Re: 질산염-아질산염-산화질소 pathway 2008 nature

[문형철]

Key Points

• The inorganic anions nitrite (NO2−) and nitrate (NO3−) are usually viewed as inert end products of nitric oxide (NO) metabolism or unwanted residues in the food chain.

• Recent studies show that nitrate and nitrite are physiologically recycled in blood and tissue to form NO and other bioactive nitrogen oxides. Thus, they should be viewed as storage pools for NO-like bioactivity, thereby complementing the NO synthase-dependent pathway.

• There are two major sources of nitrate and nitrite: the endogenous l-arginine/NO-synthase pathway and the diet. Vegetables are particularly rich in nitrate.

• The bioactivation of nitrate from dietary or endogenous sources requires its initial reduction to nitrite, and this conversion is mainly carried out by commensal bacteria inhabiting the gastrointestinal tract.

• There are numerous pathways in the body for the further reduction of nitrite to bioactive NO, involving haemoglobin, myoglobin, xanthine oxidoreductase, ascorbate, polyphenols and protons.

• The generation of NO by all these pathways is greatly enhanced during hypoxia and acidosis, thereby ensuring NO production in situations for which the oxygen-dependent NO-synthase enzyme activities are compromised.

• Nitrite reduction to NO during physiological and pathological hypoxia appear to contribute to physiological hypoxic signalling, vasodilation, modulation of cellular respiration and the cellular response to ischaemic stress.

• An expanding number of studies suggest a therapeutic potential for nitrate and nitrite in diseases such as myocardial infarction, stroke, systemic and pulmonary hypertension, and gastric ulceration.

Abstract

The inorganic anions nitrate (NO3−) and nitrite (NO2−) were previously thought to be inert end products of endogenous nitric oxide (NO) metabolism. However, recent studies show that these supposedly inert anions can be recycled in vivo to form NO, representing an important alternative source of NO to the classical l-arginine–NO-synthase pathway, in particular in hypoxic states. This Review discusses the emerging important biological functions of the nitrate–nitrite–NO pathway, and highlights studies that implicate the therapeutic potential of nitrate and nitrite in conditions such as myocardial infarction, stroke, systemic and pulmonary hypertension, and gastric ulceration.

• 무기 음이온인 아질산염(NO2−)과 질산염(NO3−)은 일반적으로 산화질소(NO) 대사의 비활성 최종 생성물 또는 먹이 사슬에서 원치 않는 잔여물로 간주됩니다.
• 최근 연구에 따르면, 질산염과 아질산염은 혈액과 조직에서 생리적으로 재활용되어 NO와 기타 생체 활성 질소 산화물을 형성합니다. 따라서, 이들은 NO 유사 생체 활성을 위한 저장 풀로 간주되어야 하며, 따라서 NO 합성 효소 의존 경로를 보완합니다.
• 질산염과 아질산염의 주요 공급원은 두 가지입니다: 내인성 l-아르기닌/NO 합성 효소 경로와 식단. 채소는 특히 질산염이 풍부합니다.
• 식이 또는 내인성 원천으로부터의 질산염의 생체 활성화는 초기 단계에서 아질산염으로 환원되어야 하며, 이 전환은 주로 위장관에 서식하는 공생 박테리아에 의해 수행됩니다.
• 아질산염을 생체 활성 NO로 추가 환원하는 데는 헤모글로빈, 미오글로빈, 크산틴 산화 환원 효소, 아스코르브산, 폴리페놀, 양성자를 포함하는 수많은 경로가 있습니다.
• 이러한 모든 경로를 통한 NO 생성은 저산소증과 산증 상태에서 크게 향상되어, 산소에 의존하는 NO 합성 효소 활성이 손상된 상황에서 NO 생성을 보장합니다.
• 생리적 및 병리학적 저산소 상태에서 아질산염이 NO로 환원되는 것은 생리적 저산소 신호, 혈관 확장, 세포 호흡 조절, 허혈성 스트레스에 대한 세포 반응에 기여하는 것으로 보입니다.
• 점점 더 많은 연구에서 질산염과 아질산염이 심근경색, 뇌졸중, 전신 및 폐 고혈압, 위궤양과 같은 질병에 치료 효과가 있을 수 있다고 주장하고 있습니다.

초록

무기 음이온인 질산염(NO3−)과 아질산염(NO2−)은

이전에 내인성 산화질소(NO) 대사의 비활성 최종 산물로 여겨졌습니다.

그러나

최근 연구에 따르면,

이러한 비활성 음이온은 생체 내에서 재활용되어

NO를 형성할 수 있으며,

특히 저산소 상태에서

기존의 l-아르기닌-NO-합성 경로에 대한 중요한 대체 NO 공급원이 될 수 있습니다.

이 리뷰에서는

질산염-아질산염-NO 경로의 새로운 생물학적 기능에 대해 논의하고,

심근경색, 뇌졸중, 전신 및 폐 고혈압, 위궤양과 같은 질환에서

질산염과 아질산염의 치료 가능성을 시사하는 연구를 강조합니다.

Nitrite (NO2 – ) and nitrate (NO3 – ) are known predominantly as undesired residues in the food chain with potentially carcinogenic effects1,2, or as inert oxidative end products of endogenous nitric oxide (NO) metabolism. However, from research performed over the past decade, it is now apparent that nitrate and nitrite are physiologically recycled in blood and tissues to form NO and other bioactive nitrogen oxides3–6. Therefore, they should now be viewed as storage pools for NO‑like bioactivity, thereby complementing the NO synthase (NOS)dependent pathway. The recognition of this mammalian nitrogen cycle has led researchers to explore the role of nitrate and nitrite in physiological processes that are known to be regulated by NO. The bioactivation of nitrate from dietary or endog‑ enous sources requires its initial reduction to nitrite, and because mammals lack specific and effective nitrate reductase enzymes, this conversion is mainly carried out by commensal bacteria in the gastrointestinal tract and on body surfaces7,8. Nitrite is unique to the nitrogen oxides in its redox position between oxidative (NO2 radical) and reductive (NO radical) signalling and its relative stability in blood and tissue9 . Once nitrite is formed, there are numerous pathways in the body for its further reduction to NO, involving haemoglobin6,10, myoglobin11,12, xanthine oxidoreductase13–15, ascorbate16, polyphenols17,18 and protons3,4 (BOX 1). The generation of NO by these pathways is greatly enhanced during hypoxia and acidosis, thereby ensuring NO production in situations for which the oxygendependent NOS enzyme activities are compromised19,20. Nitrite reduc‑ tion to NO and NO‑modified proteins during physio‑ logical and pathological hypoxia appear to contribute to physiological hypoxic signalling, vasodilation, modula‑ tion of cellular respiration and the cellular response to ischaemic stress6,11,21–26. Here, we review the metabolism and biological roles of NO within the body, and discuss the potential thera‑ peutic use of nitrate or nitrite to treat various disorders, including those associated with vasoconstriction or ischaemia–reperfusion, as well as gastric ulcers.

아질산염(NO2-)과 질산염(NO3-)은 

주로 식품 사슬에서 발암 효과가 있는 바람직하지 않은 잔류물로 알려져 있습니다1,2, 

또는 

내인성 산화질소(NO) 대사의 비활성 산화 최종 산물로 알려져 있습니다. 

그러나 

지난 10년 동안 수행된 연구에 따르면, 

질산염과 아질산염은 혈액과 조직에서 생리적으로 재활용되어 

NO와 기타 생체 활성 질소 산화물을 형성하는 것으로 밝혀졌습니다3-6. 

따라서, 

이들은 이제 NO 유사 생체 활성을 위한 저장 풀로 간주되어야 하며, 

따라서 NO 신타제(NOS) 의존 경로를 보완해야 합니다. 

이 포유류 질소 순환의 인식은 

연구자들이 NO에 의해 조절되는 것으로 알려진 생리학적 과정에서 

질산염과 아질산염의 역할을 탐구하도록 이끌었습니다. 

식이 또는 내인성 원천으로부터의 질산염의 생체 활성화는 

초기 단계에서 아질산염으로 환원되어야 하며, 

포유류는 특이적이고 효과적인 질산염 환원효소 효소가 없기 때문에, 

이러한 전환은 주로 위장관과 신체 표면에 있는 공생 박테리아에 의해 수행됩니다7,8. 

아질산염은 

산화(NO2 라디칼)와 환원(NO 라디칼) 신호 사이의 산화 환원 위치에서 

질소 산화물에 고유하며, 

혈액과 조직에서 상대적으로 안정적입니다9. 

아질산염이 형성되면, 

체내에서 NO로 환원되는 경로가 여러 가지가 있는데, 

헤모글로빈6,10, 

미오글로빈11,12, 

크산틴 산화 환원 효소13-15, 

아스코르브산염16, 

폴리페놀17,18, 

양성자3,4(박스 1) 등이 그 예입니다. 

이러한 경로에 의한 NO 생성은 

저산소증과 산증 상태에서 크게 향상되어, 

산소 의존성 NOS 효소 활성이 손상된 상황에서 

NO 생성을 보장합니다19,20. 

생리적 및 병리학적 저산소 상태에서 

아질산염이 NO로 환원되고 

NO가 단백질을 변형하는 것은 

생리적 저산소 신호, 혈관 확장, 세포 호흡 조절, 허혈 스트레스에 대한 

세포 반응에 기여하는 것으로 보입니다6,11,21-26. 

여기서는 

체내에서 NO의 대사 작용과 생물학적 역할을 검토하고, 

질산염 또는 아질산염의 잠재적 치료적 사용에 대해 논의합니다. 

여기에는 

혈관 수축 또는 허혈-재관류와 관련된 질환뿐만 아니라 

위궤양 치료도 포함됩니다.

NOS-independent NO generation

The NOS enzymes utilize l‑arginine and molecular oxygen to produce the freeradical gas • NO, a critical regulator of vascular homeostasis, neurotransmission and host defence27,28. NO is an autocrine and paracrine signalling molecule whose lifetime and diffusion gradients are limited by scavenging reactions involving haemoglobin, myoglobin and other radicals. However, as discussed here, NO can be stabilized in the blood and tissue by oxidation to nitrate and nitrite, which can be considered as endocrine molecules that are transported in the blood, accumulate in tissue and have the poten‑ tial to be converted back to NO under physiological and pathological conditions. Interestingly, the l‑arginine–NOS pathway is oxygen dependent, whereas the nitrate–nitrite–NO pathway is gradually activated as oxygen tensions falls. In this sense, NOS independent NO formation (FIG. 1) can be viewed as a backup system to ensure that there is sufficient NO formation when oxygen supply is limited, which is analo‑ gous to the complementary role of anaerobic glycolysis in energetics. The exact oxygen tension at which NOS dependent NO generation fails to signal is unknown, in part owing to uncertainties about the in vivo Km of the NOS enzymes for oxygen and the fact that the rate of NO oxidative metabolism is reduced at low oxygen. However, it is clear that at very low oxygen tensions NO generation in tissues is independent of NOS activity and dependent on nitrite5,22,26. This principle has driven hypotheses that nitrite participates in hypoxic vasodilation and in the regulation of oxygen consumption at the mitochondrial level. It also predicts a role for nitrite in cytoprotective signalling in the setting of pathological ischaemia and reperfusion.

NOS-독립적인 NO 생성

NOS 효소는 

l-아르기닌과 분자 산소를 활용하여 

혈관 항상성, 신경 전달, 숙주 방어의 중요한 조절자인 

자유 라디칼 가스 • NO를 생성합니다27,28. 

NO는 

오토크린과 파라크린 신호 전달 분자로서, 

그 수명과 확산 구배는 

헤모글로빈, 미오글로빈, 기타 라디칼과 관련된 청소 반응에 의해 제한됩니다. 

그러나, 여기서 논의된 바와 같이, 

NO는 

질산염과 아질산염으로 산화되어 

혈액과 조직에서 안정화될 수 있습니다. 

이 분자들은 

혈액으로 운반되어 조직에 축적되며, 

생리적 및 병리학적 조건 하에서 

NO로 다시 전환될 수 있는 내분비 분자로 간주될 수 있습니다. 

흥미롭게도, 

l-아르기닌-NOS 경로는 산소에 의존하는 반면, 

질산염-아질산염-NO 경로는 산소 농도가 감소함에 따라 점차 활성화됩니다. 

이러한 의미에서, 

NOS 독립적인 NO 형성(그림 1)은 

산소 공급이 제한될 때 충분한 NO 형성을 보장하는 백업 시스템으로 볼 수 있으며, 

이는 에너지 측면에서 혐기성 글리코리시스의 보완적인 역할과 유사합니다. 

NOS 의존성 NO 생성이 

신호 전달에 실패하는 정확한 산소 장력은 알려져 있지 않습니다. 

부분적으로는 

산소에 대한 NOS 효소의 생체 내 Km에 대한 불확실성과 

낮은 산소 농도에서 NO 산화 대사율이 감소한다는 사실 때문입니다. 

그러나 

매우 낮은 산소 장력에서는 

조직 내 NO 생성이 NOS 활동과 무관하며 

아질산염에 의존한다는 것은 분명합니다5,22,26. 

이 원리는 

아질산염이 저산소성 혈관 확장과 

미토콘드리아 수준에서의 산소 소비 조절에 관여한다는 가설을 뒷받침합니다. 

또한 

병리학적 허혈과 재관류 상황에서 

세포 보호 신호 전달에 아질산염이 관여한다는 것을 예측합니다.

Putting under the tongue to cause heart qi to flow freely For treating symptoms such as struck by evil, acute heart pains and cold in the hands and feet which can kill a patient in an instant. Look at the patient’s fingers and those with greenish-black nails are such cases. Take potassium nitrate (5 measures of a bi spoon) and arsenic sulphide (1 measure of a bi spoon) and combine the two into a fine powder. Lift the patient’s tongue and sprinkle 1 measure of a bi spoon under the tongue. If saliva is produced have the patient swallow it. This is a sure cure

혀 밑에 대고 혀를 움직여 심장의 기가 자유롭게 흐르도록 한다. 악한 기운에 휩쓸리거나, 심한 심장 통증, 손발이 차가워져 환자가 순식간에 죽을 수 있는 증상을 치료한다. 환자의 손가락을 보면, 손톱이 녹색을 띤 검은색인 경우가 그런 경우입니다. 질산칼륨(5스푼)과 비소황화물(1스푼)을 섞어 가루로 만듭니다. 환자의 혀를 들어 올리고 혀 밑에 1스푼을 뿌립니다. 침이 나오면 환자가 삼키게 합니다. 이것이 확실한 치료법입니다.

Figure 1 | The nitrate–nitrite–N pathway.

a | A medical recipe from Dunhuang. The nitrate–nitrite–nitric oxide (NO) pathway has been harnessed therapeutically since the medieval times as evidenced by a translation of medieval Buddhist manuscripts, which was discovered in a Buddhist grotto near the town of Dunhuang by a Daoist monk (Abbot Wang) at the beginning of the twentieth century after being hidden for 900 years. This was brought to our attention and translated by Anthony Butler, Zhou Wuzong and John Moffett. It illustrates the early appreciation of the effect of nitrate, readily available for meatcuring and gunpowder and reduced to nitrite in saliva, on cardiovascular conditions (angina and digital ischaemia). The text is written vertically beginning on the right and progressing leftwards. The term qi refers to a ‘fluid’ that, in a healthy person, flows harmoniously around the body. Its flow is disrupted during sickness. A bi spoon was a ceremonial spoon used in medicine. Chinese physicians often added realgar to a recipe as its colour is that of healthy blood. It would have had no effect because of its low solubility.

b | Two parallel pathways for the generation of bioactive NO in mammals. NO is a key signalling molecule that serves to regulate a wide range of physiological functions. It is classically produced from l‑arginine and oxygen by a family of enzymes, the NO synthases (NOSs). More recently, a fundamentally different mechanism for the generation of NO in mammals has been described. In this pathway, the inorganic anions nitrate and nitrite are reduced to form bioactive NO in blood and tissues during physiological hypoxia. Although NO generation by NOS becomes limited as oxygen levels fall, the nitrate–nitrite–NO pathway is enhanced. By the parallel action of both of these pathways, sufficient NO generation is ensured along the physiological and pathological oxygen and proton gradients.

그림 1 | 질산염-아질산염-질소 경로.
a | 둔황의 의료 처방. 

질산염-아질산염-산화질소(NO) 경로는 중세 시대부터 치료 목적으로 활용되어 왔습니다. 20세기 초, 도교 승려(왕 주지)가 900년 동안 숨겨져 있던 둔황 근처의 불교 동굴에서 발견한 중세 불교 사본의 번역본을 통해 이를 알 수 있습니다. 이 번역본은 저희의 관심을 끌었고, 앤서니 버틀러, 저우 우종, 존 모펫이 번역했습니다. 이 그림은 육류 가공과 화약 제조에 쉽게 이용되고 침 속에서 아질산염으로 환원되는 질산염이 심혈관 질환(협심증과 디지털 허혈)에 미치는 영향을 일찍이 인식하고 있었다는 것을 보여줍니다. 이 글은 오른쪽에서 시작하여 왼쪽으로 쓰여 있습니다. 기(氣)라는 용어는 건강한 사람의 몸에서 조화롭게 흐르는 '액체'를 의미합니다. 이 액체의 흐름은 질병이 있을 때 방해를 받습니다. 비숟가락은 의학에서 사용되는 의식용 숟가락입니다. 중국 의사들은 종종 레드갈을 처방에 추가했는데, 그 이유는 레드갈의 색깔이 건강한 혈액과 비슷하기 때문입니다. 그러나 레드갈의 낮은 용해도로 인해 효과가 없었을 것입니다.

b | 포유류에서 생체 활성 NO를 생성하는 두 가지 병행 경로. 

NO는 광범위한 생리 기능을 조절하는 데 중요한 신호 전달 분자입니다. NO는 일반적으로 NO 합성효소(NOS)라는 효소 군에 의해 l-아르기닌과 산소에서 생성됩니다. 최근에는 포유류에서 NO 생성에 근본적으로 다른 메커니즘이 설명되었습니다. 이 경로에서는 생리적 저산소 상태에서 무기 음이온인 질산염과 아질산염이 감소되어 혈액과 조직에서 생체 활성 NO를 형성합니다. NOS에 의한 NO 생성은 산소 수준이 떨어지면 제한되지만, 질산염-아질산염-NO 경로는 강화됩니다. 이 두 경로의 병행 작용으로 생리적 및 병리학적 산소 및 양성자 구배에 따라 충분한 NO 생성이 보장됩니다.

Sources of nitrate and nitrite

There are two major sources of nitrate and nitrite in the body: the endogenous l‑arginine–NO synthase pathway and the diet. NO, generated by NOS enzymes, is oxidized in the blood and tissues to form nitrate and nitrite27. The reaction of NO with oxyhaemoglobin produces nitrate and methaemoglobin27, whereas the oxidation of NO forms nitrite, a process that is catalysed in plasma by the multicopper oxidase and NO oxidase ceruloplasmin29. In NOS knockout mice, the circulating nitrite levels are reduced by up to 70%30, and nitrite levels are also lower in mice and humans lacking cerulo‑ plasmin29. Normal plasma levels of nitrate are in the 20–40 µ range, while nitrite levels are substantially lower (50–300 n)8,25,31,32. egular exercise increases endothelial NOS (eNOS) expression‎ and activity33, which results in higher circulating levels of nitrate33–35. In systemic inflammatory disorders such as sepsis and severe gastroenteritis, nitrate and nitrite levels are greatly increased owing to massive inducible NOS (iNOS) induction27,36. y contrast, in diseases with endothelial dysfunction and reduced eNOS activity, plasma levels of nitrate and nitrite are often low37. Dietary nitrate intake is considerable and many vegetables are particularly rich in this anion38. or example, a plate of green leafy vegetables such as lettuce or spinach contains more nitrate38 than is formed endogenously over a day by all three NOS isoforms combined39. Drinking water can also contain considerable amounts of nitrate, although in many countries the levels are strictly regu‑ lated. Nitrite can be found in some food stuffs, most notably as a preservative in cured meat and bacon. 

질산염과 아질산염의 공급원

인체 내의 질산염과 아질산염의 주요 공급원은 두 가지입니다: 

체내의 내인성 l-아르기닌-NO 합성 경로와 

식이요법입니다. 

NOS 효소에 의해 생성된 NO는 

혈액과 조직에서 산화되어 

질산염과 아질산염을 형성합니다27. 

NO가 옥시헤모글로빈과 반응하면 질산염과 메트헤모글로빈27이 생성되는 반면, 

NO의 산화는 플라즈마에서 멀티구리산화효소와 NO산화효소 세룰로플라스민29에 의해 촉매되는 과정인 

아질산염을 형성합니다. 

NOS 결핍 마우스의 경우, 

순환하는 아질산염 수치가 최대 70%까지 감소하며, 

세룰로플라스민이 결핍된 마우스와 인간에서도 아질산염 수치가 낮습니다. 

질산염의 정상 혈중 농도는 20-40 µ 범위이고, 

아질산염의 농도는 이보다 훨씬 낮습니다(50-300 n)8,25,31,32. 

규칙적인 운동은 

내피세포의 NOS(eNOS) 발현과 활성을 증가시켜33, 

그 결과 질산염의 순환 농도가 높아집니다33-35. 

패혈증이나 중증 위장염과 같은 전신 염증성 질환에서는 

유도성 NOS(iNOS)의 대규모 유도로 인해 

질산염과 아질산염 수치가 크게 증가합니다27,36. 

반면, 

내피 기능 장애와 eNOS 활성 감소가 동반되는 질병에서는 

혈장 내 질산염과 아질산염 수치가 

낮은 경우가 많습니다37. 

식이성 질산염 섭취량은 상당하며, 

많은 채소들이 특히 이 음이온이 풍부합니다38. 

예를 들어, 

상추나 시금치와 같은 녹색 잎채소 한 접시에는 

하루 동안 모든 NOS 이소형이 결합하여 체내에서 생성되는 양보다 

더 많은 양의 질산염이 함유되어 있습니다38. 

식수에도 

상당한 양의 질산염이 함유되어 있을 수 있지만, 

많은 국가에서 그 양을 엄격하게 규제하고 있습니다. 

아질산염은 

일부 식품에서 발견될 수 있으며, 

특히 소시지나 베이컨의 보존제로 사용됩니다. 

The entero-salivary circulation of nitrate

In 1994, two groups independently described intragastric NO generation from salivary nitrite in humans3,4. This process does not require NOS activity, but instead involves the enterosalivary circulation of inorganic nitrate (FIG. 2). Dietary nitrate is rapidly absorbed in the upper gastrointestinal tract. In the blood, it mixes with the nitrate formed from the oxidation of endogenous NO produced from the NOS enzymes. fter a meal rich in nitrate, the levels in plasma increase greatly and remain high for a prolonged period of time (plasma halflife of nitrate is 5–6 hours). The nitrite levels in plasma also increase after nitrate ingestion8 . lthough much of the nitrate is eventually excreted in the urine, up to 25% is actively taken up by the salivary glands and is concen‑ trated up to 20fold in saliva8,40. Once in the oral cavity, commensal facultative anaerobic bacteria use nitrate as an alternative electron acceptor to oxygen during respiration, effectively reducing salivary nitrate to nitrite by the action of nitrate reductases7,38. Human nitrate reduction requires the presence of these bacteria — suggesting a functional symbiosis relation‑ ship — as mammalian cells cannot effectively metabo‑ lize this anion. The salivary nitrate levels can approach 10 m and nitrite levels 1–2 m after a dietary nitrate load8 . When saliva enters the acidic stomach (1–1.5 l per day), much of the nitrite is rapidly protonated to form nitrous acid (HNO2 ; pKa ~3.3), which decom‑ poses further to form NO and other nitrogen oxides3,4. Nitrite reduction to NO is greatly enhanced by reducing compounds such as vitamin C and polyphenols, both of which are abundant in the diet17,18,41. The importance of oral bacteria in gastric NO generation is perhaps most clearly illustrated in experiments using germfree sterile rats, in which gastric NO formation is negligible even after a dietary load of nitrate42.

In addition to the stomach, a reductive pathway from nitrate to nitrite and then NO has also been demonstrated in the oral cavity7 , on the skin surface43 in the lower gastrointestinal tract44 and in urine45. While the scientific community had focused on the potentially harmful effects of nitrate and nitrite1 , the wellknown antibacterial effects of NO46–49 suggested a role for gastric NO in host defence3,4 (FIG. 3). Interestingly, enteropathogens can survive for a surprisingly long time in acid alone, but the combination of acid and nitrite results in effective killing3,50,51. NO and other reactive nitrogen oxides formed from acidified nitrite act on multiple bacterial targets including DN, proteins and components of the cell wall38,47. nother proposed physiological role for gastric NO is in the regulation of mucosal blood flow and mucus generation. ecent studies using the rat gastric mucosa as an in vivo bioassay tested the effects of human saliva on these two important determinants of gastric integrity. When the rat gastric mucosa was exposed to human nitriterich saliva, NO gas was immediately generated and both mucosal blood flow and mucus thickness increased in a cyclic GMPdependent manner52. urthermore, nitrate addition to drinking water for 1 week produces similar effects53. During the nitrate treatment, nitrite accumulates in the gastric mucus and when this mucus is removed, the blood flow immedi‑ ately returns to basal levels, indicating a continuous slow release of ‘NOlike’ bioactivity from nitrite trapped in the mucus53. role for salivary nitrite in regulating gastric gastrin release has also been suggested54.

침의 장내 순환을 통한 질산염의 생성

1994년, 

두 그룹이 독립적으로 

인간의 타액 중 아질산염으로부터 위내 NO 생성에 대해 설명했습니다.

이 과정은 

NOS 활동을 필요로 하지 않고, 

대신 무기질 질산염의 장내 순환을 포함합니다(그림 2). 

Figure 2 | The entero-salivary circulation of nitrate in humans. Ingested inorganic nitrate from dietary sources is rapidly absorbed in the small intestine. Although much of the circulating nitrate is eventually excreted in the urine, up to 25% is actively extracted by the salivary glands and concentrated in saliva. In the mouth, commensal facultative anaerobic bacteria effectively reduce nitrate to nitrite by the action of nitrate reductase enzymes. Nitrate reduction to nitrite requires the presence of these bacteria, as mammalian cells cannot effectively metabolize this anion. In the acidic stomach, nitrite is spontaneously decomposed to form nitric oxide (NO) and other bioactive nitrogen oxides, which regulate important physiological functions. Nitrate and remaining nitrite is absorbed from the intestine into the circulation and can convert to bioactive NO in blood and tissues under physiological hypoxia.

그림 2 | 인간의 침에서 질산염의 순환. 

식이성 무기질인 질산염은 

소장에서 빠르게 흡수됩니다. 

순환하는 질산염의 대부분은 결국 소변으로 배출되지만, 

최대 25%는 침샘에 의해 적극적으로 추출되어 침에 농축됩니다. 

입안에서 commensal  facultive 혐기성 박테리아는

질산 환원효소의 작용을 통해

질산염을 효과적으로 아질산염으로 환원합니다.

질산염을 아질산염으로 환원하려면

포유류 세포가 이 효과적으로 대사할 수 없기 때문에

이러한 박테리아가 있어야 합니다.

산성 위장에서

아질산염은 자발적으로 분해되어

중요한 생리 기능을 조절하는 산화질소(NO)와

기타 생체 활성 질소 산화물을 형성합니다.

장에서 흡수된 질산염과 잔류 아질산염은

생리적 저산소 상태에서

혈액과 조직에서 생체 활성 NO로 전환될 수 있습니다.

식이성 질산염은 상부 위장관에서 빠르게 흡수됩니다. 

혈액에서는 

NOS 효소에 의해 생성된 내인성 NO의 산화로 인해 형성된 

질산염과 혼합됩니다. 

질산염이 풍부한 식사를 한 후, 

혈장 내의 질산염 수치가 크게 증가하고 

장기간 높은 수준을 유지합니다(질산염의 혈장 반감기는 5-6시간입니다). 

질산염 섭취 후 혈장 내 

아질산염 수치도 증가합니다8. 

대부분의 질산염은 결국 소변으로 배출되지만, 

최대 25%는 침샘에 의해 적극적으로 흡수되어 

침에서 최대 20배 농축됩니다8,40. 

일단 구강 내에 들어가면, 

공생성 통성 혐기성 세균은 호흡 과정에서 

산소를 대체할 수 있는 전자 수용체로 질산염을 사용함으로써, 

질산염 환원효소7,38의 작용에 의해 

침 속의 질산염을 아질산염으로 효과적으로 환원시킵니다. 

인간이 질산염을 환원시키려면 이 세균이 있어야 하는데, 

이는 포유류 세포가 

이 음이온을 효과적으로 대사할 수 없기 때문에 

기능적 공생 관계가 있음을 시사합니다. 

식사 후 질산염 섭취량이 10m에 달하면 

침 속의 질산염 수치가 1~2m에 달합니다8. 

침이 산성 위(하루 1~1.5리터)로 들어가면, 

많은 양의 아질산염이 빠르게 양성자화되어 

아질산(HNO2; pKa ~3.3)을 형성하고, 

이 아질산은 더 분해되어 

NO와 기타 질소 산화물을 형성합니다3,4. 

NO로 전환되는 아질산염의 감소는 

비타민 C와 폴리페놀 같은 화합물을 줄임으로써 크게 향상될 수 있는데, 

이 두 가지 화합물은 식이요법에서 풍부하게 섭취할 수 있습니다17,18,41. 

위 NO 생성에 있어서 구강 세균의 중요성은 

무균 쥐를 이용한 실험에서 가장 명확하게 드러납니다. 

이 실험에서 질산염을 식이요법으로 섭취한 후에도

 위 NO의 형성은 무시할 수 있을 정도로 미미했습니다42.

위장 외에도 질산염에서 아질산염으로, 

그리고 NO로 환원되는 경로가 구강7, 피부 표면43, 하부 위장관44, 소변45에서도 입증되었습니다. 

과학계에서는 

질산염과 아질산염의 잠재적인 유해성에 초점을 맞추고 있었지만, 

NO46-49의 잘 알려진 항균 효과는 

위 NO가 숙주 방어에 중요한 역할을 한다는 것을 시사했습니다3,4(그림 3). 

Figure 3 | Pathways for nitrite reduction to NO and its proposed physiological roles.

a | In the tissues, such as the heart, there are numerous pathways for the generation of NO from nitrite, all greatly potentiated during hypoxia, including xanthine oxidoreductase (XOR), deoxygenated myoglobin (deoxyMb), enzymes of the mitochondrial chain and protons. Nitritedependent NO formation and S‑nitrosothiol formation can modulate inflammation, inhibit mitochondrial respiration and mitochondrial derived reactive oxygen species formation, and drive cyclic GMPdependent signalling under anoxia. NOdependent cytochrome c oxidase (complex IV) inhibition can also drive reactive oxygen species (ROS)dependent signalling.

b | The formation of bioactive nitric oxide (NO) from the inorganic anion nitrite is generally enhanced under acidic and reducing conditions. In the acidic gastric lumen, NO is generated nonenzymatically from nitrite in saliva after formation of nitrous acid (HNO2 ) and then decomposition into NO and other reactive nitrogen oxides. This NO helps to kill pathogenic bacteria and it also stimulates mucosal blood flow and mucus generation, thereby enhancing gastric protection. Detrimental effects have also been suggested, including nitritedependent generation of nitrosamines with potentially carcinogenic effects. c | In the blood vessels, nitrite forms vasodilatory NO after a proposed reaction with deoxygenated haemoglobin (deoxyHb) and contributes to physiological hypoxic blood flow regulation. GC, guanylate cyclase; MPT, mitochondrial permeability transition; OxyHb, oxygenated haemoglobin; PKG, protein kinase G.

그림 3 | 아질산염이 NO로 환원되는 경로와 그 생리적 역할에 대한 제안.
a | 심장 조직과 같은 조직에는 아질산염으로부터 NO를 생성하는 수많은 경로가 있으며, 저산소 상태에서 모두 크게 강화됩니다. 여기에는 크산틴 산화 환원 효소(XOR), 탈산소화 된 미오글로빈(deoxyMb), 미토콘드리아 사슬의 효소, 양성자가 포함됩니다. 질소 의존성 NO 생성 및 S-니트로소티올 생성은 염증을 조절하고, 미토콘드리아 호흡과 미토콘드리아에서 유래된 활성산소 생성을 억제하며, 무산소 상태에서 주기적 GMP 의존성 신호를 유도할 수 있습니다. NO 의존성 시토크롬 c 산화효소(복합체 IV) 억제는 활성산소(ROS) 의존성 신호 전달을 유도할 수도 있습니다.

b | 무기 음이온 아질산염으로부터 생체 활성 산화질소(NO)의 형성은 일반적으로 산성 및 환원 조건에서 강화됩니다. 산성 위 내강에서 NO는 아질산(HNO2)이 형성된 후 타액 속의 아질산염으로부터 비효소적으로 생성된 다음 NO와 다른 반응성 질소 산화물로 분해됩니다. 이 NO는 병원성 박테리아를 죽이는 데 도움이 되며, 점막의 혈류와 점액 생성을 촉진하여 위 보호 기능을 강화합니다. 또한 발암 효과가 있는 니트로사민을 생성하는 질산염 의존성 생성과 같은 유해한 영향이 제시되었습니다. 

c | 혈관 내에서, 질산염은 탈산소 헤모글로빈(deoxyHb)과 반응한 후 혈관 확장 작용을 하는 NO를 형성하고, 생리적 저산소 혈류 조절에 기여합니다. 

GC, 구아닐레이트 사이클라제; MPT, 미토콘드리아 투과성 전이; OxyHb, 산소화 헤모글로빈; PKG, 단백질 키나제 G.

흥미롭게도, 

장내 병원균은 산성 환경에서만 놀라울 정도로 오랫동안 생존할 수 있지만, 

산과 아질산염의 조합은 효과적인 살균을 가능하게 합니다3,50,51. 

NO와 산성화된 아질산염에서 형성된 기타 반응성 질소산화물은 

DN, 단백질, 세포벽 구성요소 등 

여러 세균 표적에 작용합니다38,47. 

위 NO의 또 다른 생리학적 역할은 

점막 혈류와 점액 생성을 조절하는 것입니다. 

최근 쥐의 위 점막을 생체 내 생체 검사로 사용한 연구에서 인간의 타액이 위 무결성의 두 가지 중요한 결정 요인에 미치는 영향을 테스트했습니다. 쥐의 위 점막이 인간이 가진 질산염이 풍부한 타액에 노출되었을 때, 질소 산화물이 즉시 생성되었고, 점막의 혈류량과 점액의 두께가 주기적인 GMP 의존적 방식으로 증가했습니다52. 또한, 1주일 동안 식수에 질산염을 첨가하면 유사한 효과가 나타납니다53. 

질산염 처리 과정에서 아질산염이 위 점액에 축적되고, 

이 점액이 제거되면 혈류량이 즉시 기본 수준으로 돌아오는데, 

이는 점액에 갇힌 아질산염에서 'NO와 유사한' 생체 활성이 

지속적으로 서서히 방출됨을 나타냅니다53. 

또한, 

위산 분비를 조절하는 데 

속 아질산염이 중요한 역할을 한다는 주장도 제기되었습니다54.

Vasodilatory effects of nitrite

Although the vasodilatory properties of pharmacological doses of exogenous nitrite have been known for more than half a century55–57, a physiological role of this anion in vasoregulation has been dismissed, even in more recent studies58. However, arterytovein gradients in nitrite across the human forearm, with increased consumption during exercise stress, suggests that nitrite is metabo‑ lized across the peripheral circulation25. urthermore, humans breathing NO gas exhibit increases in peripheral forearm blood flow that is associated with increases in plasma nitrite59, suggesting that nitrite could be a stable endocrine carrier and transducer of NO‑like bioactivity within the circulation60. Consistent with a potentially greater efficacy under hypoxic or metabolic stress, the potency of nitrite increases dramatically with decreases in buffer pH in aortic ring experiments24. This hypothesis was tested by infusion of sodium nitrite into the fore‑ arm brachial artery of healthy volunteers, which was surprisingly potent, increasing blood flow even at blood concentrations below 1 µ and producing substantial vasodilation6 . ore recent doseresponse experiments in normal human volunteers reveal significant vasodilation of the forearm circulation already at concentrations as low as 300 n61 and a significant decrease in blood pres‑ sure after nitrate ingestion, associated with an increase in plasma nitrite levels from 140–220 n62.

아질산염의 혈관 확장 효과

약리학적 용량의 외인성 아질산염의 혈관 확장 특성은 

반세기 이상 알려져 왔지만55-57, 

최근의 연구에서도58 이 음이온의 혈관 조절에 대한 생리적 역할은 무시되었습니다. 

그러나, 

운동 스트레스를 받는 동안에 소비량이 증가하는 아질산염의 동맥-정맥 구배는 

아질산염이 말초 순환을 통해 대사된다는 것을 시사합니다25. 

또한, 

NO 가스를 흡입하는 사람은 

말초 팔뚝 혈류량이 증가하여 

혈장 아질산염이 증가하는 것으로 나타났습니다59. 

이는 아질산염이 순환계 내에서 

NO와 유사한 생체 활성을 안정적으로 운반하고 전달할 수 있는 내분비 물질일 수 있음을 시사합니다60. 

저산소증 또는 대사 스트레스 하에서 

잠재적으로 더 큰 효능을 보이는 것과 마찬가지로, 

대동맥 고리 실험에서 완충액 pH가 감소함에 따라 

아질산염의 효능이 극적으로 증가합니다24. 

이 가설은 건강한 지원자의 전완 상완 동맥에 아질산나트륨을 주입하여 테스트되었으며, 놀랍게도 혈중 농도가 1µ 미만인 경우에도 혈류를 증가시키고 상당한 혈관 확장을 일으키는 강력한 효능을 보였습니다6. 

최근 일반인 대상의 용량-반응 실험에 따르면, 300 n61의 낮은 농도에서도 이미 팔뚝 순환의 상당한 혈관 확장이 나타나고, 질산염 섭취 후 혈압이 현저히 감소하며, 혈장 아질산염 수치가 140-220 n62로 증가하는 것으로 나타났습니다.

Pathways for systemic nitrite reduction

Haemoglobin as an allosterically regulated nitrite reductase.

The ability of nitrite to vasodilate the cir‑ culation in the presence of NO‑scavenging red blood cells under physiological conditions is unexpected and suggests new pathways to intravascular bioactivation. During infusion of nitrite into the human forearm circulation, the vasodilatory effects are associated with the formation of NO in the blood, as measured by the rate of formation of ironnitrosylated haemoglobin (NO bound to the haem of haemoglobin) during artery to vein transit6 . This rate of NO formation increases as haemoglobin oxygen saturation decreases, suggesting a hypoxiaregulated mechanism of nitrite bioactivation. These physiological findings are consistent with a nitrite reductase activity of deoxyhaemoglobin as described by rooks in 1947 and Doyle and colleagues in 1981 (Res 63,64). ccording to this chemistry, nitrite reacts with ferrous deoxyhemoglobin (Hbe2+) and a proton (H+) to generate NO and methaemoglobin (Hbe3+), which is analogous to the coupled proton and electrontransfer reactions of bacterial nitrite reductases. The NO can then bind to a second deoxyhaemoglobin to form ironnitrosylhaemoglobin (Hbe2+–NO) as outlined in equations 1 and 2.

조직적 아질산염 감소의 경로

알로스테릭 조절 아질산염 환원효소로서의 헤모글로빈. 

생리학적 조건 하에서 NO 제거 적혈구가 존재할 때 

아질산염이 혈관 확장을 일으키는 능력은 예상치 못한 것으로, 

혈관 내 생체 활성화의 새로운 경로를 제시합니다. 

아질산염이 인간의 팔뚝에 주입되는 동안, 

동맥에서 정맥으로 이동하는 동안에 

철-니트로사이드화 헤모글로빈(헤모글로빈의 헤모에 결합된 NO)의 형성 속도로 측정되는 바와 같이, 

혈관 확장 효과는 혈액 내 NO의 형성과 관련이 있습니다6. 

헤모글로빈 산소 포화도가 감소함에 따라 NO 형성 속도가 증가하는데, 

이는 아질산염 생체 활성화의 저산소 조절 메커니즘을 시사합니다. 

이러한 생리학적 발견은 

1947년 루크스(Rooks)와 1981년 도일(Doyle)과 동료 연구자들이 설명한 

데옥시헤모글로빈의 아질산 환원 효소 활성과 일치합니다(Res 63,64). 

이 화학 반응에 따르면, 

아질산염은 철분 탈옥소헤모글로빈(Hbe2+)과 양성자(H+)와 반응하여 

NO와 메트헤모글로빈(Hbe3+)을 생성하는데, 

이는 박테리아 아질산염 환원효소의 결합 양성자와 전자 전달 반응과 유사합니다. 

NO는 

두 번째 탈산헤모글로빈에 결합하여 방정식 1과 2에 설명된 바와 같이 

철-니트로실헤모글로빈(Hbe2+–NO)을 형성할 수 있습니다.

Nitrite (NO2 – ) + deoxyhaemoglobin (e2+) + H+ → NO + methaemoglobin (e3+) + OH– (1) NO + deoxyhaemoglobin (e2+) → Hbe2+NO (2)

This simple reaction has physiological implications in that it uses naturally occurring nitrite as a substrate, requires deoxygenation of haemoglobin so it has hypoxic sensor properties, requires a proton so it has pH sensor properties, and generates NO, the most potent vasodila‑ tor known. These chemical properties, and supporting physiological studies, suggest that haemoglobin may function as an allosterically regulated nitrite reductase that may contribute to hypoxic signalling and hypoxic vasodilation6,10,65,66 (FIG. 3). The chemistry of this reac‑ tion, mechanisms of NO export from the red blood cell and physiological contribution to hypoxic blood flow regulation are the subjects of active research (FIG. 4). Interestingly, recent studies suggest that NO formed from nitrite reduction (equation 1) can react with a sec‑ ond nitrite that is bound to methaemoglobin (Hbe3+)67. emarkably, when nitrite binds to methaemoglobin, it forms a nitrogen dioxide radical (• NO2 ) character (Hbe3+–NO2 – forms Hbe2+–• NO2 ), which reacts with NO in a radical–radical reaction to form N2 O3 . N2 O3 is more stable in a haemrich environment than NO and has the potential to escape from the red blood cell. The overall stoichiometry of the reaction of equation 1 and the second reaction of NO with nitrite–methaemoglobin is shown in equation 3. Note that in this reaction haemo‑ globin is catalytic and redox cycles convert two molecules of nitrite into N2 O3 .

이 간단한 반응은 자연적으로 발생하는 아질산염을 기질로 사용하고, 헤모글로빈의 탈산소화를 필요로 하므로 저산소 감지 특성을 가지며, 양성자를 필요로 하므로 pH 감지 특성을 가지며, 가장 강력한 혈관 확장제인 NO를 생성한다는 점에서 생리학적 의미를 갖습니다. 이러한 화학적 특성과 이를 뒷받침하는 생리학적 연구 결과는 헤모글로빈이 저산소 신호 전달과 저산소 혈관 확장에 기여할 수 있는 알로스테릭 조절 아질산염 환원 효소로서 기능할 수 있음을 시사합니다6,10,65,66 (그림 3). 이 반응의 화학 작용, 적혈구에서 NO가 배출되는 메커니즘, 그리고 저산소 혈류 조절에 대한 생리학적 기여는 활발한 연구의 주제입니다(그림 4). 흥미롭게도, 최근 연구에 따르면 아질산염 환원(방정식 1)으로 형성된 NO가 메트헤모글로빈(Hbe3+)에 결합된 두 번째 아질산염과 반응할 수 있다고 합니다67. 놀랍게도, 아질산염이 메트헤모글로빈에 결합하면 이산화질소 라디칼(• NO2 )이 형성됩니다(Hbe3+–NO2 – Hbe2+–• NO2 형성). 이 라디칼은 라디칼-라디칼 반응에서 NO와 반응하여 N2O3를 형성합니다. N2O3는 NO보다 적혈구 환경에서 더 안정적이며 적혈구에서 빠져나갈 가능성이 있습니다. 방정식 1의 반응과 아질산염-메트헤모글로빈과의 NO의 두 번째 반응의 전체적인 화학양론은 방정식 3에 나와 있습니다. 이 반응에서 헤모글로빈은 촉매 작용을 하며, 산화 환원 반응은 아질산염 두 분자를 N2O3로 전환합니다.

2 NO2 – + deoxyhemoglobin (e2+) + H+ → N2 O3 + deoxyhaemoglobin (e2+) + OH– (3)

Myoglobin, xanthine oxidoreductase and other pathways.

Myoglobin has a high affinity for oxygen and a low haem redox potential that contributes to rapid nitrite reduc‑ tion to NO when deoxygenated; in fact, deoxymyoglobin will reduce nitrite to NO at a rate 30times faster than haemoglobin11,66. These chemical properties suggest that when myoglobin becomes deoxygenated, such as in the subendocardium of the heart or in exercising skeletal muscle, it will rapidly convert nitrite to NO (via the same nitrite reductase reaction as described above). Indeed, myoglobin has recently been shown to convert nitrite to NO in the cardiomyocyte and in the working heart11,12. NO formed by myoglobin can bind to cytochrome c oxidase of the mitochondrial electron transport chain, reducing elec‑ tron flow and oxygen utilization11. Consistent with these studies, nitrite reduction to NO and nitritedependent modulation of cardiac consumption is abolished in the myoglobin knockout mouse12. These studies sug‑ gest that nitrite and myoglobin play an important role in regulating cardiac energetics and oxygen utilization under conditions of physiological hypoxia. Supporting this possibility, arsen and colleagues found that whole body oxygen consumption in young healthy volunteers was significantly reduced during submaximal exercise after dietary supplementation with nitrate compared with placebo treatment68. This surprising effect was associated with the metabolism of plasma nitrite. Several enzymes, including xanthine oxidoreduct‑ ase13–15,69, complexes of the mitochondrial electron transport chain70–72, cytochrome P450s73 and even the NOS enzyme74, have been shown to use nitrite as an alternative electron acceptor to molecular oxygen thereby forming NO (FIG. 3). or example, xanthine oxidoreductase is known to reduce molecular oxygen to superoxide (O2 – ), but at low oxygen tensions and pH values this enzyme can also reduce nitrite to NO at the molybdenum site of the enzyme. In terms of a potential role in vasoregulation and NO signalling, these four pathways all require low oxygen tensions to effectively generate NO and these enzymes also produce superoxide, which is expected to react rapidly with and scavenge any NO that is synthesized. It is likely that nitrite can competitively reduce vascular reactive oxygen species (OS) formation by these enzymes, by direct diversion of electrons away from oxygen, thus limiting superoxide formation. Decreases in superoxide will effectively increase vascular NO bioavailabilty. In addition to sec‑ ondary NO generation, a role of nitrite as an intrinsic signalling molecule that can directly modify target haem or thiol groups on proteins has been proposed23.

Figure 4 | state or allosteric autocatalysis of nitrite reduction by haemoglobin.

a | Allosteric nitrite reduction by haemoglobin. The rate at which haemoglobin converts nitrite into nitric oxide (NO) is maximal when it is 50% saturated with oxygen, the midpoint of the haemoglobin– oxygen dissociation curve (P50). This effect is mechanistically determined by two opposing chemistries, the availability of deoxyhaems (reaction substrate) to bind nitrite, which is maximal in T‑state or deoxygenated haemoglobin, and the amount of R‑state or oxygenated haemoglobin tetramer, which increases the intrinsic reactivity of the haem with nitrite. The latter process occurs because R‑state or oxygenated haemoglobin has a decreased haem redox potential, which is analogous to the low haem redox potential of myoglobin. This low redox potential of the R‑state ferrous haem favours an equilibrium distribution of electrons to nitrite, resulting in the increased reactivity of nitrite with the unliganded ferrous haems of R‑state haemoglobin and myoglobin. The lowered haem redox potential is kinetically manifested as an increased bimolecular rate constant for the reaction of nitrite with R‑state haemoglobin compared with T‑state haemoglobin. Because the observed rate of nitrite reduction to form NO is equal to the product of the bimolecular rate constant times the deoxyhaem (reactant) concentration, this rate is expected to be maximal at approximately 50% oxygen saturation, or the intrinsic haemoglobin P50; this allows for physiological hypoxic ‘sensing’ and NO generation. b | Physiological model. In the mammalian circulation, the allosteric state of haemoglobin tetramers is modulated primarily by oxygen ligation such that the in vivo intrinsic reactivity (bimolecular rate constant) of the deoxyhaem is therefore dictated by oxygen binding to other haems on the same tetramer. This model therefore predicts that the most effective tetrameric nitrite reductase would be the R‑state haemoglobin that rapidly deoxygenates during arterial to capillary transit. This molecule would transition through R4 (Rstate with four oxygens bound) to R3 (Rstate with three oxygens bound) to R2 intermediates and then shift to T2 (Tstate with two oxygens bound) and ultimately T1. These R3 and R2 tetramers would be the most effective nitrite reductases (bimolecular rate of 6 M–1 sec–1 for R‑haem compared with 0.03 M–1 sec–1 for T‑haem). This chemistry is consistent with experiments showing that nitrite reduction and NO signalling are most effective in systems subjected to rapid deoxygenation in the presence of oxyhaemoglobin and nitrite or when haemoglobin is 50% saturated with oxygen. A1–A5 reflect the arteriolar size, which decreases at branch points. Therapeutic opportunities Vasodilation.

Numerous studies have now confirmed the vasodilating effects of lowdose nitrite in mice, rats, sheep, dogs, primates and humans75–81. Therapeutic delivery of nitrite to vasodilate ischaemic vascular beds shows great promise in preclinical studies (FIG. 5). Patients suffering from spontaneous haemorrhage of a subarchnoidal artery aneurism are at risk for develop‑ ing delayed cerebral artery spasm. In primate models, this spasm is associated with acute depletion of cerebral spinal fluid nitrite levels. Twoweek infusions of systemic nitrite effectively prevented this complication80. Primary pulmonary hypertension of the newborn (PPHN) is a condition that is associated with a high pulmonary vascular resistance and extremely low sys‑ temic oxygenation. In sheep models of PPHN, inhaled nitrite was converted to NO gas in the lung and selec‑ tively vasodilated the pulmonary circulation78. In such diseases, which are characterized by regional ischaemia and vasoconstriction, nitrite may provide an ideal stable and naturally occurring therapeutic NO donor. The vasodilatory and biological activities of the inor‑ ganic anions nitrite and nitrate must be distinguished from the organic nitrates (that is, nitroglycerin) and nitrites (amylnitrite). Clearly, the organic nitrates and nitrites are much more potent than nitrite in terms of vasoactivity. lthough the antianginal and vasodilatory organic nitrates and nitrites are metabolized in vivo into vasodila‑ tory NO and nitrite82, this bioactivation requires metabo‑ lism by mitochondrial aldehyde dehydrogenase and other enzymes, which are all subject to induced tolerance83,84. Tolerance is characterized by a lack of nitroglycerin bio‑ logical activity with chronic drug exposure. Studies from as far back as 1930 suggest that inorganic nitrite does not induce tolerance85, implying that nitrite may represent an active metabolite of nitroglycerin that can bypass enzymatic nitroglycerin metabolism and tolerance.

Figure 5 | Therapeutic opportunities for inorganic nitrite. The inorganic anion nitrite (NO2 – ) can be metabolized in blood and tissues to form nitric oxide (NO), a pluripotent biological messenger. Nitrite reduction to NO is catalysed by various enzymatic and nonenzymatic pathways and is greatly enhanced during hypoxia and ischaemic stress, which may be of therapeutic value. In animal models, nitrite is strongly cytoprotective and protects against ischaemia– reperfusion injury. These findings suggest an opportunity for nitrite therapy for human diseases such as myocardial infarction, stroke, solidorgan transplantation and sicklecell disease. Nitrite is also cytoprotective in the stomach, where it can prevent druginduced gastric ulcers. The vasodilatory and bloodpressure lowering effects of nitrite could be useful in pulmonary and systemic hypertension, as well as in the treatment and prevention of delayed cerebral vasospasm after subarachnoidal artery aneurysm haemorrhage.

Tissue protection in ischaemia–reperfusion injury.

Systemic NOSindependent NO formation from nitrite was first demonstrated in the ischaemic heart5 . Studies in animal models of ischaemia and reperfusion have now revealed a central role of nitrite in hypoxic signal‑ ling. Physiological and therapeutic levels of nitrite exert potent cytoprotection after prolonged ischaemia and bloodflow reperfusion in liver22,86, heart22,79,87, brain88 and kidney89. These findings suggest an opportunity for nitrite therapy for human diseases associated with ischaemia–reperfusion, such as myocardial infarction, stroke, solidorgan transplantation, cardiopulmonary arrest and sicklecell disease (FIG. 5). Doseresponse studies in mice suggest a broad efficacy to safety range of nitrite of three orders of magnitude, with doses as low as 0.1 µmoles per kg to 100 µmoles per kg providing significant protection. Interestingly, the protective effect of nitrite is evident at very low plasma concentrations (less than 200 n), but is lost as plasma concentrations rise above 100–1,000 µ22. The lowest dose of nitrite given in these studies only increased the plasma levels of nitrite by 20%. Intriguingly, a similar or even greater increase in plasma nitrite is seen after ingesting a portion of spinach or lettuce8 ; this evokes pro‑ vocative questions about a putative role of nitrate as an active ingredient of the cardioprotective mediterranean diet9,38,90,91 (BOX 2). The mechanism of nitritemediated cytoprotection appears to be NO‑dependent and mitochondriatargeted. Studies using various inhibitors and genetic knockout mice provide some clues to the potentially impor‑ tant pathways. ll the published animal studies have demonstrated a loss of cytoprotection when animals were treated with the NO scavenger carboxyPTIO (2(4carboxyphenyl)4,5dihydro4,4,5,5tetramethyl 1Himidazolyl1oxy3oxide)22,79,86,88, suggesting the importance of NO in the mechanism of cytoprotection. Pretreatment of animals with a NOS inhibitor22,79 or use of eNOS knockout mice22 did not inhibit cytoprotection, proving that the nitrite effect is NOSindependent. The pathway(s) by which nitrite forms NO in hypoxic tissue remains to be determined. Two groups suggest the involvement of xanthine oxidoreductase in the reduc‑ tion of nitrite to NO on the basis of reduced efficacy after treatments with allopurinol, a xanthine oxidase inhibitor79,87,89. The fact that nitrite remains protective in isolated bufferperfused organ models, such as the angendorff heart, suggests that the haemoglobin path‑ way is not necessary for this function. We have con‑ sidered the possibility that in the heart, myoglobin can serve this function and have recently demonstrated that deoxymyoglobin has nitrite reductase activity, which can modulate mitochondrial respiration11,21. OS generation by mitochondria is a necessary component of mitochondrial signalling in cytoprotec‑ tion92–95. However, the large burst of oxidizing OS gen‑ erated after reperfusion following ischaemia can also contribute to cellular injury, necrosis and apotosis96,97. S-Nitrosation of complex I of the electron transport chain inhibits the activity of this complex98 and decreases mitochondrialderived OS formation during reper‑ fusion, an effect associated with cellular cytoprotec‑ tion99,100. Nitrite can similarly nitrosate complex I during ischaemia and reperfusion21. This modification limits complex Idependent reperfusion OS formation, acti‑ vation of the mitochondrial permeability transition pore, and cytochrome c release. Interestingly, the effects of nitrite on mitochondria and tissue cytoprotection occur both acutely (immediately before reperfusion) and remotely (if given 24 hours before reperfusion), suggesting a potential role for nitrite as an effector of ischaemic preconditioning21. The inhibitory effect of NO101–104 and nitrite on mitochondrial respiration that is associated with mitochondrialdependent cytoprotection presents an interesting paradox. The energetic cost of reversibly inhibiting mitochondrial respiration appears to be off‑ set by reduced OS generation during reperfusion. paradigm is emerging that damping electron flow to oxygen (thus limiting superoxide formation) during reperfusion, by NOdependent complex I and I inhi‑ bition98,101 or by depleting oxygen during reperfusion (postconditioning), may reduce reperfusion OS gen‑ eration and limit downstream apoptotic signalling105. Data also suggest that NO‑dependent inhibition of cytochrome c oxidase before ischaemia, that is, during normoxia, can produce the opposite effect of increas‑ ing basal OS formation, creating a preconditioning environment that is also adaptive92–95. Several recent studies of NO gas inhalation in both animals and humans suggest a transformation of NO in the lung into a more longlived bioactive NO‑species that can be transported in blood59,106. oreover, inhaled NO reduces myocardial infarction volume in mice107 and pigs108 and the extent of liver injury after orthotopic transplantation in humans109. These effects are associated with significant increases in circulating nitrite, with no significant changes in blood S‑nitrosothiol levels. NO treatment significantly reduced the overall incidence of brain injury in premature newborns with respiratory failure, an effect consistent with endocrine transport of an NO‑intermediate in blood to the central nervous system110. Thus, increasing evidence suggests that nitrite is mediating extrapulmonary effects of NO gas inhalation. The promising animal data discussed here indicate that nitrite possesses the characteristics of a useful adjunc‑ tive therapy for acute myocardial infarction, including significant cardioprotection following prolonged ischae‑ mia, simple administration and minimum associated regional and systemic side effects. ased on these con‑ siderations, a human Phase II clinical trial of intravenous nitrite for ST segment elevation myocardial infarction is currently being planned by the S National Heart, ung, and lood Institute in cooperation with uropean centres.

Gastric ulcers.

A common and potentially serious side effect of aspirinlike drugs (nonsteroidal antiinflam‑ matory drugs; NSIDs) is the development of gastric ulcers secondary to the inhibition of prostaglandin synthesis by these agents111. Similarly, in animal mod‑ els, pharmacological inhibition of the NOS enzymes increases the susceptibility to ulcerogenic compounds112. xperiments with isoformselective inhibitors suggest that the constitutive isoforms of cyclooxygenase (COX1) and NOS (eNOS and neuronal NOS; nNOS) are protective, while the opposite may be true for the inducible enzymes (COX2 and iNOS)113,114. In a recent study, rats were given sodium nitrate in the drinking water for 1 week followed by acute exposure to an NSID (diclofenac) by gastric gavage115. Dietary nitrate increased gastric NO levels and potently protected against the macroscopic injury caused by NSID exposure (FIG. 5). dditionally, nitrate pretreat‑ ment decreased mucosal myeloperoxidase activity and expression‎ of iNOS, which is indicative of reduced tissue inflammation. The protection afforded by nitrate probably relates to increased gastric mucosal blood flow and mucus generation and reduced epithelial permeability52,53. The gastroprotective effect of nitrate was abolished in rats if they were pretreated with topical antibiotics in the mouth before nitrate supplementation, thereby illustrating the importance of the oral microflora in the bioactivation of nitrate116. n additional protective effect of nitrate on ulcer development may occur through inhibition of Helicobacter pylori117. In critically ill patients, endotracheal intubation and sedation interrupt the enterosalivary nitrate cycle, which results in depleted gastric NO, nitrite and S‑nitrosothiol levels118. It has been suggested that the insufficient levels of gastric NO contribute to the gastric lesions and bacterial overgrowth commonly found in these patients118.

Antimicrobial effects.

Nitrite is used as a preservative in meat products to inhibit the growth of pathogens, most notably Clostridium botulinum, and these antibacterial effects have been attributed to NO formation119. The discovery of endogenous nitrite reduction to NO in the acidic stomach triggered researchers to explore thera‑ peutic uses for acidified nitrite as an antimicrobial agent. Indeed, acidified nitrite results in the generation of NO and other nitrogen oxides, which have potent antibac‑ terial activity against a range of pathogens, including Salmonella, Yersinia and Shigella species, H. pylori, and Pseudomonas aerguinosa3,16,117,120. These antibacterial effects of nitrite have recently been investigated in the airways. In an animal model resembling cystic fibro‑ sis, acidified nitrite successfully cleared the airways of mucoid P. aeruginosa, a pathogen commonly infecting the airways of patients with cystic fibrosis121. Infected urine typically contains considerable amounts of nitrite, owing to bacterial reduction of urinary nitrate. lthough nitrite is stable at neutral or alkaline conditions, it is reduced to NO and has potent antibacterial effects if the urine is mildly acidified (to pH 5–6); these effects are potentiated in the presence of the reducing agent vitamin C16. In fact, the in vitro antibacterial potency of nitrite and ascorbic acid is fully comparable to that of traditional antibiotics such as nitrofurantoin and trmetoprim. cidification of urine — for example, by vitamin C intake — has been used in traditional medicine for the preven‑ tion and treatment of urinary tract infections. This effect may be related to the formation of antibacterial nitrogen oxides from the acidified nitrite38,122.

Opportunities for drug development

As mentioned above, several different therapeutic indications for nitrite have been successfully tested recently both in animal models and in humans (FIG. 5). Depending on the condition to be treated, the develop‑ ment of several different nitritecontaining formulations and methods of administration are anticipated.

Topical administration of acidified inorganic nitrite.

An inorganic nitrite salt such as sodium nitrite (NaNO2 ) is combined with an acidifying agent (for example, ascorbic acid). This mixture rapidly releases NO and other nitrogen oxides and has been eval‎uated for its anti‑ microbial activity. Topical application of acidified nitrite to the skin has proved effective in various skin infec‑ tions123–125, and in the airways, acidified nitrite has been shown to kill mucoid Pseudomonas in an animal model of cystic fibrosis121. Carlsson and colleagues used the inflat‑ able retention balloon of a urinary catheter as a depot for nitrite and ascorbic acid, leading to direct intravesicular delivery of antimicrobial nitrogen intermediates126. In their in vitro studies, NO was generated in the retention balloon and diffused into the surrounding urine where it effectively killed the urinary pathogen Escherichia coli. They suggested that this could be a new approach to prevent catheterassociated urinarytract infections, the most common hospitalacquired infection.

Enteral administration of inorganic nitrite and nitrate.

It is clear that both nitrate and nitrite are readily absorbed and biologically active when given orally, and therapeutic effects have been observed in animal models of ischaemia– reperfusion injury127 and in protection against gastric ulcerations115,116,128. In addition, shortterm dietary nitrate supplementation has been shown to lower blood pressure in healthy volunteers62. combination of nitrate and nitrite salts for oral administration is theoretically attractive, as the nitrite would ensure immediate effects soon after absorption, while the nitrate would continu‑ ously provide a slow release of nitrite over a prolonged period of time via the enterosalivary recirculation described above. Similar to the recently developed NO–NSIDs, in which the active drug is combined with an organic nitrate129, the addition of inorganic nitrate to an ulcerogenic drug such as aspirin or another NSID is also a possible new composition.

Organic nitro compounds as donors of nitrite.

The bio‑ availability of nitrite after enteral administration of inorganic nitrate or nitrite can be difficult to control because of the variable metabolism of these anions within the gastrointestinal tract. However, the use of organic allylic nitro compounds as nitrite donors may overcome this potential problem130, as in vitro experi‑ ments have shown that such compounds can release nitrite and NO in the presence of thiols (lcysteine) and ascorbic acid. Traditional organic nitrates (nitroglycer‑ ine) and nitrites (amyl nitrite) used in cardiovascular medicine are also metabolized to nitrite in vivo. Whether the organic allylic nitro compounds or other donors of nitrite can offer any additional advantages over these compounds and the native inorganic anions, in terms of controlled delivery, bioavailability and tolerance, remains to be studied.

Short or long-term infusions of inorganic nitrite.

In the development of nitrite for therapeutic intravenous use, it is anticipated that the dose and duration of treatment will have to be adjusted depending on the condition and the desired effect. In animal studies, large doses of nitrite infused over a long period of time are needed to effectively alleviate the vasospasm associated with subarachnoidal haemorrhage80. In models of ischaemia–reperfusion injury, however, the dose of nitrite needed for protective effects is remarkably low21,22.

Toxicity.

The two major health concerns with inorganic nitrite and nitrate are the risk for development of meth‑ aemoglobinaemia and their potential carcinogenic effects2 . ny toxicity of the nitrate ion is thought to occur after its bioconversion to nitrite, which is considerably more reactive. ormation of methaemoglobin occurs when the oxygencarrying ferrous ion (e2+) of the haem group of the haemoglobin molecule is oxidized by nitrite to the ferric state (e3+). This converts haemoglobin to methaemoglobin, which cannot bind oxygen. Clinically significant methaemoglobinaemia with cyanosis occurs when the levels increase above a certain level (approxi‑ mately 5%). In animal studies looking at the tissue pro‑ tective and vasodilatory effects of intravenous nitrite, the increase in methaemoglobin is generally undetectable or modest even after prolonged delivery80, suggesting that methaemoglobin is not a major problem in these dose ranges. In fact, the estimated C50 for nitrite in human adults, based on methaemoglobin formation, is 1 g131, whereas calculations from animal data suggest that less than 40 mg nitrite would be necessary for the treatment of myocardial infarction in a 70 kg adult. In 2001, the S Department of Health and Human Services National Toxicology Program published extensive toxicology and carcinogenesis studies of sodium nitrite in rats and mice. Sodium nitrite was delivered in drink‑ ing water for 14week and 2‑year periods and genetic toxicology studies were conducted in Salmonella typhimurium, and in rat and mouse bone marrow and peripheral blood. Consistent with recent epidemio‑ logical studies in humans2 , there was no significant evidence of carcinogenic activity of nitrite, despite dose escalations sufficient to produce profound meth‑ aemoglobinaemia and weight loss in rodents131. recent epidemiological study eval‎uating dietary exposure of nitrite (cured meat) has suggested a possible link to the development of emphysema in atrisk subjects132; further population studies will be required to validate this observation. or most of the therapeutic indications discussed in this article, the low dose and short duration of treatment suggest that the risk of any carcinogenic effects is negligible. In fact as stated above, a large con‑ sumption of nitratecontaining vegetables may provide similar or even greater systemic loads of both nitrate and nitrite. If nitrite is to be used in much higher doses over prolonged periods of time, this issue will naturally have to be addressed.

Conclusions

The nitrate–nitrite–NO pathway may be viewed as com‑ plementary to the classical l‑arginine–NOS pathway. These pathways work partly in parallel, but when oxy‑ gen availability is reduced and NOS activity is decreased, nitrite reduction to NO becomes more pronounced. So, in pathological conditions when regional and systemic ischaemia prevail, it may be beneficial to support the nitrate and nitrite stores pharmacologically or by dietary intervention. We must now revise our longstanding view that nitrate and nitrite are only harmful substances in our diet or inert metabolites of endogenous NO. Instead, accumulating evidence suggests that the nitrate–nitrite– NO pathway critically subserves physiological hypoxic NO signalling, providing an opportunity for novel NO‑based therapeutics.

결론 

질산염-아질산염-NO 경로는 

기존의 l-아르기닌-NOS 경로와 보완적인 것으로 볼 수 있습니다. 

이 두 경로는 부분적으로 병행적으로 작용하지만, 

산소 가용성이 감소하고

 NOS 활동이 감소하면 

아질산염이 NO로 환원되는 현상이 더욱 두드러집니다. 

따라서 

국소 및 전신 허혈이 우세한 병리학적 상황에서 

질산염과 아질산염의 축적을 약리적으로 또는 

식이 중재를 통해 지원하는 것이 도움이 될 수 있습니다. 

우리는 이제 

질산염과 아질산염이 식단에서 유해한 물질이거나 

내인성 NO의 비활성 대사 산물이라는 오랜 견해를 수정해야 합니다. 

대신, 

축적된 증거는 

질산염-아질산염-NO 경로가 

생리적 저산소 NO 신호 전달에 결정적으로 기여하여 

새로운 NO 기반 치료법을 위한 기회를 제공한다는 것을 시사합니다.