Nitric Oxide 7 (2002) 24–29
`
`NITRIC
`OXIDE
`
`Biology and Chemistry
`
`www.academicpress.com
`
`The ingestion of inorganic nitrate increases gastric
`S-nitrosothiol levels and inhibits platelet function in humans
`
`G. Richardson,a S.L. Hicks,a S. O’Byrne,a M.T. Frost,b K. Moore,c N. Benjamin,a,*
`and G.M. McKnighta
`
`a Department of Clinical Pharmacology, St. Bartholomew’s Hospital, Barts and the London, Queen Mary School of Medicine and Dentistry,
`Charterhouse Square, London EC1M 6BQ, UK
`b International Antioxidant Research Centre, Guy’s, King’s and Thomas’s School of Biomedical Sciences (MTF), London SE1 9RT, UK
`c Centre for Hepatology, Royal Free and University College Medical School, Royal Free Campus, University College London, London NW3 2PF, UK
`
`Received 27 August 2001; received in revised form 27 February 2002
`
`Abstract
`
`Platelets play an important role in the development of vascular disease, while vegetarian diets, which are rich in inorganic nitrate,
`protect against it. This study was performed to assess the effect of potassium nitrate (KNO3) ingestion on platelet function in
`humans. Oral KNO3 (2 mmol) was given to healthy volunteers and its effect on platelet function assessed by measuring the aggregant
`effect of collagen. Blood samples were taken for measurement of plasma S-nitrosothiols (RSNO) and platelet cyclic GMP and
`nitrotyrosine levels. Gastric juice samples were taken for measurement of RSNO. In a separate study, the effect of oral KNO3 on
`portal RSNO levels in patients with intrahepatic porto-systemic shunts was assessed. KNO3 caused a significant increase in gastric
`RSNO levels, from 0:46  0:06 to 3:62  2:82 lM (tmax 45 min; P < 0:001), and significantly inhibited platelet function (tmax 60 min;
`P < 0:001). There was no effect on systemic or portal RSNO, platelet cGMP or platelet nitrotyrosine levels. Oral KNO3 inhibits
`platelet aggregation. The time course suggests that gastric RSNO production may be involved in this effect. The protection against
`vascular events associated with a high intake of vegetables may be due to their high nitrate content. Ó 2002 Elsevier Science (USA).
`All rights reserved.
`
`Keywords: Platelets; Nitric oxide; S-nitrosothiols; Inorganic nitrate; Gastric
`
`leafy vegetables [7]. Approximately 2 mmol of
`green,
`inorganic nitrate are contained in half a British flat let-
`tuce, or a Ploughman’s lunch [8].
`it is rapidly
`When inorganic nitrate is swallowed,
`absorbed and is concentrated in the salivary glands by
`an as-yet-uncharacterized mechanism, so that the ni-
`trate concentration of saliva is at least 10 times that
`of plasma [9]. The nitrate is then rapidly reduced to
`) in the mouth by nitrate reductase-con-
`nitrite (NO
`taining bacteria on the posterior surface of the tongue
`(Eq. (1)), as we have previously described [10]. Once
`swallowed, this nitrite is exposed to the strongly acidic
`milieu of the stomach, where the resting pH is 1.5–2.
`Here it forms nitrous acid (HNO2) (Eq. (2)), which
`decomposes to form oxides of nitrogen, including ni-
`tric oxide (NO) (Eqs. (3)–(5)). In the stomach this
`process is catalyzed by reducing substances derived
`
` 2
`
`It is clear that platelets influence the development
`and progression of occlusive vascular disease [1] and
`that the activation of platelets is involved in the clinical
`presentation of acute coronary syndromes [2]. There are
`now data to suggest that the incidence of coronary
`artery disease is at least partly dependent on diet, with
`those who eat diets with low saturated fat and high
`vegetable content being at lower risk of developing the
`disease than those who do not [3,4]. The protective
`component of vegetarian diets remains to be identified,
`with vitamin E [5] and folic acid [6] being two possible
`candidates.
`Another component of the vegetarian diet is inor-
`), which is found in large quantities in
`ganic nitrate (NO
`
` 3
`
`* Corresponding author. Fax: +44-0-20-7882-3408.
`E-mail address: n.benjamin@mds.qmw.ac.uk (N. Benjamin).
`
`1089-8603/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
`PII: S 1 0 8 9 - 8 6 0 3 ( 0 2 ) 0 0 0 1 0 - 1
`
`Human Power of N Company
`EX1017
`Page 1 of 6
`
`

`

`Nitric Oxide 7 (2002) 24–29
`
`NITRIC
`OXIDE
`
`Biology and Chemistry
`
`www.academicpress.com
`
`The ingestion of inorganic nitrate increases gastric
`S-nitrosothiol levels and inhibits platelet function in humans
`
`G. Richardson,a S.L. Hicks,a S. O’Byrne,a M.T. Frost,b K. Moore,c N. Benjamin,a,*
`and G.M. McKnighta
`
`a Department of Clinical Pharmacology, St. Bartholomew’s Hospital, Barts and the London, Queen Mary School of Medicine and Dentistry,
`Charterhouse Square, London EC1M 6BQ, UK
`b International Antioxidant Research Centre, Guy’s, King’s and Thomas’s School of Biomedical Sciences (MTF), London SE1 9RT, UK
`c Centre for Hepatology, Royal Free and University College Medical School, Royal Free Campus, University College London, London NW3 2PF, UK
`
`Received 27 August 2001; received in revised form 27 February 2002
`
`Abstract
`
`Platelets play an important role in the development of vascular disease, while vegetarian diets, which are rich in inorganic nitrate,
`protect against it. This study was performed to assess the effect of potassium nitrate (KNO3) ingestion on platelet function in
`humans. Oral KNO3 (2 mmol) was given to healthy volunteers and its effect on platelet function assessed by measuring the aggregant
`effect of collagen. Blood samples were taken for measurement of plasma S-nitrosothiols (RSNO) and platelet cyclic GMP and
`nitrotyrosine levels. Gastric juice samples were taken for measurement of RSNO. In a separate study, the effect of oral KNO3 on
`portal RSNO levels in patients with intrahepatic porto-systemic shunts was assessed. KNO3 caused a significant increase in gastric
`RSNO levels, from 0:46  0:06 to 3:62  2:82 lM (tmax 45 min; P < 0:001), and significantly inhibited platelet function (tmax 60 min;
`P < 0:001). There was no effect on systemic or portal RSNO, platelet cGMP or platelet nitrotyrosine levels. Oral KNO3 inhibits
`platelet aggregation. The time course suggests that gastric RSNO production may be involved in this effect. The protection against
`vascular events associated with a high intake of vegetables may be due to their high nitrate content. Ó 2002 Elsevier Science (USA).
`All rights reserved.
`
`Keywords: Platelets; Nitric oxide; S-nitrosothiols; Inorganic nitrate; Gastric
`
`It is clear that platelets influence the development
`and progression of occlusive vascular disease [1] and
`that the activation of platelets is involved in the clinical
`presentation of acute coronary syndromes [2]. There are
`now data to suggest that the incidence of coronary
`artery disease is at least partly dependent on diet, with
`those who eat diets with low saturated fat and high
`vegetable content being at lower risk of developing the
`disease than those who do not [3,4]. The protective
`component of vegetarian diets remains to be identified,
`with vitamin E [5] and folic acid [6] being two possible
`candidates.
`Another component of the vegetarian diet is inor-
`ganic nitrate (NO
`3 ), which is found in large quantities in
`
`* Corresponding author. Fax: +44-0-20-7882-3408.
`E-mail address: n.benjamin@mds.qmw.ac.uk (N. Benjamin).
`
`leafy vegetables [7]. Approximately 2 mmol of
`green,
`inorganic nitrate are contained in half a British flat let-
`tuce, or a Ploughman’s lunch [8].
`it is rapidly
`When inorganic nitrate is swallowed,
`absorbed and is concentrated in the salivary glands by
`an as-yet-uncharacterized mechanism, so that the ni-
`trate concentration of saliva is at least 10 times that
`of plasma [9]. The nitrate is then rapidly reduced to
`nitrite (NO
`2 ) in the mouth by nitrate reductase-con-
`taining bacteria on the posterior surface of the tongue
`(Eq. (1)), as we have previously described [10]. Once
`swallowed, this nitrite is exposed to the strongly acidic
`milieu of the stomach, where the resting pH is 1.5–2.
`Here it forms nitrous acid (HNO2) (Eq. (2)), which
`decomposes to form oxides of nitrogen, including ni-
`tric oxide (NO) (Eqs. (3)–(5)). In the stomach this
`process is catalyzed by reducing substances derived
`
`1089-8603/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved.
`PII: S 1 0 8 9 - 8 6 0 3 ( 0 2 ) 0 0 0 1 0 - 1
`
`Page 1 of 6
`
`

`

`G. Richardson et al. / Nitric Oxide 7 (2002) 24–29
`
`25
`
`from the saliva and diet, such as ascorbate, chloride,
`iodide, and thiocyanate ions.
`ð1Þ
`NO3 ! NO2
`ð2Þ
`NO2 þ H
`þ ! HNO2ðpKa ¼ 3:2 3:4Þ
`ð3Þ
`3HNO2 ! H2O þ H
`þ þ 2NO þ NO3
`ð4Þ
`2HNO2 ! H2O þ N2O3
`ð5Þ
`N2O3 ! NO þ NO2
`The nitrate produced is reabsorbed and again concen-
`trated in the salivary glands, completing the enterosali-
`vary circulation.
`In humans, ingestion of inorganic nitrate results in
`remarkably high concentrations of NO in the stomach
`[11], the fate of which remains unknown. It is possible
`that this NO affects blood supplying the stomach or that
`it is absorbed into the portal or systemic circulation in
`the form of an NO donor. NO has many important
`biological actions, including the inhibition of platelet
`function [12], which is due to activation of the enzyme
`soluble guanylate cyclase, to produce cyclic guanosine
`monophosphate (cGMP)1 [13]. Another mechanism by
`which NO can inhibit platelet function is the formation
`of nitrotyrosine. NO reacts with superoxide (O2 ) to
`form peroxynitrite (ONOO) [14]. ONOO reacts with
`tyrosine residues of proteins to form nitrotyrosine [15],
`which, in platelets, inhibits their response to an aggre-
`gant stimulus [16], although the physiological relevance
`of this remains unclear.
`S-Nitrosothiol (RSNO) formation from the combi-
`nation of thiol with nitrite or NO directly is another
`possible fate of inorganic nitrate. Thiols are ingested in
`the diet in some vegetable and in meats and have been
`shown to be present in the gastric mucosa in animals [17]
`and man [18]. Under the acidic conditions of the stom-
`ach, thiols may combine with nitrosating species such as
`nitrite [19] (Eq. (6)), or nitrous anhydride (Eq. (7)) to
`produce RSNOs.
`ð6Þ
`HNO2 þ RSH ! RSNO þ H2O
`ð7Þ
`N2O3 þ 2RSH ! 2RSNO þ H2O
`This nitrosation of thiols, favored by the acid conditions
`in the stomach (pH < 4), drives the decomposition of
`nitrous acid to nitric oxide and other nitrogen oxides.
`RSNO formation has been shown to be first order
`with respect to thiol, nitrous acid, and the hydrogen ion
`[20]. S-Nitrosothiols have a complex pH dependent
`stability profile and may undergo transnitrosation re-
`actions (Eq. (8)). This may be an important mechanism
`
`1 Abbreviations used: cGMP, cyclic guanosine monophosphate;
`RSNO, S-nitrosothiol; PRP, platelet-rich protein; PPP, plate-poor
`protein; NEM, N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluo-
`ride.
`
`for the formation of S-nitrosothiols in biological sys-
`tems:
`ð8Þ
`RSNO þ R SH ! RSH þ R SNO
`Like NO, RSNOs are also potent inhibitors of platelet
`function [21] by both cGMP-dependent [22] and inde-
`pendent mechanisms [23,24].
`Thus it would appear possible that the ingestion of
`inorganic nitrate would generate RSNOs in the stomach
`and that the generation of either NO or an NO donor
`should influence platelet function.
`We therefore undertook studies to investigate the
`effect of the ingestion of dietary equivalent amounts of
`oral inorganic nitrate on gastric RSNO generation and
`platelet function in healthy human volunteers. We also
`measured levels of the mediators of platelet function,
`cGMP and nitrotyrosine. In a separate study we meas-
`ured portal blood RSNO levels following KNO3 in-
`gestion in three patients with permanent intrahepatic
`portosystemic shunts.
`
`Methods
`
`Subjects
`
`The healthy human volunteers (ages 18–44) were
`taking no regular medications and were instructed to
`avoid taking aspirin and nonsteroidal anti-inflammatory
`drugs for at least 1 week prior to the study. They were
`maintained on a low-nitrate diet for 24 h prior to, and
`fasted for 8 h on the day of each study. Fully informed,
`written consent and approval from the subjects and the
`local ethics committee were obtained.
`Gastric RSNOs were measured following the ad-
`ministration of KNO3 or KCl in five healthy volunteers
`(three males and two females).
`The effect of oral KNO3 on platelet function was
`assessed in two separate studies. In the first, the effect of
`a single dose of KNO3 was compared to a control, KCl.
`In the second, the effect of different doses of KNO3 was
`assessed and an attempt made to determine the mech-
`anism for its anti-platelet effects. In the first study, seven
`healthy volunteers (four males and three females) were
`recruited and studied on two separate occasions, and in
`the second, six volunteers (three males and three fe-
`males) were studied on three separate occasions.
`The measurement of portal blood S-nitrosothiols was
`performed on samples taken from well-compensated
`cirrhotic patients, Child’s grade A or B, who had previ-
`ously for clinical reasons (portal hypertension and its
`complications) had transjugular intrahepatic portosys-
`temic shunts inserted. Three male patients were studied at
`the time of routine shunt checks when the shunt patency
`and pressure was assessed by transjugular shunt cannu-
`lation under local anesthesia and radiological guidance.
`
`Page 2 of 6
`
`

`

`26
`
`G. Richardson et al. / Nitric Oxide 7 (2002) 24–29
`
`Nitrate/control administration
`
`The experimental protocol on each of the study days
`was identical. Subjects swallowed either 2 mmol of
`(Thornton and Ross, Huddersfield, UK) or
`KNO3
`2 mmol of KCl (Thornton and Ross). In the second
`platelet study the subjects were also given 0.5 mmol
`KNO3. Each was diluted to a volume of approximately
`150 mL in deionized water. For portal blood samples,
`KNO3 was administered just prior to the procedure.
`Samples were taken 45 min after ingestion.
`
`Sampling procedures
`
`Gastric juice sampling procedure
`A fine bore nasogastric feeding tube (ET03; Medi-
`cina, Berkshire, UK) was introduced into the stomach of
`the volunteer. A baseline sample of gastric juice (1–2 ml)
`was taken before the administration of KNO3 or KCl,
`and further samples were then collected at 15-min in-
`tervals over a 90-min period. RSNO content was ana-
`lyzed within 4 h of sampling.
`
`Blood sampling procedure
`Volunteers were recumbent for the duration of the
`study period. A 19G butterfly (Abbot, Sligo, Republic
`of Ireland) was inserted into the antecubital vein and
`patency maintained between sampling times by flushing
`with 0.9% sodium chloride (Baxter, Thetford, UK).
`Venous blood samples were obtained, without the use of
`a tourniquet, at baseline and subsequently at 15-min
`intervals for 2 h following administration of the test
`substance. A final sample was taken at 2.5 h.
`Blood samples were collected into:
`1. Polystyrene tubes (A444; Bibby Sterilin Ltd., Stone,
`UK) containing 1 mL 3.8% sodium citrate. The sam-
`ple (9 mL blood) was centrifuged at 250g for 10 min
`to prepare platelet-rich plasma (PRP), which was re-
`moved. Platelet-poor plasma (PPP) was then prepared
`by further centrifugation of the original sample at
`1500g for 15 min. The PRP was used for platelet ag-
`gregometry studies and platelet cGMP measurements.
`2. Vacutainer EDTA tubes (4.5 mL; Becton Dickinson,
`Franklin Lakes, NJ) also containing 0.1 mL 250 mM
`N-ethylmaleimide (NEM; Sigma, Poole, UK). The
`sample (4.9 mL blood) was centrifuged at 1500g to
`prepare plasma for S-nitrosothiol determination.
`3. Vacutainer lithium heparin tubes (6 mL; Becton
`Dickinson). The sample (6 mL blood) was centrifuged
`at 1500g and then further prepared for platelet nitro-
`tyrosine analysis.
`
`Gastric S-nitrosothiol measurements
`
`Aliquots of gastric juice (500 lL) were added to
`100 lL of 35% sulfosalicyclic acid (Sigma) and mixed by
`
`vortexing for 20 min and then centrifuging at 16,000 g
`for 10 min. The supernatant (100 lL) was injected into a
`reaction flask containing 4 mL saturated cuprous chlo-
`ride (CuCl) (Sigma) solution and 2 mL of 2 mM gluta-
`thione (Sigma). The NO released was measured by
`chemiluminescence using a Sievers nitric oxide analyzer
`(NOA 280; Sievers Instruments Inc., Boulder, CO) and
`the concentration of low-molecular-weight RSNOs cal-
`culated.
`
`Platelet aggregation
`
`Platelet aggregometry studies were performed within
`3 h of collecting samples. A Chronolog dual-channel
`whole-blood lume-aggregometer (560VS) was used, and
`the tests carried out at 37 °C. The optical method [25]
`was used to determine the platelet response 3 min after
`the addition of collagen reagent (2 mg/mL; Sigma).
`Using each subject’s baseline samples,
`the lowest
`concentration of collagen needed to achieve maximum
`aggregation was determined, and this was then used on
`all test samples.
`
`Platelet cGMP measurements
`
`Platelet cGMP were measured at 15-min intervals for
`a 2-h sampling period. PRP (200 lL) was microcentri-
`fuged in an Eppendorf tube containing 50 lL of 50 mM
`EDTA (Sigma) to produce a platelet pellet. The super-
`natant was removed and the pellet frozen in liquid ni-
`trogen and then stored at )80 °C.
`Platelet cGMP levels were determined using a
`radioimmunoassay technique (Amersham International,
`Amersham, UK). Each platelet pellet was resuspended
`in a total volume of 250 lL (195 lL phosphate-buffered
`saline (Sigma), 5 lL of 50 mM phenylmethylsulfonyl-
`fluoride (PMSF) (Sigma), and 50 lL of 50 mM EDTA
`(Sigma)). The platelets were ultrasonicated for 3 s and
`then a 200 lL sample to was added to 10 lL of the
`acetylation reagent supplied with the kit prior to per-
`forming the radioimmuno assay.
`
`Platelet nitrotyrosine measurements
`
`Aliquots of plasma (1 mL) were microcentrifuged in
`Eppendorf tubes for 30 s to produce a platelet pellet.
`The supernatant was removed, and the pellet frozen in
`liquid nitrogen and then stored at )80 °C.
`Platelet nitrotyrosine levels were measured by a stable
`isotope dilution gas chromatography mass spectrometry
`method following extraction of tissue proteins and al-
`kaline hydrolysis. The platelet pellet from 1 mL of
`plasma was hydrolyzed in 1 mL 4 M sodium hydroxide
`at 120 °C overnight, following the addition of 10 ng
`[13C9]nitrotyrosine and 10 lg [D4]tyrosine as an internal
`standard. Samples were then extracted and analyzed by
`
`Page 3 of 6
`
`

`

`G. Richardson et al. / Nitric Oxide 7 (2002) 24–29
`
`27
`
`gas chromatography mass spectrometry as previously
`described [26],
`following purification by LC18 and
`ENV+ cartridge extraction and derivatization to the
`heptafluorobutyric amide and tertbutyldimethylsilyl
`derivatives. This method has an intraassay and inter-
`assay variation of less than 5%. All results are expressed
`in relation to dry weight of pellet from 1 mL plasma.
`
`S-Nitrosothiol measurements
`
`Plasma (980 lL) was placed in an Eppendorf tube
`containing 20 lL of 250 mM NEM (Sigma), and the
`samples mixed and then stored on ice. Total RSNO
`content of the samples was measured on the same day
`using a chemiluminescence-based assay as previously
`described [27].
`
`Statistics
`
`The data were analyzed using three-way ANOVA
`with subject, time, and treatment as factors, and Dun-
`nett’s test was used to compare differences with control
`(KCl) and changes from baseline with time. A P value of
`< 0:05 was taken to indicate statistical significance. The
`software package used was Minitab 13.2.
`
`Results
`
`Gastric S-nitrosothiols
`Baseline gastric RSNO levels were 0:46  0:42 lM.
`There was a significant increase in RSNO concentration
`ingestion of KNO3 but not KCl
`following the
`(P < 0:001). The peak level of gastric RSNO was
`3:62  0:53 lM at 45 min (Fig. 1).
`
`Platelet aggregation
`
`In the first study, there was a significant inhibition of
`the platelet response to collagen to 2 mmol KNO3, but
`not to KCl (P < 0:001). The total dose effect was a
`
`30.9% reduction (95% CI, )39.2 to )22.7%) in aggre-
`gation and the maximum effect was seen between 40 and
`80 min following ingestion (Fig. 2).
`In the second study, both doses of oral KNO3 in-
`hibited collagen-induced platelet aggregation, whereas
`KCl had no effect (Fig. 3). Although the degree of in-
`hibition was the same for both doses, 2 mmol KNO3
`15.9% (95% CI, 6.1 to 25.7%) and 0.5 mmol 11.3% (95%
`CI, 1.5 to 21.1%), the tmax for 2 mmol of KNO3 was
`significantly shorter, by 30 min, than that for 0.5 mmol
`(95% CI, )60 to )22.5 min).
`
`Platelet cGMP
`
`Platelet cGMP was measured following the ingestion
`of 2 mmol of KNO3. No significant change from the
`baseline level of 1:26  0:18 pmol=109 platelets was seen
`over the 2 h sampling period.
`
`Platelet nitrotyrosine
`
`Platelet nitrotyrosine was measured following the
`ingestion of 2 mmol of KNO3. Baseline nitrotyrosine
`levels were 0:142  0:056  103 ng/mg of tyrosine and
`
`Fig. 2. Time course curves for platelet responses to a collagen stimulus
`to aggregation following ingestion of KNO3 2 mmol (j; n ¼ 7), or KCl
`2 mmol (N; n ¼ 5). The data are expressed as a percentage of the
`baseline response at t ¼ 0. Each point represents mean  SE of mean.
`
`Fig. 1. Time course curve for gastric RSNO levels following the in-
`gestion of 2 mmol KNO3 (j; n ¼ 5) or 2 mmol KCl (N; n ¼ 5). Each
`point represents mean  SE of mean.
`
`Fig. 3. Time course curves for platelet responses to a collagen stimulus
`to aggregation following ingestion of KNO3 2 mmol (j; n ¼ 6), KNO3
`0.5 mmol (d; n ¼ 6) or KCl 2 mmol (N; n ¼ 4). The data are expressed
`as a percentage of the baseline response at t ¼ 0. Each point represents
`mean  SE of mean.
`
`Page 4 of 6
`
`

`

`28
`
`G. Richardson et al. / Nitric Oxide 7 (2002) 24–29
`
`did not significantly change following the ingestion of
`2 mmol of KNO3.
`
`Plasma S-nitrosothiols
`Baseline plasma RSNO levels were 27:7  4:7 nM.
`Following the ingestion of 2 mmol of KNO3, no signifi-
`cant change from baseline was seen over the 2 h sam-
`pling period.
`
`Portal blood S-nitrosothiols
`
`Portal blood samples were taken at 45 min postin-
`gestion. The was no detectable RSNO in any of the
`samples.
`
`Discussion
`
`In this study, the ingestion of KNO3 was associated
`with a rise in gastric low-molecular-weight RSNO con-
`centrations. The time course of this change in RSNO
`levels is compatible with the time course of changes in
`NO and nitrate levels in the stomach and plasma, re-
`spectively, following the ingestion of inorganic nitrate
`[11].
`The formation of RSNO in the stomach is shown in
`this study and, indeed, is inevitable from the reaction
`shown in Eq. (6).
`Orally administered inorganic nitrate in amounts
`equivalent to those found in a healthy diet (0.5–2 mmol)
`also significantly impaired the ability of platelets to re-
`spond to a collagen stimulus in healthy volunteers. The
`timing of this effect was dependent on the dose of KNO3
`administered, with the higher dose (2 mmol) achieving a
`maximum effect at 60 min after ingestion. The lower
`dose (0.5 mmol) achieved the same magnitude of effect
`at a later time.
`The increase in gastric RSNO concentration is
`therefore associated with inhibition of platelet function.
`RSNOs can inhibit platelet function, at low doses, by
`cGMP-dependent
`[22] and independent mechanisms
`[23,24]. Among other mediators of platelet function with
`which RSNOs may be involved are copper [23], ni-
`trotyrosine [16,28,29], and a cell-mediated mechanism
`[30].
`However, it cannot be determined from this study
`that RSNOs are the direct mediators of this effect. We
`found no increase in levels of platelet cGMP or ni-
`trotyrosine and no increase in total systemic levels of
`RSNOs. The reason for the delayed effect of the lower
`dose of KNO3 is difficult to explain without exact
`knowledge of the synthesis, absorption, and metabolism
`of RSNOs or other NO donors synthesized in the
`stomach via nitrosation reactions. At present little is
`known about RSNO metabolism.
`
`In the same way that low-dose aspirin exerts its anti-
`platelet effects [31], we considered that exposure of
`platelets to RSNOs in the portal circulation might ex-
`plain the effect of KNO3 as we found no increase in
`systemic RSNO levels. However, we found no increase
`in portal RSNO concentrations in a small number of
`patients with stable liver function and permanent intra-
`hepatic porto-systemic shunts. Clearly, healthy vol-
`unteers may absorb and metabolize gastric RSNOs
`differently to but there is no feasible access to their
`portal circulation.
`Thus the anti-platelet effect of KNO3 could be a re-
`sult of platelet exposure to RSNO levels which are below
`the detection limits of the assay. An alternative explan-
`ation is that rather than the gastric RSNOs acting as a
`source of NO, nitrite derived from nitrate could be
`converted to NO by xanthine oxidase, a ubiquitous en-
`zyme found in the vascular endothelium [32,33].
`If the inhibition of platelet function shown in this
`study by inorganic nitrate in amounts readily ingested in
`a healthy diet is related to a reduction in the incidence of
`cardiovascular disease, then this data has widespread
`implications for the importance of dietary nitrate in the
`primary prevention of coronary artery disease and per-
`ipheral vascular disease and for secondary prevention
`of acute vascular events. It may also have implications
`for the control or limitation of nitrate content in the diet
`of patients who have been admitted to hospitals with
`bleeding diatheses and gastrointestinal bleeding.
`However, there are limitations to this study. All the
`platelet experiments were performed on blood samples
`taken from healthy volunteers. We have no data as yet
`on patients with vascular disease taking long-term low-
`dose aspirin.
`Future studies will include the study of the chemistry
`of RSNO formation in the stomach and further inves-
`tigation into the exact mechanism of
`inhibition of
`platelet function.
`
`References
`
`[1] J.F. Mustard, S. Moore, M.A. Packham, R.L. Kinlough-Rath-
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