Nitric Oxide 15 (2006) 359–362
1089-8603/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
Cardioprotective eVects of vegetables: Is nitrate the answer?
Jon O. Lundberga,¤, Martin Feelischb, Håkan Björnea,
Emmelie Å. Janssona, Eddie Weitzberga
a Department of Physiology and Pharmacology, Karolinska Institutet 171 77, Stockholm, Sweden
b Department of Medicine, Boston University School of Medicine, Boston, MA, USA
Received 6 December 2005; revised 20 January 2006
Available online 24 March 2006
A diet rich in fruits and vegetables is associated with a lower risk of certain forms of cancer and cardiovascular disease, but the mech-
anisms behind this protection are not completely understood. Recent epidemiological studies suggest a cardioprotective action aVorded
speciWcally by green leafy vegetables. We here propose that these beneWcial eVects are related to the high content of inorganic nitrate,
which in concert with symbiotic bacteria in the oral cavity is converted into nitrite, nitric oxide, and secondary reaction products with
vasodilating and tissue-protective properties.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Nitric oxide; Nitrite; Cardiovascular; Nitrate reductase; Hypertension; Gastric cancer
Our diet exerts important long-term eVects on vital body
functions and thereby makes an important contribution to
health and disease. While high intake of cholesterol, satu-
rated fat, salt, and sugar are generally associated with a
greater risk for cardiovascular disease conventional wis-
dom has it that the opposite is true for abundant consump-
tion of fruits and vegetables . However, surprisingly few
studies have evaluated the relationship between fruit and
vegetable intake and cardiovascular disease. Recently, a
number of large epidemiological studies have attempted to
address this issue [1–4]. Joshipura et al. [3,5] found that a
high intake of fruits and vegetables was indeed associated
with a reduced risk for coronary heart disease and ischemic
stroke. The large study population also allowed for analysis
of the protection aVorded by speciWc types of foods, and the
strongest protection against coronary heart disease was
seen with high intake of green leafy vegetables. In another
study, Appel et al.  looked at the eVects of dietary supple-
mentation with vegetables on blood pressure in subjects
with borderline hypertension. They found that intake of
vegetables decreased blood pressure almost to the same
extent as monotherapy with a standard antihypertensive
drug. The speciWc nature of the active constituent(s)
responsible for the cardioprotective eVects of vegetables is
still unknown although Wber, minerals, and antioxidants
have all been proposed as viable candidates [1,2]. In the
midst of the current hype about the possible signiWcance of
polyphenolic antioxidants in protecting organs from the
sequelae of oxidative stress, we here wish to put forward an
alternative and disarmingly simple hypothesis: We propose
that the high content of inorganic nitrate is a major factor
contributing to the positive health eVects of certain vegeta-
bles via bioconversion to nitrite, nitric oxide (NO), and
other secondary reaction products (nitroso/nitrosyl com-
pounds), all of which may exert protective eVects on the
An alternative pathway for NO generation
Continuous generation of NO is essential for the integrity
of the cardiovascular system, and a decreased production
*Corresponding author. Fax: +46 8 332 278.
E-mail address: firstname.lastname@example.org (J.O. Lundberg).
J.O. Lundberg et al. / Nitric Oxide 15 (2006) 359–362
and/or bioavailability of NO is central to the pathogenesis of
cardiovascular disorders including atherosclerosis, hyperten-
sion, and ischemic heart disease [7,8]. The classical pathway
for generation of NO in mammals is via NO synthases pres-
ent, for example, in the vascular endothelium. These enzymes
produce NO from the precursor amino acid, L-arginine, and
molecular oxygen. More recently, a fundamentally diVerent
pathway for NO generation was discovered in humans [9–11]
that occurs via simple reduction of nitrite NO2
that does not involve NO synthases. This Wnding was highly
surprising since the general belief had been that both nitrate
and nitrite are biologically inert waste products from the oxi-
dation of endogenous NO. It is now clear that several alter-
native routes exist for the in vivo generation of NO from
nitrite [12–14]. These include reduction of nitrite by deoxyhe-
moglobin in blood  and reaction with xanthine oxidore-
ductase , enzymes of the mitochondrial respiratory chain
, and a yet unknown heme- and thiol-containing enzyme
activity  in tissues. Even vitamin C and simple protons
can catalyze this reaction [12,19]. Interestingly, nitrite reduc-
tion to NO is greatly enhanced during hypoxia/ischemia,
conditions under which the oxygen-dependent L-arginine/
NO synthase pathway is malfunctioning . To this end,
nitrite reduction can be regarded as a back-up system for the
generation of NO in situations of limited oxygen availability.
These Wndings may also have important therapeutic implica-
tions since nitrite is a product of the metabolic breakdown of
organic nitrates (such as the antianginal drug nitroglycerin)
in tissues, where it arises in amounts far higher than those for
NO and related nitroso/nitrosyl species .
¡, a reaction
Nitrite protects the cardiovascular system
While the notion that nitrite and nitrate may be beneW-
cial rather than detrimental to human health is not entirely
new [9,10,21–24], much of these discussions evolved around
direct antimicrobial eVects of acidiWed nitrite in the GI tract
and on the skin. Although long-term toxicological studies
in rats have not conWrmed that nitrite or nitrate are carcin-
ogenic, and epidemiological studies have failed to provide a
causal link between nitrate intake and cancer [25,26], these
considerations have not diminished concerns of the broader
public and public health authorities about current levels in
drinking water and food. During the past few years, how-
ever, an impressive amount of new data supporting a role
for nitrite in the regulation of cardiovascular function
appeared in the literature. Nitrite is now emerging as a
physiological regulator of hypoxic vasodilation and mito-
chondrial respiration, and also a modulator of ischemia-
reperfusion tissue injury and infarction [13,14,27]. Cosby
et al.  showed that infusion of nitrite can cause
vasodilation in humans and suggested a role for nitrite in
blood Xow regulation. They furthermore suggested a role
for deoxyhemoglobin in intravascular conversion of nitrite
to NO. Duranski et al.  studied the cytoprotective eVects
of nitrite in an animal model of cardiac and hepatic ische-
mia. By treating mice with low doses of sodium nitrite
systemically, they could reduce infarct size dramatically. In
higher doses, nitrite can prevent delayed cerebral vaso-
spasm after subarachnoid hemorrhage , and attenuate
pulmonary hypertension when inhaled . While most
studies on nitrite appear to show that its biological eVects
occur via generation of NO, one very recent study indicates
that nitrite may act as a signaling molecule in its own right
. Naturally, the substrate nitrite needs to be readily
available for this system to operate, which requires at least
one available source and an eVective uptake and transport
system. In humans, there are two large sources of nitrite
. One is endogenous formation of NO, which is sponta-
neously oxidized in tissues and blood to form nitrite, and
the second is the diet. In the latter, it exists largely in the
form of the precursor nitrate. Only a minor portion is taken
up directly as nitrite via ingestion of, e.g., cured meats such
as bacon and sausages where it serves, often in conjunction
with vitamin C, as a food preservative.
Bioconversion of dietary nitrate to nitrite and NO
Nitrate has been used for food preservation purposes
since centuries. Largely out of safety concerns in relation to
the formation of potentially carcinogenic nitrosamines and
the formation of methemoglobin in infants these days max-
imally allowable concentrations of nitrite and nitrate in
drinking water are strictly regulated in Europe, the US, and
many other countries. However, the by far dominating die-
tary source (>80%) of nitrate is the ingestion of vegetables
. Green leafy vegetables such as spinach and lettuce, but
also cauliXower and celery, are especially rich in nitrate as
are strawberries, grapes, and a few other fruits . Total
intake of nitrate is subject to seasonal variations, fertilizer
use and cooking procedures, and varies greatly between
individuals and regions. Vegetarians consume up to 10
times more nitrate than non-vegetarians, and a typical
Mediterranean diet, for example, is likely to contain consid-
erably more nitrate than the average Western diet.
Ingested nitrate is rapidly absorbed in the small intestine
and readily distributed throughout the body via the circula-
tion. For yet unknown reasons as much as 25% is actively
taken up from the blood by the salivary glands to be
excreted in the saliva . A substantial portion (»20%) of
this nitrate is then reduced to nitrite by commensal bacteria
in the oral cavity . These facultative anaerobes use
nitrate as an alternative electron acceptor to produce
energy. Without the enterosalivary circulation of nitrate
and the oral microXora, nitrate would leave the body
unmodiWed as this chemically stable anion cannot be
metabolized by mammalian enzymes. We could recently
show that plasma levels of nitrite increase greatly after an
oral load of sodium nitrate in an amount corresponding to
about 300g of spinach . The increase in plasma nitrite
was completely prevented if the test individuals avoided
swallowing for a certain period after nitrate intake thereby
illustrating its enterosalivary origin. It is intriguing to com-
pare the systemic nitrite load provided by a nitrate-rich
J.O. Lundberg et al. / Nitric Oxide 15 (2006) 359–362
meal with the amount of nitrite needed to protect tissues in
the setting of ischemia-reperfusion. In those studies, the
maximal protective eVects of exogenous nitrite are seen
already at a very modest dose . In fact, a similar or even
higher systemic load of nitrite is achieved by ingestion of no
more than 100g of lettuce or spinach. A recent animal
study by Bryan et al. further supports a role for dietary
nitrate in the regulation of cardiovascular function. In this
study, we could show that by limiting the intake of nitrate
and nitrite with the diet, the tissue levels of nitrite were
eVectively depleted within 2 days and these changes were
accompanied by a concomitant decrease in signaling path-
ways typically ascribed to be triggered by NO (i.e., the
depletion in tissue nitrite resulted in a measurable reduction
in cGMP). Taken together, these data suggest that dietary-
derived nitrate can be converted into bioactive nitrite and
NO in amounts suYcient to have profound eVects on the
It is possible that other constituent of our diet may work
in concert to enhance the beneWcial eVects of nitrate. One
such example is vitamin C which is abundant in many fruits
and vegetables. Interestingly, this vitamin greatly enhances
NO generation from nitrite [12,35]. Another intriguing pos-
sibility is the reaction between nitrite/NO and unsaturated
fatty acids (FAs, e.g., linoleic and oleic acid) which then
may become nitrated (NO2-FA) . Recent studies by
Freeman and co-workers [37,38] elegantly show that
nitrated FAs possess antiinXammatory activity in vitro
which may theoretically be of importance in protection
against cardiovascular diseases such as atherosclerosis. The
gastric milieu seems ideal for generation of nitrated FAs as
the combination of nitrite and acid enhances nitration
Testing the hypothesis
A combination of experimental studies and clinical trials
will allow us to determine if dietary nitrate does indeed pro-
vide the purported protection against cardiovascular dis-
ease. Several animal models of cardiovascular disease may
readily lend themselves to intervention studies with nitrate.
In this context, the use of germ-free animals may prove very
useful. In theory, such animals should have no beneWt from
nitrate supplementation as they cannot convert dietary
nitrate into bioactive nitrogen oxides . Animal studies
will also be useful to Wnd the optimal dose of nitrate needed
for cardioprotection. There is a possibility that the beneW-
cial eVects of nitrate are lost if the intake is too high. In
patients, the physiological and biochemical aspects of car-
diovascular function could be easily evaluated by the mea-
surement of, e.g., endothelial function in combination with
dietary manipulations, either by comparing individuals
with a regular and a low content of nitrite/nitrate or by sim-
ple supplementation with nitrate. In such investigations, it
is important to recognize and avoid possible confounding
factors such as the nitrate content of the drinking water,
vitamin supplementation, smoking habits, and other
factors. Further insight could be provided by epidemiologic
studies comparing cardiovascular disease burden with
nitrate intake. Since vegetables contain many other constit-
uents with potential cardioprotective properties, the predic-
tive power of such epidemiological studies is limited and
fails to prove causality. Thus, large prospective trials with
supplementation of inorganic nitrate on top of a standard
diet will be necessary to unequivocally test the validity of
We here propose that the protective eVect of certain veg-
etables on the cardiovascular system is related to their high
content of nitrate. The mechanism involves reduction of
dietary nitrate to nitrite, nitric oxide, and possibly other
biologically active reaction products in a process that
requires cooperation with symbiotic bacteria in the oral
cavity. A continuous intake of nitrate-containing food such
as green leafy vegetables may ensure that tissue levels of
NO and other nitrogen/nitrosyl species are maintained at a
level suYcient to compensate for any disturbances in
endogenous NO synthesis. Naturally, this provocative
hypothesis needs to be carefully tested in clinical trials. If
proven true, however, these considerations could have a
profound impact on our view of the role of diet and com-
mensal bacteria in the regulation of normal physiological
processes and prevention of cardiovascular disease.
The authors have received grants from the European
Commission 6th Framework Program (Eicosanox
LSHM-CT-2004-005033), the Swedish Heart and Lung
Foundation, the Swedish Research Council, the Ekhaga
Foundation and the National Institutes of Health
(NHLB). These organizations had no role in designing or
writing of this paper.
 W.C. Willett, Diet and health: what should we eat? Science 264 (1994)
 H.C. Hung, K.J. Joshipura, R. Jiang, F.B. Hu, D. Hunter, S.A. Smith-
Warner, G.A. Colditz, B. Rosner, D. Spiegelman, W.C. Willett, Fruit
and vegetable intake and risk of major chronic disease, J. Natl. Can-
cer Inst. 96 (2004) 1577–1584.
 K.J. Joshipura, A. Ascherio, J.E. Manson, M.J. Stampfer, E.B. Rimm,
F.E. Speizer, C.H. Hennekens, D. Spiegelman, W.C. Willett, Fruit and
vegetable intake in relation to risk of ischemic stroke, J. Am. Med.
Assoc. 282 (1999) 1233–1239.
 F.B. Hu, W.C. Willett, Optimal diets for prevention of coronary heart
disease, J. Am. Med. Assoc. 288 (2002) 2569–2578.
 K.J. Joshipura, F.B. Hu, J.E. Manson, M.J. Stampfer, E.B. Rimm, F.E.
Speizer, G. Colditz, A. Ascherio, B. Rosner, D. Spiegelman, W.C. Wil-
lett, The eVect of fruit and vegetable intake on risk for coronary heart
disease, Ann. Intern. Med. 134 (2001) 1106–1114.
 L.J. Appel, T.J. Moore, E. Obarzanek, W.M. Vollmer, L.P. Svetkey,
F.M. Sacks, G.A. Bray, T.M. Vogt, J.A. Cutler, M.M. Windhauser,
P.H. Lin, N. Karanja, A clinical trial of the eVects of dietary patterns
J.O. Lundberg et al. / Nitric Oxide 15 (2006) 359–362
on blood pressure, DASH Collaborative Research Group, N. Engl. J.
Med. 336 (1997) 1117–1124.
 L.J. Ignarro, Nitric oxide as a unique signaling molecule in the vascu-
lar system: a historical overview, J. Physiol. Pharmacol. 53 (2002)
 A.G. Herman, S. Moncada, Therapeutic potential of nitric oxide
donors in the prevention and treatment of atherosclerosis, Eur. Heart
J. 26 (2005) 1945–1955.
 J.O. Lundberg, E. Weitzberg, J.M. Lundberg, K. Alving, Intragastric
nitric oxide production in humans: measurements in expelled air, Gut
35 (1994) 1543–1546.
 N. Benjamin, F. O’Driscoll, H. Dougall, C. Duncan, L. Smith, M.
Golden, H. McKenzie, Stomach NO synthesis, Nature 368 (1994) 502.
 J.L. Zweier, P. Wang, A. Samouilov, P. Kuppusamy, Enzyme-inde-
pendent formation of nitric oxide in biological tissues, Nat. Med. 1
 E. Weitzberg, J.O. Lundberg, Nonenzymatic nitric oxide production
in humans, Nitric Oxide 2 (1998) 1–7.
 J.O. Lundberg, E. Weitzberg, NO generation from nitrite and its role
in vascular control, Arterioscler. Thromb. Vasc. Biol. 5 (2005) 915–
 M.T. Gladwin, Haldane, hot dogs, halitosis, and hypoxic vasodila-
tion: the emerging biology of the nitrite anion, J. Clin. Invest. 113
 K. Cosby, K.S. Partovi, J.H. Crawford, R.P. Patel, C.D. Reiter, S.
Martyr, B.K. Yang, M.A. Waclawiw, G. Zalos, X. Xu, K.T. Huang, H.
Shields, D.B. Kim-Shapiro, A.N. Schechter, R.O. Cannon, M.T. Gla-
dwin, Nitrite reduction to nitric oxide by deoxyhemoglobin vasodi-
lates the human circulation, Nat. Med. 9 (2003) 1498–1505.
 A. Webb, R. Bond, P. McLean, R. Uppal, N. Benjamin, A. Ahluwalia,
Reduction of nitrite to nitric oxide during ischemia protects against
myocardial ischemia-reperfusion damage, Proc. Natl. Acad. Sci. USA
101 (2004) 13683–13688.
 H. Nohl, K. Staniek, B. Sobhian, S. Bahrami, H. Redl, A.V. Kozlov,
Mitochondria recycle nitrite back to the bioregulator nitric monox-
ide, Acta Biochim. Pol. 47 (2000) 913–921.
 N.S. Bryan, B.O. Fernandez, S.M. Bauer, M.F. Garcia-Saura, A.B.
Milsom, T. Rassaf, R.E. Maloney, A. Bharti, J. Rodriguez, M. Feel-
isch, Nitrite is signalling molecule and regulator of gene expression in
mammalian tissue, Nat. Chem. Biol. 1 (2005) 290–297.
 H.H. Bjorne, J. Petersson, M. Phillipson, E. Weitzberg, L. Holm, J.O.
Lundberg, Nitrite in saliva increases gastric mucosal blood Xow and
mucus thickness, J. Clin. Invest. 113 (2004) 106–114.
 D.R. Janero, N.S. Bryan, F. Saijo, V. Dhawan, D.J. Schwalb, M.C.
Warren, M. Feelisch, DiVerential nitros(yl)ation of blood and tissue
constituents during glyceryl trinitrate biotransformation in vivo,
Proc. Natl. Acad. Sci. USA 101 (2004) 16958–16963.
 D.L. Archer, Evidence that ingested nitrate and nitrite are beneWcial
to health, J. Food Prot. 65 (2002) 872–875.
 C. Duncan, H. Li, R. Dykhuizen, R. Frazer, P. Johnston, G. Mac-
Knight, L. Smith, K. Lamza, H. McKenzie, L. Batt, D. Kelly, M.
Golden, N. Benjamin, C. Leifert, Protection against oral and gastroin-
testinal diseases: importance of dietary nitrate intake, oral nitrate
reduction and enterosalivary nitrate circulation, Comp. Biochem.
Physiol. A: Physiol. 118 (1997) 939–948.
 G. McKnight, Dietary nitrate in man: friend or foe? Br. J. Nutr. 81
 J.L. L’Hirondel, Nitrate and Man: Toxic, Harmless or BeneWcial,
CABI Publishing, Wallingford, UK, 2002.
 D. Forman, S. Al-Dabbagh, R. Doll, Nitrates, nitrites and gastric can-
cer in Great Britain, Nature 313 (1985) 620–625.
 S. Al-Dabbagh, D. Forman, D. Bryson, I. Stratton, R. Doll, Mortality
of nitrate fertiliser workers, Br. J. Ind. Med. 43 (1986) 507–515.
 M.T. Gladwin, A.N. Schechter, NO contest: nitrite versus S-nitroso-
hemoglobin, Circ. Res. 94 (2004) 851–855.
 M.R. Duranski, J.J. Greer, A. Dejam, S. Jaganmohan, N. Hogg, W.
Langston, R.P. Patel, S.F. Yet, X. Wang, C.G. Kevil, M.T. Gladwin,
D.J. Lefer, Cytoprotective eVects of nitrite during in vivo ischemia-
reperfusion of the heart and liver, J. Clin. Invest. 115 (2005) 1232–
 R.M. Pluta, A. Dejam, G. Grimes, M.T. Gladwin, E.H. OldWeld,
Nitrite infusions to prevent delayed cerebral vasospasm in a primate
model of subarachnoid hemorrhage, J. Am. Med. Assoc. 293 (2005)
 C.J. Hunter, A. Dejam, A.B. Blood, H. Shields, D.B. Kim-Shapiro,
R.F. Machado, S. Tarekegn, N. Mulla, A.O. Hopper, A.N. Schechter,
G.G. Power, M.T. Gladwin, Inhaled nebulized nitrite is a hypoxia-
sensitive NO-dependent selective pulmonary vasodilator, Nat. Med.
10 (2004) 1122–1127.
 J.O. Lundberg, E. Weitzberg, J.A. Cole, N. Benjamin, Nitrate, bacteria
and human health, Nat. Rev. Microbiol. 2 (2004) 593–602.
 C.L. Walters, in: M. Hill (Ed.), Nitrates and Nitrites in Food and
Water, Woodhead Publ. Ltd.
 B. Spiegelhalder, G. Eisenbrand, R. Preussman, InXuence of dietary
nitrate on nitrite content of human saliva: possible relevance to
in vivo formation of N-nitroso compounds, Food Cosmet. Toxicol. 14
 J.O. Lundberg, M. Govoni, Inorganic nitrate is a possible source for
systemic generation of nitric oxide, Free Radic. Biol. Med. 37 (2004)
 A. Modin, H. Bjorne, M. Herulf, K. Alving, E. Weitzberg, J.O. Lund-
berg, Nitrite-derived nitric oxide: a possible mediator of ‘acidic-meta-
bolic’ vasodilation, Acta Physiol. Scand. 171 (2001) 9–16.
 D.G. Lim, S. Sweeney, A. Bloodsworth, C.R. White, P.H. Chumley,
N.R. Krishna, F. Schopfer, V.B. O’Donnell, J.P. Eiserich, B.A. Free-
man, Nitrolinoleate, a nitric oxide-derived mediator of cell function:
synthesis, characterization, and vasomotor activity, Proc. Natl. Acad.
Sci. USA 99 (2002) 15941–15946.
 P.R. Baker, Y. Lin, F.J. Schopfer, S.R. Woodcock, A.L. Groeger, C.
Batthyany, S. Sweeney, M.H. Long, K.E. Iles, L.M. Baker, B.P. Bran-
chaud, Y.E. Chen, B.A. Freeman, Fatty acid transduction of nitric
oxide signaling: multiple nitrated unsaturated fatty acid derivatives
exist in human blood and urine and serve as endogenous peroxisome
proliferator-activated receptor ligands, J. Biol. Chem. 280 (2005)
 V.B. O’Donnell, J.P. Eiserich, P.H. Chumley, M.J. Jablonsky, N.R.
Krishna, M. Kirk, S. Barnes, V.M. Darley-Usmar, B.A. Freeman,
Nitration of unsaturated fatty acids by nitric oxide-derived reactive
nitrogen species peroxynitrite, nitrous acid, nitrogen dioxide, and
nitronium ion, Chem. Res. Toxicol. 12 (1999) 83–92.
 T. Sobko, C. Reinders, E. Norin, T. Midtvedt, L.E. Gustafsson, J.O.
Lundberg, Gastrointestinal nitric oxide generation in germ-free and
conventional rats, Am. J. Physiol. Gastrointest. Liver Physiol. 287