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

.. .. .... .. .

. ...... .

. ...... .

. ...... .

. ....

1-2 h 24 h

.. .. .... .. .

. ...... .

. ...... .

. ...... .

. ...... .. .... .. .

. ...... .

. ...... .

. ...... .

. ...... .

. .... .. .. ...... .

. ...... .

. ...... .

. ...... .. .... .. .

. ...... .

. ...... .

. ...... .

. ....

.. .. .... .. .

. ...... .

. ...... .

. ...... .

. ....

.. .. .... .. .

. ...... .

. ...... .

. ...... .

. ...... .. .... .. .

. ...... .

. ...... .

. ...... .

. ....

.. .. .... .. .

. ...... .

. ...... .

. ...... .

. ....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