Download - The Nitrate-Nitrite-NO Pathway Jon Lundberg Eddie Weitzberg Karolinska Institutet, Stockholm, Sweden
The Nitrate-Nitrite-NO Pathway
Jon Lundberg
Eddie Weitzberg
Karolinska Institutet, Stockholm, Sweden
NOS
L-arginine
NO.
sGCGTP
cGMP↑
O2
NO2-
Oxy-HbRed blood cell
NO3-
blood
smooth muscle
endothelium
The truth about nitrate (NO3-)
• Inert end product of NO metabolism
• Unwanted residue in the food chain
• Unwanted residue in the food chain
• Inert end product of NO
Desirable
Substra
te fo
r
The truth about nitrate (NO3-)
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
Background
Measurements of exhaled NON
O (
ppb)
?
NO Analyser
CarbonatedWater
EXPERIMENTAL SETTINGS
+5 min
NO levels in expelled stomach gas
Lundberg et al Gut 1994
Control
Lettu
ce
Om
epra
zole
Om
epra
zole
+ le
ttuce
0
1000
2000
3000N
O (
pp
b)
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)
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
Duncan Nature Med 1995
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
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
Does intragastric NO have any biological effects?
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!
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)
Exploring beneficial effects of dietary nitrate/nitrite
Antibacterial effects of gastric NO
Benjamin et al. Nature -94
HCl HCl
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1-2 h 24 h
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. ....E.coli
Antibacterial effects of gastric NO
Benjamin et al. Nature -94
HCl +nitrite
HCl +nitrite
.. .. .... .. .
. ...... .
. ...... .
. ...... .
. ....
1-2 h 24 h
”No growth”
E.coli
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)
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
Are these effects associated with gastroprotection?
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
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
Effects of dietary nitrate on NSAID-induced gastric ulcer
Diclofenac 30 mg/kg
0
25
50
75
100
125
Ulc
er In
dex
(mm
)
EA Jansson et al Free Radic Biol Med -07
What happens if we remove the oral bacteria?
Effect of an antibacterial mouthwash on intragastric NO in rats
Petersson Free Radic Biol Med 2009
Antibacterial mouthwash and nitrate-mediated gastroprotection
Petersson Free Rad Biol Med 2009
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
(p
pb
)
L-NAME had no effect!
Method for measurement of gastric NO in humans
CLD
Bjorne et al. Crit Care Med 2005
Bjorne et al. Crit Care Med 2005
Saliva-dependent gastric NO in healthy volunteers
100uM, 50 ml
healthysubjects
intubatedpatients
Stomach NO in intubated patients
Intragastric nitrite administration in intubated patients
Plasma nitrite (uM) 0.17+0.03 1.28 +0.2 0.26+ 0.03
2mM (100ml)
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
Will the nitrite formed in the oral cavity survive GI passage and enter the systemic
circulation?
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
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
-50 0 50 100 150 200
-500
0
500
1000
1500
2000
2500
3000
3500
4000
N
itrite
Co
nc
(µM
)
Time (min)
Control Mouth Wash
Change in salivary nitrite after an oral nitrate load
Δ N
itri
te (
µM
)
Govoni NO Biol Chem, in press
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
co
nc
(nM
)
Time (min)
Control Mouth Wash
Δ N
itri
te (
nM
)
Govoni NO Biol Chem, in press
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)
So, why is it interesting that dietary nitrate increases systemic nitrite
levels?
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.
Is nitrite doing anything?
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
Cosby et al. Nature Med -03
Nitrite vasodilates the human circulation at nearphysiological concentrations
Deoxy-Hb
NO2
- NO
Oxy-Hb
NO/NO2
- NO3
-
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
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 ↑↑
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
Nitrite and Stroke
Jung et al. Stroke 2006
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)
Lundberg, Weitzberg, GladwinNature Rev Drug Discov 2008
Nitrite interactions with mitochondria
How does nitrite protect?
Xanthine oxidase (XO)
Duranski J Clin Invest 2005
Nitrite and cardioprotection- In vivo
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!
NO3
-
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”
So, does dietary nitrate affect
cardiovascular function?
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
B
Placebo Nitrate 50
60
70
80
90
Dia
sto
lic
blo
od
pre
ssu
re(m
mH
g)
Larsen et al N Engl J Med 2006
Effects of Dietary Nitrate on Blood Pressure
- 4 mmHg
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.
Webb et al. Hypertension 2008
Beetroot juice and blood pressure
- 14 healthy volunteers
- 500 ml fresh beetroot juice (45 mM nitrate)
Beetroot juice and blood pressure
- 6 healthy volunteers -spit/swallow
- 500 ml fresh beetroot juice (45 mM nitrate)
Webb et al. Hypertension 2008
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 A
AR
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)
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
Placebo Nitrate
3.00
3.25
3.50
3.75
4.00
4.25
VO
2 (
l/m
in)
Oxygen consumption at 85 % of VO2 max
HR - No differenceLactate - No difference
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!
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
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.
Lundberg, Weitzberg, GladwinNature Rev Drug Discov 2008
Two parallel systems for NO generation in mammals