1. The Saga of Sodium Nitrite Nitrite and nitrate have been used for some 5000 years or more to cure and preserve meat 1891 – first publication showing that nitrate-to-nitrite conversion by bacteria was the source of preservative effects 1899 – first suggestion of nitrite as the source of cured meat color 1901 – discovery that nitric oxide from nitrite was source of cured meat color 1925 – first regulatory (USDA) limits for nitrate and nitrite were established

2. The Saga of Sodium Nitrite (cont’d) 1920’s-1950’s – Industry and university researchers refined curing processes - role of nitrite in cured meat clarified -potent antimicrobial agent -powerful antioxidant -distinctive cured meat flavor -unique, attractive color that is stable

3. The Saga of Sodium Nitrite (cont’d) 1956 – a problem emerges…; first report of carcinogenic nitrosamines found in fish meal cured with very high nitrite concentrations 1960’s – intensive studies of the relationship between nitrite, nitrosamines and human cancer… …nitrosamines were found in some cured meats and the perceptions of cured meats as a contributor to human cancer became firmly entrenched

4. The Saga of Sodium Nitrite (cont’d) Mid 1970’s – Extensive research in meat curing showed that nitrosamines were not an issue with proper control of nitrite concentrations - use of reductants and improved understanding of time/temperature relationships in meat curing reduced residual nitrite to very low concentrations and eliminated nitrosamines

5. The Saga of Sodium Nitrite (cont’d) Late 1970’s – discovery of endogenous nitrate/nitrite in the human intestine and in saliva begins to suggest a biological role for these compounds -leads to discovery of nitric oxide as a very important biological molecule -beginning of the turnaround in scientific perceptions of nitrite

6. 1986 – Nitric oxide found to be “the body’s messenger” resulting in explosion of research in biology and physiology

7. Nitric oxide… -regulates blood pressure and blood flow -provides neurotransmission/brain function -kills bacteria and other immune system challengers -promotes healing (also provides critical role in the function of the drug Seldinafil )

8. 2002 – Importance of nitric oxide for blood transfusions suggested -25% greater heart attacks/strokes observed following transfusions - collected blood loses NO within hours of collection to 30% of initial after one day; 17% after 7 days and virtually none at 42 days - work with dogs showed decreased heart attacks with NO-treated transfusions Reynolds et al., Proc. Nat. Acad. Sci. 104:17058(2007).

9. 2005 – Protective role of nitric oxide in endothelial function, cardiovascular disease and heart attacks suggested - first reports of nitrite as major storage form of NO in blood and tissue - role for dietary nitrate and nitrite suggested for cardiovascular health

11. Source of nitric oxide (NO) in biology is nitric oxide synthase (NOS) conversion of arginine to citrulline L-arginine + O2 → L-citrulline + NO + H2O Nitrite serves as a low-oxygen source of NO thus nitrite may be a critical protective component for increasing blood flow when oxygen is limited Current questions –role of blood and tissue nitrite concentrations? –role of dietary nitrite?

13. Dietary nitrite in mice Insufficiency of dietary nitrite (7 days) decreased steady state nitric oxide and increased injury from heart attacks Dietary supplementation with nitrite restored NO homeostasis within 7 days, reversed injury and improved survival from heart attacks Dietary nitrite intake can restore NO homeostasis and blood and tissue nitrite to a greater extent (emphasis mine) than change in NOS activity Dietary supplementation reduced plaque formation in genetically modified, atherosclerotic-susceptible mice Bryan et al., Proc. Nat. Acad. Sci. 104:19144(2007)

14. Human Daily NO x Intake Varies with Diet NO x (mg)/Serving Western Menu Mediterranean Menu Breakfast Bagel w/ Cr Cheese Black Coffee (12 oz) Toast w/ Jam & Butter Cappuccino (Espresso + Milk) AM Snack Carrot Nut Muffin Diet Coke (12 oz) Yogurt (Strawberry) Carrot Juice (12 oz) Lunch Big Mac Large French Fried Diet Coke (12 oz) Mediterranean Wrap Garden Veg. Soup Mineral Water (12 oz) PM Snack Snickers Black Coffee (12 oz) Trail Mix Orange Juice (12 oz) Dinner Cheese Pizza (4 slices) Diet Coke (12 oz) Salmon (Smoked) Red Wine (12 oz) from Bryan, MIRC (2007)

15. Importance of Dietary Nitrate/Nitrite Reduction of blood pressure by 5%-10%, and even attenuates sodium-induced hypertension Mean arterial pressures in animals treated with high salt (4% of diet) and nitrate (0.08 or 0.8 g/day). Carlstom et al., Cardio. Res. 89:574 (2011).

16. Importance of Dietary Nitrate/Nitrite Improves exercise performance –200 mg beetroot consumption 60 minute prior to improved running performance (speed in second half of trial) by 5% Murphy et al., J. Acad. Nutr. Diet. 112:548 (2012) Many competitive-class athletes now utilize beetroot supplements to enhance performance Cermak et al., Int. J. Sports Nutr. Exer. Met. 22:64 (2012)

17. Importance of Dietary Nitrate/Nitrite Metabolic Activity and Circulatory Responses to Maximal Exercise After Dietary Supplementation with Nitrate Max O 2 Max HR O 2 Pulse Time/Exh. Max Lac. (ml/beat) (seconds)____________ Nitrate 3.62* 183 19.8 563* 10.9 Placebo 3.72 183 20.5 524 10.5___ *significantly different Conclusion: reduced oxygen consumption and improved endurance = improved muscle efficiency Larsen et al., Free Rad. Biol. Med. 48:302 (2010)

18. Importance of Dietary Nitrate/Nitrite Nitrite concentration in human breast milk (colostrum) –Increases dramatically for the first three days after birth Declines by 60% at 4 th day, 90% after 8 days –Hypothesized to be important to infants before G.I. tract bacteria are established –Documented to prevent newborn necrotizing enterocolitis resulting from limited intestinal blood flow by nursing mothers and by administration of nitrite as therapy for NEC

19. “Most provocatively, these studies suggest that the cardioprotective effects of leafy green vegetables could derive from nitrite, in addition to the often cited antioxidant effects of these food groups.” Gladwin et al. Nat. Chem. Biol. 1:308-314(2005) “The solution to these problems should…lead to the potential consideration of nitrite as an “essential nutrient”…” Gladwin et al. Nat. Chem. Biol. 1:308-314(2005)

20. “Could it be that the stringent regulations on nitrite/nitrate in drinking water and in foods contribute to the contemporary diseases of today due to inadequate nitrite or nitrate in the diet and NOS activity unable to supply enough nitrite to maintain cellular homeostasis?” Bryan, Free Rad. Biol. and Med. 41:691-701(2006) “Therefore, nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a treatment modality for cardiovascular disease.” Bryan et al., Proc. Nat. Acad. Sci. Early Ed. (2007). www.pnas.org/cgi/doi/10.1073/pnas.0706579104

21. Nitrite: Has profound effects on human physiology Dietary intake may be protective and maintain/restore endothelial function Dietary supplementation may inhibit onset and progression of atherosclerosis Dietary levels of nitrite and nitrate should be reconsidered for optimal health Increasing steady state NO x concentrations is a natural physiological response in humans and highlights the physiological justification and safety of such intervention from Bryan, MIRC (2007)

22. In addition, very recent work shows that dietary nitrite results in: Faster wound healing (revascularization) Reduced blood triglycerides - sunlight effect (see handout), besides blood pressure decrease, may also partially explain winter weight gains, summer weight losses (or lesser gains!)

23. There are several human clinical trials currently underway on the use of nitrite 1.Nitrite Infusion Studies Condition: healthy 2.Nitrite Infusion in Healthy Volunteers Condition: healthy 3.The Role of Nitrite in Preconditiioning Mediated Tolerance to Ischemic Stress Conditions: ischemic stress; transient non-lethal ischemia 4.Dietary Nitrate and Nitrite to Increase Nitric Oxide in Patients With Coronary Artery Disease Condition: coronary arteriosclerosis 5.Biomarkers for Cardiovascular Disease Condition: coronary artery disease 6.Endothelial Cell Dysfunction in Pulmonary Hypertension Condition: hypertension, pulmonary 7.Heart Disease in Sickle Cell Anemia Conditions: sickle cell anemia; pulmonary hypertension

24. Nitric Oxide: Biology and Chemistry. Although one of the simplest biological molecules in nature, nitric oxide has found its way into nearly every phase of biology and medicine ranging from its role as a critical endogenous regulator of blood flow and thrombosis to a principal neurotransmitter mediating erectile function to a major pathophysiological mediator of inflammation and host defense. These major discoveries have stimulated intense and extensive research into a vast array of fields including chemistry, molecular biology, and gene therapy. Nitric Oxide: Biology and Chemistry provides a forum for the rapid publication of scientific investigation of the highest quality across the broad field of nitric oxide research. Research Areas Include basic and clinical topics in: Cell Biology Molecular biology Biochemistry Immunology Pathology Genetics Physiology Pharmacology Disease processes Editor-in-Chief: J.R. Lancaster J.R. Lancaster

25. Nitric Oxide Dietary Supplement Commercially available as “NEO40 Daily” (www.neogenis.com) –Composed of proprietary blend of beetroot (nitrate), Hawthorn berry (nitrate reductase), citrulline and nitrite –Promoted for individuals over 40 when endogenous NO begins to decline –Daily use shown to increase plasma nitrite and nitrate, and decrease blood tryglycerides

27. For a long time in meat curing, nitrite has been MAGIC! Now for human health, nitrite is TRULY MAGIC!

28. Is nitrate/nitrite the newly discovered “Vitamin N”? Nitrate/nitrite offer new preventative or therapeutic strategies and new dietary guidelines for good health relative to diseases characterized by NO deficiency Definition of a vitamin = a group of organic substances essential in small quantities to normal metabolism, found in minute amounts in natural foods, deficiencies of which produce specific disorders. N. Bryan, Institute of Molecular Medicine for Prevention of Human Diseases, University of Texas. Encyclopedia of Meat Sciences (in press)