The importance of nitrite in our diet can hardly be overstated. These dietary sources include cured meats even though it is by no means the largest source. The challenge is to understand the factors which prevent cured meats from being seen as a superfood and address these. The presence of nitrites does not seem to be one of these!
In Part 5. Nitrite – the Misunderstood Compound we looked at the protective effects of dietary Nitrate/Nitrite on lifestyle-related diseases mainly from the work of Kobayashi (2015). We also looked at work done which shows the adverse effect of the lack of nitrites on the body. Here I list more health benefits, this time mainly from the work of Rassaf (2014).
By way of overview, let’s briefly list again the sources of nitrite for the human body.
– Three Sources of Nitrite
1. NO -> produced endogenously from L-arginine by NO-synthases (NOSs)
In the body, nitric oxide (NO) is oxidised to nitrite. (Rassaf, 2014)
NO rapidly reacts with oxyhaemoglobin to form methemoglobin and nitrate. (Rassaf, 2014)
On the other hand, several pathways exist in the body that provides the reduction of nitrite to NO, with haemoglobin, myoglobin, neuroglobin, cytoglobin, xanthine oxidoreductase, eNOS and mitochondrial enzymes being involved (for reviews see: van Faassen et al. 2009; Lundberg et al., 2009). The extent of contribution of the different pathways depends on the tissue, the pH, oxygen tension and redox status (Feelisch et al., 2008).
2. Nitrite reduced from nitrate
3. Dietary sources
Cured meat, baked goods, beets, corn, spinach etc. are major sources of nitrite. (Rassaf, 2014)
Reference list below for nitrite dietary contributions.
Benefits of Nitrate
As I said, I now list more health benefits of nitrite.
-> Contribute to protection against UV-induced cell damage.
The presence of nitrite, but not nitrate, reduced the extent of apoptosis, or the death of cells which occurs as a normal and controlled part of an organism’s growth or development, in cultured endothelial cells during UVA-irradiation in a concentration-dependent manner by inhibiting lipid peroxidation. (Rassaf, 2014) Endothelial cells form the inner lining of a blood vessel and provide an anticoagulant barrier between the vessel wall and blood.
The protective effect described above was abolished by simultaneous administration of a NO scavenger (Suschek et al., 2003) suggesting that nitrite-derived NO may contribute to protection against UV-induced cell damage (Suschek et al., 2006). (Rassaf, 2014)
-> Protection of gastric mucosa from hazardous stress.
We look at this when we considered the work of Kobayashi (2015) but due to the importance, I mention the point again. Nitrite, generated from nitrate by oral bacteria ‘the so called enterosalivary cycle’, and then converted to NO (Benjamin et al., 1994; Lundberg et al., 1994; 2009; 2006; 2008; Kapil et al., 2010a) in the stomach was also suggested to play an important role in the protection of gastric mucosa from hazardous stress (Miyoshi et al., 2003). (Rassaf, 2014)
-> Cardiovascular Benefits
Since the rate of NO generation from nitrite depends on the reduction in oxygen and pH, nitrite could be reduced to NO in ischaemic tissue or tissue lacking oxygen and exert protective effects (for review, see van Faassen et al., 2009). Nitrite-mediated protection was independent of endothelial nitric oxide synthase (Webb et al., 2004; Duranski et al., 2005).
-> The Brain
Depending on the timing of application nitrite might not only reduce irreversible brain injury following ischaemia/reperfusion but also vasospasm following cerebral haemorrhage. (Rassaf, 2014) Ischaemia/ reperfusion refers to the paradoxical exacerbation of cellular dysfunction and death, following restoration of blood flow to previously ischaemic tissues which refers to the demand of tissue for energy, for example from oxygen, and this demand is not matched by supply moslty due to to a lack of blood flow.
-> Protection of the Liver
Nitrite exerted profound dose-dependent protective effects on cellular necrosis which refers to the loss of cell membrane integrity as a result of exposure to a noxious stimulus and apoptosis which refers to a form of programmed cell death that occurs in multicellular organisms. Nitrite has a highly significant protective effect observed at near-physiological nitrite concentrations. (Rassaf, 2014)
-> Protection of the Lungs
In a mouse model of pulmonary arterial hypertension, inhaled nebulized nitrite has been demonstrated to be a potent pulmonary vasodilator that can effectively prevent or reverse pulmonary arterial hypertension. (Rassaf, 2014)
-> Protection of the kidneys
In rats subjected to 60 min of bilateral renal ischaemia and 6 h of reperfusion sodium nitrite administered topically 1 min before reperfusion significantly attenuated renal dysfunction and injury. (Rassaf, 2014)
Renal ischemia associated with renal artery stenosis (RAS) which is the narrowing of one or more arteries that carry blood to your kidneys is the most frequent condition occurring with renin-dependent hypertension. Renovascular hypertension (RVH) results from occlusion (the blockage or closing of a blood vessel or hollow organ) of blood flow to either kidney, which stimulates renin release. Increased renin leads to a series of actions that rapidly leads to increased systemic blood pressure or hypertension or abnormally high blood pressure. (Rassaf, 2014)
Similarly, in mice subjected to bilateral renal ischaemia for 30 min and 24 h reperfusion, renal dysfunction, damage and inflammation were increased; these effects were all reduced following nitrite treatment 1 min prior to reperfusion. (Rassaf, 2014)
-> Crush syndrome and shock
Limb muscle compression and subsequent reperfusion are the causative factors in developing a crush syndrome. In rats subjected to bilateral hind limb compression for 5 h followed by reperfusion for 0 to 6 h, nitrite administration reduced the extent of rhabdomyolysis markers such as potassium, lactate dehydrogenase and creatine phosphokinase. Nitrite treatment also reduced the inflammatory activities in muscle and lung tissues, finally resulting in a dose-dependent improvement of survival rate. (Rassaf, 2014)
Similarly, in a mouse shock model induced by a lethal tumour necrosis factor challenge, nitrite treatment significantly attenuated hypothermia, mitochondrial damage, oxidative stress and dysfunction, tissue infarction and mortality. (Rassaf, 2014)
Nitrite could also provide protection against toxicity induced by Gram-negative lipopolysaccharide. (Rassaf, 2014)
Rassaf (2014) concluded that “taken together, the nitrate-nitrite-NO pathway appears to play a crucial role in protecting the heart, vessel, brain, kidney and lung against ischaemia/reperfusion injury. Nitrite treatment may be advantageous in well-known NO deficient states such as, for example, hyperlipidaemia. Timing and dose of nitrite application as well as the potential to convert nitrite to NO in the tissue are important to obtain a reduction in injury.
That nitrite is not a compound to be avoided at all costs is clear. It is essential to our health and dealing with the stress and strain of living life and mediating the effects of the many injuries we incur. The mass hysteria against the use of nitrites in cured meat is unfounded. The discussion about adapting our formulations to include the latest science related to diet and nutrition needs to take place as it is true for every food group in existence but lumping the meat industry into the same group as producers of cigarettes, for example, is unjustified and dangerous. A far more balanced and responsible discussion is called for and I hope that this series contributes to the discussion.
The accusation is widespread in the media, sensation-seeking documentaries and celebrity chefs alike that nitrite, derived from ammonia, nitrate (Salpeter) or added in the form of sodium nitrite in meat curing is tantamount to poisoning consumers and inviting cancer into your lives. I am a meat curing professional. My interest in the truth about nitrites is in the first place to be certain that I am not engaged in an action where harmful products are produced. To state this slightly differently, what steps can I take to ensure the safest possible product is made available?
The issue of nitrites is complex and to develop even a rudimentary understanding of all the issues requires that we work through a lot of technical information. Despite this, the basic evaluation is simple and well within the grasp of the general public. Here I desire to share with you what I discovered about this remarkable compound!
It is part of a short series I’ve put together on the matter entitled, The Truth About Meat Curing: What the popular media do NOT want you to know! After preliminary discussions, we now place the spotlight squarely on nitrite. We discover that instead of poison, even though this is true in large dosages and under certain conditions, it is a vitally important compound for the normal functioning of our bodies. That the sources are mostly from vegetables and not cured meat, and that any possible harmful effect is removed through the simultaneous consumption of vitamins A, C, E, etc.
What I discovered is that an entirely different (and positive) world exists related to nitrites generally and dietary nitrites in particular. The evidence is clear, overwhelming and available to anybody with an honest interest in the matter that nitrites are beneficial to human health and essential for the optimal functioning of our bodies. We will discover that there is a seemingly unresolved issue in that while nitrites, in balanced concentrations, have overwhelmingly beneficial results in the human body (may I even call it essential?!), there is seemingly contradictory information which shows that nitrites are involved, under certain conditions in the generation of N-Nitrosamines which can be cancer-causing. Parallel to this is the indication of many studies that there seems to be a relationship between the consumption of cured meats and cancer and even though the exact reason has not been elucidated, it begs the question as to possible reasons for this. How do we deal with this seemingly contradictory information, that n-nitrosamines which are the obvious culprit for any possible link between cured meat and cancer on the one hand come from nitrites and on the other hand, nitrites play an vital part in our general health and the resolution of many common diseases and ailments? Can it be that nitrosamines are not the culprit of what seems to be a link between cancer and cured meat? Can it be that lifestyle or general nutritional habits alter the nature of an important chemical in our bodies from beneficial to harmful and if this is the case, what are those factors? Is it fair to label bacon as possibly cancer-causing? When it comes to the full array of reactive nitrogen species of which nitrite is a part, is it possible to have the one without the other, especially in light of the fact that the curing molecule is nitric oxide, also one of the reactive nitrogen species? Is the statement that curing was done with no nitrite even a sensical one in light of the oxidation of nitric oxide to nitrite and nitrite to nitrate in the curing environment? It begs the question if no nitrite curing which has been the goal of meat scientists for so many years even valid question to ask or is this something that will sell products without any real benefit to the consumer as far as the removal of the real risk of n-nitrosamine formation. This is an extremely timely question as we stand at the dawn of a time when no-nitrite curing will become a reality across the world. The emphasis is about to squarely shift to nitric oxide and in light of this future trend we have to ask, can nitric oxide contribute to nitrosamine formation as is the case with nitrites which would mean that removing nitrites from the curing system has no real benefit as far as nitrosamine formation is concerned.
We have to continue the questioning. If ingesting dietary nitrite has overwhelmingly positive effects on human physiology, should nitrite curing not rather be encouraged and embraced and should ham and bacon not be seen as a superfood instead of something to be avoided? I ask another question which is the focus of my own work and that of a small band of like-minded food professionals and scientists – how do we turn ham, bacon and the cured meats we love into superfoods in such a decisive manner that there can be no argument from any quarter about this status!? These are all valid questions and despite the mammoth task ahead, I will do my best to interact with all these questions in this document. Where I fail, please point it out to me so that I can improve on the document and evolve in my thinking, but please, do it from a position of constructive interaction and partnering with me in seeking the truth!
I will try and deal as honestly as a layman can with these complex questions, believing that I have a sacred responsibility to the consumer to do exactly this and if the evidence points away from what I would like it to say, that I should have the integrity follow the lead of the evidence. My ultimate goal is therefore the TRUTH and not to generate “likes” on social media posts. Anybody with a meaningful contribution or who wants to correct me on any point can contact me at firstname.lastname@example.org or WhatsApp me at +27 71 545 3029.
History of Nitric Oxide and the Close Link between Nitrate, Nitrite and Nitric Oxide.
Nitric oxide (NO) was discovered in 1772. Nitroglycerine (NG), a vasodilator acting via NO production, was synthesized in 1847. The effect of nitroglycerine was studied on healthy volunteers by Constantin Hering in 1849 and it was proven to cause headaches. Later in 1878, nitroglycerine was used by William Murrell for the first time to treat angina. Towards the end of the 19th century, nitroglycerine was established as a remedy for relief of anginal pain.” (Ghasemi, 2011) Angina is a type of chest pain caused by reduced blood flow to the heart. In 1916, Mitchell et al. suggested that body tissues can also produce nitrate and Richard Bodo in 1928 showed a dose-dependent increase of coronary flow in response to sodium nitrite administration. In the 1970s, it was shown that nitrite-containing compounds stimulate guanylate cyclase,” which is an enzyme that converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) and pyrophosphate. An increase of cyclic guanosine monophosphate (cGMP), also caused by the intake of nitrite containing compounds cause vascular relaxation and it is presumed that cGMP activation may occur via the formation of NO. (Ghasemi, 2011)
In 1980, Furchgott and Zawadzki showed that endothelial cells are required for acetylcholine-induced relaxation of the vascular bed which refers to the vascular system or a part thereof, through the endothelium-derived relaxing factor. Even though they could not initially pinpoint what caused the relaxation of the endothelium, scientists knew that such a relaxing factor existed and the race was on to identify it. The endothelium is the thin membrane that lines the inside of the heart and blood vessels. The breakthrough came in 1987 when it was shown that endothelium-derived relaxing factor and NO are the same or almost the same thing. Nitric oxide was the agent responsible for relaxing the endothelium. (Ghasemi, 2011)
In 1992, NO was proclaimed as the molecule of the year and in 1999, Furchgott, Ignarro, and Murad were awarded the Nobel Prize in Physiology or Medicine for studies in the NO field. Due to the proven roles played by NO physiologically and pathologically, research on NO was increased rapidly and at the end of the 20th century, the rate of NO publications was approximately 6,000 papers per year, with currently more than 100,000 references invoking NO listed in PubMed.” (Ghasemi, 2011)
In our earlier discussion of nitric oxide as the curing molecule in bacon, we referred to S. J. Haldane who was the first person to demonstrate that the addition of nitrite to haemoglobin (blood protein) produces a nitric oxide (NO)-heme bond, called iron-nitrosyl-hemoglobin (HbFeIINO). He showed that nitrite is further reduced to nitric oxide (NO) in the presence of muscle myoglobin (muscle protein key in supplying oxygen to the muscle) and forms iron-nitrosyl-myoglobin. It is nitrosylated myoglobin that gives cured meat, including bacon and hot dogs, their distinctive red colour and protects the meat from oxidation and spoiling. Discovering that Nitric Oxide (NO) is a key molecule in human physiology should not have been a surprise to meat scientists. There was, an understanding in meat science since the time of Haldane that the nitrate-nitrite-NO pathway was the curing reactions in meat from saltpetre to nitric oxide. It was later decided to use nitrite directly for reasons elucidated in a previous part of this series, Part 2: The Curing Molecule
When we say that the reduction of nitrite to nitric oxide occurs chemically, we refer to the non-enzymatic reduction of nitrite to nitric oxide. Ghasemi (211) gives us the technical details of this. “NO was found to be synthesized from L-arginine by the enzymes known as NO synthase (NOS) (EC 22.214.171.124) in two separate mono-oxygenation steps; first, L-arginine is converted to N-hydroxyarginine in a reaction requiring one O2 and one NADPH and the presence of tetrahydrobiopterin (BH4) and in the second step, by oxidation of N-hydroxyarginine citrulline and NO are formed. At least three NOS enzyme isoforms including neuronal, inducible, and endothelial (eNOS) have been identified and encoded by different genes.”
This non-enzymatic production of Nitric Oxide was suggested in 1997 by Ghafourifar and Richter. They postulated the “existence of mitochondrial NOS and in 1994, Lundberg and colleagues and Benjamin and colleagues demonstrated NOS-independent NO formation. Non-enzymatic NO production by one-electron reduction of nitrite, a blood and tissue NO reservoir, seems to be found everywhere and greatly accelerated under hypoxic conditions or conditions of low oxygen levels in your body tissues. This finding changes the general belief that nitrate and nitrite are waste products of NO.” (Ghasemi, 2011)
I want to refer as an important sidenote at this point to the work of Vanek (2022) which we will look at in much greater detail in a following discussion since they beautifully elucidates the reason for the importance of Nitric Oxide and how it binds to the meat protein we rely on in meat curing, forming the reddish/ pinkish colour of cured meat and giving muscles its characteristic red colour. The important point is that just as nitric oxide is produced through enzymes and non-enzymatic ways to react with myoglobin, in the same way and hugely important to meat curing is that myoglobin has also been shown to have enzymatic functions and is responsible for the decomposition of bioactive nitric oxide to nitrate. The importance of this point can hardly be over-stated! If we are able to convert L-arginine into Nitric Oxide in other ways besides indigenously through NO synthase (the enzymes responsible for oxidising nitrogen in L-Arginine to nitric oxide), and so cure meat, and should we find that this can be done through bacteria, then we still do not strictly speaking have meat curing with no nitrite present as the nitrate will be converted through bacteria in the meat to nitrite and albeit this being present at very low dosages, there will still be nitrite in the meat that we cured.
Allow me to state it again. If we are able to access L-arginine either through bacteria or enzymes directly (as we do in salt-only-long-term-cured-hams) and as a result of this do not start our curing process with nitrite (as is the case with long-term salt-only cured hams) and we are able to claim that we cure meat with no nitrite salts as we are indeed able to do at the present time, then we can not say that we eliminated nitrite from meat curing because there is the likelihood that some of the NO will be converted to nitrate which will be reduced to nitrite again and we are back at the beginning of the quest for nitrite-free curing. Stated a different way, it would seem that curing without nitrite is not possible. This is the heart of the conundrum of people propagating that meat has been cured with no nitrites in that we are dealing with REACTIVE nitrogen species and where you find the one, you are likely to find the others. Our nitrogen species of interest, when we refer to “we will find the one where we find the others” are nitrate, nitrite and nitric oxide, but as we will see further on, these are by no means the only nitrogen species we will encounter in the human muscle and in meat curing alike.
The extent to which what I suggest above is true, we will have to verify through experimentation. The rest of this document is dedicated to answering the following question: why would we want to eliminate a physiologically important species of nitrogen from our diet in any event!? So, on the one hand, is nitrite free curing a realistic goal and secondly, why would we want to do it? Are there other ways to overcome the health concerns associated with cured meats?
Effects of Nitrite in Human physiology.
– Sources of nitrogen for Human Physiology and the Value of Nitrite
The great discovery of the past few decades is that nitrate and nitrite have a fundamentally important role in our physiology and nitrite in particular, namely to act as a reservoir for nitric oxide (NO) which is a physiologically important molecule. Apart from nitric oxide being generated from the amino acid, L-Arginine, nitric oxide is generated through what is referred to as the nitrate-nitrite-Nitric Oxide pathway which is, as we have said before, exactly the same pathway of bacon curing. So, in order for this mechanism to work, we need a direct source of nitrates or nitrites and nature provided this for us in what we eat. The biggest source is vegetables which account for 60%–80% of the daily nitrate intake in a Western diet. As you will see from the table below, they not only supply us with nitrates but with nitrites directly as well. It has been shown that elevations in the blood plasma nitrite levels can occur by increasing the dietary nitrate intake. (Kobayashi, 2015)
Nitrate, nitrite and nitric oxide are closely linked as the difference between them is one oxygen atom. NO3– (nitrate), NO2– (nitrite) and NO (nitric oxide). Nitrate is reduced to nitrite through bacteria and nitrite to nitric oxide through chemical means (enzyme and non-enzyme driven). NO can be oxidized back to nitrite again and nitrite to nitric oxide. Nitric oxide, in the presence of myoglobin, can be converted directly back to nitrate. As a result of this, where one finds nitrate and bacteria such as in the mouth or digestive tract, you will always find nitrite and nitric oxide and where you have nitric oxide, one can find nitrite and nitrate. This is true in meat curing and true in the human body. “In humans and other mammals, about one-quarter of all circulating inorganic nitrate (NO3−), derived from diet or oxidation of endogenous (within the body) nitric oxide (NO), is actively taken up by the salivary glands and excreted in saliva. As a result, salivary nitrate levels are 10–20 times higher than those levels found in our blood. The mechanism behind this massive nitrate accumulation in saliva has remained elusive. The work by Qin et al. reports that the protein sialin can function as an effective nitrate transporter.” (Lundberg, 2012)
With these brief remarks, we are then thrust into the domain of the nitrate-nitrite-NO cycle in the human body. Nitrite is no longer viewed as something to be avoided at all cost, but as a chemical essential for human life and cured meat becomes by far, not the biggest contributor of nitrate and nitrite to our system, but the possibility exists for it to become an important one as we can use the same basic principles that gave us cured meat, reduce the fat and salt and find ways to introduce essential goodness of plant matter and we are confronted with the amazing opportunity to change processed food into a superfood! In this one statement, I seek to address the unfounded negative perception of nitrite, give a clue as to the possible real reason behind the health concerns related to processed meat (fat, salt, phosphates, etc) and give a roadmap for future work by imaginative food scientists in the incorporation of healthy plant matter into the sought after food group, allowing for all the conveniences that make processed-meats a well-loved and very convenient food for our era!
Have a look at the table below which gives the main dietary sources for nitrate and nitrites. Pay close attention to where hot dogs and bacon feature on the list!
Sindelar (2012), as quoted by (Kobayashi, 2015)
Hord (2009) as quoted by (Kobayashi, 2015)
When we ingest nitrates from leafy green vegetables or cured meat, it is absorbed in the upper gastrointestinal tract which comprises the mouth, salivary glands, oesophagus, stomach, and small intestine. The levels in the blood reach the highest level around 30–60 min after the nitrates have been swallowed. Approximately 25% of nitrate absorbed by the body reappears in our mouth through our salivary glands which pump it back into our mouths. Here it is reduced by the bacteria on our tongue from nitrate to nitrite. As it reaches our stomach, a part of the nitrites which we swallow is what we call protonated (adding hydrogen to the nitrite) and nitrous acid is formed which is the form that nitrite takes on when diluted into water (NO2− + H+ → HNO2). This reaction is similar to what happens to nitrite when we dilute it into the curing brine and inject it into meat which is also a more acidic environment like the stomach. Similar to meat curing, the nitrite we ingested now decomposes to form a variety of nitrogen oxides such as Nitric Oxide, the curing molecule, nitrogen dioxides (NO2), and dinitrogen trioxide (N2O3) (2 HNO2 → N2O3 + H2O, N2O3 → NO + NO2). These nitrogen oxides form additional bioactive adducts, such as S-nitrosothiols and N-nitrosamines. S-nitrosothiols sound very intimidating but are not. They are proteins discovered in the 90s and have since been shown to be key in many biochemical processes in our body. Specifically, S-nitrosothiols play a key role in the total system encompassing our heart and blood vessels, for example, the widening of blood vessels as a result of the relaxation of the blood vessel’s muscular walls and preventing thrombosis. N-nitrosamines are known to us by now as formed by the reaction of nitrite with secondary amines which can be cancer-causing.” (Kobayashi, 2015)
The next point requires us to know what gastric mucosa refers to. It is the mucous membrane layer of the stomach, which contains the glands and the gastric pits. Blood flow plays an important role in the protection of normal gastric mucosa and in the protection and healing of damaged mucosa. “Nitric Oxide production in the stomach is greatly enhanced in the presence of micronutrients that naturally occur in plants called dietary polyphenols and vitamin C or ascorbic acid, whereas because of its lower stability and shorter half-life relative to S-nitrosothiols, the released Nitric Oxide in the stomach is thought to locally contribute to increasing the gastric mucosal blood flow and mucous thickness to ensure the normal gastric physiology, and serves as the first-line host defence against harmful bacteria which we swallowed with our food. However, not all the nitrite reacts with H+(escapes the protonation) in the acidic milieu of the stomach and enters the systemic circulation, and then reaches the peripheral organs, including skeletal muscles, where it acts in an endocrine manner (like hormones) to exert NO-like activity. An interesting side note is that because the levels of nitrite in the blood are depends to a large degree on the amount of nitrate in the saliva and its reduction to nitrite, the use of antibacterial mouthwash and frequent spitting of saliva consequently decrease the plasma levels of nitrite.” (Kobayashi, 2015) We just said that Nitric Oxide production in the stomach is greatly enhanced in the presence of micronutrients that naturally occur in plants called dietary polyphenols and vitamin C or ascorbic acid. As we will see later, these substances and in particular vitamin A, C and E plays an important role as “blocking” agents by reacting with the partially digested amino acids called amines, and with secondary amines in particular called N-Nitrosamones denoting a reaction between the amine and nitroso component in nitrite, binding nitrogen and nitrogen (therefore the name, N-Nosotros-amines), blocking the formation of n-nitrosamines. Let me state it again. If we ingest nitrite with vitamins a, c, e, etc., these vitamins react with the secondary amines before nitrite can react with it, therefore blocking nitrosamine formation. This is something to look at on its own and we will not spend much more time on this important point. Here, my goal is to show that nitrite is NOT the harmful cancer-causing entity we believed it was, but turns out to be indispensable for healthy living! We can, therefore, for the moment, suspend the concerns about N-nitrosamine formation but rest assured that we will return to this in great detail! For now, let us continue with our focus on nitrites and the diagram below shows the main way we get nitrates and nitrites into our body.
“The plasma nitrite which reaches peripheral tissues is stored in various organs. Although there have been few reports dealing with the tissue levels of nitrate/nitrite following dietary nitrate supplementation in humans, animal studies show that dietary nitrate certainly increases the tissue levels of nitrate/nitrite following an increase in the plasma levels of nitrate/nitrite, which accordingly exerts therapeutic efficacy for animal models of various disease conditions. Interestingly, while acute dietary nitrate intake increases the plasma levels of nitrite in rodents and humans, chronic dietary nitrate intake does not always increase the plasma and tissue levels of nitrite but increases the tissue levels of nitrate and S-nitrosylated products. Although the mechanism underlying this finding is yet to be clarified, there might be some redox equilibrium of nitrate-nitrite-NO after chronic dietary nitrate intake, resulting in oxidation or reduction of the tissue nitrite to form nitrate or S-nitrosylated species, respectively. On the other hand, animal models chronically fed a diet deficient in nitrate/nitrite exhibit significantly diminished plasma and tissue levels of nitrate/nitrite, resulting in increased ischemia-reperfusion injuries in the heart and liver compared with the animal models fed a normal diet. Ischaemia-Reperfusion injury (IRI) is defined as the paradoxical exacerbation of cellular dysfunction and death, following the restoration of blood flow to previously ischaemic tissues. Ischemia or ischaemia is a restriction in blood supply to any tissues, muscle group, or organ of the body, causing a shortage of oxygen. These results suggest that dietary nitrate intake is important in the maintenance of steady-state tissue levels of nitrate/nitrite for NO-mediated cytoprotection. Cytoprotection is a process by which chemical compounds provide protection to cells against harmful agents. (Kobayashi, 2015) The key point is the importance of nitrate and nitrate in our diets and the possible harmful effect of nutrition deficiency in these compounds.
“Historically, the fact that nitrate and nitrite are present in human saliva has received little attention, because no one could attribute any kind of function to these anions. However, this lack of interest ceased in the 1970s, when researchers formulated a pathophysiological model for gastric cancer based on the accumulation of nitrate in saliva. Commensal bacteria in the mouth reduce parts of the salivary-derived nitrate to nitrite (NO2−), and when swallowed into the acidic stomach, this nitrite yields reactive intermediates that can react with dietary compounds to promote the formation of N-nitrosamines (a versatile class of carcinogens in rodents). With the emergence of this theory, nitrate immediately fell into deep disgrace, and ever since that time, authorities worldwide have put strict regulations on allowable levels of nitrate in our food and drinking water.” (Lundberg, 2012)
In the 1990s, research on nitrate took an unexpected turn when two research groups independently showed that salivary nitrate was a substrate for the formation of NO, and we looked at the development of our understanding of the importance of this molecule in our lives earlier on. It was revealed that NO plays “a key role in virtually every aspect of human physiology, including regulation of cardiovascular function, cellular energetics, immune function, neurotransmission, and more. The newly described alternative means of NO generation from nitrate was fundamentally different from the NO synthase pathway; it did not use arginine as a substrate, and it was independent of NO synthases. After the discovery that nitrate could be a substrate for the formation of a potentially beneficial biological messenger, the interest in nitrate shifted away from only being focused on carcinogenesis, and instead, researchers started to study potential NO-like physiological effects of this anion. From intense research performed during the past 15 y, it is now clear that administration nitrate or nitrite has robust NO-like effects in humans and other mammals. These effects include vasodilation, reduction in blood pressure, protection against experimental ischemia-reperfusion injury, reduction in cellular oxygen consumption, reversal of metabolic syndrome, reduction in oxidative stress, stimulation of mucosal blood flow and mucus formation in the gastrointestinal tract, and more.” We will spend time further on many of these in particular looking at lifestyle diseases.
“Intriguingly, most of these nitrate effects occur at dietary doses easily achievable through a normal diet rich in vegetables. Bioactivation of nitrate requires initial reduction to the more reactive nitrite anion, and this reaction is mainly carried out by commensal bacteria in the oral cavity and to a lesser degree, the tissues by mammalian enzymes. Salivary-derived nitrite is partly reduced to NO in the acidic stomach as described above, but much nitrite also survives gastric passage and enters the systemic circulation, which is evident from the marked nitrite increase in plasma seen after ingestion of nitrate. In blood and tissues, nitrite can undergo additional metabolism to form NO and other bioactive nitrogen oxides, including S-nitrosothiols. A number of enzymes and proteins have been shown to act as nitrite reductases, including deoxygenated haemoglobin, myoglobin, xanthine oxidase, mitochondrial respiratory chain enzymes, and more.” (Lundberg, 2012)
This matter of nitrate-nitrite-Nitric Oxide as the reaction sequence from nitrate in saliva becomes very interesting to us in the meat curing industry for one specific reason. When we surveyed the approach taken by the industry and the US government in particular, we noted in Part 3: Steps to secure the safety of cured meat, of our series that the direct application of nitrite was seen as a way to bypass the first bacteria mediated reduction step of nitrate to nitrite. The reasons given by industry and scientists alike was that it would yield better control in the curing process amongst others, as it relates to the lowest possible dosage of nitrite to effect curing since the dose dependency of the toxicity of nitrites was recognised from very early.
Lundberg (2012) surveyed the work of Qin in identifying sialin as the nitrate transporter to the saliva. This is relevant to curing. Lundberg describes a disorder which leads to ineffective transport of nitrate as follows, “Mutations in the sialin gene cause Salla disease and infantile sialic acid storage disorder, which are serious autosomal recessive lysosomal storage disorders characterized by early physical impairment and mental impairment.”
A fibroblast is a type of cell that contributes to the formation of connective tissue. It secretes collagen proteins that help maintain the structural framework of tissues. “Fibroblasts from patients with infantile sialic acid storage disorder show a lower nitrate transport activity compared with healthy controls. The work by Qin et al. also tested the importance of sialin for nitrate transport in the pig in vivo. Interestingly, adenovirus-dependent expression of a sialin mutant vector (sialinH183R) in the salivary gland decreases NO3− secretion in saliva after ingestion of a nitrate-rich diet compared with control animals.” (Lundberg, 2012)
“Sialin is expressed not only in the salivary glands but also in the brain, heart, lung, kidney, and liver, although seemingly at lower levels. The functional importance of nitrate transport into cells in these tissues would be of interest to study. In this context, it is interesting to note that nitrate metabolism does, indeed, occur in mammalian cells, although to a much lesser degree than in bacteria. The work by Jansson et al. reported on a functional mammalian nitrate reductase in numerous tissues, including liver, kidney, and intestines. Xanthine oxido reductase was identified as the major mammalian nitrate reductase, but the study indicated the presence of other unidentified nitrate reductases as well.” (Lundberg, 2012) The observation that nitrate metabolism occurs in mammalian cells, although to a much lesser degree than in bacteria should not escape our notice. I discussed the matter with a collaborator on key projects, Richard Bosman and we speculated that the reason for the curing in long-term salt-only-dry-cured hams probably has more to do with the relaxing of the muscles as a result of early cell breakdown and the accompanying invasion of bacteria able to oxidize L-arginine than with the endogenous oxidants in the meat. This fact possibly further points to a symbiotic evolution of humans with oral cavity bacteria positioned to fulfil this vital role of reducing nitrate to the more reactive nitrite.
“The work by Qin et al. proposes that sialin functions as the major NO3− uptake system in salivary gland cells; however, a remaining question is how this nitrate is further transported to saliva through the apical portion of the cells. Sialin seems to be a versatile anion transporter that also mediates H+-dependent transport of NO2−, aspartate, and glutamate. Previously, antagonism between nitrate, perchlorate, iodine, and thiocyanate for secretion in human saliva was shown, but in the work by Qin et al., these anions are not studied. It will be of interest to study if sialin also transports these anions. Definitive evidence for a functional role of sialin in nitrate transport and systemic nitrite/NO homeostasis in humans is lacking, but with the identification of this protein as an important nitrate transporter, it now seems possible to study this area. One approach could be to study the nitrate–nitrite–NO pathway in genetically engineered mice or perhaps, patients with Salla disease. Are salivary and plasma levels of nitrate/nitrite different in these patients? Do these animals or the patients exhibit any signs of systemic NO deficiency, including increased blood pressure, altered blood flow responses, different cellular energetics, or others? In the case that NO homeostasis is disturbed in Salla disease, would the patients benefit from substitution with nitrite?” (Lundberg, 2012)
This is the relevant question. Look at the possible suggestions. Is it possible to bypass nitrate and the bacterial reduction to nitrite and instead, would a solution be to administer nitrite directly as happens when we ingest nitrate which is transported to the saliva glands and in the mouth, are converted to nitrite, which, in the mouth and in the reducing environment in the stomach is changed to the physiologically vital nitric oxide? Lundberg (2012) puts his finger on the issue when he asks, “By giving nitrite instead of nitrate, one could bypass the initial nitrate transport step that might be disturbed in these patients, and NO and other bioactive nitrogen oxides would form directly from nitrite in blood and tissues.” He points to the fact that this therapeutic approach “was recently successfully tested in another genetic disorder involving a disturbed NO homeostasis.” Homeostasis refers to a self-regulating process by which biological systems maintain stability while adjusting to changing external conditions. “Another approach could be to study the proposed negative consequences of nitrate transport. If salivary nitrate transport promotes nitrosamine formation, which has been believed for a long time, are nitrosamine levels and occurrence of gastric malignancies lower in subjects lacking the transporter?” (Lundberg, 2012)
Huizing reports by 2021 that “plasma-membrane nitrate transport in salivary gland acinar cells, remains enigmatic.” (Huizing, 2021) Our hiatus into this question has, however, not been without reward.
We have seen the widespread distribution of nitrate to physiologically vital sites in the body;
We glimpsed at the key role of nitrite in the blood plasma, mainly derived from ingested nitrate and nitrates.
We see how other scientists in other fields of study came to the same conclusion as food scientists in the early 1900 namely that a direct application of nitrite, bypassing the time and bacteria dependant reduction step of nitrate has beneficial consequences.
In the discussion about possible negative effects of nitrite, one very important point to remember is that our overall natural design favours an adequate intake of nitrites. This can be seen by its presence in our blood. Here, nitrite is reduced to nitric oxide.
Gladwin (2008) that “recently, multiple physiologic studies have surprisingly revealed that nitrite represents a biologic reservoir of NO that can regulate hypoxic vasodilation, cellular respiration, and signalling.” They summarise that “studies suggest a vital role for deoxyhemoglobin- and deoxymyoglobin-dependent nitrite reduction. Biophysical and chemical analysis of the nitrite-deoxyhemoglobin reaction has revealed unexpected chemistries between nitrite and deoxyhemoglobin that may contribute to and facilitate hypoxic NO generation and signalling. The first is that haemoglobin is an allosterically regulated nitrite reductase, such that oxygen binding increases the rate of nitrite conversion to NO, a process termed R-state catalysis. The second chemical property is oxidative denitrosylation, a process by which the NO formed in the deoxyhemoglobin-nitrite reaction that binds to other deoxyhemes can be released due to heme oxidation, releasing free NO. Third, the reaction undergoes a nitrite reductase/anhydrase redox cycle that catalyzes the anaerobic conversion of 2 molecules of nitrite into dinitrogen trioxide (N2O3), an uncharged molecule that may be exported from the erythrocyte. We will review these reactions in the biologic framework of hypoxic signalling in blood and the heart.”
It is interesting that nitric oxide produced in the endothelium is oxidised to nitrite. In this instance, one could say that it “bypasses” the intestinal section where it could react with amino acids to form n-nitrosamines which some of them can cause cancer. Rassaf (2014) states that Nitric Oxide is produced in the body from the amino-acid L-arginine by the NO-synthases (NOSs). Three different NOSs exist: the endothelial NOS (eNOS, NOS III), the inducible NOS (iNOS, NOS II) and the neuronal NOS (nNOS, NOS I). This may be one way that the body uses to “manage” the possible harmful effects of nitrite but there are others as we have already eluded to and will look at in greater detail further on, namely ways to “block” nitrite through ingested vitamins. Still, there is another important mechanism which we will discuss in the future when we focus on n-nitrosamines and ways to mediate its possible harmful effect. Note that making it mandatory to include vitamin C in cured meats has been a strategy employed by the industry and regulated by governments from very early on. I will say a bit more about this at the end of this article.
Let’s return to the endothelial. The endothelial is the largest organ system in the body. I repeat the definition as I realise that these concepts may be new to many of the readers and repetition aids learning! It refers to a single layer of cells, called endothelial cells which lines the inside of all blood vessels (arteries, veins and capillaries). Inductable NOS is expressed after cell activation only and then produces NO for comparatively long periods of time (hours to days) in response to autoimmune and chronically inflammatory diseases in humans and neurodegenerative diseases and heart infarction, during tumour development, after transplantation, during prostheses failure and myositis. (Kröncke, 1998) Neuronal or nNOS relates to the brain. “Brain nNOS exists in particulate and soluble forms and the differential subcellular localization of nNOS may contribute to its diverse functions and has been implicated in modulating physiological functions such as learning, memory, and neurogenesis, as well as being involved in a number of human diseases.” (Zhou, 2009)
Let’s return to Gladwin (2008) who now describes a fascinating cycle of Nitric Oxide in the blood which relies on its conversion to nitrite. As we have seen above, Nitric Oxide is produced in endothelium and then diffuses to adjacent smooth muscle to activate soluble guanylyl cyclase that produces cGMP, and ultimately produces smooth muscle relaxation. Nitric oxide is subject to rapid inactivation reactions with haemoglobin that greatly limit its lifetime in blood, however recent studies suggest that NO formed from endothelial NO synthases is also oxidized by oxygen or plasma ceruloplasmin to form nitrite. Nitrite transport in blood provides an endocrine (from glands) form of NO that is shuttled from the lungs to the periphery while limiting the exposure of authentic NO to the scavenging red cell environment. Then during the rapid haemoglobin deoxygenation from artery to vein, the nitrite is reduced back to NO. Such a cycle conserves NO in the one-electron oxidation state. In this model, the nitrite pool represents the “live payload,” only one electron away from NO.”
If the body then generates enough Nitric Oxide, is there a requirement for additional dietary intake of nitrate or nitrite? “It has been suggested that the nitrate-nitrite-NO pathway serves as a backup system to ensure sufficient NO generation under hypoxic conditions when NOS may be malfunctioning.” (Ghasemi, 2011)
“It has been shown that 3-day dietary supplementation with sodium nitrate (0.1 mmol/kg/day) could reduce significantly diastolic blood pressure in non-smoking healthy volunteers. Recently, a large cohort study of 52,693 patients from 14 countries with acute coronary syndrome, of whom 20% were on chronic nitrate, demonstrated that chronic nitrate therapy (medication routinely taken at home and started at least 7 days prior to index event) was associated with reduced severity of myocardial injury in response to acute coronary events. The result showed that the proportion of these subjects with ST-segment elevation myocardial infarction was 41% in nitrate-naïve patients compared to only 18% in nitrate users and conversely a higher percent of nitrate users (82%) presented with non-ST-segment elevation acute coronary syndrome compared to 59% in nitrate-naïve patients.” (Ghasemi, 2011)
“Increasing nitrate or nitrate dietary intake provides significant cardioprotection against ischemia-reperfusion (I/R) injury in mice and it has been proposed that nitrite-/nitrate-rich foods may provide protection against cardiovascular conditions characterized by ischemia. It has been suggested that the nitrate-nitrite-NO pathway serves as a backup system to ensure sufficient NO generation under hypoxic conditions when NOS may be malfunctioning.” (Ghasemi, 2011)
“Abundant consumption of fruits and vegetables, especially green leafy vegetables, is associated with lower risk of cardiovascular disease. It has been proposed that inorganic nitrate, which is found in vegetables with a high concentrations, i.e. >2000-3000 mg/nitrate/kg, is the major factor in contributing to the positive health effects of vegetables via bioconversion to nitrite, NO, and nitroso-compounds, NOx intake now being considered as a dietary parameter for assessing cardiovascular risk.” (Ghasemi, 2011)
“Any intervention that increases blood and tissue concentration of nitrite may provide cardioprotection against I/R injury because it serves as a NOS-independent source of NO and reacts with thiols to form S-nitrosothiols. Nitrate-nitrite-NO pathway can be boosted by exogenous administration of nitrate or nitrite and this may have important therapeutic as well as nutritional implications. However, additional studies are required to clarify the protective roles of nitrate, considering the medical status of subjects, concomitant use of inhibitors of endogenous nitrosation (e.g. vitamin C and E), or foods containing high levels of nitrosatable precursors (e.g. fish). Some individuals, including those with high blood pressure and atherosclerosis, may benefit from increased nitrate while those with oesophagal dysplasia should avoid foods with high concentration of nitrate.” (Ghasemi, 2011)
The value of nitrite in the human body, however, goes far beyond only a reservoir of Nitric Oxide. We have eluded time and time again to many of the benefits and we now drill down on some of the different benefits or tahre, its role in resolving some of the negative lifestyle diseases prevalent in our modern era. “Nitrite-induced transnitrosylation in organs might be an alternative in vivo nitrite signalling for the mammalian biology including protection of protein thiols from irreversible oxidation, transcriptional modulation, and posttranslational regulation of most classes of proteins present in all cells, and also that changes in plasma nitrite levels even within the physiological ranges (e.g., postprandial and fasting) can affect tissue levels of S-nitrosothiol and subsequent cellular biology.” (Kobayashi, 2015)
-> Protective Effects of Dietary Nitrate/Nitrite on Lifestyle-Related Diseases
Kobayashi (2015) reviewed nitrites’ protective effect on lifestyle-related diseases. They write: “Lifestyle-related disease is a chronic disease characterized by oxidative and proinflammatory state with reduced NO bioavailability. The cellular redox balance in these patients shifts toward a more oxidizing state which affects a number of protein functions at the transcriptional and posttranslational levels, consequently disrupting the cellular homeostasis. However, increased NO bioavailability can improve the intracellular redox environment by S-nitrosylation-mediated modulation of most classes of proteins present in all cells. Recently, accumulating evidence has suggested that dietary nitrate/nitrite improves the features of lifestyle-related diseases by enhancing NO availability, and thus provides potential options for prevention and therapy for these patients. Based on the recent evidence, the beneficial effects of a diet rich in these components are discussed below, focusing on insulin resistance, hypertension, cardiac ischemia/reperfusion injury, chronic obstructive pulmonary disease (COPD), cancer, and osteoporosis.”
“The insulin receptor shares a signalling pathway with the activation of endothelial NOS (eNOS) to regulate the postprandial blood flow and efficient nutrient disposition to peripheral tissues. Therefore, insulin resistance is always associated with impaired NO availability, suggesting that a reciprocal relationship exists between insulin activation and endothelial function. Insulin resistance is improved by NO at various levels including insulin secretion, mitochondrial function, modulation of inflammation, insulin signalling and glucose uptake. For example, insulin-stimulated NO production has physiological consequences resulting in capillary recruitment and increased blood flow in skeletal muscle, leading to efficient glucose disposal.” (Kobayashi, 2015)
However, the most important mechanism to improve insulin resistance might be at the post-receptor level of insulin signalling. In diabetic states, increased adiposity releases free fatty acids and produces excessive reactive oxygen species (ROS) through a toll-like receptor 4 (TLR4)-mediated mechanism, which activates a number of kinases and phosphatases, and then disrupts the balance of protein phosphorylation/dephosphorylation associated with insulin signalling. The mechanisms underlying the NO-mediated beneficial effects on insulin resistance are as follows: First, NO suppresses the TLR4-mediated inflammation and ROS production by inactivating IkB kinase-β/nuclear factor-κB (IκκB/NF-κβ), the main trigger for the induction of a number of proinflammatory cytokines. Second, Wang et al., indicated that NO mediates the S-nitrosylation of protein-tyrosine phosphatase 1B (PTPB1) and enhances the effects of insulin. Because PTPB1 dephosphorylates the insulin receptor and its substrates, attenuating the insulin effect, its phosphatase activity tends to be suppressed by eNOS-mediated S-nitrosylation. In contrast, when the vascular eNOS activity is impaired, PTPB1 suppresses the downstream signalling to PI3K/Akt, leading to insulin resistance. Therefore, NO might act as a key regulatory mediator for the downstream signalling linking glucose transporter 4 (GLUT4) translocation and glucose uptake. Third, Jiang recently reported that NO-dependent nitrosylation of GLUT4 facilitates GLUT4 translocation to the membrane for glucose uptake, and improves insulin resistance. Fourth, excess nutrients also overproduce superoxide in the mitochondrial respiratory chain, leading to the subsequent formation of ROS. NO can inhibit mitochondrial ROS production through the S-nitrosylation of mitochondrial respiratory chain complex 1 enzyme and by improving the efficiency of oxidative phosphorylation in the mitochondria.” (Kobayashi, 2015)
“Indeed, the therapeutic potential of dietary nitrate/nitrite has been supported by recent studies demonstrating the improvements of insulin resistance in humans and animals as a result of its enhancing the NO availability in plasma and tissues. As mentioned above, insulin resistance always accompanies metabolic and endothelial dysfunction, which leads to hypertension and atherosclerosis. Enhancement of the availability of NO might therefore be a promising strategy for the prevention and treatment of patients with not only insulin resistance but also endothelial dysfunction.” (Kobayashi, 2015)
-> Cardiac Ischemia/Reperfusion Injury
“During heart ischemia, ATP is progressively depleted in cardiac muscle cells, which impairs ion pumps, leads to the accumulation of calcium ions, and consequently damages the cell membrane stability. On reperfusion, the cardiac muscle cells are further injured, because in the mitochondria, ROS are produced in large quantities due to massive electron leaks and the formation of superoxide with the resupplied oxygen, which denatures cytosolic enzymes and destroys cell membranes by lipid peroxidation. ROS-mediated dysfunction of the sarcoplasmic reticulum also induces massive intracellular calcium overload, leading to the opening of the mitochondrial permeability transition pore and causing cell apoptosis or necrosis, depending on the intracellular ATP levels. The availability of vascular NO would thus be expected to be impaired due to the reduced NOS activity in ischemia and subsequent consumption by superoxide during reperfusion, resulting in severe ischemia/reperfusion injury.” (Kobayashi, 2015)
“Nitrite, nitrate, and NO-related compounds (e.g., S-nitrosothiols) are constitutively present in blood and tissues. The nitrite level in cardiac tissue is a couple of times higher than that in plasma due to an unknown form of active transport from blood to tissues or due to the oxidation of endogenously generated-NO to nitrite by ceruloplasmin, and serves as a significant extravascular pool for NO during tissue hypoxia. Carlström et al., showed that dietary nitrate increased the tissue levels of nitrite and S-nitrosothiols in the heart, and attenuated oxidative stress and prevented cardiac injury in Sprague-Dawley rats subjected to unilateral nephrectomy and a high-salt diet. Shiva et al., recently showed that the nitrite stored in the heart and liver via systemic and oral routes augmented the tolerance to ischemia/reperfusion injury in the mouse heart and liver.” (Kobayashi, 2015)
“Although the genetic overexpression of eNOS in mice attenuates myocardial infarction, in general, the protective effects of NO on cardiac ischemia/reperfusion injury depend on the local stock of nitrite and its subsequent reduction to NO at the critical moment when NOS activity is lacking under hypoxic conditions. Indeed, the tissue levels of S-nitrosothiols (NO-mediated signalling molecules) are enhanced through the nitrite reduction due to NOS inhibition, hypoxia, and acidosis, suggesting that the tissue nitrite stores can be regarded as a backup and on-demand NO donor. There are a number of factors that have been demonstrated to reduce nitrite in the tissues, including deoxyhemoglobin, deoxymyoglobin, xanthine oxidoreductase, heme-based enzymes in the mitochondria and acidosis during ischemia. In patients with coronary heart disease, the different consequences of myocardial infarction may depend on the patient’s daily intake of nitrate/nitrite. Indeed, Bryan et al., showed that dietary nitrite (50 mg/L) or nitrate (1 g/L) supplementation in drinking water for seven days maintained higher steady-state levels of nitrite and nitroso compounds, as well as nitrosyl-heme, in mouse cardiac muscle, and these mice exhibited a smaller cardiac infarct size after ischemia/reperfusion injury compared with control mice fed a diet deficient in nitrate/nitrite for seven days. These findings suggest that this protective nitrate/nitrite may be derived at least in part from dietary sources.” (Kobayashi, 2015)
“Shiva et al., demonstrated that the cytoprotective effects of nitrite on ischemia/reperfusion injury are mediated by post-translational S-nitrosylation of complex 1 in the mitochondrial respiratory chain, which consequently inhibits the overall mitochondrial ROS formation and apoptotic events. Another possible cytoprotective effect of nitrite may be mediated by the effects of S-nitrosylation on the intracellular Ca2+ handling, which decreases Ca2+ entry by inhibiting L-type Ca2+ channels and increasing the sarcoendoplasmic reticulum (SR) Ca2+ uptake by activating SR Ca2+ transport ATPase (SERCA2a) . These effects will lead to an attenuation of the increase in cytosolic Ca2+ during ischemia and Ca2+ overload during reperfusion.” (Kobayashi, 2015)
“Intriguingly, recent large-scale epidemiological studies reported the preventive effects of antioxidant supplementations including vitamins E, C, and beta carotene rich in fruits and vegetables on cardiovascular disease, whereas no beneficial effects were shown in other studies, and in some cases, a decrease in cardiovascular protection with these supplementations was observed. On the other hand, a number of epidemiological studies have shown the preventive effects of fruits and vegetables on coronary heart disease. It should be noted that the consumption of an appropriate amount of fruits and vegetables, which might contain balanced doses of nitrate/nitrite and vitamins, might be more effective with regard to health maintenance and improvement than antioxidant supplementation alone.” (Kobayashi, 2015) It is this finding in particular that gives direction to my work with two collaborators Richard Bosman and Dr Jess Goble. Whether we will succeed in our quest, time will tell but we have some impressive early breakthoughs and with solid support of scientists, industry professionals and inventors of new technology which has the potential to unluck the application of these fruits and vegetables to meat, we are hopeful and extremely motivated!
-> Chronic Obstructive Pulmonary Disease (COPD)
“COPD is considered to be a lifestyle-related disease because long-term tobacco smoking and subsequent chronic bronchitis are causally associated with this disease. Varraso et al., recently reported the importance of a healthy diet in multi-interventional programs to prevent COPD. They showed that high intake of whole grains, polyunsaturated fatty acids, nuts, and long chain omega-3 fats, and low intake of red/processed meats, refined grains and sugar-sweetened drinks, were associated with a lower risk of COPD in both women and men.” (Kobayashi, 2015)
“Because cured meats such as bacon, sausage and ham contain high doses of nitrite for preservation, antimicrobial and colour fixation, epidemiological studies have demonstrated that the consumption of cured meats is positively linked to the risk of newly diagnosed COPD. Nitrite generates reactive nitrogen species, which may cause nitrosative damage to the lungs, eventually leading to structural changes like emphysema. This is supported by an animal study in which rats chronically exposed to 2000 and 3000 mg/L of sodium nitrite in their drinking water for two years showed distinct lung emphysema. However, the dose of nitrite used in that study was 250–350 mg/kg/day, which was too high to compare with those achieved in standard human diets.
In fact, cured meats have been reported to generally comprise only 4.8% of the daily nitrite intake, and 93% of the total ingestion of nitrite is derived from saliva, suggesting that cured meats provide minimal contributions to the human intake of nitrite, even if they are frequently consumed. In addition, the recent nitrite levels in processed meats have been approximately 80% lower than those in the mid-1970s in the US. Therefore, discussions encompassing all ingested sources of nitrite should consider whether or not the nitrite derived only from the consumption of cured meats might be responsible for the development of COPD.” (Kobayashi, 2015)
“On the other hand, a number of epidemiological studies have shown the beneficial effects of n-3 fatty acids, vitamins, fruits and vegetables on lung functions and the risk of COPD. Although it may be difficult to isolate the specific effects of these dietary nutrients, as discussed above, the nitrate and nitrite derived from vegetables and fruits are reduced to NO, which is followed by the formation of S-nitrosothiols, rather than the formation of nitrosamines especially in the presence of reducing agents such as vitamin C and E in the stomach. It has been shown that high dietary nitrate intake does not cause the expected elevation of the gastric nitrite concentrations or appreciable changes in the serum nitrite concentrations.” (Kobayashi, 2015) As I stated previously, these findings do not cause the industry to sit back and proclaim, “you see, consumption of cured meat is safe” even though the validation is encouraging – in the case of me and my collaborators it energises us to do even better and work to turn cured meat into a superfood.
“As mentioned above, different from the effects of the direct elevation of nitrite concentration in the plasma, the entero-salivary route of dietary nitrate/nitrite might enhance the availability of NO through the formation of S-nitrosothiols and its transnitrosylation to the other thiol residues of proteins, suggesting that, depending on the tissues and organs, separate metabolic pathways might exist for NO availability in this entero-salivary route. Consistent with this idea, Larsen et al., recently demonstrated that acute intravenous infusion of nitrite enhanced the plasma levels of nitrite, whereas it did not affect the oxygen consumption (VO2) or the resting metabolic rate (RMR) in humans. Instead, dietary nitrate significantly reduced the VO2 and RMR by improving the mitochondrial respiratory chain function and enhancing efficient O2 consumption, suggesting that rather than direct nitrite infusion to enhance the plasma nitrite levels, biologically active nitrogen oxide (including the S-nitrosothiols produced in the stomach) might be an important molecule for the transfer of biological NO activity for cardiopulmonary function . Because COPD is a state of protein-energy malnutrition due to an increased resting metabolic rate and VO2, the effects of dietary nitrate on the reduction of the RMR and VO2 might be advantageous for patients with COPD.” (Kobayashi, 2015)
“Whether the role of NO in COPD is protective or pathogenic depends on the origin and concentration range of NO. NO activity derived from dietary nitrate and constitutive NOS might be protective against COPD largely through the S-nitrosothiol-mediated mechanism including inhibition of the noncholinergic nonadrenergic nerve activity, bronchial smooth muscle relaxation, reduction of airway hyperresponsiveness, downregulation of the proinflammatory activity of T lymphocytes, and antimicrobial defence. However, the deleterious effects of NO on the development of COPD might be derived from iNOS-mediated pro-inflammatory signalling, which is consequently (not causally) reflected by the huge amount of NO in the exhaled air of patients with COPD.” (Kobayashi, 2015)
“Recent human studies have demonstrated that dietary nitrate (beetroot juice containing approximately 200–400 mg of nitrate) improved the exercise performance and reduced blood pressure in COPD patients. However, large-scale epidemiological evidence of the impact of nitrate is still lacking.” (Kobayashi, 2015)
-> Lowering Blood Pressure
An obvious benefit of nitrite is its role as a reservoir of Nitric Oxide which is a key molecule which blood pressure. The blood pressure-lowering and performance-enhancing effects of nitrites have been known for many years. (Keller, 2017) This is due to the fact that the nitrite anion (NO–2) acts as an endogenous nitric oxide source. (Keszler, 2008) Nitrite is reduced to nitric oxide (NO). “One major mechanism of nitrite reduction is the direct reaction between this anion and the ferrous heme group of deoxygenated haemoglobin.” The oxidation reaction of nitrite with oxyhemoglobin (oxyHb) which is formed by the combination of haemoglobin with oxygen, is also well established and generates nitrate and methemoglobin (metHb). (Keszler, 2008)
“Increased consumption of fruits and vegetables is associated with a reduction of the risk of cardiovascular disease. The DASH studies recommended the consumption of diets rich in vegetables and low-fat dairy products to lower blood pressure, and these effects are thought to be attributable to the high calcium, potassium, polyphenols and fiber and low sodium content in these food items. However, vegetable diets containing high nitrate levels increase the plasma levels of nitrate and nitrite, which are the physiological substrates for NO production. Accumulating evidence has recently indicated that the nitrate/nitrite content of the fruits and vegetables could contribute to their cardiovascular health benefits in animals and humans.” (Kobayashi, 2015)
“A number of publications have demonstrated that dietary nitrate reduces blood pressure in humans. Larsen et al., reported that the diastolic blood pressure in healthy volunteers was reduced by dietary sodium nitrate (at a dose of 0.1 mmol/kg body weight per day) corresponding to the amount normally found in 150 to 250 g of a nitrate-rich vegetable, such as spinach, beetroot, or lettuce. Webb et al., studied the blood pressure and flow-mediated dilation of healthy volunteers, and showed that the vasoprotective effects of dietary nitrate (a single dose of 500 mL of beetroot juice containing 45.0 ± 2.6 mmol/L nitrate), were attributable to the activity of nitrite converted from the ingested nitrate . Kapil et al., also showed a similar finding that consuming 250 mL of beetroot juice (5.5 mmol nitrate) enhanced the plasma levels of nitrite and cGMP with a consequent decrease in blood pressure in healthy volunteers, indicating that there was soluble guanylate cyclase-cGMP-mediated vasodilation following a conversion of the nitrite to bioactive NO. They later presented the effects of dietary nitrate on hypertension, and showed the first evidence that daily dietary nitrate supplementation (250 mL of beetroot juice daily) for four weeks reduced the blood pressure, with improvements in the endothelial function and arterial stiffness in patients with hypertension. Because arterial vascular remodelling is the major histological finding associated with ageing, these vascular structural changes represent vascular wall fibrosis with increased collagen deposits and reduced elastin fibers, which result in arterial stiffening and subsequent hypertension in elderly patients. Sindler et al., recently demonstrated that dietary nitrite (50 mg/L in drinking water) was effective in the treatment of vascular ageing in mice, which was evidenced by a reduction of aortic pulse wave velocity and normalization of NO-mediated endothelium-dependent dilation. They showed that these improvements were mediated by reduction of oxidative stress and inflammation, which were linked to mitochondrial biogenesis and health as a result of increased dietary nitrite. These beneficial effects were also evident with dietary nitrate in their study, suggesting that dietary nitrate/nitrite may be useful for the prevention and treatment of chronic age-associated hypertension.” (Kobayashi, 2015)
“In addition, hypertension is also a major cause of ischemic heart and cardiac muscle remodelling, which lead to congestive heart failure. Bhushan et al., reported that dietary nitrite supplementation in drinking water (50 mg/L sodium nitrite, for nine weeks) increased the cardiac nitrite, nitrosothiol, and cGMP levels, which improved the left ventricular function during heart failure in mice with hypertension produced by transverse aortic constriction. They also showed that dietary nitrite improved the cardiac fibrosis associated with pressure-overloaded left ventricular hypertrophy through NO-mediated cytoprotective signalling. Although a number of studies on the acute effects of dietary nitrate have been conducted using animal models and healthy humans, more evidence in patients with hypertension, as well as additional studies on the long-term effects of dietary nitrate, will be needed in the future.” (Kobayashi, 2015)
“In the stomach, swallowed nitrite is decomposed to form a variety of nitrogen compounds, including N-nitrosoamines. In the 1950s, Magree et al., first reported that N-nitrosodimethylamine caused malignant primary hepatic tumours in rats. After this report, a number of studies followed in relation to the carcinogenic effects of N-nitroso compounds in animal models. In particular, the dietary intake of red and cured meats was found to be associated with an increased risk of certain types of cancer due to the relatively large amounts of nitrite added. However, the methodological aspects have been challenged concerning the high dose of nitrosatable amines, and the physiological difference between animals and humans.” (Kobayashi, 2015)
“In the stomach, the nitrosonium ion (NO+) derived from nitrite can bind to thiol compounds (R-SH) and amines (especially secondary amines: R1-NH-R2), forming S-nitrosothiol and N-nitrosamine, respectively. However, while N-nitrosamine formation occurs even at neutral or basic pH, S-nitrosothiol formation tends to occur only under acidic conditions. In addition, this reaction kinetically occurs much more easily than N-nitrosamine formation, particularly in the presence of vitamins C and E and polyphenols, which are highly present in fruits and vegetables, which also eliminate potent nitrosating agents such as the N2O3 formed from nitrite by decomposing them to NO. This might partly explain why patients with achlorhydria and non-vegetarians eating large amounts of cured meats are at risk of developing gastric cancer.” (Kobayashi, 2015)
“However, this idea appears to be inconsistent with the belief that dietary nitrite is a major cause of cancer. This is because, according to the average nitrate/nitrite intake of adults in the US, most of the daily nitrate intake (around 90%) comes from vegetables, and the nitrite intake is primarily derived from recycled nitrate in the saliva (5.2–8.6 mg/day nitrite), with very little coming from cured meats (0.05–0.6 mg/day nitrite in 50g/day cured meats) and other dietary sources (0–0.7 mg/day nitrite) , suggesting that the entero-salivary route may be the more important source of nitrosamine exposure than exogenous intake including cured meats, that is, spitting out saliva all day long might prevent cancer development more effectively than cutting cured meats. However, recent experimental and epidemiological studies could not demonstrate a positive relationship between nitrate consumption and the risk of cancer, and the Joint Food and Agriculture Organization/World Health Organization Expert Committee on Food Additives concluded in 2008 that there was no evidence that nitrate was carcinogenic in humans. Consistent with this, recent studies have found no link between dietary nitrate and cancer.” (Kobayashi, 2015)
“Bradbury et al., reported a large-scale study (>500,000 participants) of the associations between fruit, vegetable, or fiber consumption and the risk of cancer at 14 different sites. They showed that there was an inverse association between fruit intake and the risk of upper gastrointestinal tract and lung cancer, as well as an inverse association between fiber intake and liver cancer. The dietary intake of vegetables, as well as fruits and fiber, was inversely associated with the risk of colorectal cancer, suggesting that there is little evidence that vegetable intake is associated with the risk of any of the individual cancer sites reviewed.” (Kobayashi, 2015)
“However, chronic inflammation, including inflammatory bowel disease and Helicobacter pylori-induced gastritis induce inducible NOS (iNOS) and generate large quantities of NO, forming nitrosating and oxidant species such as N2O3 and peroxynitrite, which might cause mutagenesis through deamination, nitration of DNA, or inhibition of the DNA repair system. Depending on the sites and amounts of NO generation, NO might represent a double-edged sword in the sense that it confers both protective and deleterious effects on cancer development.” (Kobayashi, 2015)
“Meta-analyses of primary and secondary cancer prevention trials of dietary antioxidant supplements, such as beta carotene, vitamins A, C, and E, showed a lack of efficacy, and on the contrary, an increased risk of mortality. Although the general role of NO in carcinogenesis is complicated, and many unknown mechanisms remain to be resolved, the dietary nitrate/nitrite (at least that obtained from plant-based foods such as fruits and vegetables) has obvious inhibitory effects on cancer risk by playing some synergistic role with other nutrients in these foods.” (Kobayashi, 2015) It is again findings like these that give direction to our product developments.
“Lifestyle habits, such as smoking, alcohol intake, little or no exercise, and an inadequate amount of calcium intake all influence the calcium-vitamin D metabolism and bone mineral density, in some cases leading to osteoporosis, particularly in postmenopausal women. The implications of NOS-mediated NO in the regulation of bone cell function have been well described in a number of publications. For example, iNOS-induced NO production following stimulation with proinflammatory cytokines, such as interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α), inhibits bone resorption and formation, resulting in osteoporosis in patients with inflammatory diseases such as rheumatoid arthritis. On the other hand, eNOS, a constitutive NO synthase, plays an important role in regulating osteoblast activity and bone formation, because eNOS knockout mice exhibit osteoporosis due to defective bone formation, and eNOS gene polymorphisms were reported to be causally linked to osteoporosis in postmenopausal women.” (Kobayashi, 2015)
“In addition, Wimalawansa et al., showed that some of the beneficial effects of estrogen on bone metabolism are mediated through a NO-cGMP-mediated pathway, suggesting that NO donor therapy might provide a promising alternative to estrogen therapy. In this context, it has been shown that organic nitrate NO donors, such as glycerol trinitrate, isosorbide dinitrate and mononitrate all have beneficial effects on experimental and clinical osteoporosis, and a number of epidemiological studies also indicated that a high fruit and vegetable intake appears to have a protective effect against osteoporosis in men and pre- and postmenopausal women. However, few studies have been conducted to evaluate the detailed mechanism by which inorganic nitrate/nitrite prevents osteoporosis at the molecular level, and thus further basic research will be needed for this purpose.” (Kobayashi, 2015)
-> Methemoglobinemia (MetHb)
A negative effect of nitrite in the body relates to its link with methemoglobinemia. It is historically this link which contributed to cast nitrite in a negative light and day plays a dominant role in establishing what the WHO regards as safe levels of ingested nitrites.
“Methemoglobinemia (MetHb) is a blood disorder which the US National Institute of Health defines as occurring when “an abnormal amount of methemoglobin is produced.” They explain that “hemoglobin is the protein in red blood cells (RBCs) that carries and distributes oxygen to the body. Methemoglobin is a form of hemoglobin. Inherited (congenital) methemoglobin occurs when the disorder “is passed down through families.” Our interest is in what is referred to as acquired MetHb which is “more common than inherited forms and occurs in some people after they are exposed to certain chemicals and medicines.” One such chemical is nitrites. (National Libary of medecine) “Elevated levels of nitrite in the blood can trigger the oxidation of hemoglobin, leading to methemoglobinemia.” Keszler (2008) suggests a simplified model of the kinetics involved where the end products of the reaction are methemoglobin (metHb) and nitrate.
The “World Health Organization (WHO) used data based on the risk of methemoglobinemia to set an acceptable daily intake (ADI) for nitrate of 3.7 mg/kg body weight per day, equivalent to 222 mg nitrate per day for a 60-kg adult, and nitrite of 0.07 mg/kg body weight per day, equivalent to 4.2 mg nitrite per day for a 60-kg adult. (Keller, 2017)
The upper limit represented by the WHO ADI corresponds to the concentration of dietary nitrate that lowers blood pressure in normotensive and hypertensive adults. (Keller, 2017)
Very high concentrations of nitrate in drinking water may cause methemoglobinemia, particularly in infants (blue baby syndrome). “In the 1940s, Comly first reported cases of cyanotic infants who received formula prepared with well water containing a high nitrate content. Based on the subsequent analyses of the infantile cases of methemoglobinemia, the US Environmental Protection Agency (EPA) set a Maximum Contaminant Level (MCL) for nitrate of 44 mg/L (equal to 10 mg/L nitrogen in nitrate). However, it is now thought that methemoglobinemia per se was not caused by nitrate itself, but by faecal bacteria that infected infants and produced NO in their gut. A recent report by Avery has argued that it is unlikely that nitrate causes methemoglobinemia without bacterial contamination, and also that the 40–50 mg/L limit on nitrate in drinking water is not necessary.” (Kobayashi, 2015)
However, there are now legal limits to the concentrations of nitrate and nitrite in both food and drinking water. The WHO showed that the Acceptable Daily Intake for humans (ADI) for nitrate and nitrite were 3.7 and 0.07 mg/kg body weight/day, respectively, which were based on the calculations from the doses of <500 mg of sodium nitrate/kg body weight that were harmless to rats and dogs. The international estimates of nitrate intake from food are 31–185 mg/day in Europe and 40–100 mg/day in the United States. However, the Ministry of Health, Labour and Welfare of Japan reported that the average intake of nitrate in the Japanese population is around 200–300 mg/day, which is one and a half times to two times the ADI. Furthermore, according to a report by Hord, in which the daily nitrate and nitrite intakes were calculated based on the variations using the vegetable and fruit components of the DASH (Dietary Approaches to Stop Hypertension) dietary pattern, the level easily exceeds 1,200 mg/day nitrate. This is more than five-fold higher than the WHO’s ADI of 3.7 mg nitrate/kg body weight/day, and more than two-fold the US Environmental Protection Agency’s level of 7.0 mg nitrate/kg body weight/day for a 60 kg individual. Furthermore, as indicated in Figure 2, approximately 25% of the ingested nitrate is secreted in saliva, and 20% of the secreted nitrate in the saliva is converted to nitrite by commensal bacteria on the tongue, indicating that about 5% of the originally ingested nitrate is swallowed into the stomach. Therefore, for a DASH diet containing 1200 mg nitrate, an individual would be expected to swallow approximately 45 mg of nitrite a day, which easily exceeds the ADI of nitrite. Therefore, a comprehensive reevaluation of the health effects of dietary sources of nitrate/nitrite might be required in the near future.” (Kobayashi, 2015)
– Other International Views on Nitrite/ Nitrate from Dietary Sources besides from the USA and Europe
The Food Standards Australia New Zealand and the European Food Safety Authority concluded that the major sources of estimated nitrate and nitrite exposure, across different population groups, were vegetables and fruits (including juices).Processed meats only accounted for 10% of total dietary exposure to nitrite in the European survey.Consumption and exposure to dietary nitrate and nitrite is not considered an ‘‘appreciable health and safety risk’’, according to the Australian agency. (Keller, 2017)
Given the established vasoprotective, performance-enhancing, blood pressure lowering effects of dietary nitrates in humans, specific recommendations to encourage plant-based, nitrate-rich foods may produce significant public health benefits. (Keller, 2017)
Is vitamin C and E the crucial link that saves bacon’s bacon?
Three important nitrosamines, namely N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and N-nitrosomorpholine (NMOR), are classified as probably carcinogenic to humans (Group 2B) by the International Agency for Research on Cancer (IARC) (IARC 2000). (Erkekoglu, 2010)
Intrinsic antioxidant systems, such as protective enzymatic antioxidants as well as antioxidants available in the human diet, provide an extensive array of protection that counteract potentially injurious oxidizing agents. (Erkekoglu, 2010)
It was found that antioxidants protected the cells against nitrite and nitrosamines. (Erkekoglu, 2010) Dietary antioxidants can be a saviour when exposure to dietary genotoxic/carcinogenic compounds is the case. (Erkekoglu, 2010)
Erkekoglu, 2010 confirmed the DNA damaging effect of nitrosamines as shown in other studies (Robichová et al. 2004b; Arranz et al. 2006; 2007; Garcia et al. 2008a; b). Additionally, they used sodium nitrite to show the genotoxic effects of nitrite alone. They showed that antioxidants supplementation was capable of reducing both tail intensity and tail moment in all of the nitrosamine treatments, particularly in NDMA. They proposed that this may be related to antioxidants’ reduction of CYP2E1 and CYP2A6. They write, “CYP2E1 is responsible for α-hydroxylation of N-alkylnitrosamines with short alkyl chain, whereas cyclic nitrosamines like NPYR, NPIP, and NMOR may be activated by CYP2A6 and by CYP2E1 to a lesser extent (Kamataki et al. 2002). Furthermore, inhibition of CYP450 enzymes may not be the only mechanism underlying the protection of antioxidants. Alternative mechanisms by antioxidants may be as follows: ROS scavenging capacity, the conversion of reactive compounds to less toxic and easily excreted compounds, alteration of cell proliferation, stimulation of DNA-repair induced by nitrosamines, induction of Phase II enzymes, and NAD(P): quinine oxidoreductase activity (Roomi et al. 1998; Chaudière and Ferrari-Iliou 1999; Gamet-Payrastre et al. 2000; Surh et al. 2001; Surh 2002).” (Erkekoglu, 2010)
It is obvious that the overwhelming weight of evidence is that nitrite is not the destructive chemical that it was made out to be and that the negative media frenzy is completely misguided, to put it mildly. The health benefits of nitrate, nitrite and nitric oxide are clear. An obvious path for improving the geneneral healt and nutritional status associated with cured meats is the incorporation of vegetable and plant matter into its formulation. The fact that nitrire-free curing may possibly never be achieved has been raised and warrants further investigation. The next two segments will focus on N-nitrosamines and why the protein myaglobin evolved in such a way that it wants to react with oxygen and nitric oxide.
Gladwin, M. T. and Kim-Shapiro, D. B.. (2008) The functional nitrite reductase activity of the heme-globins. ASH Publication, Blood. Review in Translational Hematology. Blood (2008) 112 (7): 2636–2647. https://doi.org/10.1182/blood-2008-01-115261
Lundberg JO. Nitrate transport in salivary glands with implications for NO homeostasis. Proc Natl Acad Sci U S A. 2012 Aug 14;109(33):13144-5. doi: 10.1073/pnas.1210412109. Epub 2012 Jul 31. PMID: 22851765; PMCID: PMC3421160.
Rassaf T, Ferdinandy P, Schulz R. Nitrite in organ protection. Br J Pharmacol. 2014 Jan;171(1):1-11. doi: 10.1111/bph.12291. PMID: 23826831; PMCID: PMC3874691.
Sindelar, J.J.; Milkowski, A.L. Human safety controversies surrounding nitrate and nitrite in the diet. Nitric Oxide 2012, 26, 259–266.
I present a complete paper by Anthony R. Butler and Martin Feelisch where they trace the benefits and therapeutic uses of nitrate and nitrates. It forms part of a segment in EarthwormExpress, The Truth About Meat Curing: What the popular media do NOT want you to know! Having studied the matter of the potential detrimental addition of nitrites to curing brines from a human health perspective and having examined thousands of scientific articles on the subject I came to the conclusion that most of the negative press in the popular media on the subject is irrational and based on a partial evaluation of the salient points related to the issue.
Volume 117, Issue 16, 22 April 2008; Pages 2151-2159
Potential carcinogenic effects, blue baby syndrome, and occasional intoxications caused by nitrite, as well as the suspected health risks related to fertilizer overuse, contributed to the negative image that inorganic nitrite and nitrate have had for decades. Recent experimental studies related to the molecular interaction between nitrite and heme proteins in blood and tissues, the potential role of nitrite in hypoxic vasodilatation, and an unexpected protective action of nitrite against ischemia/reperfusion injury, however, paint a different picture and have led to a renewed interest in the physiological and pharmacological properties of nitrite and nitrate. The range of effects reported suggests that these simple oxyanions of nitrogen have a much richer profile of biological actions than hitherto assumed, and several efforts are currently underway to investigate possible beneficial effects in the clinical arena. We provide here a brief historical account of the medical uses of nitrite and nitrate over the centuries that may serve as a basis for a careful reassessment of the health implications of their exposure and intake and may inform investigations into their therapeutic potential in the future.
The presence of nitrite (NO2−) and nitrate (NO3−) in bodily fluids has been known for some time. Dietary studies carried out by Mitchell et al1 at the beginning of the 20th century established that the amounts of nitrate excreted in the urine are higher than those ingested with the food, suggesting that the excess nitrate must be a product of endogenous biosynthesis. Later metabolic balance studies by Green et al2,3 showed that this assumption was correct and provided unequivocal evidence for mammalian nitrate biosynthesis. Griess,4 using his eponymous chemical test, showed that human saliva contains small quantities of nitrite, and the detection of very high levels of nitrite in the urine of a volunteer, who happened to have contracted a fever, was the first indication that endogenous production of nitric oxide (NO) is part of the immune response. Nitrite is not normally present in urine, and it was Cruickshank and Moyes5 who realized that it originated from bacterial reduction of urinary nitrate, an observation that forms the basis of today’s dipstick tests for urinary tract infection. Shortly after the discovery by Palmer et al6 that vascular endothelial cells produce NO from l-arginine, Marletta et al7 reported that the same pathway accounts for the production of nitrite and nitrate by activated macrophages, and countless investigators have since used nitrite and nitrate to assess NO production in basic and translational research studies. More recently, the ease with which nitrate is reduced to nitrite and nitrite is converted into NO has occasioned interest in the role of plasma nitrite in vascular smooth muscle relaxation,8 the control of blood pressure and flow,8 and possible therapeutic uses of nitrite.9,10 Subsequent animal experimental studies revealed that a number of organs are protected against ischemia/reperfusion-related tissue injury after systemic application of small amounts of nitrite,11 suggesting further therapeutic uses. Strangely, this renewed interest in nitrite/nitrate, together with emerging data suggesting possible new roles for these anions in physiology, coincides with the conclusion by the International Agency for Research on Cancer that “ingested nitrate or nitrite under conditions that result in endogenous nitrosation is probably carcinogenic for humans.”12 The purpose of this review is neither to consider the physiological role of naturally occurring nitrite and nitrate in organs and bodily fluids or their usefulness as biomarkers of NO activity nor to discuss their possible role as carcinogens; rather, it is to explore the uses of inorganic nitrite and nitrate in medicine, not only modern medicine but also medicine of the past. It transpires that medical interest in these oxyanions of nitrogen is not new.
Discovery and Chemical Properties
Nitrates, particularly potassium nitrate (known also as niter or nitre and saltpeter), have been known since prehistoric times, and in the Middle Ages, natural deposits were commercially exploited. The Chinese invented gunpowder around 800 CE, and with its appearance in Europe during the 13th century, potassium nitrate became strategically important. Demand increased further with the Agricultural Revolution of the 19th century and the use of nitrates as fertilizers. Natural sources were eventually supplemented by synthetically produced nitrate at the beginning of the last century.13
Nitrite is present at trace levels in soil, natural waters, and plant and animal tissues. In pure form, nitrite was first made by the prolific Swedish chemist Scheele14 working in the laboratory of his pharmacy in the market town of Köping. He heated potassium nitrate at red heat for half an hour and obtained what he recognized as a new “salt.” The 2 compounds (potassium nitrate and nitrite) were characterized by Péligot15 and the reaction established as 2KNO3→2KNO2+O2.
The release of oxygen from a substance known to alchemists as “aerial niter” since the times of Paracelsus explains the role of nitrates in gunpowder, rocket propellants, and other explosives.16 Sodium nitrite rapidly gained importance in the development of organic chemistry during the 19th century, when it was discovered that nitrous acid (HNO2) reacts with aromatic amines (ArNH2) to produce diazonium ions,17 a highly important synthon for the dyestuffs industry and for synthetic organic chemistry generally: ArNH2+HNO2+H+→ArN=N++2H20.
The mechanism of such diazotization reactions has been subject to extensive study.18 Diazotization may be responsible, in part, for the carcinogenic role of nitrite under certain conditions, particularly in the context of drug-nitrite interactions.19
Nitric acid (HNO3) is a strong acid that is completely ionized at all biologically interesting pHs. Although nitrous acid (HNO2) is a weak acid, with a pKa of 3.15 (pKa is the pH at which the acid is 50% dissociated), it is also, at physiological pHs, completely dissociated, except in the stomach, on the surface of airways, within select cellular compartments (eg, the mitochondrial intermembrane space, endosomes, secretory vesicles, lysosomes, and other acidic organelles), and on the skin.
Nitrite as a Vasodilator
The scope of this review is limited to inorganic nitrite and nitrate, but interest in a medical role for inorganic nitrite was first aroused because of the spectacular success of organic nitrites and related compounds in the treatment of angina pectoris. Butter,20 writing about the treatment of angina in 1791, gave no drug treatment and had little more to offer than the recommendation of a tranquil lifestyle. However, while working at the Edinburgh Royal Infirmary in the 1860s, Brunton21 noted that the pain of angina could be lessened by venesection and wrongly concluded that the pain must be due to elevated blood pressure. As a treatment for angina, the reduction of circulating blood by venesection was inconvenient. Therefore, he decided to try the effect on a patient of inhaling amyl nitrite, a recently synthesized compound and one that his colleague had shown lowered blood pressure in animals (A. Gamgee, unpublished observation). The result was dramatic.21 Pain associated with an anginal attack disappeared rapidly, and the effect lasted for several minutes, generally long enough for the patient to recover by resting. For a time, amyl nitrite was the favored treatment for angina, but its volatility made it troublesome to administer, and it was soon replaced by chemically related compounds that had the same effect but were less volatile. The most popular replacement was glyceryl trinitrate (GTN), an organic nitrate better known as nitroglycerin.22 The fact that this compound is highly explosive and a component of dynamite appears not to have been a problem. In his 1894 textbook, Phillips23 lists a number of chemically related compounds that can be used in the treatment of angina. The list includes not only amyl nitrite but also propyl, ethyl, and isobutyl nitrites, as well as GTN. A similar list is provided by White24 in his 1899 textbook. GTN, a drug introduced into allopathic medicine thanks to extensive homoeopathic studies by Hahnemann,25 occasioned greatest favour among practising physicians, and by 1956, in a symposium on hypotensive drugs,26 it was the only drug of this type that was listed. GTN was first synthesized by Sobrero at the University of Torino in 1812, and considering the way in which he handled it, he was fortunate not to cause a fatal accident.27 He thought it too explosively violent to have any practical use. Nobel, the highly successful Swedish entrepreneur, was able to moderate its action by incorporating it into kieselguhr to form dynamite. It is largely from this invention that the Nobel family fortune is derived. Tragically, Nobel’s younger brother Emil was killed while working with GTN, a dark episode in Nobel’s life. Sobrero bitterly resented Nobel’s commercial success with what he saw as his invention, although Nobel always acknowledged his debt to Sobrero.28 It is a curious coincidence that by 1895 Nobel had developed angina and was prescribed GTN as treatment, but it is a happier coincidence that the 1998 Nobel Prize for Physiology or Medicine was awarded for the discovery of the role of NO as a signalling molecule in the cardiovascular system. Now that NO is known to be an important vasorelaxant, it is easy to see why drugs of this type act the way they do. Each is a substrate for ≥1 enzyme systems, possibly located in the vascular wall, that convert it into nitrite and subsequently to NO. One such enzyme, a mitochondrial aldehyde dehydrogenase, has been purified and partially characterized.29 However, the contribution of this or other enzyme systems to the overall vasodilation by these drugs is difficult to assess because multiple metabolic pathways appear to act in concert.30
In view of the range of organic nitrites and related compounds that act as vasodilators, it is not surprising that potassium and sodium nitrites were tested in this regard. In 1880, Reichert and Mitchell31 published a very full account of the biological action of potassium nitrite on humans and animals. At that time, the value of amyl nitrite in the treatment of angina was severely compromised by the short duration of its effect, so the search for an improved drug had begun. The effect of potassium nitrite on the nervous system, brain, spinal cord, pulse, arterial blood pressure, and respiration of healthy human volunteers was noted, as was the variability between individuals. The most significant observation was that even a small dose of <0.5 grains (≈30 mg) given by mouth caused, at first, an increase in arterial blood pressure, followed by a moderate decrease. With larger doses, pronounced hypotension ensued. They also noted that potassium nitrite, however administered, had a profound effect on the appearance and oxygen-carrying capacity of the blood. They compared the biological action of potassium nitrite with that of amyl and ethyl nitrites and concluded, rather interestingly, that the similarity of action depends on the conversion of organic nitrites to nitrous acid. Observations similar to those of Reichert and Mitchell were made by Atkinson32 and Densham.33 Practicing physicians, including Hay34 and Leech,35 examined the therapeutic value of inorganic nitrites as hypotensive drugs and noted that, although of slower onset, their therapeutic effect lasts much longer, and they might be seen as superior drugs. They soon appeared in the Materia Medica of the time. In 1906, the drug supplier Squibb sold a 1-lb bottle of sodium nitrite (sodii nitris) for $1,36 and by the mid-1920s, an injectable solution of sodium nitrite became available (Nitroskleran, E. Tosse & Co, Hamburg, Germany) for the treatment of hypertension and vasospasm.37 Instructions for using sodium nitrite to treat angina are given in Martindale’s Additional Pharmacopoeia and in the US National Standard Dispensatory of 1905.38 A textbook on Materia Medica for medical students in 1921 gives details of the appropriate dose,39 but by the middle of the 20th century, its medicinal use had essentially ceased, largely because of adverse side effects. Blumgarten40 noted that sodium and potassium nitrites often caused nausea, belching, stomachache, and diarrhoea. Although these side effects may have caused physicians to hesitate in prescribing sodium nitrite for angina, another event precipitated the fall of inorganic nitrite from favour (see below).
Interest in the vasodilator properties of nitrite enjoyed a renaissance with the notion that nitrite may be involved in the regulation of local blood flow after conversion to NO by nonenzymatic mechanisms41,42 and an oxygen-sensitive nitrite-reductase43 and S-nitrosothiol–synthase44 function of haemoglobin. Like NO, inhaled nebulized nitrite has been shown to be an effective pulmonary vasodilator45 and, along with organic nitrites,46 suggested for potential use in neonatal pulmonary hypertension. Although there is no doubt that appropriate pharmacological doses of nitrite can normalize elevated blood pressure,47 the question of whether physiological concentrations of nitrite are vasodilator active is still a matter of debate.48,49
Conversion of Nitrite Into NO and NO-Related Products
In view of the close chemical connection between nitrite and NO, it is tempting to assume that nitrite acts as a source of NO when functioning as a vasodilator. However, such conversion requires either strongly acidic conditions or enzymatic catalysis. At low pH, nitrous acid can give rise to the spontaneous generation of NO: 2HNO2→H2O+N2O3 and N2O3→NO+NO2.
Solutions of acidified nitrite have been used successfully to generate NO and to induce vasorelaxation in isolated blood vessel studies,50 and the same reaction mechanism has been proposed to explain the biological action of nitrite.51,52 However, pHs at which this occurs are generally not found within living systems,53 with the exceptions mentioned above. On the other hand, the enzyme xanthine oxidoreductase converts nitrite into NO when oxygen levels are low, and this is a more likely course of action54 in the vascular system, at least under ischemic conditions. In fact, recent data suggest that hypoxic NO formation from nitrite is carried out by multiple enzyme systems10 and occurs in virtually all tissues and organs (Feelisch et al, unpublished data, 2006). Independently of its reduction to NO, nitrite is converted into NO-related products, including S-nitrosothiols and NO-heme species, at normal physiological pH and oxygen levels.55 Although it cannot be excluded that some of the biological effects of nitrite may be mediated by nitrite itself, it is fair to assume that most of the physiological and therapeutic actions of nitrite that require conversion into NO and NO-related products involve enzymatic catalysis.
Nitrite as an Antidote for Cyanide and Hydrogen Sulfide Poisoning
In popular literature, cyanide (CN−) is considered the acme of human poisons. In fact, it is by no means the most poisonous substance generally available, but it acts very rapidly, and it is on this rapid action that its reputation rests. Large doses cause instant death; even with low doses, the characteristic symptoms of cyanide poisoning (loss of consciousness, motionless eyes, dilated pupils, cold skin, and sluggish pulse and respiration) appear within seconds. Despite the catastrophic consequences of an overdose, potassium cyanide was used in medicine for many years as a treatment for chest complaints,56 particularly a dry cough.57 It was not removed from the British Pharmacopoeia until 1945.
By the end of the 19th century, it was established that the toxicity of cyanide was due to interference with the process of cellular respiration.58 Keilin59 showed that cyanide reacts with the ferric heme of the enzyme cytochrome c oxidase, a vital link between the tricarboxylic acid cycle and formation of metabolic water causing inhibition of mitochondrial respiration. Because cyanide also reacts with methemoglobin,60 it should be possible to prevent the reaction of cyanide with cytochrome c oxidase by massively increasing the concentration of methemoglobin in the blood. Nitrite oxidizes the central iron atom of haemoglobin from the ferrous (Fe2+) to the ferric (Fe3+) state, producing methemoglobin, and is, therefore, a potential antidote for cyanide poisoning. The clinical use of nitrite in this setting was first proposed by Hug61 and is now universally used. Sodium thiosulfate also is included in the antidote to provide a source of sulfur to aid the conversion of cyanide into thiocyanate by rhodanese. The first cases of acute cyanide poisoning in humans to be treated with nitrite and thiosulfate were reported in 1934. One patient had ingested 5 g potassium cyanide but recovered after being given 1.5 g sodium nitrite and 18 g sodium thiosulfate.62 In many countries, nitrite is part of the cyanide antidote kit. Nowadays, patients are given an ampoule of amyl nitrite by inhalation or an intravenous injection of 3% sodium nitrite, followed by a slow injection of 50% sodium thiosulfate.63
Although formation of methemoglobin is generally accepted as the explanation of the efficacy of nitrite as an antidote, evidence suggests that this is not the complete explanation.64,65 There may be alternative or additional routes whereby nitrite detoxifies, but no details are available.66 Compounds that promote NO release in vivo (like bradykinin) modify cyanide toxicity. Whether this is an alternative mode of action of nitrite in detoxification or just another source of nitrite from endogenous NO is, at this time, difficult to assess.
Nitrite also is an efficacious antidote to poisoning by hydrogen sulfide (H2S), an occupational hazard with high lethality and long-term neurological sequelae in survivors. Like NO and CO, low concentrations of H2S are produced endogenously and have vasodilator properties, but the physiological significance of its formation is currently unknown.67 Supraphysiological concentrations of sulfide, as experienced after H2S inhalation, lead to rapid inhibition of mitochondrial respiration by reversible binding to the central iron atom of cytochrome c oxidase in place of oxygen, explaining why H2S poisoning shares many similarities with cyanide intoxication.68 Nitrite administration, which is superior to that of oxygen alone69 and often is combined with hyperbaric oxygen therapy, is most effective when given immediately after H2S exposure.70 It is thought to act via induction of methemoglobinemia and subsequent binding of hydrosulfide anions (HS−) to the oxidized blood pigment, leading to inhibition of cytochrome c oxidase and reinstitution of aerobic respiration in the tissues. Although this mode of action appears reasonable, the rather slow rate of methemoglobin formation by nitrite is inconsistent with the rapid recovery typically observed in the clinical setting, suggesting, as with the treatment of cyanide poisoning, the involvement of additional mechanisms. Although nitrite has been known for many years to have protective and antidotal effects against experimental sulfide poisoning in rodents,71 nitrite administration for H2S intoxication was introduced into human therapy only in the mid-1970s.72 The recommended dosage regimen for nitrite in sulfide intoxication is identical to that established for the treatment of cyanide poisoning, ie, initiation with inhalations of amyl nitrite followed by intravenous injection of 10 mL of a 3% solution of sodium nitrite.73
Other Medical Uses of Inorganic Nitrite
In view of the success of nitrite with angina, it was tried for the treatment of other medical conditions. Law74 recommended the administration of very large doses (20 grains or 1.3 g) of sodium nitrite to treat epilepsy. Other physicians tried this dose and found that the side effects were far too serious to continue the treatment, with considerable consequences for the therapeutic use of inorganic nitrite. The toxic nature of such high doses was confirmed by Ringer and Murrell,75 who concluded that Law had been using an impure sample of sodium nitrite that was largely sodium nitrate. They attempted to establish a safe dose, but the reputation of sodium nitrite had suffered, and because of the success of GTN, nitrite disappeared from widespread use. The final blow came when Magee and Barnes19 reported that certain nitrosamines, which could be formed in the stomach by reaction between nitrite and naturally occurring secondary amines in food, are strongly carcinogenic in rodents. Although these findings were quickly confirmed by others and have been extended to other animal species, a causal relationship between nitrite and nitrate exposure and human cancer has not been unequivocally demonstrated.76 Nevertheless, further medical use of nitrite ceased for decades, except as an antidote in emergencies, and maximal contaminant levels of nitrite and nitrate levels in drinking water and foods soon became strictly regulated in most countries worldwide. In light of the negative image, nitrite has acquired over the years, it is somewhat surprising that the use of nitrite as an antibacterial agent in canned food has continued. More recently, the antimicrobial properties of nitrite that form the basis for its use in food preservation have been explored for potential benefit in lung and skin diseases.
Acidification is a prerequisite for nitrite to act as an antimicrobial agent, suggesting (albeit not proving) that the active principle is NO. It has been known for some time that the nitrite found in human saliva originates from nitrate that is actively secreted into the oral cavity and gets partially reduced there by the local commensal bacterial flora.77 After swallowing, nitrite ends up in the acidic environment of the stomach, and the NO thus produced is thought to contribute to the antibacterial effects of gastric juice. Similarly, the nitrite produced from nitrate in sweat is believed to exert antimicrobial effects on the surface of the skin.78 Thus, acidified nitrite may be a component of innate immunity at several locations on and within the body. Some attempts to capitalize on this insight point in potentially promising therapeutic directions, although few of these findings have made their way into the clinic.
The effectiveness of acidified nitrite in killing antibiotic-resistant Pseudomonas bacteria might offer a possibility to eradicate a major cause for chronic lung infections in cystic fibrosis patients,79 provided a safe mode of administration can be found. The antimicrobial properties of NO can be exploited by dermal application of creams containing nitrite and an acidifying agent, eg, ascorbic acid, to treat a number of skin diseases.80 The same concept has been demonstrated to increase microcirculatory blood flow in Raynaud patients81,82 and to accelerate wound healing.83 Although the effects of acidified nitrite are typically ascribed to the generation of NO, the possibility that part of the nitrite applied is absorbed and converted into NO-related products in the tissue cannot be excluded.
Use of Inorganic Nitrate in Medicine
Although modern manuals of Materia Medica and pharmacopeias state that potassium nitrate has no drug action other than as a diuretic (see below), historical records show that it has been used extensively in medicine over the years to treat a number of conditions. In view of the close chemical relationship between nitrite and nitrate, we suggest that the value of inorganic nitrate in medicine is due, at least in part, to its conversion into nitrite during administration or contamination with nitrite because of the manner in which it was manufactured.
Niter occurs in natural deposits in desert regions. Fairly large amounts are found in the northwestern provinces of China, and it was well known to early Chinese alchemists. They called it xiao shi (solve stone), and it was first recognized in the 4th century CE. It was a component of some of the elixirs of immortality concocted by Daoist savants as they searched for a means of realizing the Daoist ideal of life without death.84 Entirely by chance, they mixed it with sulfur and charcoal and thus created gunpowder, which was used by the Chinese not only for fireworks but also for civil engineering and warfare. The first printed formula for gunpowder occurs in a Chinese manual of war that appeared in 1044 CE.
One of the oldest accounts of the use of niter in Chinese medicine is as a treatment for what appears to be angina in an 8th century Chinese manuscript uncovered at the Buddhist grotto of Dunhuang.85 The patient is instructed to take niter, hold it under the tongue for a time, and then swallow the saliva. The significance of the instructions is that under the tongue, even in a healthy mouth, nitrate-reducing bacteria convert some of the nitrate into nitrite.77,86 So, if the patient follows the physician’s instructions fully, he or she will be taking in nitrite, known to be a treatment for the alleviation of anginal pain.
Arab physicians were among the most advanced of the medieval period, but there is no mention of niter in a book on cardiac drugs by Avicenna, born 980 CE. The first extant Arabic mention of niter occurs in a book by Kitab al-Jami’fi al-Adwiya al-Mufrada (Book of the Assembly of Medical Simples) finished by Abu-Muhammad al-Malaqi Ibn al-Baitar around CE 1240. Niter was called Thalji al-Sin (Chinese snow), indicating the contact between Chinese and Arab civilizations. It was about this time that Arabs started to use niter in gunpowder and as a component of prescriptions.
Niter does not occur naturally to any great extent in Europe, and the efficacious use of niter in early European medicine is easier to understand if one realizes how the niter was produced. When gunpowder became known in Europe (Roger Bacon mentions it in 1240 CE), there was enormous demand for niter, and much was shipped to Europe from India, where it occurs in natural deposits. But, the demand outstripped supply, and indigenous manufacture was started. It was made in plantations or “nitriaries,” particularly in France and Germany. Natural conditions were simulated by exposing heaps of decaying organic matter mixed with lime to atmospheric action.87 Nitrates appeared as efflorescences and were converted into potassium salt by reaction with potassium carbonate (potash). Two groups of bacteria are responsible for this process: Nitrosomonas convert ammonia into nitrite, and Nitrobacter convert nitrite into nitrate.88 It is quite possible that niter from nitriaries contained some nitrite, thus giving it medicinal value. This is unlikely in niter from natural deposits because they are old and aerial oxidation will, over time, convert all the nitrite into nitrate. So, the 8th century Chinese physician mentioned previously had to instruct the patient on how to generate nitrite, but European physicians of the 14th to 17th centuries, using niter from a different source, could prescribe it without further refinement because nitrite was there already.
However, such a prescription was rather hit-or-miss in that the amount of nitrite present was a matter of chance. In one of the most comprehensive accounts of the use of niter, methods of making it more effective are described. The book, by Challoner, was printed in London in 1584 and entitled A Short Discourse of the Most Rare and Excellent Vertue of Nitre.89 The spelling of the English is idiosyncratic (rather like that of modern students) because spelling was not fully standardized until the publication of Johnson’s dictionary in 1775. Challoner’s book is concerned mainly with the value of niter in treating various dermatological conditions (“diseases of the skinne”), including “tawnie steynings, freckles, duskness and flegmatike evaporations.” It will, he claims, “restore the skinne and complexion to the native bewtie.” The key to understanding this claim lies in the first section of the book in which the author tells his readers how to make niter more effective (“yet more sharpe and subtile”). He describes 3 ways, all involving heating (called “calcination” by Challoner). Heat, of course, converts some of the niter into potassium nitrite, and so, without realizing it, Challoner anticipated the discovery of potassium nitrite by Scheele by nearly 200 years. As discussed above, nitrite has an antibacterial effect and accelerates wound healing, hence its effectiveness on infected skin blemishes (“skales, scrabbes, skurffe, dandruffe, pimples, tetters, bytes” and so on). Naturally occurring nitrite in saliva has the same effect and explains, in part, why most animals instinctively lick a wound.90
Challoner does not stop with the application of niter to the skin. He claims that it can be used “for uncumbring and clensing of the lunges” and for the “remedie of hoarnesses, olde coughe and toughe coughe, weising in the windpipes,” and so on. For this use, he suggests making the niter into a pill and then “hold one of those pilles lounge under the tongue, to mixe thereof as much as may be with the moisture of the mouth … and lastlie swallow it,” a procedure curiously reminiscent of the Chinese prescription and anticipating some of the work of Lundberg et al.77
Nitrate and the Treatment of Lung Diseases
For a time, amyl nitrite was used for relieving patients suffering an asthma attack. In an article91 in 1891, other nitrites, including sodium nitrite, were suggested for this purpose. The author remarks that the use of nitrites is not the treatment of choice but that it is said to be beneficial, probably by virtue of its smooth muscle–relaxing effects. However, relief could be delivered even better by a procedure using nitrate rather than nitrite. Blotting paper was soaked in a solution of niter and allowed to dry. Squares of the paper were burnt in a jar, and the patient inhaled the fumes. Apparently, this procedure was frequently successful in relaxing a bronchial spasm. It was first published as a patent in 1867,92 is described in detail in the Encyclopedia Britannica of 1911,93 and occurred as recently as 1926 in the US Dispensatory.94 The products of thermal decomposition of niter include NO, NO2, and O2.95 Because NO is a poor bronchodilator and NO2 is toxic, it is difficult to see how inhalation of this mixture brings relief. The combination possibly has an effect that is greater than the sum of its parts.
In addition to its use in asthma, sodium nitrate was given orally to treat chronic bronchitis.96 It is unclear whether the apparent effectiveness of this treatment was secondary to its conversion to nitrite causing bronchial relaxation and antibacterial effects or due to an effect of nitrate itself.
Nitrates as Diuretics
Nitrates have been used as diuretics for centuries. One of the first descriptions of the medical use of potassium nitrate for the treatment of dropsy (edema) is found in Thomas Willis’ Pharmaceutice Rationalis of 1674.97 Although it was long known that relatively large amounts (grams) were required to achieve effective diuresis, the dose-response relationship was first established in systematic “homeopathic provings” in 1825.98 Clear differences in potency exist between various nitrate salts,99 with ammonium nitrate being the most effective. Their mode of action was revealed by studies in dogs demonstrating an enhanced excretion of urinary chloride and sodium, resulting in a net loss of salt and water caused by increased glomerular filtration without an equivalent increase in tubular reabsorption.100,101 Whether these effects are mediated by formation of nitrite or NO is unknown.
Extensive animal and human studies by Keith et al102 confirmed the superiority of the ammonium over the sodium salt of nitrate. They also demonstrated that nitrates can potentiate the effects of other diuretics and that toxic symptoms are remarkably rare, even when administered in doses of 10 to 15 g daily for several weeks. Thus, ammonium nitrate was introduced as a new, more effective diuretic in 1926 and was used with great success to treat various forms of edema in North America, particularly at the Mayo Clinic. After a time of exaggerated emphasis on possible toxic effects of nitrates during the preceding 2 decades, which led physicians to use lower, inadequate doses, it looked as though ammonium nitrate was here to stay as the diuretic of choice. What had triggered the fear of inducing severe cyanosis when potassium or sodium nitrate was used as a diuretic before was the toxicity associated with the use of massive amounts of bismuth subnitrate for diagnostic purposes,103 which is somewhat surprising because the toxicity of large amounts of nitrate was well known for a long time.104 Concerns about the safety of nitrates reached a new height with the appearance of case reports about transient methemoglobinemia after administration of ammonium nitrate.105,106 The reasons for these rare complications (which disappeared on discontinuation of nitrate therapy in most cases) remain unclear but may have been due to contamination of the nitrate salt with nitrite, renal insufficiency causing elevated circulating levels of nitrate, or gastrointestinal disorders with enhanced reduction of nitrate to nitrite by the bacterial gut flora.107 With alternative diuretics in the form of organic mercurials available, the therapeutic use of nitrates as diuretics was abandoned by the mid-1930s.
Nitrate in Other Medicinal Preparations
The fact that most nitrate salts are readily water soluble has been exploited to produce medicines that require quick dissolution or application in liquid form. Although the effects of most of these drugs (eg, cerium and silver nitrate) have little to do with the amounts of nitrate they contain, application of large enough quantities can cause methemoglobinemia.108 Presumably, the same holds true for the excessive use of toothpastes aimed at treating dental hypersensitivity, some of which contain up to 10% potassium nitrate, although no intoxication from this source is documented in the literature.
Conclusions and Outlook
Despite the widespread use of sometimes astonishing amounts of nitrite and nitrate for different indications in medicine of the past, little use is made of them in contemporary medicine (except as antidote and solubility enhancer). This is a result of several factors, some of which we have described in this review. Apart from the replacement by more modern and effective medicines in some cases, the major driving force for this development appears to have been the fear fostered by discussions, in both the lay press and scientific literature, about the purported health risks of exposure to nitrite and nitrate. Reports about methemoglobinemia in infants caused by drinks or food prepared with nitrate-rich (and bacterially contaminated) well water and vegetables such as spinach, celery, and carrots (“blue baby syndrome”), intentional and occupational intoxications in adults, increasing nitrate levels in soil and lakes as a result of fertilizer overuse, and the formation of potentially carcinogenic N-nitrosamines all contributed to the negative image that nitrite and nitrate have held in recent years. As a result, major efforts have been made to remove as much nitrite and nitrate as possible from our drinking water, to advocate replacement of nitrite by other (often less effective) food preservatives, and to establish cultivation conditions that result in crops with reduced levels of nitrate. Although possible long-term consequences of a chronically reduced intake of nitrite and nitrate on human health are unknown, doubts have been raised about the general health risk of nitrite/nitrate intake.76,109–112 Interestingly, the average dietary intake of nitrate roughly equals that produced by the endogenous production of NO.113 Thus, if nitrite truly were of concern to human health because of its propensity to form carcinogenic nitrosamines, then the human body would have a significant evolutionary design flaw because ≈5% of all ingested and endogenously produced nitrate eventually ends up as nitrite in the stomach, as pointed out by Archer109 (so far about “intelligent design”). Despite the critical voices, the image of nitrite and nitrate remains stigmatized.
What appears to have the greatest potential to change our current perception of the risk and value of nitrite and nitrate is the most recent emergence of data on the physiological and pharmacological effects of relatively low concentrations/doses of nitrite. Previously considered a biologically inert oxidative decomposition product of NO, nitrite has been proposed to be a signalling molecule in its own right.55 Given its propensity for conversion into NO and related species, unequivocal evidence for this role may be difficult to provide unless nitrite-specific signalling pathways are identified. Although speculative, it is possible that the nitrite-based reaction channels of contemporary mammalian cells are a vestige of earlier bacterial pathways and that the evolutionarily more recent l-arginine/NO pathway uses signalling cascades originally evolved for nitrite, not the other way round. Regardless, surprisingly low amounts of nitrite have been demonstrated to exert potent cytoprotective effects against ischemia/reperfusion-related tissue damage in vivo,10,11 an action possibly mediated by modulation of mitochondrial function.113 Nitrate, which has been proposed to contribute to the health-promoting effects of the Mediterranean diet,114 has been demonstrated to inhibit platelet aggregation,115 to mildly lower blood pressure,116 to enhance gastric mucosal defence mechanisms,107 and to reduce the oxygen cost of exercise.117 The last is perhaps one of the most surprising of the more recent findings across the spectrum of nitrate actions. This particular observation may explain why an enhanced production of NO, which not only elevates blood flow and thus oxygen transport to tissues but leads to increased levels of circulating nitrite and nitrate, is crucial for the adaptation of life to the chronic hypoxia experienced at high altitude.118 Taken together, these results have shifted the attention away from toxic and vasodilator properties to a focus on metabolic effects. Moreover, they make one wonder to what extent inorganic nitrate may contribute to the effectiveness of organic nitrates in the setting of heart failure, for example.
Although efforts are underway to assess the potential usefulness of inorganic nitrite in a number of clinical research studies at the US National Institutes of Health, none of these are likely to whet the appetite of the pharmaceutical industry to invest substantial amounts of money into drug development because not only are intellectual property claims related to simple inorganic compounds legally difficult to defend but the material itself is cheap and readily available. The situation may change if medicinal chemists come up with new prodrugs that allow targeted delivery of nitrite to specific tissues or organs or if nitrite/nitrate is intelligently used as an adjuvant to current therapeutics. Which of the many facets of nitrite and nitrate action is likely to form the basis for future pharmaceutical exploitation is difficult to predict at present. Although rational approaches to the pharmacological treatment of medical problems have a tendency to ridicule the wisdom of century-old folk medicine and to condemn the alchemist’s doing as quackery, there is much to learn from the past. In reviewing the therapeutic use of nitrite and nitrate over centuries, it appears that some of the potential that these simple compounds may hold for medical use has not been realized, often because the basis for some unwanted drug effects was not understood and thus could not be controlled at the time. But, even if the scare factor continues to dominate mainstream thinking, there is an obvious need for a careful reassessment of the health risks of nitrite and nitrate. If initiated soon, such activity may provide the necessary “activation energy” to overcome the fear and to stimulate the development of new therapeutic principles that use pathways regulated by nitrite and nitrate.
Sources of Funding
This work was supported by funds from the Guthrie Trust (a travel grant for visiting the Wellcome History of Medicine Library in London to Dr Butler) and the Medical Research Council (Strategic Appointment Scheme to Dr Feelisch).
Correspondence to Anthony R. Butler, Bute Medical School, University of St. Andrews, St. Andrews, Fife, KY16 9ST, Scotland (e-mail email@example.com); or Martin Feelisch, Clinical Sciences Research Institute, Warwick Medical School, Gibbet Hill Rd, Coventry, CV4 7AL, England (e-mail firstname.lastname@example.org).
1 Mitchell HH, Shonle HA, Grindley HS. The origin of nitrates in urine. J Biol Chem. 1916; 24: 461–490.CrossrefGoogle Scholar
3 Green LC, Ruiz de Luzuriaga K, Wagner DA, Rand W, Istfan N, Young VR, Tannenbaum SR. Nitrate biosynthesis in man. Proc Natl Acad Sci U S A. 1981; 78: 7764–7768.CrossrefMedlineGoogle Scholar
4 Griess P. Über Metadiamidobenzol als Regens auf Saltpetrige Säure. Chem Ber. 1878; 11: 624–627.CrossrefGoogle Scholar
5 Cruickshank J, Moyes JM. The presence and significance of nitrites in urine. BMJ. 1914; 2: 712–713.Google Scholar
6 Palmer RM, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature. 1988; 333: 664–666.CrossrefMedlineGoogle Scholar
7 Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS. Macrophage oxidation of l-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry. 1988; 27: 8706–8711.CrossrefMedlineGoogle Scholar
8 Gladwin MT, Shelhammer JH, Schechter AN, Pease-Fye ME, Waciawiw MA, Panza JA, Ognibene FP, Cannon RO. Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans. Proc Natl Acad Sci U S A. 2000; 97: 11482–11487.CrossrefMedlineGoogle Scholar
9 Dejam A, Hunter CJ, Tremonti C, Pluta RM, Hon YY, Grimes G, Partovi K, Pelletier MM, Oldfield EH, Cannon RO, Schechter AN, Gladwin MT. Nitrite infusion in humans and nonhuman primates: endocrine effects, pharmacokinetics, and tolerance formation. Circulation. 2007; 116: 1821–1831.LinkGoogle Scholar
10 Lundberg JO, Weitzberg E, Gladwin MT. The nitrate-nitrite-nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008; 7: 156–167.CrossrefMedlineGoogle Scholar
11 Dezfulian C, Raat N, Shiva S, Gladwin MT. Role of the anion nitrite in ischemia-reperfusion cytoprotection and therapeutics. Cardiovasc Res. 2007; 75: 327–338.CrossrefMedlineGoogle Scholar
27 Bellamy A. The development of nitroglycerine as an explosive. Atti del convegno in celerazione del centenario della morte di Ascanio Sobrero. Turín, Italy: University of Turín; 1989.Google Scholar
28 Fant K. Alfred Nobel: En Biografi. Stockholm, Sweden: Norstedts Förlag; 1991: 302.Google Scholar
29 Chen Z, Zhang J, Stamler JS. Identification of the enzymatic mechanism of nitroglycerine bioactivation. Proc Natl Acad Sci U S A. 2002; 99: 8306–8311.CrossrefMedlineGoogle Scholar
30 Janero DR, Bryan NS, Saijo F, Dhawan V, Schwalb DJ, Warren MC, Feelisch M. Differential nitros(yl)ation of blood and tissue constituents during glyceryl trinitrate biotransformation in vivo. Proc Natl Acad Sci U S A. 2004; 101: 16958–16963.CrossrefMedlineGoogle Scholar
31 Reichert ET, Mitchell SW. On the physiological action of potassium nitrite. Am J Med Sci. 1880; 156: 158–180.Google Scholar
32 Atkinson GA. The physiology of the nitrites and nitro-glycerine. J Anat Physiol. 1888; 22: 225–239, 351–371.MedlineGoogle Scholar
33 Densham B. The adjuvant action of the lactate ion on the vaso-dilator effect of sodium nitrite. J Physiol. 1927; 63: 175–179.CrossrefMedlineGoogle Scholar
34 Hay M. Nitrite of sodium in the treatment of angina pectoris. Practitioner. 1883; 30: 170–194.Google Scholar
36 Squibb’s Materia Medica. New York, NY: E.R. Squibb & Sons; 1906.Google Scholar
37 Deusch G, Liepelt A. Die Hautkapillaren beim arteriellen Hochdruck und ihre Beeinflussung durch Nitrite. Deutsches Arch Klin Med. 1928; 160: 207–211.Google Scholar
38 Hare HA, Caspari C, Rusby HH. National Standard Dispensatory. Philadelphia, Pa: Lea Bros & Co; 1905.Google Scholar
39 Bennett RG. Materia Medica and Pharmacy for Medical Students. London, UK: HK Lewis; 1921.Google Scholar
40 Blumgarten AS. Textbook of Materia Medica and Therapeutics. New York, NY: MacMillan; 1934.Google Scholar
41 Modin A, Bjorne H, Herulf M, Alving K, Weitzberg E, Lundberg JO. Nitrite-derived nitric oxide: a possible mediator of “acidic-metabolic” vasodilation. Acta Physiol Scand. 2001; 171: 9–16.MedlineGoogle Scholar
42 Zweier JL, Wang P, Samouilov A, Kuppusamy P. Enzyme-independent formation of nitric oxide in biological tissues. Nat Med. 1995; 1: 804–809.CrossrefMedlineGoogle Scholar
43 Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK, Waclawiw MA, Zalos G, Xu X, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO, Gladwin MT. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med. 2003; 9: 1498–1505.CrossrefMedlineGoogle Scholar
44 Angelo M, Singel DJ, Stamler JS. An S-nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate. Proc Natl Acad Sci U S A. 2006; 103: 8366–8371.CrossrefMedlineGoogle Scholar
45 Hunter CJ, Dejam A, Blood AB, Shields H, Kim-Shapiro DB, Machado RF, Tarekegn S, Mulla N, Hopper AO, Schechter AN, Power GG, Gladwin MT. Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat Med. 2004; 10: 1122–1127.CrossrefMedlineGoogle Scholar
46 Moya MP, Gow AJ, Califf RM, Goldberg RN, Stamler JS. Inhaled ethyl nitrite gas for persistent pulmonary hypertension of the newborn. Lancet. 2002; 360: 141–143.CrossrefMedlineGoogle Scholar
47 Beier S, Classen HG, Loeffler K, Schumacher E, Thöni H. Antihypertensive effect of oral nitrite uptake in the spontaneously hypertensive rat. Arzneimittelforschung. 1995; 45: 258–261.MedlineGoogle Scholar
48 Lauer T, Preik M, Rassaf T, Strauer BE, Deussen A, Feelisch M, Kelm M. Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action. Proc Natl Acad Sci U S A. 2001; 98: 12814–12819.CrossrefMedlineGoogle Scholar
49 Dalsgaard T, Simonsen U, Fago A. Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities. Am J Physiol Heart Circ Physiol. 2007; 292: H3072–H3078.CrossrefMedlineGoogle Scholar
50 Furchgott RF, Bhadrakom. Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs. J Pharmacol Exp Ther. 1953; 108: 129–143.MedlineGoogle Scholar
51 Farrari R, Cargoni A, Bernocchi P, Pasini E, Curello S, Ceconi C, Ruigrok TJC. Metabolic adaptation during a sequence of no-flow and low-flow ischemia. Circulation. 1996; 94: 2587–2596.CrossrefMedlineGoogle Scholar
52 Samlouilov A, Kuppusamy P, Zweier JI. Evaluation of the magnitude and rate of nitric oxide production from nitrite in biological systems. Arch Biochem Biophys. 1998; 357: 1–7.CrossrefMedlineGoogle Scholar
53 Butler AR, Ridd JH. Formation of nitric oxide from nitrous acid in ischemic tissue and skin. Nitric Oxide. 2004; 10: 20–24.CrossrefMedlineGoogle Scholar
54 Webb A, Bond R, McClean P, Uppal R, Benjamin N, Ahluwalia A. Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemic-reperfusion damage. Proc Natl Acad Sci U S A. 2004; 101: 13683–13688.CrossrefMedlineGoogle Scholar
55 Bryan NS, Fernandez BO, Bauer SM, Garcia-Saura MF, Milsom AB, Rassaf T, Maloney RE, Bharti A, Rodriguez J, Feelisch M. Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues. Nat Chem Biol. 2005; 1: 290–297.CrossrefMedlineGoogle Scholar
56 Granville AB. Further Observations on the Internal Use of the Hydro-cyanic (Prussic) Acid. London, UK: Butgess & Hill; 1820.Google Scholar
57 Magendie F. Sur l’emploi de l’acide prussique dans le traitement de plusieurs maladies de poitrine, et particulièrement dans la phtisie pulmonaire. Ann Chim Phys. 1817; 6: 347–360.Google Scholar
58 Commoner B. Cyanide inhibition as a means of elucidating the mechanism of cellular respiration. Bio Rev (Cambridge Phil Soc). 1940; 15: 168–201.CrossrefGoogle Scholar
59 Keilin D, Hartree EF. Cytochrome c and cytochrome oxidase. Proc Royal Soc London Series B. 1939; 127: 167–191.CrossrefGoogle Scholar
60 Kobert R. Über Cyanmethämoglobin und den Nachweis der Blausäure. Stuttgart, Enke: Germany; 1891; Cited by: Keilin D. The History of Cell Respiration and Cytochrome. Cambridge, UK: CUP; 1966.Google Scholar
61 Hug E. Accion del nitrite de sodio del hiposulfate de sodio en el tratamiento de la intoxication provocada por el ciannuro de potassio en al conejo. Rev Soc Argent Biol. 1932; 3: 270–276.Google Scholar
62 Viana C, Cagnoli H, Cendan J. L’action du nitrite de sodium dans l’intoxication par les cyanures. C R Seances Soc Biol Fil. 1934; 115: 1649–1651.Google Scholar
63 Matthew H, Lawson AAH. Treatment of Common Acute Poisonings. 4th ed. Edinburgh, Scotland: Churchill Livingstone; 1979.Google Scholar
64 Cucinell SA, Groff WA, Vick JA, Weger N. Treatment of cyanide poisoning. Fed Proc. 1974; 33: 234. Abstract.Google Scholar
65 Isom GE, Way JL. Lethality of cyanide in the absence of inhibition of liver cytochrome oxidase. Biochem Pharmacol. 1976; 25: 605–608.CrossrefMedlineGoogle Scholar
66 Baskin SI, Froehlich HL, Groff WA. The dissociation of reversal of cyanide (CN) toxicity and methemoglobin formation by nitrite (N) in the isolated heart. Fed Proc. 1986; 45: 196. Abstract.Google Scholar
67 Ali MY, Ping CY, Mok YY, Ling L, Whiteman M, Bhatia M, Moore PK. Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br J Pharmacol. 2006; 149: 625–634.CrossrefMedlineGoogle Scholar
74 Law WT. Sodium nitrite in the treatment of epilepsy. Practitioner. 1882; 28: 420–424.Google Scholar
75 Ringer S, Murrell W. Nitrite of sodium as a toxic agent. Lancet. 1883; 2: 766–767.Google Scholar
76 van Grinsven HJM, Ward MH, Benjamin N, de Kok TM. Does the evidence about health risks associated with nitrate ingestion warrant an increase of the nitrate standard for drinking water? Environ Health. 2006; 5: 26–30.CrossrefMedlineGoogle Scholar
78 Weller R, Pattullo S, Smith L, Golden M, Ormerod A, Benjamin N. Nitric oxide is generated on the skin surface by reduction of sweat nitrate. J Invest Dermatol. 1996; 107: 327–331.CrossrefMedlineGoogle Scholar
80 Weller R, Price RJ, Ormerod AD, Benjamin N, Leifert C. Antimicrobial effect of acidified nitrite on dermatophyte fungi, Candida and bacterial skin pathogens. J Appl Microbiol. 2001; 90: 648–652.CrossrefMedlineGoogle Scholar
81 Tucker AT, Pearson RM, Cooke ED, Benjamin N. Effect of nitric-oxide-generating system on microcirculatory blood flow in skin of patients with severe Raynaud’s syndrome: a randomised trial. Lancet. 1999; 354: 1670–1675.CrossrefMedlineGoogle Scholar
82 Khan F, Pearson RJ, Newton DJ, Belch JJF, Butler AR. Chemical synthesis and microvascular effects of new nitric oxide donors in humans. Clin Sci. 2003; 195: 577–584.Google Scholar
83 Weller R, Finnen MJ. The effects of topical treatment with acidified nitrite on wound healing in normal and diabetic mice. Nitric Oxide. 2006; 15: 395–399.CrossrefMedlineGoogle Scholar
84 Ts’ao TC, Ho PY, Needham J. An early mediaeval Chinese alchemical text on aqueous solutions. Ambix. 1959; 7: 122–155.Google Scholar
85 Butler AR, Moffett J. In: Lo EY, Cullen C, eds. Medieval Chinese Medicine: The Dunhuang Medical Manuscripts. London, UK: Routledge; 2004.Google Scholar
86 Duncan C, Dougall H, Johnson P, Green S, Brogan R, Leifert C, Smith L, Golden M, Benjamin N. Chemical generation of nitric oxide from the enterosalivary circulation of dietary nitrate. Nat Med. 1995; 1: 546–551.CrossrefMedlineGoogle Scholar
87 Pietsch G. Dissertation sur la Generation du Nitre. Berlin, Germany: Chez Haude et Spener; 1746.Google Scholar
110 Addiscott TM, Benjamin N. Are you taking your nitrate? Food Sci Technol Today. 2000; 14: 59–61.Google Scholar
111 L’Hirondel JJ, L’Hirondel J-L. Nitrate and Man: Toxic, Harmless or Beneficial? Oxford, UK: CABI Publishing; 2002.Google Scholar
112 Suschek CV, Schewe T, Sies H, Kroncke KD. Nitrite, a naturally occurring precursor of nitric oxide that acts like a “prodrug.” Biol Chem. 2006; 387: 499–506.MedlineGoogle Scholar
113 Shiva S, Sack MN, Greer JJ, Duranski M, Ringwood LA, Burwell L, Wang X, Macarthur PH, Shoja A, Raghavachari N, Calvert JW, Brookes PS, Lefer DJ, Gladwin MT. Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer. J Exp Med. 2007; 204: 2089–2102.CrossrefMedlineGoogle Scholar
114 Lundberg JO, Feelisch M, Bjorne H, Jansson EA, Weitzberg E. Cardioprotective effects of vegetables: is nitrate the answer? Nitric Oxide. 2006; 15: 359–362.CrossrefMedlineGoogle Scholar
115 Richardson G, Hicks SL, O’Byrne S, Frost MT, Moore K, Benjamin N, McKnight GM. The ingestion of inorganic nitrate increases gastric S-nitrosothiol levels and inhibits platelet function in humans. Nitric Oxide. 2002; 7: 24–29.CrossrefMedlineGoogle Scholar
116 Larsen FJ, Ekblom B, Sahlin K, Lundberg JO, Weitzberg E. Effects of dietary nitrate on blood pressure in healthy volunteers. N Engl J Med. 2006; 355: 2792–2793.CrossrefMedlineGoogle Scholar
117 Larsen FJ, Weitzberg E, Lundberg JO, Ekblom B. Effects of dietary nitrate on oxygen cost during exercise. Acta Physiol. 2007; 191: 59–66.CrossrefMedlineGoogle Scholar
118 Erzurum SC, Ghosh S, Janocha AJ, Xu W, Bauer S, Bryan NS, Tejero T, Hemann C, Hille R, Stuehr DJ, Feelisch M, Beall CM. Higher blood flow and circulating nitric oxide products offset high-altitude hypoxia among Tibetans. Proc Natl Acad U S A. 2007; 104: 17593–17598.CrossrefMedlineGoogle Scholar