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The Nitrate-Nitrite-NO Pathway Jon Lundberg Eddie Weitzberg

The Nitrate-Nitrite-NO Pathway Jon Lundberg Eddie Weitzberg Karolinska Institutet , Stockholm, Sweden. blood. Red blood cell. Oxy-Hb. NO 3 -. NO 2 -. O 2. sGC. GTP. cGMP ↑. L-arginine. NO . NOS. endothelium. smooth muscle. The truth about nitrate (NO 3 - ).

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The Nitrate-Nitrite-NO Pathway Jon Lundberg Eddie Weitzberg

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  1. The Nitrate-Nitrite-NOPathway Jon Lundberg Eddie Weitzberg KarolinskaInstitutet, Stockholm, Sweden

  2. blood Red blood cell Oxy-Hb NO3- NO2- O2 sGC GTP cGMP↑ L-arginine NO. NOS endothelium smooth muscle

  3. The truth about nitrate (NO3-) • Inert end product of NO metabolism • Unwanted residue in the food chain

  4. The truth about nitrate (NO3-) • Unwanted residue in the food chain • Inert end product of NO Desirable Substrate for

  5. Nitric oxide: Role in the cardiovascular system L-arginine + O2 Platelet aggregation ↓ NO NO synthase Leucocyte adhesion ↓ Vasodilation Mitochondrialfunction Reduced NO bioavailability is a central event in mostcardiovascular disorders (Hypertension, atherosclerosis, stroke, myocardialinfarction)

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

  7. EXPERIMENTAL SETTINGS Carbonated Water +5 min N O A n a l y s e r

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

  9. Nitrite forms NO after protonation NO2- + H+ → HNO2 (pKa~3.3) 2 HNO2 → N2O3 + H2O → NO + NO2 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 (NO3-) 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. Nitrite, µM Nitrate, mM Salivary nitate/nitrite levels after an oral nitrate intake Sodium nitrate 3 mg/kg Lundberg Govoni FRBM, 2004

  13. The Entero-salivary Circulation of Nitrate and its Reduction to Nitrite and NO In the Stomach NO2- + H+ → HNO2 (pKa~3.3) 2 HNO2 → N2O3 + H2O → NO + NO2 Lundberg, Weitzberg, Gladwin Nature Rev Drug Discov 2008

  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!

  16. 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)

  17. Exploring beneficial effects of dietary nitrate/nitrite

  18. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HCl HCl Antibacterial effects of gastric NO E.coli 1-2 h 24 h Benjamin et al. Nature -94

  19. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HCl + nitrite HCl + nitrite ”No growth” Antibacterial effects of gastric NO E.coli 1-2 h 24 h Benjamin et al. Nature -94

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

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

  22. Are these effects associated with gastroprotection?

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

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

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

  26. What happens if we remove the oral bacteria?

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

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

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

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

  31. Saliva-dependentgastric NO in healthyvolunteers 100uM, 50 ml Bjorne et al. Crit Care Med 2005

  32. Stomach NO in intubated patients healthy subjects intubated patients

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

  34. The Stomach as a Bioreactor for Chemical Interactions Between Nitrate/Nitrite and Other Dietary Components Fruit Vegetables Wine Polyphenols Vitamin C Olive oil Nitrate Nitrite Polyphenols ethanol Fish/Meat Unsaturated fatty acids Protein HNO2 Nitrosamines Nitrated fatty acids? NO/S-NO Ethyl nitrite Modified from Gago et al. FRBM 2007-2008

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

  36. Swallowing from start No swallowing first 60 min 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 = start of swallowing Lundberg & Govoni Free Rad Biol Med 2004

  37. Antibacterial mouthwash and plasma nitrite • 7 healthy subjects, crossover design • Chlorhexidine (0.2 %) vs placebo mouthwash • NaNO3 10 mg/kg • Repeated plasma/saliva samples for nitrite

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

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

  40. 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) Lundberg & Govoni Free Rad Biol Med 2004 Govoni et al NO Biol Chem, in press

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

  42. 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.

  43. Is nitrite doing anything?

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

  45. Deoxy-Hb Oxy-Hb NO NO3- NO2- NO/NO2- Nitrite vasodilates the human circulation at near physiological concentrations Cosby et al. Nature Med -03

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

  47. 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 ↑↑

  48. Nitrite and myocardial Infarction Figure 2A Figure 2C Murine in vivo Myocardial Ischemia-Reperfusion Protocol Nitrite or Nitrate (48 nmoles, i.v.) * Reperfusion Control LCA Ischemia 0 0.2 0 0.5 0 1 24 2 Time (hours) * In Vivo area-at-risk - Evans Blue injection Infarct Size - 1.0% 2,3,5-TTC Figure 2D Figure 2B Duransky, Gladwin, Lefer, JCI 2005

  49. Nitrite and Stroke Jung et al. Stroke 2006

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