for the detoxifica-tion of RNIs. These include indirect pathwayssuch as inhibition of uptake or productionof RNIs, repair of RNI-dependent damageand scavenging and detoxification of RNIsand related products27,68. The best character-ized are the bacterial nitrite and NO reduc-tases, but the importance of NO to humanphysiology has attracted studies of othermechanisms of NO management. Someexamples are listed in TABLE 2. There are a few

Table 2 | Examples of how pathogenic and commensal bacteria metabolize nitrite and its reactive derivatives

Reactive nitrogen species Protein Function References

Escherichia coli

Nitrite NrfA Nitrite reduction to ammonia 109NirBD Nitrite reduction to ammonia 110HCP Unknown, but possible HNO2 reductase 111

Nitric oxide NrfA Reduces NO to ammonia 55HMP NO dioxygenase activity 112,113HCP Possible NO reductase (?) Deduced from 111NorV–NorW; flavorubredoxin Alternative NO reductase 114

Peroxynitrite Peroxynitrite reductase; AhpC Reduces peroxynitrite to nitrite 79

Hydroxylamine HCP Reduces NH2OH to ammonia 80

Campylobacter jejuni

Nitrite NrfA Reduces nitrite to ammonia (?) Uncharacterized; identified fromthe genome database

Nitric oxide NrfA Reduces NO to ammonia, as in E. coli (?) Uncharacterized; identified fromthe genome database

Cgb Possibly detoxifies NO, mechanism unknown Uncharacterized; identified from the genome database

Neisseria gonorrhoeae

Nitrite AniA Reduces nitrite to NO 115

Nitric oxide NorB Converts NO to N2O 116Cytochrome c′ Binds NO at the outer membrane 117 HMP homologue Reduces NO to N2O (?) From incomplete genome

database

Hydroxylamine Cytochrome c′ 118

HMP, bacterial haemoglobin-like protein; HCP, hybrid cluster protein, also known as the prismane protein.


Final speculation and future directionsNitrate in plasma and tissues represents a sta-ble, inert end product of endogenous andexogenous nitrogen oxides and cannot be further metabolized by mammalian cells.However, as we have described, commensalbacteria can help to ‘reactivate’ this nitrate byreducing it to the more reactive nitrite. Nitritecan then form various biologically active com-pounds, including not only potentially harm-ful N-nitrosamines, but also other nitrogenoxides that have possible protective effects.

The newly discovered beneficial effects ofnitrite could potentially have therapeuticimplications — for example, to selectivelyincrease the blood flow in ischaemic tissues orto stimulate mucosal blood flow locally, forexample, in the stomach. Indeed, the genera-tion of NO from nitrite is automaticallyenhanced in acidic or reducing environments.Another possible development is to use acidi-fied nitrite as an antimicrobial agent — RNIsgenerated locally, for example, in the stomach,oral cavity, urine94 or on the skin107, could beused to treat or prevent infections. Naturally,any beneficial effects of using nitrite or relatedcompounds therapeutically must be verifiedin controlled clinical studies.

Finally, as described here, it is now clearthat there are two pathways for the genera-tion of NO and other RNIs in mammals:the NOS-dependent pathway in phagocytesand other cells and the newly described

Several other recent animal studies indi-cate that dietary nitrate has gastroprotectiveactivity through the generation of NO in thestomach86–88. Miyoshi et al. examined theeffects of oral nitrate supplementation onstress-induced gastric injury in rats86. Pre-treatment with inorganic nitrate was stronglyprotective and the effects were paralleled byintragastric generation of NO. Interestingly,NO generation and the protective effects ofdietary nitrate were abolished when the oralmicroflora was removed by topical antibiotictreatment before the experiment.

Taken together, these studies clearly indi-cate that dietary nitrate has important gas-troprotective effects. The crucial step in the bioactivation of inorganic nitrate is thereduction to nitrite, which is carried out bythe oral microflora.

Oral cavity. As well as acidification of salivarynitrite once it has been swallowed into thestomach, acidification can also occur in theoral cavity itself, which results in the local gen-eration of NO and other RNIs21,89. Cariogenicbacteria such as Streptococcus mutans and lac-tobacilli cause dental damage by convertingsugars into organic acids. It has now beenshown that these microorganisms are suscepti-ble to nitrite and will ‘self-destruct’ if allowed toproduce acid in a nitrite-rich environment90.This might explain some of the protectiveeffects of normal saliva against dental caries.

Skin. Normal human skin continually releasesNO. Although this could result from NOSthat is present in dermal vascular endothelialcells, Weller et al. showed that complete inhi-bition of this enzyme with L-NMMA (a NOSinhibitor) had no effect on the production ofNO by the skin91. This evidence, together withthe observation that antibiotic therapydecreases skin NO formation and appliednitrite enhances NO formation, led to theproposal91 that commensal bacteria are pro-ducing NO by the reduction of sweat nitrateto nitrite and its subsequent conversion toNO by the acidic environment (pH 5.5) onhealthy skin. Skin commensal bacteria such ascoagulase-negative staphylococci commonlyexpress a nitrate reductase enzyme (M. Wilks,personal communication). The amount ofNO and other nitrogen oxides is clearly notsufficient to kill normal skin commensals, butmight be useful in suppressing the growth offungal pathogens, which are more likely tocause disease in this organ92.

Urine. Most plasma nitrate is eventuallyexcreted in the urine, as discussed above. Asurine is sterile, the concentrations of nitrite

are usually very low; however, during a lower-urinary-tract infection bacteria can producenitrite from the surplus nitrate source. Infact, a urinary test-strip for nitrite is routinelyused in the clinic to detect urinary-tractinfections. Because the pH of infected urineis usually quite high (values of about 6–8),there is normally no further reduction to NOand other RNIs. However, if the pH value ofnitrite-containing urine is decreased, largeamounts of RNIs are formed. As describedabove, these nitrogen oxides are bactericidalfor several microorganisms. The antibacterialeffects of nitrite in urine have been studied invitro25,93. All urinary pathogens that havebeen tested are sensitive to acidified nitrite,including E. coli, Pseudomonas aeruginosaand Staphylococcus saprophyticus, and theaddition of ascorbic acid to urine furtherenhances the antibacterial effects93. Theeffects of nitrite are seen when the pH valueof urine is decreased to 5.5 or below, whichcan be achieved by the ingestion of vitamin Cor other acidifying agents93. Interestingly,acidification of urine is used in traditionalfolk medicine for the treatment and preven-tion of urinary-tract infections, although themechanism of action is poorly understood93.It has been proposed that the generation ofbactericidal nitrogen oxides in acidified urinecontributes to these beneficial effects93,94.

Nitrite reduction to NO in the systemic circula-tion. The generation of NO from nitrite occursspontaneously in highly acidic or reducingenvironments, as we have discussed. Interest-ingly, such non-enzymatic generation of NOcan also occur systemically. In ischaemic tis-sues in which the pH value is decreased, NOis formed from nitrite by similar mecha-nisms26,95,96. In addition, recent research indi-cates that nitrite can be converted to NO byseveral other pathways, which involve mam-malian enzymes or proteins54,97–104 (TABLE 3). Ithas now been shown that physiological con-centrations of nitrite can dilate blood vesselsthrough conversion to NO96,99. With this newknowledge, nitrite might be considered animportant vascular storage pool of NO.Interestingly, it was recently found that the lev-els of nitrite in plasma increase 4–5-fold afteringestion of inorganic nitrate105. This increasewas abolished if the test subject avoided swal-lowing after the nitrate intake, thereby illus-trating its salivary origin. By extrapolation,this could in fact indicate that the commensaloral flora contributes not only to the localregulation of gastric function, as discussedabove, but also to systemic NO-mediatedeffects, such as the regulation of vascular tone,platelet function106 and leukocyte adhesion.


10

1

0.1

0.01

5 4 3pH

Nitr

ite c

once

ntra

tion

(mM

)

2

Normal range for salivary nitrite

concentrations

2 h30 min

MIC

Bactericidal Growth continues

Figure 5 | The antibacterial effects of salivarynitrite in gastric juice. The graph shows theminimum bactericidal concentration for 30 minand 2 h and the minimum inhibitory concentration(MIC), all in µmol ml–1, for Salmonella entericaserovar Enteritidis exposed to varyingconcentrations of acid and nitrite. At typicalstomach pH values, increasing the nitriteconcentration from a low normal salivaryconcentration to a high normal value renders theacidified nitrite mix bactericidal rather thanbacteriostatic. Data taken from REF. 22.



10. Iijima, K. et al. Dietary nitrate generates potentiallymutagenic concentrations of nitric oxide at thegastroesophageal junction. Gastroenterology 122,1248–1257 (2002).

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27. Nathan, C. & Shiloh, M. U. Reactive oxygen and nitrogenintermediates in the relationship between mammalianhosts and microbial pathogens. Proc. Natl Acad. Sci.USA 97, 8841–8848 (2000).

Jon O. Lundberg is at the Department ofPhysiology & Pharmacology, Karolinska Institute,

171 77 Stockholm, Sweden.

Eddie Weitzberg is at the Department ofAnaesthesiology and Intensive Care,

Karolinska Hospital, 171 76 Stockholm, Sweden.

Jeff A. Cole is at the School of Biochemistry,University of Birmingham, PO Box 363,Edgbaston, Birmingham B15 2TT, UK.

Nigel Benjamin is at the Peninsula Medical School,St. Luke’s Campus, Heavitree Road,

Exeter EX1 2LU, UK.

Correspondence to J.O.L.e-mail: [email protected]

doi:10.1038/nrmicro929

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NOS-independent pathway involving thereduction of nitrite. Although not yet studiedspecifically, it is possible that these pathwaysinteract in vivo. One such interaction canoccur in phagocytic cells. The phagosomes ofmacrophages are very acidic (pH <5), a situa-tion in which nitrite is rapidly converted totoxic RNIs26,108. Interestingly, cellular oxygenconcentrations affect the two systems differ-ently. Although hypoxia and acidosis inhibitNO generation from iNOS (molecular oxy-gen is a co-substrate), these conditions greatlyenhance the production of RNIs from nitrite.So, the reduction of nitrite by acidification orby other pathways could be a back-up systemfor the NOS-driven generation of RNIs whenconditions for NO production by iNOS areunfavourable. The same could be true for thelow-grade generation of NO by constitutiveenzymes in blood vessels. When the oxygensupply is insufficient for the NOS to operate(for example, during ischaemia), the nitrite-derived NO pathway automatically takes over.

In mammals, the generation of NO by NOSand its biological significance has been studiedintensely over the past two decades. The NOS-independent generation of NO from nitrite ismuch less well characterized and we have prob-ably only witnessed the start of this fascinatingnew field.As we have described, bacteria have apivotal role in this pathway. With the recentidentification of potentially important physio-logical effects of nitrite, the current view of thenitrate-reducing commensals as being onlyharmful might have to be reconsidered.

Table 3 | Proposed pathways for the generation of NO from nitrite in mammals

Location Source of nitrite Proposed effects References

Acidic reduction*

Stomach Bacterial reduction of salivary nitrate Beneficial — host defence, mucosal protection 19–21,24,119Harmful —carcinogenesis

Oral cavity Bacterial reduction of salivary nitrate Host defence against peridontal and cariogenic 90,120bacteria

Skin Bacterial reduction of sweat nitrate Host defence against skin pathogens 107,121

Lower urinary tract Bacterial reduction of urinary nitrate Host defence 93,94,122

Ischaemic tissue Oxidized NO Hypoxic vasodilation, ischaemia-reperfusion injury 95,96

Deoxyhaemoglobin

Blood Oxidized NO Vasodilation 97–99

Xanthine oxidoreductase

Ischaemic tissue Oxidized NO Vasodilation 100–102

Milk Reduction of nitrate in milk Host defence 103

Cytochrome P450

Hepatocytes, smooth muscle cells Oxidized NO, organic nitrates Final step in NO generation from organic nitrates 98,104

Mitochondrial cytochromes

Hepatocytes, other cells Oxidized NO, organic nitrates Final step in NO generation from organic nitrates 98,104

Bacterial nitrite reductases

Gastrointestinal tract Nitrate reductase Anti-inflammatory activity? 54,123

*With the exception of NO, a variety of different reactive nitrogen intermediates are generated from acidified nitrite (see text and BOX 1 for details).


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AcknowledgementsThe authors wish to thank the Ekhaga Foundation, the SwedishResearch Council, the EU 6th Framework Programme and theSwedish Heart and Lung Foundation for generous support.

Competing interests statementThe authors declare competing financial interests: see Web version for details.

Online links

DATABASESThe following terms in this article are linked online to:Entrez: http://www.ncbi.nlm.nih.gov/Entrez/Bacillus subtilis | Clostridium difficile | Escherichia coli |Helicobacter pylori | Mycobacterium tuberculosis |Pseudomonas aeruginosa | Salmonella enterica serovarEnteritidis | Salmonella enterica serovar Typhi | Salmonellaenterica serovar TyphimuriumSwissProt: http://www.ca.expasy.org/sprot/NarG | NirK | NirS | NrfAccess to this links box is available online.

ERRATUM

NITRATE, BACTERIA AND HUMAN HEALTHJon O. Lundberg, Eddie Weitzberg, Jeff A. Cole and Nigel Benjamin

Nature Rev. Microbiol. 2, 593–602 (2004)

Reference 105 was erroneously deleted from the reference list of this article and should have appeared as follows:

105. Lundberg, J. O. & Govoni, M. Inorganic nitrate is a possible source for systemic generation of nitric oxide. Free Radic. Biol. Med. 37, 395–400 (2004).

The enhanced text version of this article has been corrected.

Nitrate and Human Health (1)

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