J Pharmacol Sci 96, 395 – 400 (2004)
`
`Forum Minireview
`
`Journal of Pharmacological Sciences
`©2004 The Japanese Pharmacological Society
`
`Malfunction of Vascular Control in Lifestyle-Related Diseases:
`Formation of Systemic Hemoglobin-Nitric Oxide Complex (HbNO)
`From Dietary Nitrite
`
`Koichiro Tsuchiya1,*, Yoshiharu Takiguchi1, Masumi Okamoto2, Yuki Izawa2, Yasuhisa Kanematsu2,
`Masanori Yoshizumi2, and Toshiaki Tamaki2
`
`1Department of Clinical Pharmacology, Subdivision of Clinical Pharmaceutical Sciences, Institute of Health Biosciences,
`The University of Tokushima Graduate School, 1-78 Sho-machi, Tokushima 770-8505, Japan
`2Department of Pharmacology, Institute of Health Biosciences, The University of Tokushima Graduate School,
`3-18-15, Kuramoto, Tokushima 770-8503, Japan
`
`Received October 13, 2004; Accepted October 29, 2004
`
`Abstract. Nitric oxide (NO) has many physiological functions. It is believed to be produced
`from L-arginine by nitric oxide synthase (NOS), and nitrite and nitrate are waste forms of it. By
`the way, nitrate and nitrite are abundant in vegetables and fruits, especially leafy vegetables and
`pickled vegetables. Orally-ingested nitrate is changed to nitrite by micro-organelles living in the
`hypopharynx area, and nitrite is expected to change to NO in the stomach due to its low pH.
`Indeed, some researchers reported that NO is produced in the gastric cavity, although few reports
`mentioned the physiological meanings of this NO formation. Therefore, we investigated whether
`the nitrite-derived NO can shift to the circulation and acts like NOS-derived NO does in tissues.
`We adopted a stable isotope of nitrite (15NO2
`-) in order to distinguish between the endogenous
`nitrite and the exogenously administered one and measured nitrosyl hemoglobin (HbNO) as an
`index of circulating NO using electron paramagnetic resonance spectroscopy. It appeared that
`the oral administration of 15N-nitrite formed the Hb15NO in rat blood and decreased the blood
`pressure of chronic L-NAME treated rats. Our findings suggest that the intake of nitrite (or
`nitrate)-rich foods such as vegetables and fruits would alter the systemic HbNO dynamism,
`resulting in the improvement of cardiovascular diseases.
`
`Keywords: nitric oxide (NO), hemoglobin-NO adduct, electron paramagnetic resonance, nitrite
`
`Introduction
`
`Nitric oxide (NO), a free radical molecule, has
`numerous roles in various physiological functions,
`such as regulation of the blood pressure (1), immune
`response to bacterial infection (2), and nervous systems
`(3). It is believed that nitric oxide synthase (NOS) makes
`NO by catalyzing the oxygen-, tetrahydrobiopterin-, and
`NADPH-dependent oxidation of L-arginine, and nitrite
`and nitrate are recognized as a waste forms of NO.
`However, an alternative pathway for NO production in
`biological systems has been found in the last decade.
`Catalysis by xanthine oxidoreductase was found to serve
`
`*Corresponding author. FAX: +81-88-633-9516
`E-mail: tsuchiya@ph.tokushima-u.ac.jp
`
`395
`
`as an alternative enzymatic NO production pathway
`from organic- and inorganic nitrates under hypoxic
`conditions (4). In addition to enzymatic production of
`NO, non-enzymatic nitrite-derived mechanisms for NO
`generation has been recognized to occur by the follow-
`ing reactions (5):
`(i)
`(pKa = 3.3)
`- + H+
`NO2
`HNO2
`NO+ + H2O
`HNO2 + H+
`(ii)
`H2NO2
`+ + NO2
`(iii)
`H2NO2
`N2O3 + H2O
`(iv)
`NO + NO2
`N2O3
`These reactions are favorable under the acidic
`condition due to the low pKa value of reaction (i) (6),
`and nitrite-derived NO formation seems to occur in
`acidic environments such as the stomach (7, 8), oral
`cavity (9), and acidic urine (10).
`By the way, it has been believed that vegetarian diets
`
`+
`
`-
`
`Copyright © 2004. Production and Hosting by Elseiver B.V. On behalf of Japanese Pharmacological Society.
`This is an open access article under the CC BY-NC-ND License (http://creativecommons.org/licenses/by-nc-nd/ ).
`
`Human Power of N Company
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`have hypotensive effects (11 – 14). And recently, the
`DASH (Dietary Approaches to Stop Hypertension) diet,
`which is rich in fruits and vegetables and low in
`saturated and total fats, showed hypotensive effects in
`randomized controlled trials (15, 16). It has not been
`determined which components of the DASH diet are
`responsible for the hypotensive effect. Nitrate and nitrite
`are abundant in vegetables and fruits (17, 18), especially
`leafy vegetables and Japanese pickled vegetables.
`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 (19). The remaining ingested nitrate
`(almost equal
`to 25%) undergoes entero-salivary
`recirculation, and it is concentrated in the salivary glands
`and then secreted in the saliva (20, 21). Orally-ingested
`and salivary gland-derived nitrate changes to nitrite by
`micro-organelles living in the hypopharynx area. The
`rate of microbial reduction of nitrate to nitrite in the
`oral cavity is reported to be around 10% to 20% of
`total ingested nitrate (22, 23), and the nitrite is moved
`into the stomach by swallowing.
`Indeed, NO is formed from nitrite in the gastric cavity
`(19), and blood pressure was lowered in a dose-depen-
`dent manner by oral nitrite uptake in spontaneously
`hypertensive rats (24, 25) and normotensive rats (26),
`although few reports mentioned what the physiological
`meaning of this nitrite-derived NO was. Therefore, we
`investigated 1) whether the nitrite-derived NO dis-
`tributes to the circulation, and 2) whether nitrite-derived
`NO acts like NOS-derived NO does in vivo. We
`
`employed a stable isotope of nitrite (15NO2-) in order to
`distinguish between endogenous nitrite and the exo-
`genously administered one and measured nitrosyl hemo-
`globin (HbNO) as an index of circulating NO in whole
`blood using electron paramagnetic resonance spectros-
`copy (27).
`
`HbNO measurement by EPR spectroscopy
`
`It was reported that endotherium-derived NO diffuses
`into blood and binds to hemoglobin to form the rela-
`tively stable HbNO in erythrocytes (28), which means
`the amount of HbNO may reflect the blood NO
`concentration. Systemic HbNO concentration is reported
`to be 0.8 m M in rats (29) and 0.3 – 3 m M in humans
`(30, 31). EPR spectroscopy can detect the HbNO
`because HbNO is a paramagnetic species, although there
`are still some difficulties in obtaining fine HbNO signals
`because of the existence of paramagnetic compounds
`other than HbNO, such as ceruloplasmin, other heme
`proteins, and molybdenum enzymes, which give a
`strong EPR signal overlapping the same region of HbNO
`(32 – 37). Therefore, we developed an improved method
`
`Fig. 1. Representative EPR spectra of whole blood of 15N-nitrite
`(A) and 14N-nitrite (B) treated rat. Nitrite (1 mg / kg body weight)
`was administered orally, and blood was obtained from the vena cava
`after 60 min under anesthesia. EPR spectral conditions were same as
`the typical EPR conditions described in the text. The arrow in the
`figure indicates the position of doublet splitting of Hb15NO species
`(A) and of triplet splitting of Hb14NO (B), and their constants were
`18 and 24 gauss.
`
`of detecting the HbNO signal in whole blood by EPR
`spectroscopy (EPR subtraction method) (27). In
`addition, we adopted a stable isotope of nitrite (15N-
`nitrite) because the Hb15NO EPR signal is different
`from the Hb14NO signal (Fig. 1: A and B), and using
`15N-nitrite enabled us to clarify whether the source of
`NO is nitrite.
`
`Sample preparation and EPR measurements
`
`Male Sprague-Dawley rats (12 weeks of age, weigh-
`ing 350 – 400 g) were used for the present study. Venous
`blood was taken from the vena cava with a 1-ml plastic
`syringe under anesthesia (pentobarbital sodium (40 mg
`/ kg body weight)). The kidney tissue was transferred to
`a 5-cm length of EPR quartz tubing by puncturing.
`Obtained samples were stored in liquid nitrogen until
`the EPR measurement.
`EPR measurements were carried out in liquid nitro-
`gen. The frozen sample was directly transferred to a
`liquid nitrogen-filled quartz finger dewer, which was
`placed in the cavity of the EPR instrument. A JES
`TE-300 EPR spectrometer (JEOL Co., Ltd., Tokyo) with
`an ES-UCX2 cavity (JEOL Co.) was utilized to collect
`EPR spectra at the X-band (9.5 GHz). Typical EPR
`conditions were as follows: power, 20 mW; frequency,
`9.045 GHz; magnetic field, 3200 ± 250 gauss; modula-
`tion width, 6.3 gauss; sweep time, 15 min; and time
`constant 0.3 s. Spectra ware processed using software
`ESPRIT 432 (JEOL Co.). The EPR signal subtraction
`was accomplished as reported previously (27).
`
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`Systemic HbNO Formation From Nitrite
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`397
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`Appearance of HbNO by bolus treatment of nitrite
`
`In the control animal, no Hb15NO (AZ = 23.4 gauss,
`gZ = 2.01 (38))-derived EPR signal was observed
`because of its low abundancy (data not shown). When
`1 mg Na15NO2/ kg body weight was orally administered
`to the rat, marked Hb15NO-derived doublet EPR signals
`and methemoglobin-derived EPR signals were observed
`in the blood and it gradually augmented with time
`(Fig. 2: B and C). This means that nitrite can be a source
`of circulating NO, and it binds with systemic Hb,
`forming HbNO. It is still unclear how NO produced in
`the stomach gets directly into erythrocytes and then
`scavenged by hemoglobin to form HbNO. One explana-
`tion is that NO diffuses through the epithelial barrier
`(39) to reach hemoglobin since NO is a hydrophobic
`molecule. Another proposed pathway for HbNO forma-
`tion from nitrite is that orally ingested nitrite is rapidly
`absorbed from stomach to the blood stream (40) and
`
`Fig. 2. Changes in EPR-active species with time after oral
`administration of 15N-nitrite in blood. Spectrum A: control rat
`(-nitrite). Spectra B and C: rats were subjected to 15N-nitrite (1 mg
`NaNO2/ kg body weight, p.o.) and then blood was collected from
`the vena cava 15 min (B) and 60 min (C) after treatment. EPR
`spectral conditions were same as Fig. 1 but the magnetic field was
`2500 ± 2500 gauss (spectrum A – C) or 3200 ± 250 gauss (spectrum
`D); modulation width, 6.3 gauss; sweep time, 8 min; and time
`constant 0.1 s. The arrow in the figure indicates the position of
`high-spin iron (III) (methemoglobin).
`
`then interacts with deoxyhemoglobin to form methe-
`moglobin and HbNO (41). It seems that the latter mecha-
`nism is in accord with the observed formation of both
`Hb15NO and methemoglobin-derived EPR signals.
`However, further research will be required to clarify
`the mechanisms.
`
`Physiological role of nitrite: hypotensive effects and
`ischemia-reperfusion-related NO formation in kidney
`
`Nitrite is known as a vasodilator at high concentra-
`tions in vitro (42 – 47) and ex vivo (48). However, its
`hypotensive effects in vivo are still under debate (49,
`50). Therefore, we used chronic L-NAME-treated rats
`as an animal model of hypertension and examined the
`effect of nitrite on the level of HbNO as an index of
`NO. The oral administration of L-NAME (1 g / liter, in
`drinking water for 3 weeks) induced hypertension (168 ±
`11 mmHg) with reduction of blood HbNO concentration
`(43% of the control). However, co-administration of
`nitrite with L-NAME dose-dependently lowered the
`systemic blood pressure (0.1 g NaNO2/ liter: 151 ± 10
`mmHg, 1.0 g NaNO2/ liter: 141 ± 17 mmHg) and re-
`stored the HbNO level (90% and 93%). Although the
`orally administered nitrite concentration was high, our
`results demonstrated that nitrite treatment attenuated
`the decrease of blood NO in L-NAME-treated rats.
`Next, we demonstrated that the nitrite can be an
`alternative source of NO in ischemic kidney and that
`it binds with hemoglobin and then is spread by the
`circulation after reperfusion (51).
`Renal ischemia-reperfusion injury is a critical patho-
`logic condition occurring as a result of kidney trans-
`plantation (52), and NO may play an important patho-
`logical role in ischemic renal injury. By the way, it has
`been believed that NO is synthesized from L-arginine,
`NADPH, tetrahydrobiopterin, and molecular oxygen
`catalyzed by NOS. In other words, molecular oxygen is
`obligatory component for NO production by NOS. If so,
`how does NOS form NO under the hypoxic condition?
`In 2000, Hirabayashi et al. reported that the NO produc-
`tion from renal cortex tissue increased sharply during
`ischemia and that it was independent of L-arginine
`administration in rats (53), which may imply that NO
`is produced in an NOS-independent manner under
`ischemic conditions. Furthermore, Zweier et al. pro-
`posed that nitrite could be a source of NO in the event
`of ischemic heart (54). Taken together, it seems that
`NO is produced in an NOS-independent manner under
`ischemic conditions and that nitrite can be a candidate
`for the source of NO. It seems likely that endogenous
`nitrite in the kidney can be a source of NO under
`ischemic conditions because the kidney has relatively
`
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`high nitrite content (69 m M). Then we investigated
`whether nitrite can be a source of NO in rat kidney
`during and after in vivo renal ischemia induced by renal
`artery and vein occlusion using the EPR subtraction
`method.
`We adopted a stable isotope of nitrite (15N-nitrite)
`because using 15N-nitrite enabled us to clarify whether
`the source of NO is nitrite. When 3 m mol / kg of 15N-
`nitrite was intravenously injected into rats and then
`followed by 40 min of ischemia, marked 15NO formation
`was observed with the appearance of a large doublet
`Hb15NO signal in kidney (Fig. 3B) in comparison to
`sham-operated rats (Fig. 3A). At 40-min ischemia and
`
`1-min reperfusion in the kidney of the 15NO2--admin-
`istered rat, the Hb15NO signal in the kidney was
`decreased (Fig. 3C) compared to that of a 40-min-
`ischemia rat (Fig. 3B) and circulating Hb15NO was
`increased instead (data not shown). To investigate
`whether NOS contributes to NO generation from nitrite,
`the effects of L-NAME was examined in rats by 40-min
`occlusion. Rats were orally administered L-NAME
`(1 g / liter drinking water) for 1 week to induce NOS
`dysfunction (55, 56) and then received i.v. injection of
`15N-nitrite (3 m mol / kg) followed by 40-min ischemia.
`
`Fig. 3. Changes in Hb15NO signal intensity in kidney of 15N-nitrite
`infused rats. Spectrum A: rat was subject to 15N-nitrite infusion
`(3 m mol / kg) from the femoral vein and then given a sham operation
`without renal ischemia. Spectrum B: 40-min ischemia. Spectrum C:
`40-min ischemia followed by 1-min reperfusion. Spectrum D: same
`as B, but the rat was orally administered L-NAME (1 g / liter in
`drinking water) for 1 week. EPR spectral conditions were same as
`Fig. 1. The arrow in the figure indicates the position of doublet
`splitting of Hb15NO species. Modified from Ref. 51.
`
`As shown in Fig. 3D, L-NAME treatment did not affect
`the Hb15NO formation in the ischemic kidney of 15N-
`nitrite-treated rats compared to the control (Fig. 3B).
`These results suggest that the nitrite can be an alternative
`source of NO in ischemic kidney.
`
`Concluding remarks
`
`In conclusion, in this review we described that 1)
`orally administered nitrite appears in the circulation as
`HbNO using a stable isotope of nitrogen and EPR
`spectroscopy, 2) nitrite treatment attenuates L-NAME
`induced hypertension in a dose-dependent manner, and
`3) nitrite may be an alternative source of NO during
`renal ischemia. These results may explain, at least in
`part, the mechanism of the DASH diet-induced hypo-
`tensive and organ protective effects. Further research is
`needed to investigate the interaction between nitrite-
`nitrate intakes and human health.
`
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