Re: 신생아의 질산염, 아질산염 섭취 1014년 nature

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Dietary intake and bio-activation of nitrite and nitrate in newborn infants

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• Published: 14 October 2014

Dietary intake and bio-activation of nitrite and nitrate in newborn infants

• Jesica A. Jones, 
• Andrew O. Hopper, 
• Gordon G. Power & 
• Arlin B. Blood 

Pediatric Research volume 77, pages173–181 (2015)Cite this article

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Abstract

Nitrate and nitrite are commonly thought of as inert end products of nitric oxide (NO) oxidation, possibly carcinogenic food additives, or well-water contaminants. However, recent studies have shown that nitrate and nitrite play an important role in cardiovascular and gastrointestinal homeostasis through conversion back into NO via a physiological system involving enterosalivary recirculation, bacterial nitrate reductases, and enzyme-catalyzed or acidic reduction of nitrite to NO. The diet is a key source of nitrate in adults; however, infants ingest significantly less nitrate due to low concentrations in breast milk. In the mouth, bacteria convert nitrate to nitrite, which has gastro-protective effects. However, these nitrate-reducing bacteria are relatively inactive in infants. Swallowed nitrite is reduced to NO by acid in the stomach, affecting gastric blood flow, mucus production, and the gastric microbiota. These effects are likely attenuated in the less acidic neonatal stomach. Systemically, nitrite acts as a reservoir of NO bioactivity that can protect against ischemic injury, yet plasma nitrite concentrations are markedly lower in infants than in adults. The physiological importance of the diminished nitrate→nitrite→NO axis in infants and its implications in the etiology and treatment of newborn diseases such as necrotizing enterocolitis and hypoxic/ischemic injury are yet to be determined.

질산염과 아질산염은

일반적으로 산화질소(NO)의 불활성 최종 산물로 간주되며,

발암 가능성이 있는 식품 첨가물 또는 우물물 오염 물질로 간주됩니다.

그러나 최근 연구에 따르면

질산염과 아질산염은

장-타액 재순환,

박테리아 질산 환원 효소,

효소 촉매 또는 산성 환원을 통해

NO로 다시 전환되는 생리적 시스템을 통해

심혈관 및 위장 항상성에 중요한 역할을 합니다.

식단은

성인의 질산염 섭취의 주요 원천입니다.

그러나

유아는 모유의 질산염 농도가 낮기 때문에

질산염을 훨씬 적게 섭취합니다.

입안에서 박테리아가

질산염을 위장 보호 효과가 있는 아질산염으로 전환합니다.

그러나

이러한 질산염을 환원하는 박테리아는

유아의 경우 상대적으로 활동이 저조합니다.

삼킨 아질산염은

위장의 산에 의해 NO로 환원되어

위 혈류, 점액 생성, 위 미생물총에 영향을 미칩니다.

이러한 효과는

신생아의 산성도가 낮은 위장에서 약화될 가능성이 큽니다.

전신적으로,

아질산염은 허혈성 손상을 방지할 수 있는 NO 생체 활성의 저장소 역할을 하지만,

유아의 혈장 아질산염 농도는 성인보다 현저히 낮습니다.

유아의 질산염→아질산염→NO 축의 감소가 갖는 생리학적 중요성과

괴사성 장염, 저산소/허혈성 손상 등

신생아 질환의 병인 및 치료에 미치는 영향은 아직 밝혀지지 않았습니다.

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Main

The discovery that nitric oxide (NO) is produced endogenously by NO synthases initiated a paradigm shift from thinking of NO as a toxic gas to the realization that it is a key regulator of vascular homeostasis, amongst many other physiological roles. One of the greatest clinical impacts of this discovery has been the use of inhaled NO gas for the treatment of persistent pulmonary hypertension in newborns, where it has significantly reduced the need for extracorporeal oxygenation (1,2). Initially, it was thought that the effects of inhaled NO would be confined to the lungs, as free NO gas in blood is scavenged by reactions with hemoglobin in only a few milliseconds (3). However, it is now widely reported that inhaled NO has an array of extrapulmonary effects, suggesting that one or more of its metabolites serve as reservoirs of NO bioactivity capable of circulating from the lungs to the peripheral organs. The existence of such an endocrine mediator of NO and its effects would be of great physiological relevance and much recent research has focused on identifying metabolites of NO that may serve in that role.

Nitrate (NO3−) and nitrite (NO2−) are the two major end products of NO metabolism. Historically, these compounds were considered to be relatively inert at physiological concentrations but environmental pollutants that posed potential health risks to humans at high concentrations. However, similar to the turnabout course of our understating of NO in biology, evidence now indicates that while nitrate and nitrite may be toxic at high concentrations, they play an important physiological role as they can be converted back into NO via a system involving enterosalivary recirculation, bacterial nitrate reductases, and enzyme-catalyzed or acidic reduction of nitrite to NO. This review will summarize our current knowledge regarding the bioactivity of nitrate and nitrite in adults and will outline known differences in infants (summarized in Figure 1 and Supplementary Table S1 online).

메인

산화질소(NO)가

NO 합성효소에 의해 체내에서 생성된다는 사실이 밝혀지면서,

NO가 독성 가스로만 여겨지던 관념에서 벗어나,

혈관 항상성의 핵심 조절자라는 사실과 더불어,

그 밖의 여러 가지 생리학적 역할이 있다는 사실이 밝혀졌습니다.

이 발견이 임상적으로 미친 가장 큰 영향 중 하나는

신생아의 지속적인 폐고혈압 치료를 위해 흡입된 NO 가스를 사용함으로써

체외 산소 공급의 필요성을 크게 줄였다는 점입니다(1,2).

처음에는 흡입된 NO의 효과가 폐에만 국한될 것이라고 생각했습니다.

혈액 내의 유리 NO 가스는

단 몇 밀리초(3) 만에 헤모글로빈과의 반응으로 제거되기 때문입니다.

그러나 현재

흡입된 NO가 폐 외의 다양한 효과를 가지고 있다는 사실이 널리 보고되고 있으며,

그 대사 산물 중 하나 이상이 폐에서 말초 기관으로 순환할 수 있는

NO 생체 활성의 저장소 역할을 한다는 것을 시사하고 있습니다.

NO의 내분비 매개체의 존재와 그 영향은 생리학적으로 매우 중요하며,

최근 많은 연구가 이러한 역할을 할 수 있는 NO의 대사 산물을 확인하는 데 초점을 맞추고 있습니다.

질산염(NO3−)과 아질산염(NO2−)은

NO 대사의 두 가지 주요 최종 산물입니다.

역사적으로, 이러한 화합물은

생리학적 농도에서는 상대적으로 비활성 상태인 것으로 간주되었지만,

고농도에서는 인간에게 잠재적인 건강 위험을 초래하는 환경 오염 물질로 간주되었습니다.

그러나

생물학에서 NO를 과소평가하는 것과 마찬가지로,

현재 증거에 따르면 질산염과 아질산염은

고농도에서는 독성이 있을 수 있지만,

장내 타액 재순환, 세균성 질산염 환원효소, 효소 촉매 또는 아질산염의 산성 환원을 통해

NO로 다시 전환될 수 있기 때문에 중요한 생리적 역할을 합니다.

이 리뷰는

성인에서 질산염과 아질산염의 생체활성에 관한 현재의 지식을 요약하고,

유아의 알려진 차이점을 간략하게 설명합니다(그림 1 및 온라인 보충표 S1에 요약되어 있음).

Figure 1

Schematic summary showing major differences in the supply and handling of nitrite and nitrate by the adult and infant. Multiple deficiencies in the infant lead to diminished nitric oxide (NO) bioactivity in the stomach and lower circulating nitrite concentrations in the blood.

성인과 유아의 아질산염과 질산염의 공급과 처리의 주요 차이점을 보여주는 도식적 요약. 

유아의 여러 가지 결핍은 위장에서 산화질소(NO)의 생체 활성을 감소시키고 혈액 내 순환하는 아질산염 농도를 낮춥니다.

Diet

Dietary Nitrate and Nitrite

Nitrate is the most preval‎ent nitrogen oxide species in the body. Although some of it is derived as an end product of the oxidation of endogenous NO and nitrite, nitrate concentrations are also heavily influenced by dietary intake. Nitrate itself is inert in mammalian tissues, but it can be reduced to nitrite by symbiotic bacteria that are part of the normal flora in the mouth and gastrointestinal (GI) tract (discussed below). Thus, by the action of these bacteria, dietary nitrate contributes to the body’s pool of nitrite. Vegetables are the most common source of dietary nitrate with particularly high concentrations (>2500 mg/kg) in beets, radishes, celery, and green leafy vegetables such as lettuce, kale, and spinach. Although daily nitrate ingestion can vary significantly dependent upon the types and amount of vegetables eaten, it is estimated that a typical adult ingests approximately 0.7–3.0 mg/kg body weight of nitrate per day (4).

Compared with nitrate, the amount of nitrite ingested in a normal adult diet is relatively small. In fact, it is likely that more of the nitrite in the body is derived from the bacterial reduction of nitrate and oxidation of endogenously-produced NO than from the diet (5,6). The biggest source of nitrite in the diet is cured and processed meats, where it is used as an additive to prevent bacterial growth and enhance the color. A typical adult ingests about 0.1 mg/kg body weight of dietary nitrite daily (7).

Although there are some discrepancies in the reported concentrations of nitrate and nitrite in breast milk and artificial milk (perhaps due to differing assay methodologies), we and others have recently shown that newborn infants ingest markedly lower amounts of nitrate and nitrite than adults on a per kg body weight basis. This is true regardless of whether they are receiving breast milk, artificial milk, or parenteral nutrition (8,9,10). Based on a breast milk intake of 150 ml/kg/day and our measurements of nitrate and nitrite concentrations (13 and 0.13 μmol/l, respectively), we have estimated that infants ingest approximately 0.12 ml/kg/day of nitrate and 0.0007 ml/kg/day of nitrite from fresh breast milk, which equates to only 5% and 0.6% of the nitrate and nitrite intake of adults (10). A comparison of the average dietary nitrate intake in newborn infants and adults is shown in Figure 2 .

식이요법

식이요법 질산염과 아질산염

질산염은

체내에서 가장 흔하게 발견되는 질소 산화물입니다.

일부는

체내 NO와 아질산염의 산화 과정에서 생성되지만,

질산염의 농도는 식이요법에 의해 크게 영향을 받습니다.

질산염 자체는

포유류 조직에서 비활성이지만,

입과 위장관(GI)의 정상적인 식물상(아래에서 설명)의 일부인 공생 박테리아에 의해

아질산염으로 환원될 수 있습니다.

따라서, 이러한 박테리아의 작용에 의해,

식이성 질산염은 체내의 아질산염 저장고에 기여합니다.

채소는 식이 질산염의 가장 일반적인 공급원이며,

특히 비트, 무, 셀러리, 상추, 케일, 시금치와 같은 녹색 잎채소에

특히 높은 농도(2500mg/kg 이상)가 함유되어 있습니다.

매일 섭취하는 질산염의 양은 섭취하는 채소의 종류와 양에 따라 크게 달라질 수 있지만,

전형적인 성인의 경우 하루에 체중 1kg당 약 0.7-3.0mg의 질산염을 섭취하는 것으로 추정됩니다(4).

질산염과 비교했을 때,

정상적인 성인의 식단에서 섭취하는 아질산염의 양은 상대적으로 적습니다.

사실,

체내의 아질산염은 식단에서 섭취하는 것보다

질산염의 박테리아 환원 작용과 체내에서 생성된 NO의 산화 작용을 통해

더 많이 생성될 가능성이 큽니다(5,6).

식단에서 아질산염이 가장 많이 생성되는 식품은

숙성육과 가공육인데,

이때 아질산염은 박테리아의 성장을 억제하고 색을 더 선명하게 만드는 첨가제로 사용됩니다.

일반적인 성인은 매일 체중 1kg당 0.1mg의 식이 아질산염을 섭취합니다(7).

모유와 인공 우유에 함유된 아질산염과 아질산염의 농도에는 약간의 차이가 있지만(아마도 분석 방법의 차이로 인한 것일 수 있음), 최근에 저희와 다른 연구자들은 신생아가 체중 1kg당 성인보다 훨씬 적은 양의 아질산염과 아질산염을 섭취한다는 사실을 밝혀냈습니다. 모유 수유, 인공 수유, 비경구 영양 공급(8,9,10)을 받는지에 관계없이 마찬가지입니다. 모유 섭취량 150ml/kg/일 및 질산염과 아질산염 농도 측정 결과(각각 13 및 0.13μmol/l)를 바탕으로, 영아는 하루에 약 0.12ml/kg/일의 질산염과 0. 신선한 모유에서 0.0007ml/kg/day의 아질산염이 검출되는데, 이는 성인의 질산염과 아질산염 섭취량의 5%와 0.6%에 불과합니다(10). 신생아와 성인의 평균 식이 질산염 섭취량을 비교한 것이 그림 2 에 나와 있습니다.

Figure 2

Dietary nitrate and nitrite levels for newborns and adults. (a) Daily dietary nitrate ingestion, normalized for body weight, is shown for newborns and adults, based on a mean (±SEM) of reported concentrations in breast milk and formula (for newborns) (8,9,10) and a typical adult diet (4). (b,c) Nitrate and nitrite concentrations in total parenteral nutrition (TPN), fresh and freeze-thawed breast milk, freeze-thawed colostrum, and a convenience sample of artificial milk formulas. (Figure adapted from Jones et al., 2014 (10).)

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We have also shown that nitrite is oxidized to nitrate in breast milk by an enzyme normally present in milk, lactoperoxidase, leading to even lower levels in milk that has been allowed to sit at room temperature or which has been freeze-thawed (10). Breast milk nitrite concentrations also fall during the first few weeks of life, with the highest levels found in colostrum and decreasing to nearly undetectable amounts in milk collected after the third week postpartum (9,10). The levels of nitrate and nitrite in artificial milk vary widely across a range that extends above and below concentrations measured in breast milk, averaging 43 and 0.3 μmol/l, respectively (10) ( Figure 2 ). The recently increased use of nutritional additives for caloric and protein enhancement raises the possibility of an additional source of dietary nitrate and nitrite, although to our knowledge concentrations in these additives have not yet been reported. Whether the newborn deficiency of dietary nitrite and nitrate serves an important physiological role, or whether supplementation of breast milk with these anions would be beneficial or problematic remains to be studied.

Until recently, a majority of research related to dietary nitrate and nitrite was in the context of toxicology. It has been known since 1945 that unusually high nitrate concentrations in vegetables and drinking water, often due to contamination with fertilizer, can cause cyanosis due to oxidation of hemoglobin to methemoglobin by nitrite derived from bacterial nitrate reductases, a problem often referred to as “blue baby syndrome.” Newborn infants are particularly susceptible to this problem as they have ~25% lower methemoglobin reductase activity than adults (11). It is also proposed that dietary nitrate and nitrite are associated with GI cancer due to the formation of carcinogenic N-nitroso compounds. Although a definitive causal link between dietary nitrate and cancer has not been identified (12), the associations between the intake of nitrite-treated meats and gastric cancer are more established (13). This is consistent with evidence that nitrite added to meats as a preservative can be converted to harmful N-nitrosamines in the meat itself before ingestion or in the body after it has been ingested (14). In an effort to protect against toxicity, the Environmental Protection Agency has set limits on inorganic nitrate and nitrite levels in drinking water and the World Health Organization has put forward acceptable daily intakes for nitrate at 3.7 mg/kg of body weight and for nitrite at 0.06 mg/kg of body weight. These levels are easily exceeded, however, with a high vegetable diet, and some have called for a resetting of these limits based on recent advances in our understanding of the roles of dietary nitrate and nitrite (9). The upper limits of toxicity of dietary nitrate in newborns have been investigated. Phillips et al. found that up to 21 mg/kg of nitrate per day was well tolerated by seven newborn infants, with six infants showing no increase in methemoglobin and the other one only a slight increase, and not enough to produce detectable cyanosis. Likewise, no symptoms of cyanosis occurred even when 100 mg nitrate kg/day was given to an infant for 8 d (15).

In contrast to the evidence of the toxic effects of nitrate and nitrite, the data increasingly indicate that a diet rich in nitrate is beneficial to overall cardiovascular health. In adults, raising dietary nitrate intake has been shown to improve exercise tolerance (13,16,17), decrease blood pressure (5,18), inhibit platelet aggregation (5), decrease risk of cardiovascular disease (19), and improve vascular compliance (20) (see Weitzberg and Lundberg, 2013 for a comprehensive review) (21). In addition, nitrite supplementation ameliorates microvascular inflammation and endothelial dysfunction in mice fed a high-cholesterol diet (22). Dietary nitrate also appears to have beneficial effects in the GI tract of adult rats, where it has been shown to protect against nonsteroidal anti-inflammatory drug (NSAID)-induced ulcers (23). Weighing the beneficial effects of increasing dietary nitrate and nitrite against the potential risks of methemoglobinemia and carcinogenicity is the focus of ongoing studies.

또한 아질산염이 우유에 존재하는 효소인 락토페록시다아제에 의해 모유에서 질산염으로 산화되어 실온에 방치되거나 냉동-해동된 우유에서 더 낮은 수준으로 감소한다는 사실도 밝혀졌습니다(10).

모유 내 아질산염 농도는 생후 첫 몇 주 동안 감소하는데, 초유에서 가장 높은 수치가 발견되고 산후 3주 후에 채취한 모유에서는 거의 검출할 수 없는 수준으로 감소합니다(9,10).

인공 모유의 아질산염과 아질산염 농도는 모유에서 측정된 농도보다 높거나 낮은 범위에서 매우 다양하며, 평균적으로 각각 43μmol/l와 0.3μmol/l입니다(10) ( 그림 2 ). 최근에 열량과 단백질 강화를 위해 영양 첨가제의 사용이 증가하면서 식이 질산염과 아질산염의 추가 공급원이 될 가능성이 높아졌지만, 이러한 첨가제의 농도는 아직 보고된 바가 없습니다. 신생아의 식이 아질산염과 질산염 결핍이 중요한 생리학적 역할을 하는지, 아니면 모유에 이러한 음이온을 보충하는 것이 유익한지 아니면 문제가 되는지는 아직 연구가 필요합니다.

최근까지 질산염과 아질산염에 관한 연구의 대부분은 독성학에 관한 것이었습니다. 1945년부터 비료로 인한 오염으로 야채와 식수에 비정상적으로 높은 농도의 질산염이 함유되어 있을 경우, 세균성 질산 환원 효소에 의해 아질산염이 생성되어 헤모글로빈이 메트헤모글로빈으로 산화되어 청색증(블루 베이비 신드롬)을 유발할 수 있다는 사실이 알려져 왔습니다.

신생아는 성인보다 메트헤모글로빈 환원효소 활성이 25% 정도 낮기 때문에 이 문제에 특히 취약합니다(11). 또한, 식이성 질산염과 아질산염은 발암성 N-니트로소 화합물의 형성으로 인해 위장암과 관련이 있는 것으로 알려져 있습니다. 식이성 질산염과 암의 명확한 인과관계는 밝혀지지 않았지만(12), 아질산염 처리된 육류 섭취와 위암의 연관성은 더 잘 알려져 있습니다(13). 이는 육류 보존제로 첨가된 아질산염이 섭취 전이나 섭취 후 체내에서 육류 자체에 유해한 N-니트로사민으로 전환될 수 있다는 증거와 일치합니다(14). 환경 보호국은 독성으로부터 보호하기 위해 식수의 무기 질산염과 아질산염 수치를 제한하고 있으며, 세계보건기구는 질산염의 일일 허용 섭취량을 체중 1kg당 3.7mg, 아질산염의 일일 허용 섭취량을 체중 1kg당 0.06mg으로 규정했습니다. 그러나 채소 위주의 식사를 하면 이 한도를 쉽게 초과할 수 있으며, 일부 사람들은 식이성 질산염과 아질산염의 역할에 대한 최근의 이해를 바탕으로 이 한도를 재설정해야 한다고 주장하고 있습니다(9). 신생아의 식이성 질산염의 독성 상한선이 조사되었습니다. Phillips et al.은 7명의 신생아가 하루에 최대 21mg/kg의 질산염을 섭취해도 잘 견디는 것으로 나타났습니다. 6명의 신생아는 메트헤모글로빈 증가가 없었고, 다른 신생아는 약간의 증가만 보였으며, 검출 가능한 청색증을 유발할 만큼의 증가가 아니었습니다. 마찬가지로, 100mg의 질산염을 8일 동안 유아에게 투여했을 때에도 청색증 증상이 나타나지 않았습니다(15).

질산염과 아질산염의 독성 영향에 대한 증거와는 대조적으로,

질산염이 풍부한 식단이 전반적인 심혈관 건강에 도움이 된다는 데이터가 점점 늘어나고 있습니다.

성인에서 식이성 질산염 섭취량을 늘리면 운동 내성이 향상되고(13,16,17),

혈압이 낮아지고(5,18),

혈소판 응집이 억제되고(5),

심혈관 질환의 위험이 감소하며(19),

혈관 순응도가 향상되는 것으로 나타났습니다(20) (Weitzberg and Lundberg, 2013을 참조하여 종합적인 검토를 확인하십시오) (21).

또한, 아질산염 보충제는 고콜레스테롤 식단을 섭취한 쥐의 미세혈관 염증과 내피 기능 장애를 개선합니다(22). 식이 질산염은 비스테로이드성 항염증제(NSAID)로 인한 궤양(23)을 예방하는 것으로 밝혀진 성인 쥐의 위장관에 유익한 영향을 미치는 것으로 보입니다. 메트헤모글로빈혈증과 발암 가능성에 대한 잠재적 위험을 고려하여 식이 질산염과 아질산염의 증가가 미치는 유익한 효과를 평가하는 것이 현재 진행 중인 연구의 초점입니다.

Saliva

In addition to dietary intake of nitrate and nitrite, the levels of these anions in swallowed saliva also have a significant impact on the amount of nitrate and nitrite that is ingested. As discussed in this section, this appears to be another point of significant difference between adults and newborns, thus compounding the effects of low dietary nitrate and nitrite ingestion in newborns.

Bacterial Conversion of Salivary Nitrate to Nitrite

Fasting nitrate concentrations average about 200 µmol/l in the saliva of adults but can reach as high as 10 mmol/l after a nitrate-rich meal (24). These concentrations are approximately 10-fold higher than the concentrations measured in plasma due to active transport of nitrate from the blood into the saliva by the salivary glands. The transport of nitrate has been suggested to be mediated by the enzyme sialin via an adenosine 59-triphosphate-dependent electrogenic NO3−/H+ transport mechanism in the salivary acinar cells (25). The nitrate concentration in the saliva of newborns is approximately 200 µmol/l, similar to that of adults (26). As in adults, this concentration is many-fold higher than in blood (16–40 µmol/l). Thus, the active transport mechanisms in the salivary glands of newborn infants are present with a concentrating power comparable with that of adults (27,28). That the body expends energy to actively concentrate nitrate into the saliva suggests that nitrate is not just an inert end product of NO metabolism, but has potential bioactivity in the body.

Although nitrate itself appears to be inert in mammalian tissues, it is made physiologically relevant after reduction to nitrite by bacteria residing in the crypts of the dorsal posterior surface of the tongue. These bacteria utilize nitrate as the terminal electron acceptor in the respiratory chain, rather than oxygen and reduce about 20% of salivary nitrate in adults (13,29). A true symbiotic relationship between these bacteria and the human host exists as humans lack the requisite enzymes to bring about this conversion independently but provide nitrate to the bacteria that then perform nitrate reduction via respiration. As discussed below, these nitrate-reducing bacteria are critical to the beneficial effects of dietary nitrate.

The primary bacteria that mediate nitrate reduction in the mouth are obligate anaerobes of the Veillonella species and facultative anaerobes of the Actinomyces, Rothia, and Staphylococcus species, all of which possess nitrate reductase enzymes that allow them to respire nitrate and rapidly produce nitrite (30). Veillonella and Actinomyces species have been found in saliva collected from infants in the first 2 mo of life and appear to be some of the first bacteria to colonize the mouths of newborns (26,31,32,33). Despite the presence of these bacteria, oral nitrate reductase activity is markedly lower in newborn infants when compared with adults, as shown in Figure 3 . It is unknown whether this difference comes from insufficient numbers of bacteria, whether the bacteria do not possess sufficient nitrate-reducing capacity, or whether the mouth of newborns lack some cofactor for nitrate reduction or some other necessary element.

타액

질산염과 아질산염의 섭취와 더불어, 삼킨 타액에 포함된 음이온의 양도 섭취되는 질산염과 아질산염의 양에 상당한 영향을 미칩니다. 이 섹션에서 논의된 바와 같이, 이것은 성인과 신생아 사이의 또 다른 중요한 차이점으로 보이며, 따라서 신생아의 낮은 질산염과 아질산염 섭취의 영향을 더욱 복잡하게 만듭니다.

타액 속의 질산염이 아질산염으로 전환되는 세균 작용

금식 시 성인 타액의 질산염 농도는 평균 약 200 µmol/l이지만, 질산염이 풍부한 식사를 한 후에는 10 mmol/l까지 올라갈 수 있습니다(24). 이러한 농도는 타액선에 의해 혈액에서 타액으로 질산염이 활발하게 수송되기 때문에 혈장에서 측정된 농도보다 약 10배 더 높습니다. 질산염의 수송은 타액선 세포에서 아데노신 59-인산 의존성 전기적 NO3−/H+ 수송 메커니즘을 통해 시알린 효소에 의해 매개되는 것으로 제안되었습니다(25). 신생아의 타액 내 질산염 농도는 성인의 농도와 비슷한 약 200 µmol/l입니다(26). 성인의 경우와 마찬가지로, 이 농도는 혈액(16-40 µmol/l)보다 훨씬 더 높습니다. 따라서 신생아의 타액선에는 성인과 비슷한 농축 능력을 가진 활성 수송 메커니즘이 존재합니다(27,28). 몸이 에너지를 소비하여 질산염을 타액에 적극적으로 농축한다는 사실은 질산염이 NO 대사의 비활성 최종 산물일 뿐 아니라 신체에서 잠재적인 생체활성을 가지고 있음을 시사합니다.

질산염 자체는 포유류 조직에서 비활성 물질로 보이지만, 혀의 등쪽 뒤쪽 표면에 있는 구멍에 있는 박테리아에 의해 아질산염으로 환원된 후 생리적으로 관련이 있는 물질이 됩니다. 이 박테리아는 질산염을 산소 대신 호흡 사슬의 최종 전자 수용체로 활용하여 성인의 타액 내 질산염을 약 20% 감소시킵니다(13,29). 인간은 이러한 전환을 독립적으로 수행하는 데 필요한 효소가 부족하지만, 호흡을 통해 질산염 환원을 수행하는 박테리아에 질산염을 공급하기 때문에 박테리아와 인간 사이에는 진정한 공생 관계가 존재합니다. 아래에서 설명하듯이, 이러한 질산염 환원 박테리아는 식이 질산염의 유익한 효과에 매우 중요합니다.

입안에서 질산염 환원을 매개하는 주요 박테리아는 Veillonella 종의 의무 혐기성 세균과 Actinomyces, Rothia, Staphylococcus 종의 선택적 혐기성 세균이며, 이들 모두 질산염 환원효소를 가지고 있어서 질산염을 호흡하고 아질산염을 빠르게 생성할 수 있습니다(30). 베일론넬라와 액티노마이세스 종은 생후 2개월 미만의 유아의 타액에서 발견되었으며, 신생아의 입에 처음으로 서식하는 박테리아 중 하나인 것으로 보입니다(26,31,32,33). 이러한 박테리아가 존재함에도 불구하고, 그림 3 에 나타난 바와 같이 신생아의 구강 내 질산염 환원효소 활성은 성인과 비교했을 때 현저히 낮습니다. 이러한 차이가 박테리아의 수가 부족해서인지, 박테리아가 질산염을 충분히 환원할 수 있는 능력이 부족해서인지, 아니면 신생아의 입에 질산염 환원을 위한 보조 인자가 부족해서인지, 아니면 다른 필요한 요소가 부족해서인지 여부는 알 수 없습니다.

Figure 3

Nitrate-reducing activity, normalized for saliva weight, in swab samples collected from the mouths of preterm (born at <35 wk gestation) and term (born at >36 wk) infants in the neonatal intensive care unit (NICU) or from healthy infants in an outpatient clinic between 2 and 6 wk after birth, or from normal healthy adults. Mammalian cells lack the enzymes required for nitrate reduction, but bacteria dwelling in crypts of the tongue bring about the reaction. Note that the rate in infants is ~10% of that in adults. (Figure adapted from Kanady et al., 2012 (26).)

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By measuring the nitrate-reducing capacity of bacteria in oral swabs collected from infants, we have shown that there is essentially no detectable nitrite production from nitrate in the first 5 d of life. While there is measurable nitrate-reducing capacity in swabs (normalized to the saliva content in the swab) collected from infants at 2–8 wk of age, the rate of nitrite production is only ~10% of that of adults (26), as illustrated in Figure 3 . Notably, infants also produce relatively small volumes of saliva during the first few weeks of life, which may attenuate bacterial growth and may also result in less swallowed salivary nitrite compared with the adult (26). The developmental time point at which the nitrate-reducing capacity of the infant mouth becomes comparable to the adult is unknown.

The diminished bacterial nitrate-reducing capacity in infants may be of physiological relevance because salivary nitrite impacts both GI and cardiovascular function in adults. Blockade of salivary nitrate secretion by ligation of the submandibular gland duct in rats results in decreased gastric nitrate, nitrite, and NO concentrations and exacerbates stress-induced gastric ulcers (34). The severity of gastric ulcers in these rats is reduced upon supplemental nitrate treatment (34). These gastroprotective effects appear to be mediated through the action of increased salivary nitrite, as nitrite-rich saliva results in increased gastric mucosal blood flow, a thicker mucus layer, and attenuation of the inflammatory response associated with NSAID administration in rats (23,35,36). These effects of nitrite on the stomach are likely due to its conversion to NO, as discussed below.

In addition to the effects in the GI tract, increasing dietary nitrate, and concomitant increases in salivary nitrite, have been shown to decrease arterial blood pressure, protect against ischemia–reperfusion (I/R)-induced endothelial dysfunction, and decrease platelet aggregation (5,18). The importance of salivary nitrite production by oral bacteria is again highlighted by the finding that if subjects refrain from swallowing saliva after a dietary nitrate-load or are given antibacterial mouthwash to decrease bacterial nitrate-reducing activity, the hypotensive effects of nitrate are attenuated and there is no inhibition of platelet aggregation (5,37). Increasing dietary nitrate also leads to increased circulating nitrite concentrations (5), which is associated with a host of beneficial effects ranging from improved exercise tolerance to protection against I/R injury (discussed below).

Thus, considering the beneficial GI and cardiovascular effects of dietary nitrate and subsequent salivary nitrite production by oral nitrate-reducing bacteria in adults, the lack of the critical bacterial nitrate reduction in infants is noteworthy and deserves investigation. Moreover, the lack of bacterial nitrate-reducing activity in infants will compound the already low levels of nitrate and nitrite in their diet, ultimately leading to significantly lower nitrite delivery to the infant stomach. The potential benefit of adding a mother’s oral bacteria to an infant’s mouth is untested.

GI Tract

Intragastric Conversion of Nitrite to NO

In 1994, two independent studies showed that NO was generated from nitrite in the stomach of human adults (38,39). These studies showed a novel nitric oxide synthase (NOS)-independent in-vivo mechanism by which NO could be generated from nitrite. Since then, there has been great interest in nitrite as not merely an inert NO metabolite but as a physiologically relevant source of NO bioactivity. The chemical reaction by which NO is generated in the acidic stomach of adults involves protonation of nitrite to form nitrous acid (pKa 3.3), which rapidly decomposes to several highly reactive nitrogen oxides, including NO free radical, NO2, N2O3, and peroxynitrite (38,40). In the stomach, these nitrogen oxides can form new stable products through nitration and nitrosylation of amines, amides, thiols, and fatty acids. These products have wide-ranging bioactivities, which include modulation of inflammatory signaling pathways, inhibition of platelet aggregation, vasodilation, mucus production, and bacterial colonization, among many other functions (40,41,42,43).

Via gastric conversion to NO, ingested nitrite has been shown to kill many different enteropathogens including Salmonella, Shigella, Helicobacter pylori, Escherichia coli, Yersinia enerocolitica, Clostridium difficile, and Candida albicans, establishing nitrite as a key player in host defense (44,45,46,47). In addition to acting as a bactericidal agent, NO plays a key role in host defense in the GI tract by stimulating mucus and fluid secretion, regulating the epithelial barrier, mediating vascular smooth muscle tone, diminishing leukocyte adherence to the endothelium, modulating mucosal repair, and influencing the release of inflammatory mediators (21,48). While it is now apparent that nitrite-derived NO plays many protective roles in the stomach and GI tract, it is important to note that nitrite in the stomach (via conversion to nitrous acid and other nitrogen oxides) can also act as a nitrosating agent, converting ingested amines into their carcinogenic N-nitroso derivatives (13). However, while the nitrosating ability of acidified nitrite is clear, there is still no direct evidence that increased ingestion of nitrate, and subsequent conversion to salivary nitrite and gastric NO, causes increased risk of gastric cancer (29).

NO generation from nitrite in the stomach is highly pH dependent and is effectively attenuated with proton pump inhibitors (39). Consequently, the protective effects of swallowed nitrite appear to be highly dependent upon gastric acidity as increasing the pH above a value of 4 effectively prevents nitrite-induced increases in blood flow (35) and reduction in pathogenic bacteria (38,47) and blocks nitrite’s hypotensive effects (49).

The pH dependence of nitrite-derived gastric NO is of particular interest in the newborn, as the newborn stomach has a relatively high pH compared with the adult stomach (50,51,52). Figure 4 illustrates the effect of pH on the rate of NO production from nitrite using previously calculated rate constants (53). As shown in Figure 4 , the less acidic environment of the newborn stomach would attenuate the generation of NO from nitrite delivered to the stomach, compounding the already low nitrite ingestion from the saliva and diet. High gastric pH in newborns has been associated with an increased risk of necrotizing enterocolitis (NEC) (54,55), whereas low gastric pH protects against bacterial translocation across the gut wall in neonatal rabbit pups (56). Considering that acidified nitrite kills bacteria, improves mucus secretion and mucosal blood flow, and is protective against I/R injury (discussed below), it is worth speculating that enhancing NO generation from nitrite would be protective against NEC.

Figure 4

Nomogram showing the rate of nitric oxide (NO) generation in gastric fluid for various pH and nitrite concentrations. The adult and newborn ranges of nitrite concentrations are shown as box and whisker (min to max) plots of measurements of saliva (26) and placed at reported typical ranges of gastric pH (

 pH 2; 

 pH 3; 

 pH 4; 

 pH 5; 

 pH 6) for adults and newborns (50). The infant rate is estimated to be about 100-fold slower than the adult rate due to less acidity and lower nitrite levels in the newborn stomach. Curves were constructed using rate constants and equations given by Zweier et al. (53).

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Intestine

In contrast to the NOS-independent generation of NO in the stomach, NO generation in the colon appears to be mediated by NOS-dependent mechanisms, as demonstrated by the finding that rats treated with the NOS inhibitor NG-nitro-l-arginine methyl ester have significantly less NO generation in the colon while NO generation in the stomach is unaffected (57). In rats, NO concentrations in the stomach (>4000 ppb) are orders of magnitude higher than in the small intestine (<20 ppb), cecum (~200 ppb), or colon (<25 ppb) (57). Interestingly, germ-free rats have markedly lower NO generation in all areas of the GI tract, including the stomach, indicating an important role for bacteria in NO production throughout the GI tract. Germ-free rats provide a useful comparison to newborn infants, as diminished gastric NO in germ-free rats is thought to be due to the lack of oral nitrate-reducing bacteria since gastric NO production is dependent on substrate (ingested nitrite) availability (57). In the cecum, NO is in part formed via reduction of nitrate and nitrite by strains of Lactobacilli and Bifidobacteria (58) and stimulation of the mucosal NOS enzymes by GI bacteria (57). NO generation in the intestine, either by Lactobacilli farciminis or by administration of an NO-donor, has been demonstrated to have anti-inflammatory effects in an animal model of colitis (59), highlighting the potential protective effects of increased NO generation in the intestine.

Given the beneficial effects of NO derived from ingested nitrite on the GI microbiota, blood flow, and mucus production (described above), it is interesting to speculate about nitrite’s role in the context of NEC. NEC is the most common GI disease to afflict premature infants. It is a disease characterized by intestinal barrier failure (60) most likely subsequent to an ischemic insult. The main factors contributing to the regulation of GI blood flow in the preterm infant are poorly defined, and it is not known whether a deficiency in NO contributes to the dysregulation of GI blood flow that is thought to precede NEC. Translocation of bacteria across the compromised GI wall leads to activation of an inflammatory response characterized by pronounced up-regulation of NO production by inducible NOS. In this inflammatory stage of the disease, overproduction of NO by inducible NOS results in toxic levels of peroxynitrite, further damaging the integrity of the gut wall by inducing enterocyte apoptosis and necrosis, or by disrupting tight junctions and gap junctions that normally maintain epithelial monolayer integrity (61,62). Thus, a vicious cycle characteristic of severe NEC is created by bacterial invasion, immune activation, uncontrolled inflammation with production of reactive oxygen species and nitrogen species, vasoconstriction followed by I/R injury, gut barrier failure, intestinal necrosis, sepsis, and shock (63). It is reasonable to hypothesize that NO plays a dichotomous role in NEC, with deficient levels of NO contributing to an increased vascular resistance during the initiating ischemic event, and subsequent overproduction of NO during the inflammatory stage of the disease leading to propagation of the injury. Interestingly, Yazji et al. have recently reported that nitrite/nitrate-deficient formula predisposes newborn mice to NEC, and that both the incidence and severity of NEC was ameliorated by nitrite/nitrate supplementation to the formula to achieve levels comparable with that of breast milk (64). However, the clinical relevance of this finding is uncertain given the fact that nitrite and nitrate concentrations of many commercially available formulas are already higher than those found in breast milk (10). Whether manipulation of the decreased levels of ingested and circulating nitrite in the preterm infant would prevent or alter the course of NEC is an area worthy of study.

Circulation

Circulating Nitrite

Shortly after the discovery that NO is generated from nitrite in the acidic environment of the stomach, Zweier et al. showed that NO could also be generated from nitrite in ischemic heart tissue (65). Nitrite reduction to NO in hypoxic tissues appears to be mediated either by acidic disproportionation (similar to the mechanism in the acidic stomach) or by the activity of metal-containing proteins with nitrite-reducing activity. These proteins include the heme-associated globins in their deoxygenated state such as deoxygenated myoglobin, hemoglobin, cytoglobin, and neuroglobin as well as mitochondrial enzymes like complex III; molybdenum metalloenzymes such as xanthine oxidoreductase (66); cytochrome P450 enzymes; and endothelial NOS (see the review by Kim-Shapiro and Gladwin (67)). While the initial report by Zweier et al. suggested that nitrite reduction to NO in acidic tissues exacerbates post-ischemic injury (65), nitrite has since been consistently shown in experimental animals to be protective against I/R injury in the heart, brain, liver, and kidney (29,68). The mechanism by which nitrite confers protection against I/R injury is not well understood, but is thought to involve reduction to NO which modulates the function of the mitochondria, leading to more efficient oxygen utilization, decreased reactive oxygen species formation, and the inhibition of apoptotic signaling (69). The therapeutic potential of nitrite against I/R injury in newborns has not yet been studied. Considering that neonates are particularly at risk for hypoxic and ischemic insults, further research is needed to address the therapeutic and preventative potential for nitrite supplementation in the neonatal population.

While nitrite supplementation has yet to be studied in neonatal populations, it has been shown that treatment of persistent pulmonary hypertension of the newborn with inhaled NO (iNO) increases nitrite levels in the blood at least twofold (27,70). Although the resulting nitrite concentrations reached only ~300 nmol/l after iNO administration, similar increases in circulating nitrite concentrations have been shown to protect mice against hepatic infarct (71), increase blood flow in the human forearm (72), and decrease systolic blood pressure in adults (5,18,73). Thus, increases in circulating nitrite levels resulting from iNO treatment may be enough to cause significant systemic effects (27). Indeed, there are reports demonstrating protective effects of iNO therapy in a mouse model of myocardial infarction (74), adult human liver transplant patients (75), and in children following cardiopulmonary bypass (76). Whether the protective effects of iNO are due to elevations in circulating nitrite remains to be determined.

Given that nitrite could theoretically improve an infant’s ability to withstand ischemic stress, it is important to discuss the mounting evidence that normal newborn infants appear to have numerous mechanisms in place that decrease systemic nitrite levels during the first few weeks of life. As shown in Figure 1 , these mechanisms include low dietary nitrite and nitrate intake, the lack of bacterial nitrate reduction in the mouth, and the relatively high pH in the stomach. It has now become evident that plasma nitrite concentrations fall at birth and remain lower than adult levels for the first few weeks of life. We have recently found (data not published) that circulating nitrite concentrations decrease markedly after birth, falling from approximately 0.18 ± 0.02 μmol/l in umbilical cord plasma to 0.08 ± 0.02 μmol/l in plasma collected from term infants on their first day of life. Interestingly, plasma collected from preterm infants has even lower nitrite concentrations (0.04 ± 0.01 μmol/l). Plasma nitrite concentrations are significantly lower in infants than those measured in adults, which typically range from 0.05 to 0.30 μmol/l (44). These findings are consistent with previous reports that adult plasma nitrite levels are significantly higher than those of newborn infants (27), but are similar to those in umbilical cord blood (77). The relevance of the dramatic fall in circulating nitrite levels immediately after birth is uncertain, but may be an important part of the circulatory changes that occur at birth.

There are many factors that contribute to plasma nitrite concentrations. In adults, a majority of plasma nitrite is derived from the oxidation of NO produced by endothelial NOS (6). This oxidation depends on enzyme-catalyzed reactions in the plasma (78), which are significantly attenuated in the newborn (79). In addition, endothelial NOS activity may be diminished in newborns due to low levels of l-arginine (80) and increased levels of asymmetric dimethylarginine, the endogenous inhibitor of NOS (81,82,83). Furthermore, the rapid increase in tissue PO2 that occurs at birth may result in increased superoxide levels, particularly in preterm infants who are likely to have low antioxidant defenses (84,85,86,87). This superoxide can rapidly scavenge NO to produce peroxynitrite instead of nitrite. Another potential cause of relatively low plasma nitrite in infants could be the lack of significant oral bacterial nitrate reduction, as discussed above and illustrated in Figure 3 . We have recently shown that adults given antibacterial mouth rinse have significantly reduced plasma nitrite concentrations (26), highlighting the importance of the oral nitrate-reducing bacteria to the amount of circulating nitrite. Thus, with the confluence of all of these factors, it appears that nitrite bioavailability is diminished in the newborn by a system of concerted mechanisms, as evidenced by the sharp fall in nitrite concentrations at birth. The physiological relevance of this decrease remains to be elucidated and should be more fully understood before efforts are made to study the potentially therapeutic use of nitrite in this patient population.

Summary

Before birth, nitrite concentrations in fetal blood are similar to those in maternal blood due to rapid passive exchange of the anions across the placenta. Within hours of birth, however, the nitrite concentration in the newborn falls sharply in association with increases in blood pressure, increases in pulmonary blood flow, and many other adaptations to increasing oxygen tensions. In the early weeks of life, nitrate and nitrite levels remain low for several reasons. There is limited ingestion of nitrate and nitrite because their concentrations are low in milk and formula. There is little reduction of nitrate to the physiologically active nitrite by oral bacteria. Finally, there is little generation of NO in the newborn stomach because the pH is high. The net result is that the recirculation of nitrate and nitrite as bioactive sources of NO is markedly lower in the newborn than in the adult.

In recent decades, the many serious concerns that nitrite in the diet would cause cancer and methemoglobinemia have lessened and been replaced by new findings of cardiovascular benefits. In the newborn period, there arises the prospect of protecting the GI tract from bacterial invasion by supplementation with nitrite, thereby increasing NO bioactivity and its protective actions. However, careful investigation must be done weighing the risks against the benefits before supplementation with nitrite can be undertaken safely in newborn infants.

Statement of Financial Support

The research was supported by intramural funding from the John Mace Pediatric Research Grant Fund of the Loma Linda University School of Medicine, Department of Pediatrics, Loma Linda, CA, and the National Institutes of Health, Bethesda, MD, grant HL095973 (awarded to Arlin B. Blood).

Disclosure

The authors have no financial ties to the products in the review or any potential or perceived conflicts of interest to disclose.

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