the nitrate-nitrite-no pathway jon lundberg eddie weitzberg
DESCRIPTION
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 - ). - PowerPoint PPT PresentationTRANSCRIPT
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The Nitrate-Nitrite-NO Pathway
Jon LundbergEddie WeitzbergKarolinska Institutet, Stockholm, Sweden
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NOS
L-arginine
NO.
sGCGTP
cGMP↑
O2
NO2-
Oxy-HbRed blood cell
NO3-
blood
smooth muscle
endothelium
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The truth about nitrate (NO3-)
• Inert end product of NO metabolism
• Unwanted residue in the food chain
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• Unwanted residue in the food chain
• Inert end product of NO
Desirable
Substrate fo
r
The truth about nitrate (NO3-)
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Nitric oxide: Role in the cardiovascular system
NO synthase
L-arginine + O2
NO
Platelet aggregation ↓
Reduced NO bioavailability is a central event in most cardiovascular disorders
(Hypertension, atherosclerosis, stroke, myocardial infarction)
Leucocyte adhesion ↓
Vasodilation
Mitochondrial function
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Background
Measurements of exhaled NONO
(ppb
)
?
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NO Anal yser
CarbonatedWater
EXPERIMENTAL SETTINGS
+5 min
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NO levels in expelled stomach gas
Lundberg et al Gut 1994
Control
Lettuce
Omepraz
ole
Omepraz
ole + le
ttuce
0
1000
2000
3000N
O (p
pb)
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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)
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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
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Duncan Nature Med 1995
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Nitr
ate,
mM
Nitr
ite, µ
M
Salivary nitate/nitrite levels after an oral nitrate intake
Lundberg Govoni FRBM, 2004
Sodium nitrate 3 mg/kg
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The Entero-salivary Circulation of Nitrate and its Reduction to Nitrite and NOIn the Stomach
Lundberg, Weitzberg, Gladwin Nature Rev Drug Discov 2008
NO2- + H+ → HNO2 (pKa~3.3)
2 HNO2 → N2O3 + H2O
→ NO + NO2
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Does intragastric NO have any biological effects?
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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!
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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)
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Exploring beneficial effects of dietary nitrate/nitrite
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Antibacterial effects of gastric NO
Benjamin et al. Nature -94
HCl HCl
.. .. .... .. .. ...... .. ....
.. .. ...... .. ....
1-2 h 24 h.. .. .... .. .. ...... .. ....
.. .. ...... .. ...... .. .... .. .. ...... .. ....
.. .. ...... .. ...... .. .... .. .. ....
.. .. ...... .. ....
.. .. ...... .. .... .. .. ...... .. ....
.. .. ...... .. ....
.. .. .... .. .. ...... .. ....
.. .. ...... .. ....
.. .. .... .. .. ...... .. ....
.. .. ...... .. ...... .. .... .. .. ....
.. .. ...... .. ....
.. .. ....
.. .. .... .. .. ...... .. ....
.. .. ...... .. ....
E.coli
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Antibacterial effects of gastric NO
Benjamin et al. Nature -94
HCl +nitrite
HCl +nitrite
.. .. .... .. .. ...... .. ....
.. .. ...... .. ....
1-2 h 24 h
”No growth”
E.coli
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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)
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Salivary nitrite increases gastric mucosal blood flow and mucus generation
Mucus thickness
Mucosal 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
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Are these effects associated with gastroprotection?
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Dietary nitrate, aspirin-like drugs and ulcers
Nitrate 0,1/1 mmol/kg
7 days 4 hours
Diclofenac 30 mg/kg
Start of experiment Gavage with Diclofenac SacrificeCollection of materials
Nitrate dose corresponds to 100-500 gr nitrate-rich vegetable/dayin a human
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Diclofenac, 30 mg/kg, 4 hours
Control Nitrate, 1mmol/kg, 7 days
Dietary nitrate and NSAID-induced Gastric Ulcers
EA Jansson et al Free Radic Biol Med -07
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Effects of dietary nitrate on NSAID-induced gastric ulcer
Diclofenac 30 mg/kg
0
25
50
75
100
125U
lcer
Inde
x (m
m)
EA Jansson et al Free Radic Biol Med -07
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What happens if we remove the oral bacteria?
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Effect of an antibacterial mouthwash on intragastric NO in rats
Petersson Free Radic Biol Med 2009
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Antibacterial mouthwash and nitrate-mediated gastroprotection
Petersson Free Rad Biol Med 2009
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Role of bacteria and dietary nitrate in intragastric NO generation
Sobko et al Am J Physiol 2005
10
100
1000
10000
Conventional Germ free
***
ns
control
nitrate
NO
(ppb
)
L-NAME had no effect!
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Method for measurement of gastric NO in humans
CLD
Bjorne et al. Crit Care Med 2005
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Bjorne et al. Crit Care Med 2005
Saliva-dependent gastric NO in healthy volunteers
100uM, 50 ml
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healthysubjects
intubatedpatients
Stomach NO in intubated patients
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Intragastric nitrite administration in intubated patients
Plasma nitrite (uM) 0.17+0.03 1.28 +0.2 0.26+ 0.03
2mM (100ml)
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The Stomach as a Bioreactor for Chemical Interactions Between Nitrate/Nitrite and Other Dietary Components
Olive oil
Vegetables FruitWine
Unsaturated fattyacids
NitrateNitrite
PolyphenolsVitamin C
Polyphenolsethanol
HNO2
Nitrated fatty acids? NO/S-NO Ethyl nitriteNitrosamines
Fish/Meat
Protein
Modified from Gago et al. FRBM 2007-2008
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Will the nitrite formed in the oral cavity survive GI passage and enter the systemic
circulation?
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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
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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
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-50 0 50 100 150 200
-500
0
500
1000
1500
2000
2500
3000
3500
4000
N
itrite
Con
c (µ
M)
Time (min)
Control Mouth Wash
Change in salivary nitrite after an oral nitrate load
Δ Ni
trite
(µM
)
Govoni NO Biol Chem, in press
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Change in plasma nitrite after an oral nitrate load
-50 0 50 100 150 200
0
50
100
150
200
250
300
350
N
itrite
con
c (n
M)
Time (min)
Control Mouth Wash
Δ Ni
trite
(nM
)
Govoni NO Biol Chem, in press
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Lundberg & Govoni Free Rad Biol Med 2004Govoni 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)
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So, why is it interesting that dietary nitrate increases systemic nitrite
levels?
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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.
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Is nitrite doing anything?
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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
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Cosby et al. Nature Med -03
Nitrite vasodilates the human circulation at nearphysiological concentrations
Deoxy-Hb
NO2- NO
Oxy-Hb
NO/NO2- NO3
-
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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 2005Lundbergf et al Nature Rev Microbiol 2004Gladwin et al Nature Chem Biol 2005
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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 ↑↑
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0 0 00.2 0.5 1 2 24
Time (hours)
LCA IschemiaControl Reperfusion
Nitrite or Nitrate (48 nmoles, i.v.)
Murine in vivo MyocardialIschemia-Reperfusion Protocol
* In Vivo area-at-risk - Evans Blue injectionInfarct Size - 1.0% 2,3,5-TTC
*
Figure 2A Figure 2C
Figure 2D
0
10
20
30
40
50
60
70
Nitrate 2.4 4.8 48 960 1920
Nitrite Dose (nmoles)
11 6666 8
0
10
20
30
40
50
60
Nitrate
******
**
Nitrite Dose (nmoles)
2.4 4.8 48 960 1920
11 6666 8
Figure 2B
Duransky, Gladwin, Lefer, JCI 2005
Nitrite and myocardial Infarction
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Nitrite and Stroke
Jung et al. Stroke 2006
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Low Dose Nitrite Prevents Ischemia-Reperfusion Injury in Several Organs
Organ Ref
Heart Webb, PNAS-04 Duranski, JCI-05
Liver Duranski, JCI-05Kidney Tripatara, J Am Soc
Nephrol-07Stroke Jung, Stroke-06Pulmonary embolism Dias-Junior, Free Rad Biol
Med-07
Systemic administration, (rats, mice, dogs)
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Lundberg, Weitzberg, GladwinNature Rev Drug Discov 2008
Nitrite interactions with mitochondria
How does nitrite protect?
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Xanthine oxidase (XO)
Duranski J Clin Invest 2005
Nitrite and cardioprotection- In vivo
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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 plateof spinach!
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NO3-
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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”
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So, does dietary nitrate affect cardiovascular function?
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Dietary Nitrate and Blood Pressure
- 17 healthy volunteers age 20-25 years
- Nitrate supplementation (NaNO3 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
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B
Placebo Nitrate 50
60
70
80
90
Dia
stol
ic b
lood
pre
ssur
e(m
mH
g)
Larsen et al N Engl J Med 2006
Effects of Dietary Nitrate on Blood Pressure
- 4 mmHg
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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.
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Webb et al. Hypertension 2008
Beetroot juice and blood pressure
- 14 healthy volunteers
- 500 ml fresh beetroot juice (45 mM nitrate)
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Beetroot juice and blood pressure
- 6 healthy volunteers -spit/swallow
- 500 ml fresh beetroot juice (45 mM nitrate)
Webb et al. Hypertension 2008
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Plasma Heart0
20
40
120
140
160
180 *
Nitr
oso
[nM
]
Control 1g/L Nitrate
*11 115 5
C
AAR/LV Inf/AAR Inf/LV0
10
20
30
40
50
60
**
% L
V o
r AA
R
Control 1g/L Nitrate
**
D
10 10 10 10 10 10
Dietary Nitrate Protects from Injury from Myocardial Infarctionin mice
Bryan et al PNAS (2007)
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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
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Placebo Nitrate
3.00
3.25
3.50
3.75
4.00
4.25
VO2
(l/m
in)
Oxygen consumption at 85 % of VO2 max
HR - No differenceLactate - No difference
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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!
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NO and mitochondria
NO inhibits mitochondrial respiration Cleeter 1994, Brown 1994
Nitrite is reduced to NO Kozlov 1999, Nohl 2000
NO promotes biogenesis Nisoli 2003
Nitrite nitrosates complex 1 Shiva 2007
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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.
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Lundberg, Weitzberg, GladwinNature Rev Drug Discov 2008
Two parallel systems for NO generation in mammals