Am J Physiol Heart Circ Physiol 288: H2163–H2170, 2005.
First published December 30, 2004; doi:10.1152/ajpheart.00525.2004.
Nitrite is an alternative source of NO in vivo
Koichiro Tsuchiya,3 Yasuhisa Kanematsu,1 Masanori Yoshizumi,1 Hideki Ohnishi,3
Kazuyoshi Kirima,1 Yuki Izawa,1 Michiyo Shikishima,1 Tatsuhiro Ishida,4
Shuji Kondo,2 Shoji Kagami,2 Yoshiharu Takiguchi,3 and Toshiaki Tamaki1
1
Department of Pharmacology and 2Department of Pediatrics, The University of Tokushima School
of Medicine, and 3Department of Clinical Pharmacology and 4Department of Pharmacokinetics
and Biopharmaceutics, Institute of Health Biosciences, The University of Tokushima, Tokushima, Japan
Submitted 2 June 2004; accepted in final form 22 December 2004
nitric oxide; hypertension; electron paramagnetic resonance
derived mechanisms for generation of NO reported include the
following reactions (20):
NO 2⫺ ⫹ H⫹ º HNO2
⫹
⫹
2
(1)
⫹
HNO 2 ⫹ H º H2NO º NO ⫹ H2O
⫹
2
⫺
2
(2)
H 2NO ⫹ NO º N2O3 ⫹ H2O
(3)
N 2O3 º NO ⫹ NO2
(4)
The low pKa value (3.3) (86) of reaction 1 makes it unlikely
that nonenzymatic NO formation from nitrite (NO⫺
2 ) occurs at
physiological pH of the blood (91). Hence, researchers reported that NO⫺
2 -derived NO formation is found in acidic
environments such as the stomach (57, 59), oral cavity (18),
acidic urine (56), and ischemic rat heart (100).
It was reported that blood pressure was lowered in a dosedependent manner by oral nitrite uptake in spontaneously
hypertensive rats (4, 30). Furthermore, nitrite treatment decreased mean arterial blood pressure in normotensive rats (94).
However, it was unclear whether the orally ingested nitritederived NO was responsible for this blood pressure-lowering
effect.
In the circulation, NO exists as a relatively stable hemoglobin (Hb)-NO adduct (HbNO) (71). Because NO can be sequestered by Hb in its ␣-hemes (99) until its eventual release to
maintain physiological concentrations (⬃10⫺7 M ⬍ [NO] ⬍
⬃10⫺5 M) in the blood, the amount of HbNO may reflect the
blood NO concentration. In addition, it has been reported that
the concentration of HbNO is relatively high [0.8 ␮M in rats
(35) and 0.3–3 ␮M in humans (24, 44)] compared with the
concentration of NO. HbNO has a characteristic electron paramagnetic resonance (EPR) spectrum, and we succeeded in
measuring the blood HbNO level as an index of NO by using
the EPR HbNO signal subtraction method in a previous study
(38). Therefore, in the present study, we examined the formation of HbNO after nitrite treatment to clarify whether nitrite
acts as a systemic source of NO.
(NO) is a free radical molecule that has numerous
roles in various physiological functions, such as regulation of
the cardiovascular, immune, and nervous systems. An imbalance of NO is implicated in many diseases, such as hypertension, arteriosclerosis, septic and hemorrhagic shock, ischemiareperfusion injury, diabetes, and neurodegenerative disorders
(28, 65, 98). It has been believed that NO is synthesized from
L-arginine, NADPH, tetrahydrobiopterin (BH4), and molecular
oxygen catalyzed by NO synthases (NOSs). However, an
alternative pathway for NO production in biological systems
has been described in the last decade. In addition to NOSs,
xanthine oxidoreductase can be an alternative enzymatic pathway for NO production through reduction of the therapeutic
organic nitrate nitroglycerin as well as inorganic nitrate and
nitrite under hypoxic conditions in the presence of NADH (61).
Besides enzymatic production of NO, nonenzymatic nitrite-
Materials. N␻-nitro-L-arginine methyl ester (L-NAME) was purchased from Nacalai Tesque (Kyoto, Japan). Sodium nitrite and other
chemicals were obtained from Wako Pure Chemical Industries (Tokyo, Japan). The stable sodium nitrite isotope (Na15NO2) was obtained from Cambridge Isotope Laboratories (Andover, MA). NO gas
was obtained commercially from Sumitomo Seika Chemicals (Osaka,
Address for reprint requests and other correspondence: T. Tamaki, Dept. of
Pharmacology, The Univ. of Tokushima School of Medicine, Tokushima
770-8503, Japan (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
NITRIC OXIDE
http://www.ajpheart.org
MATERIALS AND METHODS
0363-6135/05 $8.00 Copyright © 2005 the American Physiological Society
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Tsuchiya, Koichiro, Yasuhisa Kanematsu, Masanori Yoshizumi,
Hideki Ohnishi, Kazuyoshi Kirima, Yuki Izawa, Michiyo Shikishima, Tatsuhiro Ishida, Shuji Kondo, Shoji Kagami, Yoshiharu
Takiguchi, and Toshiaki Tamaki. Nitrite is an alternative source of NO
in vivo. Am J Physiol Heart Circ Physiol 288: H2163–H2170, 2005. First
published December 30, 2004; doi:10.1152/ajpheart.00525.2004.—In
this study, we investigated whether orally administered nitrite is
changed to NO and whether nitrite attenuates hypertension in a
dose-dependent manner. We utilized a stable isotope of [15N]nitrite
(15NO⫺
2 ) as a source of nitrite to distinguish between endogenous
nitrite and that exogenously administered and measured hemoglobin
(Hb)-NO as an index of circulating NO in whole blood using electron
paramagnetic resonance (EPR) spectroscopy. When 1 mg/kg
Na15NO2 was orally administered to rats, an apparent EPR signal
derived from Hb15NO (AZ ⫽ 23.4 gauss) appeared in the blood. The
peak blood HbNO concentration occurred at the first measurement
after intake (5 min) for treatment with 1 and 3 mg/kg (HbNO: 4.93 ⫾
0.52 and 10.58 ⫾ 0.40 ␮M, respectively) and at 15 min with 10 mg/kg
(HbNO: 38.27 ⫾ 9.23 ␮M). In addition, coadministration of nitrite
(100 mg/l drinking water) with N␻-nitro-L-arginine methyl ester
(L-NAME; 1 g/l) for 3 wk significantly attenuated the L-NAMEinduced hypertension (149 ⫾ 10 mmHg) compared with L-NAME
alone (170 ⫾ 13 mmHg). Furthermore, this phenomenon was associated with an increase in circulating HbNO. Our findings clearly
indicate that orally ingested nitrite can be an alternative to L-arginine
as a source of NO in vivo and may explain, at least in part, the
mechanism of the nitrite/nitrate-rich Dietary Approaches to Stop
Hypertension diet-induced hypotensive effects.
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NITRITE IS AN ALTERNATIVE SOURCE OF NO IN VIVO
Chronic administration of nitrite with simultaneous NOS inhibition. At 13 wk of age, the rats were divided into four groups (group
1, L-NAME; group 2, L-NAME ⫹ low-dose nitrite; group 3,
L-NAME ⫹ high-dose nitrite; and group 4, control; Fig. 1). Each
group had seven rats, and all animals except controls received LNAME (1 g/l; days 0 to 21) without or with sodium nitrite (100 mg/l,
group 2; 1,000 mg/l, group 3; days 0 to 23). Both L-NAME and
sodium nitrite were dissolved in tap water. On the basis of the drug
solution intake, the effective daily consumption of L-NAME was
estimated to be ⬃65 mg 䡠 kg⫺1 䡠 day⫺1. To evaluate the endothelial
function in maintenance of blood NO levels, we stopped L-NAME
treatment 2 days before the experiments were conducted. Systolic
blood pressure (SBP) was measured every week via the tail-cuff
method using a BP-98A sphygmomanometer (Softron, Tokyo, Japan).
On the 24th day, rats were anesthetized with pentobarbital sodium,
and venous blood was collected (see Preparation of NOS-derived,
NO-depleted whole blood) for HbNO measurement.
EPR measurement. All EPR measurements were carried out according to a previous report from our laboratory (38). Briefly, the
frozen blood was directly transferred to a liquid nitrogen-filled quartz
EPR finger dewar, which was placed in the cavity of the EPR
measurement device. A JES TE 300 ESR spectrometer (JEOL, Tokyo,
Japan) with an ES-UCX2 cavity (JEOL) was utilized to collect EPR
spectra at the X band (9.5 GHz). Each sample was measured four
times, and values were normalized using ESPRIT 432 software
(JEOL) to improve the signal-to-noise ratio. Typical instrument conditions were as follows: microwave power, 20 mW; modulation
amplitude, 6.3 gauss; time constant, 1 s; scan time, 60 min; scan
range, 3,200 ⫾ 250 gauss; and microwave frequency, 9.045 GHz.
Spectra were stored on an IBM personal computer for analysis.
The HbNO signal was obtained by subtracting the EPR spectrum of
HbNO-depleted whole blood from that of each sample as described
previously (38). The relative HbNO concentration was obtained from
the peak-to-peak amplitude of the first signal of the triplet signal of the
Z factor of HbNO at g ⫽ 2.01, and the absolute HbNO concentration
was calculated by double integration of the region of the HbNO signal
(35) and then compared with that of NO-supplemented blood samples.
Statistical analysis. All data are expressed as means ⫾ SD. Data
were analyzed using two-way ANOVA, followed by the Tukey test
for comparisons between groups. P ⬍ 0.05 was accepted as statistically significant.
RESULTS
Fig. 1. Experimental design. Down arrows indicate blood drawing points.
L-NAME, N␻-nitro-L-arginine methyl ester.
AJP-Heart Circ Physiol • VOL
Bolus nitrite treatment and HbNO formation. In the control
animal, a small Hb14NO (AZ ⫽ 17.5 gauss, gZ ⫽ 2.01)-derived
EPR signal was observed due to the basal HbNO in the blood
(36) (Fig. 2A). When Na14NO2 was orally administered to rats
(1 mg/kg), triplet EPR signals due to Hb14NO2 in the blood
were increased (Fig. 2B). To further clarify the hypothesis that
exogenously administered nitrite is responsible for the augmented HbNO signal, we adopted a stable isotope of sodium
nitrite (Na15NO2) instead of naturally abundant (⬎99%) sodium nitrite (Na14NO2). The shape of the EPR signal of
Hb15NO is different from that of Hb14NO, which enabled us to
distinguish them from each other. As shown in Fig. 2C, when
1 mg/kg Na15NO2 was orally administered to rats, marked
Hb15NO-derived doublet EPR signals [AZ ⫽ 23.4 gauss (43)]
were observed in the blood, which indicated that orally administered nitrite can be a source of circulating NO as a form of HbNO.
Next, we investigated the time course of blood HbNO
concentration changes after ingestion of nitrite. As shown in
Fig. 3, oral administration of nitrite (1–10 mg/kg body wt)
produced a pronounced increase in blood HbNO. The HbNO
concentration remained considerably higher even at the end of
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Japan), and higher oxides such as NO2 and N2O3 (NOx) were removed
by passing NO gas through a trap containing 1 M KOH. A NOsaturated aqueous solution was prepared by bubbling NO gas for 15
min through water that had been previously deoxygenated by bubbling with purified argon gas for 15 min (45).
Animals. Male Sprague-Dawley rats (12 wk old, weighing 350 –
400 g) were obtained from Japan SLC (Shizuoka, Japan) and kept in
plastic cages at a controlled temperature (25°C) under controlled
lighting conditions (12:12-h light-dark cycle). The animals were fed a
commercial diet (nitrite concentration was 5.2 mg/kg or less) and had
access to tap water ad libitum until the day of the experiments. All
animal care and treatments were conducted in accordance with the
guidelines of the animal use and care committee of the University of
Tokushima. All experiments were performed according to the Guide
for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23,
Revised 1985).
Preparation of HbNO for calibration curves. Rats were anesthetized with pentobarbital sodium (40 mg/kg body wt ip). Venous blood
was collected from the vena cava, and aliquots (1 ml) were mixed with
various amounts of the NO-saturated solution at 0°C. The samples
were immediately drawn up into disposable 1-ml plastic syringes and
then immersed and stored in liquid nitrogen until EPR measurement.
Preparation of NOS-derived, NO-depleted whole blood. To prepare
the NOS-derived, NO-depleted blood (38), 12 rats were administered
tap water containing L-NAME (1 g/l) for 1 wk. The rats were
euthanized, and venous blood was taken from the vena cava with a
1-ml plastic syringe and stored in liquid nitrogen until use.
Acute administration of nitrite. At the age of 13 wk, the rats were
administered distilled water for 1 wk (Fig. 1). On the day after the
completion of treatment, sodium nitrite (1, 3, or 10 mg/kg) was
administered by oral gavage (1 ml/kg). The rats were anesthetized
with pentobarbital sodium (40 mg/kg ip), and venous blood was
obtained from the vena cava at 5, 15, 30, or 60 min after nitrite
administration (see Preparation of NOS-derived, NO-depleted whole
blood). We used sodium nitrite instead of potassium nitrite to prevent
possible effects of potassium on the cardiovascular system and blood
pressure (73, 83).
NITRITE IS AN ALTERNATIVE SOURCE OF NO IN VIVO
H2165
gZ ⫽ 2.01) was observed. Treatment with L-NAME alone
significantly reduced the blood HbNO-derived EPR signal
(P ⬍ 0.01), and nitrite treatment restored the L-NAME-induced
HbNO signal reduction in a dose-dependent manner.
DISCUSSION
the 60-min study period for the 3 and 10 mg/kg treatments. The
peak blood HbNO concentration occurred at the first measurement after intake (5 min) for 1 and 3 mg/kg treatments (4.93 ⫾
0.52 and 10.58 ⫾ 0.40 ␮M, respectively) and at 15 min for the
10 mg/kg treatment (38.27 ⫾ 9.23 ␮M). According to the
decay profiles of blood HbNO concentrations of 1 and 3 mg/kg
treatments, the half-life of HbNO was calculated to be 0.73 h⫺1.
Changes of SBP due to nitrite administration in L-NAMEtreated rats. The effects of nitrite on body weight, water
consumption, and daily intake of test compounds are summarized in Table 1, and changes in SBP are shown in Fig. 4. Body
weight increased continuously in all groups during the experimental period. The reason rats treated with L-NAME and 100
mg/l nitrite weighed significantly less than rats in the other
groups is expected to be a reduction in food utilization (26, 89)
and a decrease of liquid intake due to nitrite ingestion (89).
When the experiments were started, basal SBP was similar in
all groups. The administration of L-NAME for 3 wk induced
pronounced hypertension (170 ⫾ 11 mmHg) (37, 88, 90).
However, nitrite treatment dose dependently lowered SBP in
the L-NAME-treated rats in a concentration-dependent manner
(150 ⫾ 10 mmHg, 100 mg NO⫺
2 /l; 141 ⫾ 16 mmHg, 1,000 mg
NO⫺
2 /l; Fig. 4). After the washout period (days 21 and 22), SBP
in the L-NAME-treated hypertensive rats was slightly lower;
the hypertension was improved. Figure 5 shows representative
EPR spectra and the HbNO concentrations for each group. In
control rats, a small EPR signal of HbNO (AZ ⫽17.5 gauss,
AJP-Heart Circ Physiol • VOL
Fig. 3. Time course of blood hemoglobin-nitric oxide adduct (HbNO) concentrations after oral sodium nitrite treatment. Rats were orally treated with 1
(E), 3 (䊐), and 10 mg NaNO2/kg body wt (‚), and blood was then drawn from
the vena cava under anesthesia. The HbNO concentration was obtained by
double integration of the region of the HbNO EPR signal and compared with
that of NO-supplemented blood as described in MATERIALS AND METHODS.
HbNO concentrations are expressed as means ⫾ SD. Each point represents the
average of 3 experiments. **P ⬍ 0.01 vs. 1 mg NaNO2 treatment.
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Fig. 2. Representative electron paramagnetic resonance (EPR) spectra of
whole blood measured at 77K from an untreated rat (A), a rat given 1 mg/kg
Na14NO2 (B), and a Na15NO2-treated rat (C). Rats were orally administered
distilled water (A) or sodium nitrite (B and C) at a rate of 1 ml/kg body wt, and
blood was then taken from the vena cava at 15 min after treatment under
pentobarbital sodium anesthesia (40 mg/kg body wt ip). Spectrometer conditions are described in MATERIALS AND METHODS. AZ and gZ are EPR parameters.
We undertook the present study to assess the effect of orally
administered nitrite on circulating HbNO formation by using
the EPR technique. Using 15NO⫺
2 , we clearly showed that
orally administered nitrite is an alternative source of NO.
It was first reported in 1926 that vegetarian diets have
hypotensive effects (16), and recent studies revealed that vegetarians tend to have lower blood pressures than nonvegetarians (60, 74, 75). In addition, the Dietary Approaches to Stop
Hypertension (DASH) diet, which is rich in fruits and vegetables and low in saturated and total fats, showed hypotensive
effects in randomized controlled trials (1, 67), although these
results may be due to increased intake of fiber, unrefined
carbohydrates, polyunsaturated fatty acids, potassium, magnesium, calcium, ascorbate, and vitamin E or lower intake of
energy, total and saturated fat, cholesterol, protein, and sodium
(66). It has not been determined which components of the
DASH diet are responsible for the hypotensive effect.
Thöni and colleagues (4, 11) suggested that a nitrate-derived
NO formation pathway is a possible mechanism for the hypotensive effect of vegetable- and fruit-rich diets. Vegetables and
fruits contain large amounts of nitrate and nitrite (97) and
supply 86% of the daily nitrate intake in the general US
population (an average of 75 mg of NO⫺
3 in nonvegetarians and
268 mg of NO⫺
3 in vegetarians) and 16% of the daily nitrite
intake (an average of 0.77 mg of NO⫺
2 in nonvegetarians and
1.7 mg of NO⫺
2 in consumers of a diet in cured meat) (14).
When nitrate is ingested, it is rapidly absorbed in the upper
small intestine, and up to 75% is excreted in the urine within
24 h (70). The remaining ingested nitrate (⬃25%) undergoes
H2166
NITRITE IS AN ALTERNATIVE SOURCE OF NO IN VIVO
Table 1. Effects of nitrite with or without L-NAME on body weight, water consumption, and daily intake of test compounds in rats
⫹ Nitrite
(100 mg/l)
L-NAME
Body weight, g
13 wk old
16 wk old
Daily water consumption, ml䡠day⫺1䡠rat⫺1
Daily intake of compounds, mg/rat
L-NAME
Sodium nitrite
⫹ Nitrite
(1,000 mg/l)
L-NAME
Distilled Water
L-NAME
374⫾25
452⫾14
37⫾6
391⫾7
449⫾13
30⫾6
374⫾11
426⫾8†
27⫾4*
374⫾14
428⫾20
27⫾4*
30⫾6
27⫾4
2.7⫾0
27⫾4
27⫾4
Values are means ⫾ SD and represent effects of nitrite with or without N␻-nitro-L-arginine methyl ester (L-NAME). *P ⬍ 0.05, †P ⬍ 0.01 vs. distilled water
group.
Fig. 4. Changes in systolic blood pressure (SBP): E, control; F, L-NAME; 䊐,
low-dose nitrite (100 mg NaNO2/l); ‚, high-dose nitrite (1,000 mg NaNO2/l).
Values are expressed as means ⫾ SD. Each group had 7 rats. *P ⬍ 0.05,
**P ⬍ 0.01 vs. L-NAME treatment.
AJP-Heart Circ Physiol • VOL
spontaneous decomposition through reactions 2– 4; alternatively, several reducing species such as ascorbic acid are
expected to be good candidates for NO production from nitrous
acid or other nitrite metabolites according to reaction 5 (70, 84,
96). Nitrate and thiocyanate are secreted by the salivary glands;
25% of the nitrate is then reduced by bacteria on the dorsum of
the tongue, and these compounds move to the stomach. Thiocyanate is a powerful catalyst of nitrosation of secondary
amines by acidified nitrite, and this simultaneous delivery of
nitrite and thiocyanate to the acidic environment of the stomach therefore has been regarded as a potentially important
source of endogenous formation of carcinogenic N-nitroso
compounds (6). Incidentally, ascorbic acid is actively secreted
into gastric juice (77, 79) and plays a key role in preventing
acid-catalyzed luminal nitrosation competitively with the secondary amines and amides (62, 63), which results in the
formation of NO and dehydroascorbate in the stomach (2, 52),
N 2O3, HNO2, NOSCN ⫹ ascorbic acid 3
NO ⫹ dehydroascorbic acid
(5)
and then NO diffuses into the adjacent epithelium barrier of the
stomach (84). These hypotheses are supported by the facts that
1) omeprazole, a proton pump inhibitor, suppresses the formation of NO in the stomach with a rise in intragastric nitrite (69);
2) thiocyanate is secreted from salivary glands, and it catalyzes
the nitrosation of secondary amines by acidified nitrite (6),
although ascorbic acid, which is secreted into the gastric juice,
prevents acid-catalyzed luminal nitrosation competitively with
the secondary amines and amides (62, 63); and 3) a large
amount of NO is formed when lettuce and saliva are mixed
under acidic conditions (57); hence, lettuce contains ascorbic
acid and nitrite.
However, it is still unclear whether orally ingested nitrite is
responsible for the formation of NO in the blood and whether
the NO generated in the stomach contributes to systemic blood
pressure. Therefore, we measured the increase in HbNO concentration after oral nitrite treatment to confirm the origin of
the increased HbNO. In addition, we measured the HbNO
formation in nitrite-treated rats with L-NAME-induced hypertension by using the EPR subtraction method (38) and compared HbNO concentrations and blood pressure among the rats.
The results of bolus nitrite treatment demonstrated that
orally administered nitrite forms HbNO in vivo (Fig. 2B), and
this phenomenon was further confirmed by use of 15NO⫺
2
instead of 14NO⫺
2 (Fig. 2C) (44). The formation of HbNO by
NO donors has already been confirmed by investigators applying the EPR technique both in vivo (10, 41) and in vitro (41,
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enterosalivary recirculation, and it is concentrated in the salivary glands and then secreted in the saliva (3, 8). The rate of
microbial reduction of nitrate to nitrite in the oral cavity is
reported to be ⬃10 –20% of total ingested nitrate (19, 81), and
the nitrite is moved into the stomach by swallowing. All
combined, vegetarians ingest a maximum of ⬃14.2 mg of
nitrite per day (0.24 mg/kg body wt for a 60-kg person). In the
case of rats, 0.156 mg of nitrite seems to be supplied by rat
chow (the nitrite concentration of rat chow is 5.2 mg/kg on
average, and a rat consumes ⬃30 g/day). It had been believed
that ingested nitrite is absorbed by the gastrointestinal tract
(51) or reacts in the stomach with secondary or tertiary amines
and amides present in foods such as cheese or meat to form
N-nitroso compounds (7). However, the rate of nitrosation of
amines is dependent on the nitrite concentration to a power of
greater than unity, which means that nitrite ingested at one time
over a short period will be more active in the synthesis of
N-nitroso compounds than a continuous supply at lower concentrations over long periods (95). In addition, findings of
recent studies of nitrite metabolism suggest another reaction
mechanism rather than nitrosamine formation pathways. McKnight et al. (59) reported that a large and sustained increase in
chemically derived gastric NO concentration after an oral
nitrite load and very low gastric nitrite concentrations resulting
from nitrate would suggest that nitrosamine formation is not
likely in healthy volunteers. Once the nitrite is mixed with
stomach acids, it interacts with hydrogen to form nitrous acid
in accordance with reaction 1 (57). Nitrous acid forms NO by
NITRITE IS AN ALTERNATIVE SOURCE OF NO IN VIVO
H2167
Fig. 5. Effect of chronic L-NAME treatment in
combination with nitrite on HbNO concentration.
Left, typical EPR spectrum for each group; right,
HbNO concentrations expressed as means ⫾ SD.
The control group had 7 rats, and the other groups
had 5 rats. *P ⬍ 0.01.
AJP-Heart Circ Physiol • VOL
trite was chronically administered to L-NAME-treated hypertensive rats for 3 wk, SBP dropped [L-NAME control: 170 ⫾
13 mmHg; L-NAME ⫹ nitrite (1,000 mg/l): 149 ⫾ 10 mmHg]
(Fig. 4) as reported elsewhere (4, 11, 30, 94). In addition, the
blood HbNO concentration was augmented with the decrease
of SBP in nitrite-treated rats (Fig. 5). These results suggest the
following reaction mechanisms.
First, acid-derived disproportionation of nitrite to NO occurs
in the stomach (76). This process forms nitrous acid as the first
step, as follows:
NO 2⫺ ⫹ H⫹ 3 HNO2
(6)
The nitrous acid can exist as a resonance form, i.e., a nitrosonium ion with a hydroxide-like structure as an intermediate (2):
HNO 2 43 兵HOON其
(7)
This intermediate reacts with another nitrite ion to form dinitrogen trioxide (76), which results in the formation of NO by
decomposition:
兵HOON其 ⫹ NO2⫺ 3 N2O3 ⫹ OH⫺
(8)
N 2O3 3 NO ⫹ NO2
(9)
Alternatively, the intermediate decomposes to nitrosonium ion
and hydroxyl ion in accordance with its pKa value (3.4) (5, 59):
兵HOON其 3 OH⫺ ⫹ NO⫹
(10)
Thereafter, the nitrosonium ion interacts with reductive
compounds (R), such as flavonoids (85) and ascorbate (87), to
form NO:
NO ⫹ ⫹ R 3 NO ⫹ R䡠
(11)
and then is effectively scavenged through reaction with Hb in
erythrocytes to form HbNO (99).
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44). Kohno et al. (41) succeeded in detecting the EPR signal of
HbNO in rats treated with an intravenous injection of a large
amount of nitrite (6.9 mg NaNO2/rat), although they did not
detect the HbNO signal when ⬍0.69 mg NaNO2/rat was
injected because of the decomposition of nitrite by oxyhemoglobin (42, 80). However, our results indicate that treatment
with 0.4 mg NaNO2/rat (1 mg NaNO2/kg body wt po) produces
an apparent HbNO EPR signal with a peak concentration of
4.93 ⫾ 0.52 ␮M at 5 min after treatment (Fig. 3). A possible
explanation for the difference in the effects of oral administration and intravenous treatment on HbNO formation may be the
existence of acid decomposition pathways of nitrite in the
stomach (57), followed by the salvage of NO by Hb in
erythrocytes (99).
Nitrite has been considered a toxic compound [the oral
median lethal dose (LD50) in rats is 180 mg/kg (78)], although
it is present throughout the body in various concentrations: 500
␮M in rat lung tissue, 69 ␮M in the human kidney, 10 ␮M in
aortic tissues (72), and 12 ␮M in the rat heart (100). In
contrast, the nitrite concentration in the blood circulation is
relatively low compared with tissues levels, and its values vary
from undetectable (27) and submicromolar [0.2 ␮M (34), 0.45
␮M (48) in healthy humans] to micromolar [6.6 ␮M in human
serum (21)] and a few tens of micromoles [26 ␮M in patients
with left-to-right shunt (25)]. Recently, Kleinbongard et al.
(39) reviewed the physiologically relevant plasma nitrite concentration range in various mammals (100 – 600 nM), and they
concluded that it is 0.3 ␮M in humans and 0.2 ␮M in rats.
Nitrite is known as a vasodilator at high concentrations in
vitro (29, 32, 33, 47, 58, 68) and ex vivo (15). However, Lauer
et al. (46) demonstrated that nitrite had no vasodilatory effects
when it was infused intra-arterially into humans (the venous
plasma nitrite concentration exceeded 130 ␮M when nitrite
was infused at 36 ␮mol/min for 1 min), even at concentrations
200 times that achieved during maximal endothelial NOS
stimulation with acetylcholine. In our experiments, when ni-
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NITRITE IS AN ALTERNATIVE SOURCE OF NO IN VIVO
AJP-Heart Circ Physiol • VOL
NO production in vivo; we next need to examine the physiological role of a dietary dose of nitrite. Further research is
needed to investigate the interaction between nitrite-nitrate
intake and human health.
GRANTS
This work was supported in part by Grant-In-Aid for Scientific Research
16590196-00 from Ministry of Education, Culture, Sports, Science and Technology, Japan, and by Grants-In-Aid of Research Program of Tokushima
University.
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It is still unclear how NO produced in the stomach gets
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When nitrite is orally administered to animals, nitrite in the
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with or without regional forearm NOS inhibition caused a
dose-dependent increase in blood flow, and this phenomenon
was associated with the formation of HbNO. If this pathway
were valid, the time course of changes in the blood concentration of HbNO following oral administration would depend on
the nitrite concentration in the blood.
However, the peak blood concentration of HbNO was observed at the first measurement after intake (5 min) for 1 and
3 mg NaNO2/kg and at 15 min for 10 mg NaNO2/kg treatment
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Once HbNO has been produced, it acts as a pool of NO
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