Gut 1997; 40: 211-214
`
`211
`
`Chemical synthesis of nitric oxide in the stomach
`from dietary nitrate in humans
`
`GM McKnight, L M Smith, RS Drummond, C W Duncan, M Golden, N Benjamin
`
`Abstract
`Background/Aims—It has been suggested
`that dietary nitrate, after concentration in
`the saliva and reduction to nitrite by
`tongue surface bacteria,
`is chemically
`reducedto nitric oxide (NO)in the acidic
`conditions of the stomach. This study
`aimed to quantify this in humans.
`Methods—Ten healthy fasting volunteers
`were studied twice, after oral adminis-
`tration of 2 mmol of potassium nitrate or
`potassium chloride. Plasma, salivary and
`gastric nitrate, salivary and gastricnitrite,
`and gastric headspace NO concentrations
`were measured oversix hours.
`Results—On the control day the par-
`ameters measured variedlittle from basal
`values. Gastric nitrate concentration was
`105-3 (13) pmol/l (mean (SEM), plasma
`nitrate concentration was
`17-9
`(2-4)
`pumol/, salivary nitrate concentration 92-6
`(31-6) pmol/l, and nitrite concentration
`53-9 (22°8) pmol/l. Gastric nitrite concen-
`trations were minimal
`(<1
`pmol/l).
`Gastric headspace gas NO concentration
`was 16-4 (5:8) parts per million (ppm).
`After nitrate ingestion, gastric nitrate
`peaked at 20 minutes at 3430 (832) pmol/l,
`plasma nitrate at 134 (7-2) pmol/l, salivary
`nitrate at 15167 (280-5) pmol/l, and
`salivary nitrite at 761-5 (187-7) pmol/l after
`20-40 minutes. Gastric nitrite concen-
`trations tended to be low, variable, and
`any rise was non-sustained. Gastric NO
`concentrations rose considerably from
`148 (3-1) ppm to 89:4
`(28-6) ppm
`(p<0-0001) after 60 minutes. All par-
`ameters remained increased significantly
`for the duration of the study.
`Conclusions—A very large and sustained
`increase in chemically derived gastric NO
`concentrations after an oral nitrate load
`was shown, which may be important both
`in host defence against swallowed patho-
`gens and in gastric physiology.
`(Gut 1997; 40: 211-214)
`
`Keywords: nitric oxide, salivary nitrate and nitrite,
`stomach.
`
`Nitric oxide (NO) is an important biological
`molecule that is responsible for cell signalling
`and host defence in a number of mammalian
`tissues.'? It
`is now well established that
`mammalian cells produce NO from the amino
`acid L-arginine with a family of NO synthase
`enzymes.
`
`We have recently proposed that NO maybe
`synthesised by an alternative mechanism that
`relies on sequential
`reduction of nitrate.’
`Dietary nitrate (principally derived from green,
`leafy vegetables) is absorbed from the stomach
`and proximal small intestine into the plasma
`and subsequently concentrated in saliva.**
`Approximately 25% of dietary nitrate is re-
`circulated. The dorsal surface of the tongue
`harbours large numbers of nitrate reducing
`facultative anaerobic bacteria, which rapidly
`reduce nitrate to nitrite under hypoxic con-
`ditions.°’ There is therefore a high concen-
`tration ofnitrite in saliva, which increases with
`oral nitrate intake.* When swallowed,
`this
`nitrite is readily protonated under the acidic
`conditions of the stomach to form nitrous acid
`(acid dissociation constant pK,3-2—3-4), which
`in turn decomposesto various nitrogen oxides:
`
`NO,-+H* > HNO,(pK,~3-2-3-4)
`3HNO, —>H,0+2NO+NO,
`2HNO,
`~~ H,O+N,0,
`N,O;
`—~NO+NO,
`
`We have suggested that acidification of
`salivary nitrite is important in augmenting the
`antimicrobial effects of stomach acid and have
`shown that Candida albicans and Escherichia coli
`are much more susceptible to this combination
`than acid alone.’ Although it is not clear how
`acidified nitrite acts to kill micro-organisms,it
`is possible that NO, or a product of NO,is
`responsible as formation of this molecule from
`L-arginine has been shown to be importantin
`host defence.* The generation of NO in the
`human stomach has been demonstrated by
`measurement of NO in expelled gas after a
`carbonated drink in healthy subjects.? The
`purpose of this study was to more clearly
`quantify NO synthesis in the human stomach
`and determine
`the
`temporal
`relation of
`chemical NO productionafternitrate ingestion
`in healthy volunteers.
`
`Methods
`Ten healthy volunteers (six male; mean body
`weight 69 kg), who were not
`taking any
`medication, were invited to take part (21-43
`years). Local Ethics Committee approval was
`given for this study. Volunteers were fully
`informed and written consent obtained.
`Subjects were studied after overnightfasting on
`two separate occasions at least one week apart.
`The
`
`igs
`SF 36
`
`Human Power of N Company
`EX1038
`Page | of 4
`
`Department of
`Medicine and
`Therapeutics,
`Polwarth Building,
`Medical School,
`University of
`Aberdeen, Aberdeen
`G M McKnight
`LM Smith
`RS Drummond
`CW Duncan
`M Golden
`N Benjamin
`Correspondenceto:
`Dr G M McKnight,
`Department of Medicine and
`Therapeutics, Polwarth
`Building, Medical School,
`University of Aberdeen,
`Foresterhill,
`Aberdeen AB9 2ZD.
`
`Accepted for publication
`28 August 1996
`
`Human Power of N Company
`EX1038
`Page 1 of 4
`
`

`

`Gut 1997; 40: 211-214
`
`211
`
`Chemical synthesis of nitric oxide in the stomach
`from dietary nitrate in humans
`
`G M McKnight, L M Smith, R S Drummond, C W Duncan, M Golden, N Benjamin
`
`Abstract
`Background/Aims-It has been suggested
`that dietary nitrate, after concentration in
`the saliva and reduction to nitrite by
`tongue surface bacteria, is chemically
`reduced to nitric oxide (NO) in the acidic
`conditions of the stomach. This study
`aimed to quantify this in humans.
`Methods-Ten healthy fasting volunteers
`were studied twice, after oral adminis-
`tration of 2 mmol of potassium nitrate or
`potassium chloride. Plasma, salivary and
`gastric nitrate, salivary and gastric nitrite,
`and gastric headspace NO concentrations
`were measured over six hours.
`Results-On the control day the par-
`ameters measured varied little from basal
`values. Gastric nitrate concentration was
`105-3 (13) iimol/l (mean (SEM), plasma
`(2.4)
`nitrate
`concentration was
`17-9
`,umol/l, salivary nitrate concentration 92-6
`(31.6) ,umol/l, and nitrite concentration
`53*9 (22.8) ,umol/l. Gastric nitrite concen-
`were minimal
`(<1
`,ImolJl).
`trations
`Gastric headspace gas NO concentration
`was 16-4 (5-8) parts per million (ppm).
`After nitrate ingestion, gastric nitrate
`peaked at 20 minutes at 3430 (832) ,tmol/l,
`plasma nitrate at 134 (7.2) ,umol/l, salivary
`(280.5)
`nitrate
`,umol/l, and
`1516-7
`at
`salivary nitrite at 761 5 (187-7) ,umol/l after
`20-40 minutes. Gastric nitrite concen-
`trations tended to be low, variable, and
`any rise was non-sustained. Gastric NO
`concentrations rose considerably from
`(3.1) ppm to
`(28.6) ppm
`14-8
`89-4
`(p<0.0001) after 60 minutes. All par-
`ameters remained increased significantly
`for the duration of the study.
`Conclusions-A very large and sustained
`increase in chemically derived gastric NO
`concentrations after an oral nitrate load
`was shown, which may be important both
`in host defence against swallowed patho-
`gens and in gastric physiology.
`(Gut 1997; 40: 211-214)
`
`Keywords: nitric oxide, salivary nitrate and nitrite,
`stomach.
`
`Nitric oxide (NO) is an important biological
`molecule that is responsible for cell signalling
`and host defence in a number of mammalian
`tissues.' 2
`It
`is now well established that
`mammalian cells produce NO from the amino
`acid L-arginine with a family of NO synthase
`enzymes.
`
`We have recently proposed that NO may be
`synthesised by an alternative mechanism that
`relies on sequential reduction of nitrate.3
`Dietary nitrate (principally derived from green,
`leafy vegetables) is absorbed from the stomach
`and proximal small intestine into the plasma
`and subsequently concentrated in saliva.4
`Approximately 25% of dietary nitrate is re-
`circulated. The dorsal surface of the tongue
`harbours large numbers of nitrate reducing
`facultative anaerobic bacteria, which rapidly
`reduce nitrate to nitrite under hypoxic con-
`ditions.6 7 There is therefore a high concen-
`tration of nitrite in saliva, which increases with
`oral nitrate intake.4 When swallowed, this
`nitrite is readily protonated under the acidic
`conditions of the stomach to form nitrous acid
`(acid dissociation constant pKa3-2-3.4), which
`in turn decomposes to various nitrogen oxides:
`NO2-+H+
`- HN02 (pK-3-2-334)
`3HNO2
`-+ H20+2NO+NO3-
`2HNO2
`-+ H2O+N203
`N203
`-+ NO+NO2
`We have suggested that acidification of
`salivary nitrite is important in augmenting the
`antimicrobial effects of stomach acid and have
`shown that Candida albicans and Escherichia coli
`are much more susceptible to this combination
`than acid alone.3 Although it is not clear how
`acidified nitrite acts to kill micro-organisms, it
`is possible that NO, or a product of NO, is
`responsible as formation of this molecule from
`L-arginine has been shown to be important in
`host defence.8 The generation of NO in the
`human stomach has been demonstrated by
`measurement of NO in expelled gas after a
`carbonated drink in healthy subjects.9 The
`purpose of this study was to more clearly
`quantify NO synthesis in the human stomach
`and determine
`the
`temporal
`of
`relation
`chemical NO production after nitrate ingestion
`in healthy volunteers.
`
`Methods
`Ten healthy volunteers (six male; mean body
`weight 69 kg), who were not taking any
`medication, were invited to take part (21-43
`years). Local Ethics Committee approval was
`given for this study. Volunteers were fully
`informed
`and written
`obtained.
`consent
`Subjects were studied after overnight fasting on
`two separate occasions at least one week apart.
`The experimental protocol on each day was
`identical, other than oral administration on the
`test day of a 50 ml solution containing 2 mmol
`of potassium nitrate BP (Thornton and Ross,
`
`Department of
`Medicine and
`Therapeutics,
`Polwarth Building,
`Medical School,
`University of
`Aberdeen, Aberdeen
`G M McKnight
`L M Smith
`R S Drummond
`C W Duncan
`M Golden
`N Benjamin
`Correspondence to:
`Dr G M McKnight,
`Department of Medicine and
`Therapeutics, Polwarth
`Building, Medical School,
`University of Aberdeen,
`Foresterhill,
`Aberdeen AB9 2ZD.
`Accepted for publication
`28 August 1996
`
`Page 1 of 4
`
`

`

`McKnight, Smith, Drummond, Duncan, Golden, Benjamin
`
`UK). The chemiluminescence analyser was
`connected to a Maclab and Macintosh data
`acquisition system.
`
`Statistical analysis
`The results on the control and test days were
`compared using two way analysis of variance
`with repeated measures and post-hoc analysis
`using matched pairs
`simple and
`tests;
`t
`Spearman rank correlation analysis. A value of
`p<005 was considered significant.
`
`Results
`On the control day basal concentrations of
`plasma nitrate (17X9 (2 4)
`iimol/l), gastric
`nitrate (105-3 (23 1) [umol/l), salivary nitrate
`(92-6 (31-6) ,imol/l), and salivary nitrite (53 9
`(22-8) pumoJ/l), gastric nitrite (<1 ,umol/l) and
`headspace gas NO (16-4 (5 8) ppm) varied
`little over the six hour time course (Fig 1;
`Table).
`In contrast, after oral administration of 2
`mmol of potassium nitrate solution there were
`pronounced increases in plasma nitrate, gastric
`nitrate, salivary nitrate, salivary nitrite, and
`gastric headspace NO concentration. These
`remained considerably increased even at the
`end of the six hour study period (Fig 1; Table).
`Gastric nitrite concentrations tended to be low,
`variable, and any rise was not sustained
`although measurement was hampered by
`
`# Test (2 mmol KNO3)
`Control (2 mmol KCI)
`
`Huddersfield, UK), or on the control day,
`potassium chloride (BDH chemicals, Merck
`Ltd, Poole, Dorset, UK). After three hours, a
`low nitrate (29 ,umol total), neutral pH drink
`(Complan) was given. The experimental order
`was randomly allocated.
`After insertion of a fine bore nasogastric
`feeding tube (ET03 Medicina Ltd), blood,
`gastric headspace gas, gastric juice (5 ml), and
`unstimulated saliva'0 samples (1-3 ml) were
`taken at 20 minute intervals for two hours and
`30 minute intervals thereafter. To facilitate the
`aspiration of gastric contents, five minutes
`before sampling 50 ml ambient air (<10 ppb)
`was introduced via the nasogastric tube. The
`pH of gastric juice samples was measured and
`then the juices alkalinised with 50 RI 5M
`NaOH to prevent further reduction of NO2-.
`Nitrate and nitrite assays were performed by a
`modified Griess reaction, nitrate being first
`reduced on a copper coated cadmium column
`as previously described." Nitrite assays were
`performed in microwell plates and the diazo
`colour reaction detected at 540 nm. NO in
`aspirated headspace gas was analysed after
`dilution (10 ml gastric gas diluted to 100 ml
`total volume in NO impermeable container)
`with laboratory air to achieve final concen-
`tration less than 20 ppm using a chemilumin-
`escence meter (Thermo Electron Instruments,
`model 42A, Warrington, Cheshire, UK), which
`calibrated
`is
`standard
`with
`NO/nitrogen
`mixtures (MG Gas Products Ltd, Reigate,
`
`c0)
`
`._
`0-6hE
`C
`
`0c
`
`Time (min)
`
`0 C
`
`._
`
`0-
`C
`0
`
`0.
`
`212
`
`-C ,
`
`co E
`C -
`0 =
`
`CD
`
`0 0
`
`+,
`roE
`C)
`
`0E
`
`Z-
`
`0E
`
`C0 a
`
`)
`CC
`0
`
`C0
`
`C6
`
`1)
`co
`
`0C0
`
`C-)
`
`0
`
`20 40 60 80 100 120 150 180 210 240 270 300 330 360
`Complan
`Time (min)
`Figure 1: Effect of ingestion of nitrate on basal concentrations ofplasma nitrate, gastric and salivary nitrates/nitrites, gastric NO (all p<0 001 ANO VA),
`and alteration of intraluminal pH with time and Complan ingestion, at 182 minutes represented by solid bar. *Represents total salivary nitrate secreting by
`salivary glands before reduction on tongue (measured as nitrate plus nitrite). Data shown as mean (SEM).
`
`Page 2 of 4
`
`

`

`Gastric nitnic oxide production
`
`213
`
`EfJfect ofan inorganic nitrate load (2 mmol) on plasma salivaty and gastric nitrate, nitrite
`and nitric oxide concentrations
`
`Sample
`
`Gastric nitrate
`Plasma nitrate
`Total salivary nitrate
`Salivary nitrite
`Gastric nitrite
`Gastric NO (ppm)
`
`Control (mean (SEM))
`(,umolA)
`133-4 (21-2)
`17-9 (2-4)
`92-6 (31-6)
`53-9 (22 8)
`0-63 (0-63)
`16-4 (5 8)
`
`Test (mean (SEM))
`(,umol/l)
`3430-1 (832-1)
`134 (7 2)
`1516-7 (280 5)
`761-5 (187-7)
`105-3 (32-3)
`89-4 (28 6)
`
`Peak time
`(mins)
`20
`40
`20-40
`20-40
`40
`60
`
`Gastric NO values are parts per million (ppm). Nitrate and nitrite values are ,umol/A as denoted.
`
`Discussion
`This study shows that very large concen-
`trations of NO are generated in the stomach
`after an oral nitrate intake. The maximum
`concentration of NO was seen approximately
`60 minutes after a dose of nitrate, which
`represents the average intake of an adult over
`one to two days in the UK.12 The mean con-
`centration ofNO at this time averaged 90 ppm;
`this is about 7000 times that found in exhaled
`breath.9 The concentrations
`of stomach
`headspace gas NO were considerably higher in
`our study than those previously reported (basal
`difficulty in achieving a clear supernatant that
`gastric NO 800-6000 parts per
`expelled
`would not interfere with absorption at 540 nm.
`billion).9 This may be due to dilution of gastric
`Due to the turbidity of some gastric juice
`gas in the previous study with carbon dioxide
`samples even after repeated centrifugation,
`generated from the carbonated drink used to
`absorption readings were inappropriately high
`induce belching. The higher values measured
`although there was no visibly detectable pink
`in this study may be an underestimate of true
`colour of the diazo reaction.
`I NO concentrations in the stomach as NO at
`Peak gastric nitrate concentration occurred
`this concentration will readily combine with
`at the first measurement after intake (20 min),
`oxygen to form NO2, albeit by second order
`plasma values peaked at 40 minutes and gastric
`kinetics, which is not measured by the
`headspace NO concentration reached a peak at
`used.
`analyser
`chemiluminescence
`60 minutes (Table). All remained significantly
`we
`7
`increased for the duration of the study (p< 10'
`Furthermore, as we had to inject air into the
`stomach five minutes before sampling the con-
`ANOVA; apart from gastric
`nitrite). The
`centration of NO in headspace gas may not
`highest measured gastric headspace gas NO
`have reached equilibrium with the gastric juice.
`value was 291 ppm 60 minutes after intake of
`f
`In previous studies in vitro we found that
`the nitrate solution.
`headspace NO reaches equilibrium with the
`Gastric acidity was not significantly affected
`I
`by nitrate intake but dropped dramatically after
`aqueous phase in a shaken closed vessel at
`r
`about one hour.7
`the ingestion of Complan recovering by the
`next sampling time and thereafter decreasing
`At six hours after nitrate intake the concen-
`(Fig 1). Although gastric NO was falling by the
`tration remained considerably higher than
`baseline, suggesting a prolonged effect of
`neutral
`drink
`ingested,
`time
`the
`was
`dietary nitrate on stomach NO synthesis. The
`presumably parallel to the decline in salivary
`increased NO generation was associated with
`nitrate and nitrite concentrations, there was
`the expected rise in salivary nitrate and nitrite
`nevertheless a significant reduction in NO
`and the timecourse suggests that stomach NO
`production when gastric pH rose after the
`synthesis derives from acidification of salivary
`drink and a significant recovery when gastric
`nitrite, even though the correlation between
`pH returned to basal conditions. This negative
`stomach NO and salivary nitrite in individual
`relation between gastric juice pH and stomach
`subjects did not reach statistical significance.
`headspace gas NO concentration is shown in
`The concentration of stomach headspace NO
`Figure 2 (rs=-0 55; p<001). There was no
`was, however, associated with the degree of
`significant relation between stomach NO and
`I
`acidity in the stomach. During the course of
`salivary nitrite concentrations (control day
`(rs=0 32;
`the experiment, on both study days, stomach
`day
`(r2=0 46;
`p=0 024);
`test
`p<0-001)), nor were any of the measured
`acidity increased initially, presumably due to
`I
`the presence of a nasogastric tube. After
`variables different between sexes.
`ingestion of Complan there was a short lived
`reduction in acidity accompanied by reduction
`in nitric oxide generation even allowing for the
`expected fall in NO by this stage in the study.
`As gastric acidity recovered so NO generation
`followed and reached pre-meal levels and at the
`end of the study day NO levels were still
`significantly higher than on the control day.
`This is probably because of the persisting
`increase in plasma nitrate and thus assimilation
`into saliva even at the end of the study period.
`Previous studies in vitro have shown that
`NO is generated when nitrite is acidified. The
`amount of NO generated in the stomach in
`vivo, however, greatly exceeds that expected
`studies,
`from in
`vitro
`suggesting another
`j reducing agent such as ascorbate may be
`j
`present in saliva or gastric juice to increase
`6
`nitrite reduction to NO.7
`We have previously suggested that salivary
`concentration of nitrate and subsequent rapid
`
`PKa
`
`o Before Complan
`* 28 min after
`Complan
`* Recovery phase
`
`0
`
`A
`
`A
`
`100 r
`
`0
`
`80 K-
`
`60 F-
`
`*
`
`40 F-
`
`A
`
`0
`
`E 0 z
`
`20
`
`ni
`v
`
`..
`
`0
`
`o
`
`2
`
`3
`
`A
`
`A 9
`
`4
`
`A
`
`5
`
`.k.
`
`pH
`Figure 2: pH dependency ofgastric NO production.
`Spearman rank correlation -0.55, p<0 01.
`
`Page 3 of 4
`
`

`

`214
`
`McKnight, Smith, Drummond, Duncani, Golden, Benjamin
`
`reduction to nitrite by lingual bacteria is a
`symbiotic process designed to serve a useful
`function. The very high and sustained concen-
`trations of stomach NO generated by chemical
`reduction of salivary nitrite are likely to destroy
`or inhibit swallowed microbial pathogens.
`of
`variety
`that
`studies
`a
`suggest
`Several
`this substance.
`to
`sensitive
`organisms are
`Helicobacter pylon, which inhabits the mucus
`layer covering the mucosa (only a few microns
`thick) will be exposed to these high concen-
`trations of NO that rapidly diffused through
`the
`mediums;
`and
`lipid
`aqueous
`both
`sensitivity of this organism to NO is as yet
`unknown.
`In addition NO is known to be involved in
`the regulation of gastric mucosal blood flow'3
`of mucosal
`the
`preservation
`and hence
`integrity, gastric motility,'4 and mucus pro-
`duction. Previous studies have suggested a role
`for NO in stimulated gastrin and gastric acid
`secretion'5 16 although the high NO concen-
`trations after nitrate intake did not reduce
`gastric pH in this study.
`Although there has been continuing concern
`as regards the nitrosating potential of nitrate
`epidemio-
`constituents,
`dietary
`containing
`logical studies have failed to establish any
`association between gastrointestinal
`causal
`While we
`malignancy and dietary nitrate.
`have not attempted in this initial study to
`quantify nitrosamine production, in the normal
`acidic stomach the large amounts of NO that
`are generated and the very low gastric nitrite
`concentrations resulting from a nitrate load
`would suggest that nitrosamine formation is
`not favoured.
`In summary, this study shows that dietary
`nitrate, by bacterial, as previously described,
`and chemical reduction, may generate very
`large concentrations of NO in the stomach,
`much greater than those generated by intrinsic
`NO synthase.'8 The mechanism we describe
`may be extremely important in augmenting the
`acid and
`of gastric
`properties
`antiseptic
`fuinction.
`gastric
`physiological
`modifying
`Additionally, the formation of NO in the
`stomach from acidified nitrate may be one of
`the mechanisms by which the nitrosating
`
`potential of nitrite and nitrate containing
`constituents of the diet is minimised. We
`believe that the mechanism of enterosalivary
`circulation of nitrate is designed to produce
`large concentrations of NO in the mouth and
`stomach and thus may have beneficial as well
`effects on human
`deleterious
`possibly
`as
`health.
`
`This work was supported by a grant from the Ministry of
`Agriculture, Fisheries and Foods. The authors thank Dr N A
`G Mowat for helpful discussion, advice, and critical reading of
`the manuscript.
`
`1 Moncada S, Higgs A. The L-Arginine-Nitric oxide path -axv.
`NEniglJ7Med 1993; 329: 2002-12.
`2 Anggard E. Nitric oxide: mediator, murderer and medicine.
`Lanzcet 1994; 343: 1199-206.
`3 Benjamin N, O'Driscoll F, Dougall H, Duncan C, Smith L,
`Golden M, cl al. Stomach NO svnthesis. Nature 1994;
`368: 502.
`4 Tannenbaum SR, Weisman M, Fett D. The effect of nitrate
`intake on nitrite formation in human saliva. Food Cosniet
`Toxicol 1976; 14: 549-52.
`5 Spiegelhalder B, Eisenbrand G, Preussmann R. Influence of
`dietary nitrate on nitrite content of human saliva: possible
`relevance to inzvitro formation of N-nitroso compounds.
`Food Cosniet Toxicol 1976; 14: 545-8.
`6 Sasaki T, Matano K. Formation of nitrite from nitrate at the
`dorsum linguae. 7 Food Hyg Socjpn 1979; 20: 363-9.
`7 Duncan C, Dougall H, Johnston P, Green S, Brogan R,
`Liefert C, et al. Chemical generation of nitric oxide in the
`mouth from the enterosalivarv circulation of dietarn
`nitrate. Nature Medicinle 1995; 1: 546-51.
`8 Nathan C.
`Nitric
`secretory product
`of
`oxide
`as
`a
`mammalian cells. FASEB 1992; 6: 3051-64.
`9 Lundberg JON, Weitzberg E, Lundberg JIM, Alving K.
`Intragastric nitric oxide production in humans: measure-
`ments in expelled air. Gut 1994; 35: 1543-6.
`10 Granli T, Dahl R, Brodin P, Bockman OC. Nitrate and
`nitrite concentrations in human saliva: variations with
`salivary flow rate. Food Cheni Toxicol 1989; 27: 675-80.
`11 Green LC, WagnerDA, Glogow,skiJ, SkipperPL, WishnokJS,
`Tannenbaum SR. Analysis of nitrate, nitrite, and [15N]
`in biological fluids. Anial Biocheiii
`1982; 26:
`nitrate
`131-8.
`12 Knight TIM, Forman D, Al-Dabbagh SA, Doll R. Esti-
`mation of dietary intake of nitrate and nitrite in Great
`Britain. Food Chern Toxicol 1987; 25: 277-85.
`13 Pique JM, Whittle BJR, Esplugues JY. The X asodilator role
`of endogenous nitric oxide in the rat gastric micro-
`circulation. Eur_ Pharmacol 1989; 174: 293-6.
`14 Desai KM, Sessa WC, Vane JR. Involvement of nitric oxide
`in the reflex relaxation of the stomach to accommodate
`food or fluid. Nature 1991; 351: 477-9.
`15 Stroff T, Plate S, Respondek M, Miller KM, Peskar B.M.
`Protection by gastrin in the rat stomach involves afferent
`neurons, calcitonin gene-related peptide and nitric oxide.
`Gastroewterology 1995; 109: 89-97.
`16 Bilski J, Konturek PC, Konturek SJ, Cieszkowski M,
`Czamobilski K. Role of endogenous nitric oxide in the
`control of gastric acid secretion, blood flow and gastrin
`release in conscious dogs. Regul Pept 1994; 54: 175-84.
`17 Boeing H. Epidemiological research in stomach cancer:
`progress over the last ten vears. _7 Canicer Res Clinz Onicol
`1991;47: 133-43.
`18 Kugler P, Drenckhahn D. Intrinsic source of stomach NO.
`Nature 1994; 370: 25-6.
`
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