2 content: l-arginine metabolism nitric oxide synthases dimethylarginines dimethylarginineseffects...

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ContentContent::••L-Arginine metabolismL-Arginine metabolism••Nitric oxide Nitric oxide

••SynthasesSynthases ••DimethylargininesDimethylarginines

••EffectsEffects••Arginine SupplementationArginine Supplementation

••Infusion/ OralInfusion/ Oral••Endothelial FunctionEndothelial Function

••EpidemiologyEpidemiology

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Source of ArginineSource of Arginine

L-Arginine is considered a semi-L-Arginine is considered a semi-essential amino acid: it becomes essential amino acid: it becomes essential in growing children, during essential in growing children, during pregnancy or after injury. pregnancy or after injury.

A Western diet provides about 4-6 A Western diet provides about 4-6 g/day of which 40-50% is absorbed. g/day of which 40-50% is absorbed.

55

••The liver produces considerable The liver produces considerable amounts of arginine during the urea amounts of arginine during the urea cycle, but little is available for cycle, but little is available for synthesis.synthesis.

• • The intestines produce citrulline The intestines produce citrulline

which is converted by other tissues which is converted by other tissues (kidney, 80%) into L-arginine which (kidney, 80%) into L-arginine which is then made available to other is then made available to other tissues. tissues.

66

NADP+

2O2

The synthesis of nitric oxide (NO) is catalyzed by nitric oxide synthase (NOS). The reaction is more complicated than indicated in the figure.

NH2

C=NH2+

NH

CH2

CH2

CH2

H C NH3+

COO-

L-Arginine

NH2

C=O

NH

CH2

CH2

CH2

H C NH3+

COO-

L-Citrulline

NADPH+ H+ NADP+

2O2 NO + H2O Nitric Oxide

77

Nitric Oxide SynthaseNitric Oxide Synthase

Three IsoformsThree Isoforms

Neuronal (constitutive, calcium Neuronal (constitutive, calcium dependent)dependent)

Endothelial (constitutive, calcium Endothelial (constitutive, calcium dependent)dependent)

Macrophages (inducible, calcium Macrophages (inducible, calcium independent). Can lead to high independent). Can lead to high levels of NO being formed. levels of NO being formed.

88

Nitric Oxide Effects Nitric Oxide Effects (via formation of cGMP)(via formation of cGMP)Relaxes smooth muscleRelaxes smooth muscle

Inhibits platelet aggregation and Inhibits platelet aggregation and activationactivation

NeurotransmitterNeurotransmitter

Tumoricidal and bactericidal agent Tumoricidal and bactericidal agent from macrophages (excess can from macrophages (excess can damage healthy tissue)damage healthy tissue)

99

• • Many studies involve infusion or Many studies involve infusion or dietary supplements of L-arginine in dietary supplements of L-arginine in both animals and humans. both animals and humans.

• • The physiological effects elicited The physiological effects elicited were unexpected as the kwere unexpected as the kmm of NOS for of NOS for

L-arginine is about 2 L-arginine is about 2 μμM whereas the M whereas the circulating levels of L-arginine are circulating levels of L-arginine are about 1about 100 00 μμM. M.

1010

• • The explanation may involve the The explanation may involve the presence of naturally occurring presence of naturally occurring inhibitors of NOS (ADMA and NMA). inhibitors of NOS (ADMA and NMA). These two analogues of L-arginine These two analogues of L-arginine plus SDMA are also competitors for plus SDMA are also competitors for the ythe y+ + transport system that delivers transport system that delivers L-arginine to NOS. L-arginine to NOS.

1111

Structure of L-arginine and Circulatory AnaloguesStructure of L-arginine and Circulatory Analogues

NH NH2

C

NH

CH2

CH2

CH2

CH

NH2 COOH

L-ARGININE L-NMA ADMA SDMA

NH NH

CH3

C

NH

CH2

CH2

CH2

CH

NH2 COOH

NH N

CH3 CH3

C

NH

CH2

CH2

CH2

CH

NH2 COOH

C

N NH

CH3 CH3

NH

CH2

CH2

CH2

CH NH2 COOH

1212

As shown in the metabolic pathway As shown in the metabolic pathway (slide 14):(slide 14):

••L-arginine is methylated while a L-arginine is methylated while a component of proteins by:component of proteins by:••PRMT (type I): occurs in nucleus, PRMT (type I): occurs in nucleus, many substrates forms ADMA and many substrates forms ADMA and NMANMA••PRMT (type II) : specific for myelin PRMT (type II) : specific for myelin basic protein, forms SDMA and NMAbasic protein, forms SDMA and NMA

••The methylated analogues are The methylated analogues are released by hydrolysis during normal released by hydrolysis during normal protein turnoverprotein turnover

1313

• • The methylated analogues are removed The methylated analogues are removed by renal excretion or catabolismby renal excretion or catabolism••DDAH type I associated with neural DDAH type I associated with neural NOSNOS••DDAH type II associated with DDAH type II associated with endothelial NOSendothelial NOS••Neither DDAH is active on SDMANeither DDAH is active on SDMA••DPT (a minor pathway) acts on all DPT (a minor pathway) acts on all three analoguesthree analogues••The enzymes are particularly active in The enzymes are particularly active in kidneykidney

1414

Protein

PRMT (types I and II)

Modified Protein Containing ADMA+ SDMA+ NMA

Hydrolysis

ADMA +SDMA +NMA

Acetylated Products α-keto

acid products

Citrulline + Methylamines

DDAH (types I and II) Renal

Excretion

PRMT: Protein arginine methyltransferaseADMA: Asymmetrical dimethylarginineSDMA: Symmetrical dimethylarginine

NMA: N-monomethylarginineDDAH: Dimethylaminohydrolase

DPT: Dimethylarginine pyruvate transferase

DPT

1616

In slides 18 and 19, results from Cooke et al (1992) are shown. The investigators fed male rabbits either (a) normal chow (control) or (b) 1% cholesterol diet; or (c) 1% cholesterol diet supplemented with drinking water containing 2.25% L-arginine HCl. After 10 weeks of dietary intervention, analyses indicated:

1717

Endothelium dependent relaxation of Endothelium dependent relaxation of the thoracic aortae elicited by the thoracic aortae elicited by acetylcholine was reduced in acetylcholine was reduced in cholesterol-fed animals and the cholesterol-fed animals and the response was significantly response was significantly ameliorated by L-arginine.ameliorated by L-arginine.L-arginine also significantly reduced L-arginine also significantly reduced the lesion surface area in the the lesion surface area in the descending thoracic aorta elicited by descending thoracic aorta elicited by cholesterol diets (intima thickness cholesterol diets (intima thickness also reduced)also reduced)

1818

0

20

40

60

80

100

120

9 8 7 6 5 4

ACh (-Log M)

% N

on

rep

inep

rin

e P

reco

ntr

acti

on

Control

Cholesterol

Arginine

Cooke, JP, et al. Journal of Clinical Investigation (1992) 90:1168-1172.

1919

0

10

20

30

40

Plaque (% of Total Surface

Area)

Cholesterol Arginine

Cooke, JP, et al. Journal of Clinical Investigation (1992) 90:1168-1172.

2020

••Candipan et al (1996) fed rabbits Candipan et al (1996) fed rabbits either normal chow (controls) or 0.5% either normal chow (controls) or 0.5% cholesterol chow for 10 weeks and cholesterol chow for 10 weeks and then the cholesterol group received then the cholesterol group received either vehicle or L-arginine (2.25% in either vehicle or L-arginine (2.25% in water) (arginine group) for an water) (arginine group) for an additional 13 weeks. additional 13 weeks.

2121

••Histomorphic measurements indicated Histomorphic measurements indicated a gradual deterioration in the a gradual deterioration in the cholesterol group (intima-media cholesterol group (intima-media thickness) and this was ameliorated thickness) and this was ameliorated by L-arginine at 18 weeks but not at by L-arginine at 18 weeks but not at 23 weeks (slide 22). 23 weeks (slide 22).

••This may indicate that the This may indicate that the effects of L-arginine are not effects of L-arginine are not sustained. sustained.

2222

Candipan, RC, et al. Arteriosclerosis, Thrombosis, and Vascular Biology (1996) 16(1): 44-50.

Intima, mm2

WeekWeek CHOLCHOL

ARG ARG   

1010 0.92±0.360.92±0.36 0.98±0.160.98±0.16

1414 1.80±0.371.80±0.37 2.33±1.362.33±1.36

1818 3.58±0.713.58±0.71 1.51±0.64*1.51±0.64*

2323    4.21±0.744.21±0.74 4.18±1.714.18±1.71

*p< 0.05*p< 0.05

2323

In human studies, Drexler et al (1994) In human studies, Drexler et al (1994) infused 18 cardiac transplant infused 18 cardiac transplant recipients with acetylcholine (10recipients with acetylcholine (10-6,-6, 10 10--

55, 10, 10-4-4 mol/L) before and after mol/L) before and after intravenous with L-arginine (10 mg/ intravenous with L-arginine (10 mg/ kg. min. for 20 minutes). (slide 24)kg. min. for 20 minutes). (slide 24)

Acetylcholine elicited a dose-Acetylcholine elicited a dose-dependent constriction of the dependent constriction of the coronary artery that was attenuated coronary artery that was attenuated by L-arginine (p <0.01 at 10by L-arginine (p <0.01 at 10-4 -4

acetylcholine)acetylcholine)

2424

-7

-6

-5

-4

-3

-2

-1

0

1

2

Response to ACh (% change from

baseline)

ach 10-6

ach 10-5

ach10-4

Before L-arginine After L-arginine

Before L-Arginine After L-Arginine

Drexler, H, et al. Circulation (1994) 89(4):1615

2525

• • BBööger et al (1998) reported that infusion ger et al (1998) reported that infusion of L-arginine ameliorated the clinical of L-arginine ameliorated the clinical symptoms of intermittent claudication in symptoms of intermittent claudication in patients with peripheral arterial occlusive patients with peripheral arterial occlusive disease. disease.

• • 13 patients received two 13 patients received two intravenous infusions of L-arginine (8 g intravenous infusions of L-arginine (8 g each) for 3 weeks. each) for 3 weeks.

2626

••13 patients received no infusions 13 patients received no infusions (control group)(control group)

••Both groups maintained normal Both groups maintained normal walking exercises. walking exercises.

••Results indicated that L-arginine Results indicated that L-arginine improved pain-free walking distance improved pain-free walking distance (slide 27) by 230± 63% (p < 0.05). (slide 27) by 230± 63% (p < 0.05). Absolute walking distance also Absolute walking distance also improved by 155 ±48% (p < 0.05). improved by 155 ±48% (p < 0.05).

2727

Pai

n-f

ree

Wal

kin

g D

ista

nce

(m

)

Böger, RH, et al. J Am Coll Cardiol (1998) 32(5): 1336-44.

2828

••Physiological effects have also been Physiological effects have also been elicited by infusion of NOS inhibitors.elicited by infusion of NOS inhibitors.

••Vallance et al (1992) infused ADMA(8 Vallance et al (1992) infused ADMA(8 μμmol/min for 5 min into 5 volunteers) mol/min for 5 min into 5 volunteers) and observed a decrease in forearm and observed a decrease in forearm blood flow (slide 30)blood flow (slide 30)

2929

••McVeigh et al (2001) infused L-NAME McVeigh et al (2001) infused L-NAME (N(NGG- nitro-L-arginine methyl ester) into - nitro-L-arginine methyl ester) into 15 healthy men and observed an 15 healthy men and observed an increase in systemic vascular increase in systemic vascular resistance (slide 31) and a decrease resistance (slide 31) and a decrease in small artery compliance (slide 32)in small artery compliance (slide 32)

••The effects were ameliorated by The effects were ameliorated by infusion of L-arginine but not by D-infusion of L-arginine but not by D-arginine. arginine.

3030

Vallance, P, et al. Lancet (1992) 339:572-575.

3131 McVeigh, GE, et al. Clinical Science. (2001)100: 387-393.

† p < 0.01 versus control

‡ p < 0.01 D-arginine versus L-arginine

3232 McVeigh, GE et al. Clinical Science. (2001) 100: 387-393.

* p < 0.05 versus controls

† p < 0.01 versus controls

‡ p < 0.01 D-arginine versus L-arginine

3434

•Epidemiological studies have observed associations between ADMA concentrations and subclinical and clinical measures of atherosclerosis.

•Miyazaki et al (1999) studied 116 subjects with no symptoms of coronary or peripheral artery disease and not taking medications. Results indicated:

3535

••Plasma ADMA levels were significantly Plasma ADMA levels were significantly correlated with intima-media correlated with intima-media thickness (slide 36)thickness (slide 36)

••Stepwise multiple regression analysis Stepwise multiple regression analysis indicated plasma ADMA was a indicated plasma ADMA was a significant determinant of the intima-significant determinant of the intima-media thickness (slide 37)media thickness (slide 37)

3636 Miyazaki, H, et al. Circulation (1999) 99(9): 1141-1146.

3737 Miyazaki, H, et al. Circulation (1999) 99(9):1141-1146.

Variable Variable CoefficientCoefficient

p p

AgeAge    0.0050.005    0.00010.0001

Plasma ADMAPlasma ADMA 0.290.29 0.030.03

Mean arterialMean arterialpressurepressure 0.160.16 0.10.1

Σ glucoseΣ glucose 0.140.14    0.160.16

SmokingSmoking -0.05-0.05 0.560.56

Total cholesterolTotal cholesterol 0.020.02 0.810.81

Positive family Positive family -0.02-0.02 0.940.94

historyhistory            

r²= 0.41.r²= 0.41.

3838

• •Zoccalli et al (2001) studied 225 Zoccalli et al (2001) studied 225 haemodialysis patients with end-haemodialysis patients with end-stage renal disease. stage renal disease.

••Plasma ADMA was significantly and Plasma ADMA was significantly and independently correlated with all-independently correlated with all-cause mortality and fatal and non-cause mortality and fatal and non-fatal cardiovascular events. No fatal cardiovascular events. No significant associations were significant associations were observed for plasma SDMA or L-observed for plasma SDMA or L-arginine (slides 40 and 41).arginine (slides 40 and 41).

3939

• • Valkonen et al (2001), in a prospective Valkonen et al (2001), in a prospective case-control study analyzed the case-control study analyzed the association between ADMA and the risk of association between ADMA and the risk of acute coronary events. acute coronary events.

• •Among non-smoking men, ADMA was a Among non-smoking men, ADMA was a significant risk factor for acute coronary significant risk factor for acute coronary events. The conclusions were dependent events. The conclusions were dependent on presence or absence of a history of on presence or absence of a history of coronary heart disease (CHD): not coronary heart disease (CHD): not significant in the absence of a history of significant in the absence of a history of CHD; significant in the presence of a CHD; significant in the presence of a history of CHD (slide 42). history of CHD (slide 42).

4040 Zoccalli, C, et al. Lancet (2001) 358: 2113-2117.

0.220.22 0.92(0.80-1.05)0.92(0.80-1.05)0.920.921.01(0.89-1.14)1.01(0.89-1.14)  10 10 mmol/L mmol/L L-arginineL-arginine

0.340.34 1.06(0.94-1.18)1.06(0.94-1.18)0.730.731.02(0.93-1.11)1.02(0.93-1.11)

11 μmol/L μmol/LSDMASDMA

0.00010.0001 1.26(1.11-1.41)1.26(1.11-1.41)

<<0.00010.00011.28(1.16-1.41)1.28(1.16-1.41)

11 μmol/L μmol/L ADMAADMA

   (95% Cl)(95% Cl)            

   hazard ratio*hazard ratio*(95%Cl)(95%Cl)  

    pp Fully adjusted Fully adjusted ppHazard ratio*Hazard ratio*      Unit of Unit of increaseincrease

        All-cause mortalityAll-cause mortality  

4141

   Hazard ratio*Hazard ratio* ppFully adjusted Fully adjusted hazardhazard pp

  Unit ofUnit ofincreaseincrease (95% Cl)(95% Cl)       ratio (95% Cl)ratio (95% Cl)   

ADMAADMA 1 μmol/L 1 μmol/L 1.21 (1.10-1.32)1.21 (1.10-1.32) 0.00010.0001 1.17 (1.04-1.33)1.17 (1.04-1.33) 0.0080.008

SDMASDMA 1 μmol/L 1 μmol/L 0.97 (0.88-1.07)0.97 (0.88-1.07) 0.610.61 1.00 (0.88-1.14)1.00 (0.88-1.14) 0.980.98

L-L-argininearginine

10mmol/10mmol/LL 1.06 (0.94-1.19)1.06 (0.94-1.19) 0.370.37 1.00 (0.87-1.15)1.00 (0.87-1.15) 0.970.97

Fatal and non-fatal cardiovascular eventsFatal and non-fatal cardiovascular events

Zoccalli, C, et al. Lancet (2001) 358: 2113-2117.

4242 Valkonen, V-P, et al. Lancet (2001) 358: 2127-8.

All (n=50)All (n=50) No history of No history of coronary heart coronary heart disease* (n=80)disease* (n=80)

History of History of coronary heart coronary heart disease* (n=70)disease* (n=70)

Odds ratio (95% Odds ratio (95% Cl)Cl)

pp Odds ratio (95% Odds ratio (95% Cl)Cl)

pp Odds ratio (95% Odds ratio (95% Cl)Cl)

pp

BaselineBaseline

characteristiccharacteristic

ADMA, highest ADMA, highest quartile quartile

((>0.62 >0.62 μμmol/L)mol/L)

3.92 (1.25-12.3)3.92 (1.25-12.3) 0.020.02 2.39 (0.54-10.5)2.39 (0.54-10.5) 0.250.25 21.8 (1.4-348.5)21.8 (1.4-348.5) 0.030.03

Family history of Family history of

coronary heart coronary heart diseasedisease

3.02 (1.14-7.96)3.02 (1.14-7.96) 0.030.03 2.53 (0.75-8.54)2.53 (0.75-8.54) 0.140.14 3.3 (0.6-19.1)3.3 (0.6-19.1) 0.190.19

4343

Are the effects of L-arginine Are the effects of L-arginine supplementation sustained?supplementation sustained?

Will L-arginine supplementation be of Will L-arginine supplementation be of clinical benefit?clinical benefit?

How general will any benefit be?How general will any benefit be?

4444

I would like to thank Ms. Meghan I would like to thank Ms. Meghan Dabkowski for her assistance in the Dabkowski for her assistance in the

preparation of this presentation. preparation of this presentation.