Nitric Oxide Biology
Nitric oxide has recently been implicated in hypoxic vasodilation in the circulation (Kim-Shapiro, D. B., M. T. Gladwin, et al. (2005). “The reaction between nitrite and hemoglobin: the role of nitrite in hemoglobin-mediated hypoxic vasodilation.” Journal of Inorganic Biochemistry 99: 237-246). As early as 1880, nitrite was described in terms of its vasodilatory abilities (Reichert, E. T. and S. W. Mitchell (1880). “On the physiological action of potassium nitrite, with a note on the physiological action on man.” Am J Med Sci 159: 158-180) and much later, Furchgott used acidified sodium nitrite to relax precontracted aortic strips in 1953 (Furchgott, R. F. and S. Bhadrakom (1953). “Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs.” J Pharcol Exp Ther 108(2): 129-143). Both studies used supra-physiological concentrations of nitrite. However, recent studies have rediscovered the vasodilatory effect of nitrite on forearm and systemic blood flow after nitrite infusion. Cosby et al. (Cosby, Partovi et al. 2003) suggested that nitrite is a large intravascular storage pool for NO and that nitrite bioactivation to NO could dilate regions with tissue oxygen debt in the human circulation. Nitrite is found in high abundance throughout the mammalian organ system (Bryan, Rassaf et al. 2004). It is normally a short-lived, highly regulated ion in the circulation (200-600 nM) with a half life in whole blood of 110 seconds (Kelm, M. (1999). “Nitric oxide metabolism and breakdown.” Biochim Biophys Acta 1411: 273-289). Two independent groups have recently demonstrated the cytoprotective effects of nitrite in ischemia-reperfusion injury (Webb, Bond et al. 2004; Duranski, Greer et al. 2005). Duranski et al attribute nitrite's protective effects to the reduction of nitrite to NO by the reductase activity of hemoglobin. The study by Webb et al using an isolated heart setup was in the absence of blood, clearly demonstrating that the myocardial tissue itself can metabolize nitrite without the need for hemoglobin. Moreover, inhalation of nitrite selectively dilates the pulmonary circulation under hypoxic conditions in vivo in sheep (Hunter, C. J., A. Dejam, et al. (2004). “Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator.” Nat Med 10: 1122-1127). Experiments in primates revealed a beneficial effect of long-term application of nitrite on cerebral vasospasm (Pluta, Dejam et al. 2005). Topical application of nitrite improves skin infections and ulcerations (Hardwick, J. B., A. T. Tucker, et al. (2001). “A novel method for the delivery of nitric oxide therapy to the skin of human subjects using a semi-permeable membrane.” Clin Sci (Lond) 100(4): 395-400). Furthermore, in the stomach, nitrite-derived NO seems to play an important role in host defense (Duncan, C., H. Dougall, et al. (1995). “Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate.” Nat Med 1(6): 546-551; Dykhuizen, R. S., R. Frazer, et al. (1996). “Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense.” Antimicrob Agents Chemother 40(6): 1422-1425) and in regulation of gastric mucosal integrity (Bjorne, H. H., J. Petersson, et al. (2004). “Nitrite in saliva increases gastric mucosal blood flow and mucus thickness.” J Clin Invest 113(1): 106-114). All of these studies together along with the observation that nitrite can act as a marker of NOS activity (Kleinbongard, Dejam et al. 2003) opened a new avenue for the diagnostic and therapeutic application of nitrite, especially in cardiovascular diseases, using nitrite as marker as well as an active agent. However, it is still not known how and to what extent nitrite reduction to NO occurs or how the NO-independent effects of nitrite contribute to the cytoprotection of ischemia/reperfusion insult.
Nitric oxide has clearly emerged as an important molecule in biology, but, historically, its effects on the endogenous NO pathway have been poorly investigated. Nitrite is also a common clinical and laboratory chemical that is used as a vasodilator (Reichert and Mitchell 1880), bronchodilator (Hunter, Dejam et al. 2004), intestinal relaxant (Kozlov, A. V., B. Sobhian, et al. (2001). “Organ specific formation of nitrosyl complexes under intestinal ischemia-reperfusion in rats involves NOS-independent mechanism(s).” Shock 15: 366-371) and used as an antidote for cyanide poisoning (Chen, K. K. and C. L. Rose (1952). “Nitrite and thiosulfate therapy in cyanide poisoning.” J Am Med Assoc 149(2): 113-119). A two year study on the carcinogenicity of nitrite by NIH has conclusively found that there was no evidence of carcinogenic activity by sodium nitrite in male or female rats or mice (Program, N. T. (2001). On The Toxicology and Carcinogenesis Studues of Sodium Nitrite. U.S. D. o. H. a. H. Services, National Institute of Health. NTP TR 495: 1-276). Early studies on nitrogen balance in humans and analyses of fecal and ileostomy samples indicated that nitrite and nitrate are formed de novo in the intestine. It was these early findings by Tannenbaum et al. (Tannenbaum, S. R., D. Fett, et al. (1978). “Nitrite and nitrate are formed by endogenous synthesis in the human intestine.” Science 200: 1487-1488) that significantly altered conceptions of human exposure to exogenous nitrite and nitrates and represented the original observations that would eventually lead to the discovery of the L-arginine:NO pathway. Prior to these studies it was thought that steady-state levels of nitrite and nitrate originated solely from the diet and from nitrogen-fixing enteric bacteria. Endogenous sources of nitrite in mammals are derived from: 1. oxidation of endogenous nitric oxide, 2. nutritional sources such as meat, vegetable and drinking water, 3. reduction of salivary nitrate by commensal bacteria in the mouth and gastrointestinal tract. The discovery of the NO pathway and the emerging biomedical applications of nitrite and nitrate necessitate a paradigm shift on the role of nitrite and nitrate in physiology.
Nitrate/Nitrite Reduction to NO
Humans, unlike prokaryotes, are thought to lack the enzymatic machinery to reduce nitrate back to nitrite. However, due to the commensal bacteria that reside within the human body it has been demonstrated that these bacteria can reduce nitrate thereby supplying an alternative source of nitrite (Goaz, P. W. and H. A. Biswell (1961). “Nitrite reduction in whole saliva.” J Dent Res 40: 355-365; Tannenbaum, Sinskey et al. 1974; Ishiwata, H., A. Tanimura, et al. (1975). “Nitrite and nitrate concentrations in human saliva collected from salivary ducts.” J Food Hyg Soc Jpn 16: 89-92; van Maanen, van Geel et al. 1996). Therefore dietary and enzymatic sources of nitrate are now a potentially large source of nitrite in the human body. Nitrate is rapidly absorbed in the small intestines and readily distributed throughout the body (Walker, R. (1996). “The metabolism of dietary nitrites and nitrates.” Biochem Soc Trans 24(3): 780-785). As much as 25% of the ingested nitrate is actively taken up by the salivary glands to be excreted in the saliva (Spiegelhalder, B., G. Eisenbrand, et al. (1976). “Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in vivo formation of N-nitroso compounds.” Food Cosmet Toxicol 14: 545-548). Approximately 20% of the salivary nitrate is then reduced to nitrite by bacteria in the mouth (Spiegelhalder, Eisenbrand et al. 1976) and then disproportionates with formation of NO after entering the acidic environment of the stomach. This nitrate pathway to NO has been shown to help reduce gastrointestinal tract infection, increase mucous barrier thickness and gastric blood flow (Pique, J. M., B. J. Whittle, et al. (1989). “The vasodilator role of endogenous nitric oxide in the rat gastric microcirculation.” Eur. J. Pharmacol 174(2-3): 293-296; Brown, J. F., P. J. Hanson, et al. (1992). “Nitric oxide donors increase mucus gel thickness in rat stomach.” Eur. J. Pharmacol 223(1): 103-104; McKnight, G. M., L. M. Smith, et al. (1994). “Chemical synthesis of nitric oxide in the stomach from dietary nitrate in humans.” Gut 40(2): 211-214; Walker 1996). The concentrations of nitrate in drinking water are usually <10 mg/L in the absence of bacterial contamination (Kross, B. C., G. R. Hallberg, et al. (1993). “The nitrate concentration of private well water in Iowa.” Am J Public Health 83(2): 270-272). Vegetables, especially beets, celery, and leafy vegetables like lettuce and spinach are rich in nitrates (Meah, M. N., N. Harrison, et al. (1994). “Nitrate and nitrite in foods and the diet.” Food Addit Contam 11(4): 519-532; Walker 1996; Vallance, P. (1997). “Dietary nitrate: poison or panacea?” Gut 40(2): 211-214). Other vegetables contain nitrate at lower concentrations, but because they are consumed in greater quantity, they may contribute more nitrate and thus nitrite from the diet. For the average population, most nitrate exposure (86%) comes from vegetables, whereas the primary contributors to nitrite intake are cured meats (39%), baked goods and cereals (34%), and vegetables (16%). The National Research Council report The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds (NRC 1981) reported estimates of nitrite and nitrate intake based on food consumption tables. They report that the average total nitrite and nitrate intake in the U.S. was 0.77 mg and 76 mg, respectively per day. Nitrite and nitrate are excreted in the kidneys. Nitrate is excreted in the urine as such or after conversion to urea (Green, L. C., K. Ruiz de Luzuriaga, et al. (1981). “Nitrate biosynthesis in man.” Proc. Natl. Acad. Sci. USA 78(12): 7764-7768). Clearance of nitrate from blood to urine approximates 20 ml/min in adults (Wennmalm, A., G. Benthin, et al. (1993). “Metabolism and excretion of nitric oxide in humans. An experimental and clinical study.” Circ Res 73(6): 1121-1127), indicating considerable renal tubular reabsorption of this ion. There is little detectable nitrite or nitrate in feces (Bednar, C. and C. Kies (1994). “Nitrate and Vitamin C from fruits and vegetables: impact of intake variations on nitrate and nitrite excretions in humans.” Plant Foods Hum Nutr 45(1): 71-80). There is some loss of nitrate and nitrite in sweat, but is not a major route of excretion (Weller, R., S. Pattullo, et al. (1996). “Nitric oxide is generated on the skin surface by reduction of sweat nitrate.” J Invest Dermatol 107(3): 327-331). Assuming the human body (70 kg) produces 1.68 mmole NO per day (based on 1 μmole/kg/hr NO production), an average daily intake of 0.77 mg of nitrite would equate to 11.1 μmoles per day and 76 mg nitrate would equate to 894 μmoles per day or roughly 1 mmole NOx per day from diet. This almost matches what the human body makes from NO, assuming most of the NO goes to stepwise oxidation to nitrite and nitrate.
The endogenous production of NO by NOS has been established as playing an important role in vascular homeostasis, neurotransmission, and host defense mechanisms (Moncada, S., R. M. J. Palmer, et al. (1991). “Nitric oxide: physiology, pathophysiology and pharmacology.” Pharmacol Rev 43(2): 109-142). The major pathway for NO metabolism is the stepwise oxidation to nitrite and nitrate (Yoshida, K., K. Kasama, et al. (1983). “Biotransformation of nitric oxide, nitrite and nitrate.” Int Arch Occup Environ Health 52: 103-115). In plasma or other physiological fluids or buffers, NO is oxidized almost completely to nitrite, where it remains stable for several hours (Kelm, M., M. Feelisch, et al. (1992). The Biology of nitric oxide. Physiological and Clinical Aspects. S. Moncada, M. A. Marletta, J. B. Hibbs and E. A. Higgs. London, Portland Press. 1: 319-322, hereby incorporated by reference herein; Grube, R., M. Kelm, et al. (1994). The Biology of Nitric Oxide. Enzymology, Biochemistry, and Immunology. S. Moncada, M. Feelisch, R. Busse and E. A. Higgs. London, Portland Press. 4: 201-204, hereby incorporated by reference herein); however, the half life of NO2 − in human whole blood is about 110 seconds (Kelm 1999).
The oxidation of NO by molecular oxygen is second order with respect to NO:
4NO2 −+4H+ (3)
whereby NO2, N2O3 and NO2 − represent nitrogen dioxide, dinitrogen trioxide and nitrite, respectively. It should be noted that N2O3 is a potent nitrosating agent by virtue of its ability to generate the nitrosonium ion (NO+). NO and nitrite are rapidly oxidized to nitrate in whole blood. As stated above, the half life of NO2 − in human blood is about 110 seconds (Kelm 1999). Nitrate on the other hand has a circulating half life of 5-8 hours (Tannenbaum, S. R. (1994). “Nitrate and nitrite: origin in humans.” Science 205: 1333-1335, hereby incorporated by reference herein; Kelm, M. and K. Yoshida (1996). Metabolic Fate of Nitric Oxide and Related N-oxides. Methods in Nitric Oxide Research. M. Feelisch and J. S. Stamler. Chichester, John Wiley and Sons: 47-58, hereby incorporated by reference herein). Although the mechanisms by which NO and NO2 − are converted to NO3 − in vivo are not entirely clear, there are several possibilities. During fasting conditions with low intake of nitrite/nitrate, enzymatic NO formation from NOS accounts for the majority of nitrite (Rhodes, P., A. M. Leone, et al. (1995). “The L-arginine:nitric oxide pathway is the major source of plasma nitrite in fasted humans.” Biochem Biophys Res Commun 209: 590-596).
NO production from nitrite has been described in infarcted heart tissue (Zweier, J. L., et al., Enzyme-independent formation of nitric oxide in biological tissues. Nature Medicine, 1995. 1(8): p. 804-809). Nitrite reductase activity in mammalian tissues has been linked to the mitochondrial electron transport system (Walters, C. L., R. J. Casselden, and A. M. Taylor, Nitrite metabolism by skeletal muscle mitochondria in relation to haem pigments. Biochim Biophys Acta, 1967. 143: p. 310-318; Reutov, V. P. and E. G. Sorokina, NO-synthase and nitrite-reductase components of nitric oxide cycle. Biochemistry (Mosc), 1998. 63(7): p. 874-884; Kozlov, A. V., K. Staniek, and H. Nohl, Nitrite reductase activity is a novel function of mammalian mitochondria. FEBS Lett, 1999. 454: p. 127-130; Nohl, H., et al., Mitochondria recycle nitrite back to the bioregulator nitric monoxide. Acta Biochim Pol, 2000. 47: p. 913-921; Tischner, R., E. Planchet, and W. M. Kaiser, Mitochondrial electron transport as a source for nitric oxide in the unicellular green algae Chlorella sorokiniana. FEBS Lett, 2004. 576: p. 151-155), protonation (Zweier, J. L., et al., Enzyme-independent formation of nitric oxide in biological tissues. Nature Medicine, 1995. 1(8): p. 804-809; Hunter, C. J., et al., Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat Med, 2004. 10: p. 1122-1127), deoxyhemoglobin (Hunter, C. J., et al., Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat Med, 2004. 10: p. 1122-1127; Cosby, K., et al., Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nature Medicine, 2003. 9: p. 1498-1505), and xanthine oxidase (Li, H., et al., Characterization of the effects of oxygen on xanthine oxidase-mediated nitric oxide formation. J. Biol Chem, 2004. 279: p. 16939-16946; Alikulov, Z. A., N. P. L'vov, and V. L. Kretovich, Nitrate and nitrite reductase activity of milk xanthine oxidase. Biokhimiia, 1980. 45(9): p. 1714-1718; Webb, A., et al., Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc Natl Acad Sci USA, 2004. 101(13683-13688)). Mitochondrial nitrite reduction has been shown to occur by ubiquinol (Kozlov, A. V., K. Staniek, and H. Nohl, Nitrite reductase activity is a novel function of mammalian mitochondria. FEBS Lett, 1999. 454: p. 127-130; Nohl, H., et al., The multiple functions of coenzyme Q. Bioorg Chem, 2001. 29(1): p. 1-13) and cytochrome c oxidase (Castello, P. R., et al., Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in eukaryotes. Cell Metab, 2006. 3(4): p. 277-87) with subsequent binding of the NO produced to cytochrome bc1 site of complex III or complex IV resulting in oxygen-dependent reversible inhibition of mitochondrial respiration (Takehara, Y., et al., Oxygen-dependent reversible inhibition of mitochondrial respiration by nitric oxide. Cell Struct Funct, 1996. 21(4): p. 251-8). The acidic reduction of nitrite requires protonation and a one-electron reduction. The relatively low pKa of nitrite (3.34) (Principles of Modern Chemistry. Third ed, ed. D. W. Oxtoby and N. H. Nachtrieb. 1996, Fort Worth: Harcourt Brace College Publishers. 848) limits this activity in physiology but it can occur in the stomach or during ischemic events when tissue pH falls. Since many different pathways have been shown to be able to reduce nitrite but require different conditions and substrates for optimal nitrite reduction, it is likely that all pathways may become relevant but at different oxygen tension, substrate availability, and perhaps even compartment specific needs.
The evolution of nitrite from a vilified substance that generates carcinogenic nitrosamines in the stomach, to a life-saving drug that liberates a protective agent (NO) during hypoxic events, as well as performs many actions independent of NO, warrants a re-evaluation of nitrite in biology. With nitrite acting as both an end product of NO synthesis and a reservoir for NO, it is therefore a critical homeostatic molecule in NO biology.
The collective evidence reviewed in this section strongly supports the notion that there is a fundamental and physiological basis for developing nitrite-based therapeutics. It is not understood how orally ingested nitrite (pKa 3.8) can survive the acidic environment of the stomach (pH 1-2). Furthermore, once nitrite is absorbed into the bloodstream it is known to be quickly oxidized to nitrate with a half life of 110 seconds. Orally administered nitrite in specific combination with nitrate can extend the therapeutic range of nitrite from seconds to tens of minutes providing an approach to treat or reduce injury from heart attack with nitrite e.g., by increasing stores, of nitrite.
The human diet exerts important long-term effects on vital body functions and thereby makes an important contribution to health and disease. While high intake of cholesterol, saturated fat, salt, and sugar are associated with a greater risk for cardiovascular disease, conventional wisdom has it that the opposite is true for abundant consumption of fruits and vegetables. A diet rich in fruits and vegetables is associated with a lower risk of certain forms of cancer and cardiovascular disease. Recent epidemiological studies suggest a cardioprotective action afforded specifically by green leafy vegetables. Green leafy vegetables such as spinach and lettuce, in addition to being rich in antioxidants are especially rich in nitrite and nitrate as are berries, grapes, and a few other fruits. The high content of nitrite and nitrate is a major factor contributing to the positive health effects of certain vegetables via bioconversion to NO which exerts protective effects on the cardiovascular system. A continuous intake of nitrite- and nitrate-containing food such as green leafy vegetables and berries may ensure that blood and tissue levels of NO are maintained at a level sufficient to compensate for any disturbances in endogenous NO synthesis.
Dietary sources of NO metabolites improve circulation and oxygen delivery and lead to better health and increased energy. This dietary pathway not only provides essential nutrients for NO production but also provides a rescue pathway for people at risk for cardiovascular disease while providing the nutrition and protection of a high vegetable diet in the form of a daily supplement formulation which renders subjects protected from injury from heart attack or other cardiovascular events, i.e. stroke, pulmonary embolism. This strategy including supplementation may serve as an inexpensive cardioprotective regimen which may delay or reduce the onset or progression of cardiovascular or heart disease and protect from myocardial infarction. Although such amounts may be found in a high vegetable diet, the time it would take to consume the required assortment of vegetables as well as the impact on the digestive system would adversely impact the absorption and/or bioavailability. Moreover, the reaction of other compounds and nutrients in the naturally occurring vegetable assortment may also adversely impact the impact the absorption and/or bioavailability. Thus, a nitric oxide supplement in e.g., daily dose formulations are advantageous because it increases the absorption and/or bioavailability.