1. The Nitrate-Nitrite-NO Pathway Jon Lundberg Eddie Weitzberg Karolinska Institutet, Stockholm, Sweden

2. NOS L-arginine NO. sGC GTP cGMP↑ O2O2 NO 2 - Oxy-Hb Red blood cell NO 3 - blood smooth muscle endothelium

3. The truth about nitrate (NO 3 - ) Inert end product of NO metabolism Unwanted residue in the food chain

4. Inert end product of NO Desirable Substrate for The truth about nitrate (NO 3 - )

5. Nitric oxide: Role in the cardiovascular system NO synthase L-arginine + O 2 NO Platelet aggregation ↓ Reduced NO bioavailability is a central event in most cardiovascular disorders (Hypertension, atherosclerosis, stroke, myocardial infarction) Leucocyte adhesion ↓ Vasodilation Mitochondrial function

6. Background Measurements of exhaled NO NO (ppb) ?

7. NO Analyser Carbonated Water EXPERIMENTAL SETTINGS +5 min

8. . NO levels in expelled stomach gas Lundberg et al Gut 1994

9. Nitrite forms NO after protonation NO 2 - + H + → HNO 2 (pKa~3.3) 2 HNO 2 → N 2 O 3 + H 2 O → NO + NO 2 Gastric pH is 1-2 in fasting humans Saliva is very rich in nitrite (mM)

10. Why is saliva so rich in nitrite? In humans 25% of circulating nitrate (NO 3 - ) is actively taken up by the salivary glands and secreted in saliva Nitrate in plasma vs saliva, ratio 1:25 Bacteria on the tongue effectively reduce nitrate to nitrite Spiegelhalder Food Cosmet Toxicol 1976

11. Duncan Nature Med 1995

12. CLOSE Nitrate, mM Nitrite, µM Salivary nitate/nitrite levels after an oral nitrate intake Lundberg Govoni FRBM, 2004 Sodium nitrate 3 mg/kg

13. The Entero-salivary Circulation of Nitrate and its Reduction to Nitrite and NO In the Stomach Lundberg, Weitzberg, Gladwin Nature Rev Drug Discov 2008 NO 2 - + H+ → HNO 2 (pKa~3.3) 2 HNO 2 → N 2 O 3 + H 2 O → NO + NO 2

14. Does intragastric NO have any biological effects?

15. The dark side of dietary nitrate/nitrite -Promotes formation of potentially carcinogenic nitrosamines (gastric cancer) Nitrate content in food and drinking water is strictly regulated!

17. However...... Epidemiological data do not support a role of dietary nitrate in gastric cancer > 80% of our nitrate intake comes from vegetables! (in particular green leafy vegetables)

18. Exploring beneficial effects of dietary nitrate/nitrite

19. Antibacterial effects of gastric NO Benjamin et al. Nature -94 HCl........................................ 1-2 h24 h................................................................................................................................................................................................................................................................................................................................ E.coli

20. Antibacterial effects of gastric NO Benjamin et al. Nature -94 HCl + nitrite HCl + nitrite........................................ 1-2 h24 h ”No growth” E.coli

21. Effects of nitrate-derived NO on the gastric mucosa Human saliva 1.Fasting saliva (low nitrite, <50 µM) 2. Saliva collected after a nitrate intake (high nitrite, 1-2 mM)

22. Salivary nitrite increases gastric mucosal blood flow and mucus generation Mucus thicknessMucosal blood flow Bjorne et al J Clin Invest 2004 - Effects were cGMP mediated (inhibited by ODQ) - Intragastric NO gas increased - L-NAME/Indomethazine had no effect

23. Are these effects associated with gastroprotection?

24. Dietary nitrate, aspirin-like drugs and ulcers Nitrate 0,1/1 mmol/kg 7 days 4 hours Diclofenac 30 mg/kg Start of experimentGavage with Diclofenac Sacrifice Collection of materials Nitrate dose corresponds to 100-500 gr nitrate-rich vegetable/day in a human

25. Diclofenac, 30 mg/kg, 4 hours ControlNitrate, 1mmol/kg, 7 days Dietary nitrate and NSAID-induced Gastric Ulcers EA Jansson et al Free Radic Biol Med -07

26. Effects of dietary nitrate on NSAID-induced gastric ulcer EA Jansson et al Free Radic Biol Med -07

27. What happens if we remove the oral bacteria?

28. Effect of an antibacterial mouthwash on intragastric NO in rats Petersson Free Radic Biol Med 2009

29. Antibacterial mouthwash and nitrate-mediated gastroprotection Petersson Free Rad Biol Med 2009

30. Role of bacteria and dietary nitrate in intragastric NO generation Sobko et al Am J Physiol 2005 L-NAME had no effect!

31. Method for measurement of gastric NO in humans CLD Bjorne et al. Crit Care Med 2005

32. Saliva-dependent gastric NO in healthy volunteers 100  M, 50 ml

33. healthy subjects intubated patients Stomach NO in intubated patients

34. Intragastric nitrite administration in intubated patients Plasma nitrite (uM) 0.17+0.03 1.28 +0.2 0.26+ 0.03 2mM (100ml)

35. The Stomach as a Bioreactor for Chemical Interactions Between Nitrate/Nitrite and Other Dietary Components Olive oil Vegetables Fruit Wine Unsaturated fatty acids Nitrate Nitrite Polyphenols Vitamin C Polyphenols ethanol HNO 2 Nitrated fatty acids?NO/S-NOEthyl nitrite Nitrosamines Fish/Meat Protein Modified from Gago et al. FRBM 2007-2008

36. Will the nitrite formed in the oral cavity survive GI passage and enter the systemic circulation?

37. Lundberg & Govoni Free Rad Biol Med 2004 Dietary nitrate as a source of systemic nitrite - 10 subjects ingested sodium nitrate 8 mg/kg (= 100-300 g spinach) - With or without swallowing saliva Swallowing from start No swallowing first 60 min = start of swallowing

38. Antibacterial mouthwash and plasma nitrite - 7 healthy subjects, crossover design - Chlorhexidine (0.2 %) vs placebo mouthwash - NaNO 3 10 mg/kg - Repeated plasma/saliva samples for nitrite

39. Change in salivary nitrite after an oral nitrate load Δ Nitrite (µM) Govoni NO Biol Chem, in press

40. Change in plasma nitrite after an oral nitrate load Δ Nitrite (nM) Govoni NO Biol Chem, in press

41. Lundberg & Govoni Free Rad Biol Med 2004 Govoni et al NO Biol Chem, in press Conclusions from these studies…. - Dietary nitrate can be a major source of systemic nitrite - This requires enterosalivary circulation of nitrate and reduction by commensal bacteria in the mouth - Nitrate can be considered a nitrite ”prodrug” with a slow release profile (T 1/2 nitrate ~ 6h)

42. So, why is it interesting that dietary nitrate increases systemic nitrite levels?

43. Non-enzymatic NO production in the ischemic heart - NO production increased 10-fold after global ischemia - This NO was not inhibited by NOS inhibitors - Using isotopically labeled nitrite it was shown that nitrite is reduced to NO in the ischemic heart JL Zweier et al Nature Med 1995.

44. Is nitrite doing anything?

45. Vasodilatation by physiological amounts of nitrite Modin et al Acta Physiol Scand -01 -Rat aorta -Neutral (pH 7.4) or acidic buffer (pH 6.7) + nitrite

46. Cosby et al. Nature Med -03 Nitrite vasodilates the human circulation at near physiological concentrations Deoxy-Hb NO 2 - NO Oxy-Hb NO/NO 2 - NO 3 -

47. Multiple pathways exist for nitrite reduction to bioactive NO - Protons - Vitamin C/polyphenols - Deoxyhemoglobin/Deoxymyoglobin - Xanthine oxido reductase (XOR) - Mitochondrial cytochromes - Cytochrome P-450’s ”All pathways greatly enhanced during hypoxia!” Lundberg Weitzberg ATVB 2005 Lundbergf et al Nature Rev Microbiol 2004 Gladwin et al Nature Chem Biol 2005

48. Nitrite as a therapeutic? It seems to convert to NO preferentially during ischemia or low oxygen tensions Oxygen supply ↓ - NO synthase dysfunctional - nitrite reduction to NO ↑↑

49. 0000.20.51 2 24 Time (hours) LCA Ischemia Control Reperfusion Nitrite or Nitrate (48 nmoles, i.v.) Murine in vivo Myocardial Ischemia-Reperfusion Protocol * In Vivo area-at-risk - Evans Blue injection Infarct Size - 1.0% 2,3,5-TTC * Figure 2A Figure 2C Figure 2D Figure 2B Duransky, Gladwin, Lefer, JCI 2005 Nitrite and myocardial Infarction

50. Nitrite and Stroke Jung et al. Stroke 2006

51. Low Dose Nitrite Prevents Ischemia- Reperfusion Injury in Several Organs OrganRef Heart Webb, PNAS-04 Duranski, JCI-05 LiverDuranski, JCI-05 KidneyTripatara, J Am Soc Nephrol-07 StrokeJung, Stroke-06 Pulmonary embolismDias-Junior, Free Rad Biol Med-07 Systemic administration, (rats, mice, dogs)

52. Lundberg, Weitzberg, Gladwin Nature Rev Drug Discov 2008 Nitrite interactions with mitochondria How does nitrite protect?

53. Xanthine oxidase (XO ) Duranski J Clin Invest 2005 Nitrite and cardioprotection- In vivo

54. How much nitrite can be made systemically from nitrate in vegetables? The dose of nitrite needed for maximal cardioprotection in mice (48 nmoles or 3 µg ) corresponds to an oral nitrate load of around 200-300 mg in a human This is less than one beetroot or half a plate of spinach!

55. NO 3 -

56. JAMA. 1999:282:1233-9. J Natl Cancer Inst. 2004:96:1577-84. Ann Intern Med. 2001:134:1106-14 Vegetables and fruit protect against stroke and coronary heart disease ”Of the food groups analyzed, green leafy vegetable intake showed the strongest inverse association with cardiovascular disease”

57. So, does dietary nitrate affect cardiovascular function?

58. Dietary Nitrate and Blood Pressure - 17 healthy volunteers age 20-25 years - Nitrate supplementation (NaNO 3 0.1 mmol/kg/day) or placebo (NaCl) - Double blind placebo-controlled cross- over design - Nitrate amount corresponds to about 150- 200 g spinach, lettuce or beetroot

59. Larsen et al N Engl J Med 2006 Effects of Dietary Nitrate on Blood Pressure - 4 mmHg

60. Dietary patterns and Blood Pressure Appel et al. N.Engl.J.Med 1991 -459 healthy adults randomly divided into three groups given different diets for 8 weeks. The control diet was a typical American diet. The Fruit and vegetables diet provided more fruits and vegetables and fewer snacks and sweets than the control diet The combination diet was rich in fruits and vegetables, and had reduced amounts total fat and cholesterol. A diet rich in fruits and vegetables can substantially lower blood pressure.

61. Webb et al. Hypertension 2008 Beetroot juice and blood pressure -14 healthy volunteers - 500 ml fresh beetroot juice (45 mM nitrate)

62. Beetroot juice and blood pressure -6 healthy volunteers -spit/swallow -500 ml fresh beetroot juice (45 mM nitrate) Webb et al. Hypertension 2008

63. Dietary Nitrate Protects from Injury from Myocardial Infarction in mice Bryan et al PNAS (2007)

64. Dietary nitrate and oxygen consumption during exercise 9 male endurance athelets, (trained cyclists) 20-25 y 3 days supplementation with sodium nitrate/placebo (0.1 mol/kg/day) Randomized, double-blind, placebo-controlled cross- over design Graded exercise on a ergometer cycle

65. Oxygen consumption at 85 % of VO 2 max HR - No difference Lactate - No difference

66. Conclusions from this study Dietary nitrate reduces oxygen cost during submaximal exercise Maximal attainable work rate was intact Near identical lactate levels indicate more efficient energy production A dietary approach to decrease oxygen cost during exercise has never been described previously Mechanism of action needs to be clarified!

67. NO and mitochondria NO inhibits mitochondrial respirationCleeter 1994, Brown 1994 Nitrite is reduced to NO Kozlov 1999, Nohl 2000 NO promotes biogenesisNisoli 2003 Nitrite nitrosates complex 1Shiva 2007

68. Overall Summary and Speculation Nitrate is bioactivated in vivo to form nitrite and then NO- like bioactivity. Symbiotic oral bacteria play a central role in bioactivation of nitrate. Dietary Nitrate has protective effects in the GI tract Nitrate also regulates cardiovascular and metabolic function, and these effects may contribute to the cardioprotective effects of a diet rich in vegetables.

69. Lundberg, Weitzberg, Gladwin Nature Rev Drug Discov 2008 Two parallel systems for NO generation in mammals