Download Asymmetrical Dimethylarginine in Idiopathic Pulmonary Arterial

Asymmetrical Dimethylarginine in Idiopathic Pulmonary
Arterial Hypertension
Jan T. Kielstein, Stefanie M. Bode-Böger, Gerrit Hesse, Jens Martens-Lobenhoffer, Attila Takacs,
Danilo Fliser, Marius M. Hoeper
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Objective—We explored the potential role of the endogenous NO synthase inhibitor asymmetrical dimethylarginine
(ADMA) in patients with idiopathic pulmonary arterial hypertension (IPAH).
Method and Results—We correlated plasma ADMA levels and cardiovascular indices from right heart catheterization in 57 patients
with IPAH. Predictors of survival in patients with IPAH were studied. Furthermore, the effect of systemic ADMA infusion on
pulmonary ventricular resistance and stroke volume was investigated in healthy volunteers using right heart catheterization. Mean
plasma ADMA concentrations were significantly higher in patients with IPAH than in control subjects (0.53⫾0.15 versus
0.36⫾0.05 ␮mol/L; P⬍0.001). ADMA plasma concentrations correlated significantly with indices of right ventricular function,
such as mixed-venous oxygen saturation (r⫽⫺0.49; P⬍0.0001), right atrial pressure (r⫽0.39; P⬍0.003), cardiac index (r⫽⫺0.35;
P⬍0.008), as well as survival (r⫽⫺0.47; P⬍0.0001). Multiple regression analysis revealed that right atrial pressure (r⫽0.31;
P⬍0.026) and ADMA (r⫽0.29; P⬍0.039) were independent predictors of mortality. Moreover, patients with supra-median plasma
ADMA levels had significantly (P⬍0.021) worse survival than patients with infra-median ADMA values. ADMA infusion in
healthy volunteers increased pulmonary vascular resistance (68.9⫾7.6 versus 95.6⫾6.3 dyne 䡠 s 䡠 cm⫺5; P⬍0.05) and decreased
stroke volume (101.1⫾6.7 mL versus 95.6⫾6.3 mL; P⬍0.05).
Conclusion—Increased ADMA plasma levels are associated with unfavorable pulmonary hemodynamics and worse
outcome in patients with IPAH. (Arterioscler Thromb Vasc Biol. 2005;25:1414-1418.)
Key Words: idiopathic pulmonary arterial hypertension 䡲 ADMA 䡲 nitric oxide
I
diopathic pulmonary arterial hypertension (IPAH) is a
disease of unknown etiology that is characterized by
progressive obliteration of small and medium-size pulmonary
arteries, elevation in pulmonary arterial pressure, and increase in pulmonary vascular resistance, eventually leading to
right heart failure and death.1 Without appropriate medical
treatment, mean survival of patients experiencing IPAH is ⬍3
years.2 In the current understanding of the pathogenesis of
IPAH, it is thought that a combination of genetically determined predisposition coupled with ill-defined exogenous
factors may contribute to damage of the pulmonary vessels,
resulting in endothelial dysfunction with proliferation of
vascular endothelial and smooth muscle cells.3–5 Endothelial
dysfunction attributable to reduced bioavailability of endogenous vasodilator substances such as NO is thought to play an
important role in the development of pulmonary hypertension.6,7 NO is synthesized in the endothelium from the amino
acid L-arginine by the action of NO synthase (NOS). Endogenous guanidino-substituted analogues of L-arginine that can
selectively inhibit NOS have been implicated in the pathogenesis of various cardiovascular diseases. The most potent
of these endogenous NOS inhibitors is asymmetrical dimethylarginine (ADMA). Elevated ADMA levels and low
L-arginine/ADMA ratios not only correlate with the severity
of atherosclerotic disease8 –11 but also predict cardiovascular
outcome and overall mortality.12,13 We demonstrated recently
that pathophysiologically relevant ADMA blood concentrations inhibited NO production and reduced cardiac output and
renal perfusion in healthy subjects.14 Experimental data from
a rat model of chronic hypoxia-induced pulmonary hypertension revealed that increased pulmonary concentrations of
ADMA contributes to pulmonary hypertension.15 On the
basis of these observations, it seems possible that increased
plasma ADMA concentrations may also contribute to endothelial dysfunction in patients with pulmonary vascular disease.16
The present study was undertaken to evaluate a potential role of
ADMA in IPAH.
Subjects and Methods
Participants and Protocols
The study protocol was approved by the local Ethics committee, and
all participants gave informed consent. We recruited 57 whites (19
Original received December 7, 2004; final version accepted April 7, 2005.
From the Divisions of Nephrology (J.T.K., G.H., D.F.) and Respiratory Medicine (A.T., M.M.H.), Department of Internal Medicine, Medical School
Hannover, Germany; and the Institute of Clinical Pharmacology (S.M.B.-B., J.M.-L.), “Otto-von-Guericke” University, Magdeburg, Germany.
J.T.K. and S.M.B.-B. contributed equally to this work.
Correspondence to J.T. Kielstein, MD, Department of Internal Medicine, Division of Nephrology, Medical School Hannover Carl-Neuberg-Strasse 1,
30625 Hannover, Germany. E-mail [email protected]
© 2005 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
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DOI: 10.1161/01.ATV.0000168414.06853.f0
Kielstein et al
ADMA in Idiopathic Pulmonary Arterial Hypertension
Downloaded from http://atvb.ahajournals.org/ by guest on May 2, 2017
males; 49⫾14 years) with severe IPAH in functional class III and IV
according to the diagnostic criteria defined by a World Health
Organizatin expert panel.17 For comparison, 22 subjects (9 males;
48⫾14 years) without relevant medical problems matched with
respect to renal function, mean arterial pressure, and cholesterol and
triglyceride levels were examined. All patients underwent a right
heart catheter examination as part of the initial diagnostic workup at
the time of the first diagnosis of pulmonary hypertension. Details of
the catheter procedure have been described previously.18 None of the
patients were under treatment with nitrates, NO donors, prostaglandins, endothelin receptor antagonists, or sildenafil at the time of
diagnostic cardiac catheterization. No other relevant medical problems were present at that time. Blood samples for measurement of
plasma ADMA concentrations were obtained during heart catheterization. Blood samples were immediately cooled on ice, centrifuged
at 1500g and 4°C for 10 minutes, and the supernatants were stored in
1 mL aliquots at ⫺80°C until further use.
After completion of the diagnostic cardiac catheterization, all
patients were further followed to assess survival. Patients were
recruited for this study between February 2000 and September 2001
and followed until September 2003 or until death. All patients were
treated with oral anticoagulants unless contraindicated. One patient
was considered a “responder” after acute vasodilator challenge and
received calcium channel blockers. The remaining patients were
nonresponders in functional class New York Heart Association III or
IV and were treated with inhaled, oral, or intravenous prostanoids, or
endothelin receptor antagonists.
ADMA Infusion
The study protocol was separately approved by the ethics committee
of the Hannover Medical School. Written informed consent was
given by all participants, who were healthy nonsmoking male
volunteers. A right heart catheter was placed under local anesthesia,
and hemodynamic measurements were performed as described
previously.18 After an appropriate equilibration period to minimize
stress after catheter placement, hemodynamics were assessed continuously during placebo infusion period (50 minutes), followed by
intravenous infusion of 0.1 mg/kg per minute ADMA for 40 minutes.
Hemodynamics were monitored continuously for another 120 minutes after discontinuation of ADMA infusion.
Measurements and Calculations
Plasma concentrations of ADMA were measured applying a recently
developed liquid chromatography-mass spectrometry method described previously.19 Briefly, after addition of the internal standard
solution (13C6-arginine and homoarginine), 250 ␮L plasma was
deproteinized by the addition of 0.5 mL acetonitril, the supernatant
was evaporated to dryness, and the residue was redissolved in
formiate buffer. Samples were automatically derivatized with
orthophthaldialdehyde/2-mercaptoethanol reagent and were analytically separated on a Merck Superspher RP-18 250⫻4 mm highperformance liquid chromatography column, applying a formiate
buffer/methanol gradient. All analytes were separated sufficiently
and detected selectively by a ThermoFinnigan LCQ mass spectrometer equipped with an electrospray ionization ion source. The
coefficient of variation is 7.5%. The method was validated according
to the guidelines for biochemical assays.20 Plasma total homocysteine concentrations were measured with a fluorescence-polarization
immunoassay. All other measurements were done with routine
laboratory tests using certified assay methods.
1415
Figure 1. Individual values of plasma ADMA concentration in
control subjects (n⫽20) and in patients with IPAH (primary pulmonary hypertension [PPH]; n⫽57).
reveal independent determinants of survival. Moreover, we analyzed
survival in patients with IPAH using a log-rank analysis and created
Kaplan–Meier survival curves. For this purpose, the group of
patients was divided into those with a supramedian ADMA level and
those with plasma ADMA concentrations below the median value.
Data are presented as mean⫾SD.
A paired t test was used to compare the intraindividual preinfusion
(minute 50) and postinfusion (minute 90) cardiovascular parameters
obtained during the right heart catheter experiments. The significance level was set at P⬍0.05.
Results
Mean plasma ADMA concentrations were significantly
higher in patients with IPAH than in control subjects
(0.53⫾0.15 versus 0.36⫾0.05 ␮mol/L; P⬍0.001␮mol/L;
Figure 1).
Hemodynamic parameters obtained during right heart catheter examination in patients with IPAH are shown in Table 1.
Results of the correlation analyses between plasma ADMA
concentration and age, right heart catheter indices, and
survival (categorized as dead/alive) are summarized in Table
2. Plasma ADMA levels significantly correlated with mixedvenous oxygen saturation (Figure 2A), right atrial pressures
(Figure 2B), pulmonary vascular resistance, stroke volume,
cardiac index, and survival (Table 2). There was no significant difference between IPAH patients and controls with
regard to cholesterol and triglyceride and blood glucose
levels; Table I, available online at http://atvb.ahajournals.org)
Although the mean homocysteine plasma level was within the
normal range in both groups, IPAH patients tended to have
higher mean serum homocysteine levels (13.2⫾4.0 versus
10.5⫾3.4 ␮mol/L; P⬍0.05).
TABLE 1. Data From Right Heart Catheter Examination in 57
Patients With IPAH
Mean arterial blood pressure, mm Hg
94⫾14
Right atrial pressure, mm Hg
9.0⫾6.2
Mean pulmonary arterial pressure, mm Hg
Statistical Analysis
Pulmonary vascular resistance, dyne 䡠 s 䡠 cm⫺5
We used SPSS for statistical analysis (SPSS 11.51 for Windows).
The normality of data distribution was confirmed with the Shapiro–
Wilk test. Comparison between groups was done using a 2-tailed t
test for comparison of random data. Pearson’s correlation analyses
were performed between plasma ADMA concentrations on the one
hand, and age, right heart catheter indices, and survival (categorized
as dead/alive) on the other. In addition, ADMA, age, and right heart
catheter indices were applied in a multiple regression analysis to
Stroke volume, mL
54⫾14
1101⫾549
49⫾18
Cardiac index, L 䡠 min 䡠 m
2⫺1
Systemic vascular resistance, dyne 䡠 s 䡠 cm⫺5
2.0⫾0.7
1945⫾726
PaO2, Torr
64⫾11
Mixed-venous oxygen saturation, %
60⫾9
All measurements were made without supplemental oxygen.
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Arterioscler Thromb Vasc Biol.
July 2005
TABLE 2. Results of Correlation Analyses With Plasma ADMA
Concentration on the One Hand, and Age, Right Heart Catheter
Indices, and Survival (categorized as dead/alive) on the Other
TABLE 3. Clinical Characteristics and Data From Right Heart
Catheter Examination of Survivors and Nonsurvivors
Mixed-venous oxygen saturation
r⫽⫺0.49
P⬍0.0001
n
Survival
r⫽⫺0.47
P⬍0.0001
Age, years
Right atrial pressure
r⫽0.39
P⬍0.003
Plasma ADMA concentration, ␮mol/L
Stroke volume
r⫽⫺0.35
P⬍0.008
Right atrial pressure, mm Hg
Cardiac index
r⫽⫺0.35
P⬍0.008
Mean pulmonary arterial pressure, mm Hg
Mean arterial blood pressure
r⫽⫺0.32
P⬍0.015
Pulmonary vascular resistance, dyne 䡠 s 䡠 cm⫺5
Pulmonary vascular resistance
r⫽0.24
P⫽0.08
Stroke volume, mL
51⫾19
43⫾16
Systemic vascular resistance
r⫽0.16
P⫽0.22
Cardiac index, L 䡠 min 䡠 m2⫺1
2.2⫾0.8
1.8⫾0.5
Survivors
Age
r⫽⫺0.13
P⫽0.34
Mean arterial blood pressure, mm Hg
PaO2
r⫽0.10
P⫽0.49
Systemic vascular resistance, dyne 䡠 s 䡠 cm⫺5
Mean pulmonary arterial pressure
r⫽⫺0.07
P⫽0.63
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The median follow-up period in patients with IPAH after
completion of the diagnostic right heart catheterization was
26 (1 to 44) months. During this follow-up period, 18 patients
died. Clinical data and results of the right heart catheter
examination from survivors and nonsurvivors are presented
in Table 3. On stepwise multiple regression analysis, only
right atrial pressure (r⫽0.31; P⫽0.026) and ADMA (r⫽0.29;
P⫽0.039) were independent predictors of mortality after
inclusion of age, mean pulmonary arterial pressure, cardiac
index, stroke volume, mean arterial blood pressure, pulmonary vascular resistance, and mixed-venous oxygen saturation. Figure 3 presents Kaplan–Meier survival curves for
patients with IPAH. In patients with supramedian values of
plasma ADMA concentrations (ie, ⬎0.51 ␮mol/L), survival
was significantly worse (P⫽0.021) than in patients with
plasma ADMA levels below the median value.
Figure 2. Scatter plots of the relationship between plasma
ADMA concentration. Mixed-venous oxygen saturation (A;
r⫽⫺0.49; P⬍0.0001) and right atrial pressure (B; r⫽⫺0.39;
P⬍0.003), in 57 patients with IPAH.
Nonsurvivors
39
18
49⫾14
48⫾13
0.48⫾0.13
0.63⫾0.14*
7.5⫾5.1
12.3⫾7.1*
54⫾13
56⫾15
1028⫾489
1259⫾648
96⫾15
89⫾12
1928⫾758
1980⫾670
PaO2, Torr
64⫾12
62⫾10
Mixed-venous oxygen saturation, %
62⫾7
56⫾11*
All measurements were made without supplemental oxygen.
Comparison between survivors and nonsurvivors (*P⬍0.05).
ADMA infusion in healthy volunteers increased pulmonary vascular resistance (68.9⫾7.6 versus 95.6⫾6.3
dyne 䡠 s 䡠 cm⫺5; P⬍0.05) and decreased stroke volume
(101.1⫾6.7 versus 95.6⫾6.3 mL; P⬍0.05; Figure IA and IB,
available online at http://atvb.ahajournals.org).
Discussion
The major novel findings of the present study are: (1) plasma
ADMA levels in patients with IPAH were significantly
higher than in matched healthy controls; (2) in patients with
IPAH, plasma ADMA concentrations correlate significantly
with predictors of survival such as right atrial pressure and
mixed-venous oxygen saturation; and (3) ADMA itself is an
independent predictor of survival in patients with IPAH.
These observations lead to several questions. Is ADMA a
pathophysiologically relevant mediator or simply an innocent
Figure 3. Kaplan–Meier survival curves in patients with IPAH
and supramedian values of plasma ADMA concentrations
(n⫽28) and inframedian plasma ADMA values (n⫽29). In
patients with supramedian values of plasma ADMA concentrations (ie, ⬎0.51 ␮mol/L), survival was significantly worse
(P⬍0.021).
Kielstein et al
ADMA in Idiopathic Pulmonary Arterial Hypertension
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bystander in this disease, and do these findings have potential
therapeutic implications?
Is ADMA a relevant factor in the pathogenesis of IPAH?
Firm conclusions from our data would be premature in this
respect. However, the results of the infusion study suggest
that acute administration of ADMA increases pulmonary
vascular resistance and decreases stroke volume. A decrease
in cardiac output by ADMA infusion had been shown
previously.14 This finding is in line with data on the multifacet involvement of NO in cardiac (patho)physiology; small
increments in NO concentration have a positive inotrop
effect, and conversely, inhibition of NOS may result in a
negative inotropic effect.21 Above that, a very recent study
showed that long-term (4-week) application ADMA can
indeed cause significant microvascular lesions in mice.22
ADMA is released from proteins that have been posttranslationally methylated and subsequently hydrolyzed. To
some extent, ADMA is renally excreted, but the major
metabolic pathway is degradation by the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which hydrolyzes ADMA to dimethylamine and L-citrulline.23 A recent
study in transgenic mice that overexpress DDAH provided
compelling evidence that the metabolism of ADMA plays an
important role in regulation of NOS activity.24 A relevant role
of ADMA in the pathogenesis of IPAH could derive from
reduced activity of DDAH with increasing severity of the
disease, eventually as a result of increased oxidant stress as
well as hypoxia.9 This assumption is corroborated by findings
from several studies. Hypoxia in severe hemorrhagic shock
has been shown to lead to an increase in ADMA in a pig
model.25 More specifically, in a hypoxia-induced pulmonary
hypertension rat model, chronic hypoxia leads to an increase
in ADMA caused by a reduced pulmonary expression and
activity of DDAH.15 The patients in the present study had a
mean mixed venous oxygen saturation of 60%, reflecting
some degree of tissue hypoxia, which could have contributed
to increased ADMA levels. Furthermore, in a pig model of
pulmonary hypertension of the newborn, it was shown that
the underlying mechanism for ADMA elevations is suppression of DDAH expression and activity.26
Another possible mechanism of reduced DDAH activity
might be related to plasma homocysteine concentrations.
Recently, Stuehlinger et al reported that homocysteine in
concentrations ⬎30 ␮mol/L inhibits the activity of DDAH,
leading to an accumulation of ADMA in vitro.27 Consistent
with a previous study,28 plasma homocysteine levels in IPAH
were within the normal range (ie, between 5 and 15 ␮mol/L)
but significantly elevated compared with controls. It is
possible that concentrations as low as 15 ␮mol/L might
influence DDAH activity and subsequently increase ADMA
concentrations because already physiological increments in
plasma homocysteine have been shown to induce vascular
endothelial dysfunction in normal human subjects.29
Finally, an alternative explanation for increased ADMA
blood concentrations in IPAH patients with higher mortality
would be that they simply reflect severe injury of the
pulmonary vascular bed because endothelial cells can produce large amounts of ADMA.30
1417
Our findings that ADMA is an independent predictor of
mortality in patients with IPAH is reminiscent of the results
of 2 landmark studies showing that increased ADMA predicts
cardiovascular and overall mortality in patients with endstage renal disease as well as in patients with coronary heart
disease.12,13 Furthermore, it fits into a rapidly growing number of clinical studies documenting a strong correlation
between ADMA and cardiovascular morbidity and mortality
in different populations.10,31–35 Several studies in different
populations have yielded ample evidence that even small
increases in plasma ADMA levels are associated with deterioration of endothelial function and a significant increase in
the rate of cardiovascular events.12,13,36 Accordingly, we
found a significant correlation between ADMA and right
atrial pressure as well as cardiac index, both of which are
important prognostic parameters in IPAH. All in all, the
pathophysiological role of ADMA in IPAH still remains to be
unfold in detail, even more so because the relationship
between the ADMA/NO system is incompletely understood
and probably much more complex than currently known.
Is the ADMA/NO system a potential therapeutic target in
IPAH? Because L-arginine is a potent NO donor, it is
tempting to speculate that raising the plasma L-arginine
concentration might attenuate the detrimental effects of
ADMA in patients with IPAH. Two studies have addressed
L-arginine supplementation in patients with IPAH with conflicting results. Short-term infusion (ie, 15 minutes) of highdose L-arginine had no hemodynamic effect in 5 patients with
IPAH.37 In contrast, in a placebo-controlled study in 19
patients with IPAH, 1-week oral L-arginine supplementation
resulted in improved exercise capacity and hemodynamics.38
A multicenter trial to establish the potential therapeutic role
of L-arginine supplementation in IPAH has been concluded
recently but, apparently, results have been negative (these
data have not yet been published). Thus, there is not enough
evidence to recommend L-arginine supplements for patients
with IPAH.
In conclusion, the significant correlation of plasma ADMA
concentrations with important indices of pulmonary hemodynamics, together with the predictive power of ADMA for
survival of patients with IPAH as well as the short-term effect
of ADMA infusion on the pulmonary circulation in healthy
men, suggests that ADMA may be a relevant factor in the
pathogenesis of IPAH.
Acknowledgments
Part of this study was presented at the 58th annual fall conference
and scientific sessions of the Council for High Blood Pressure
Research in association with the Council on the Kidney in Cardiovascular Disease, October 9 –12, 2004.
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Asymmetrical Dimethylarginine in Idiopathic Pulmonary Arterial Hypertension
Jan T. Kielstein, Stefanie M. Bode-Böger, Gerrit Hesse, Jens Martens-Lobenhoffer, Attila
Takacs, Danilo Fliser and Marius M. Hoeper
Arterioscler Thromb Vasc Biol. 2005;25:1414-1418; originally published online April 28, 2005;
doi: 10.1161/01.ATV.0000168414.06853.f0
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PVR (dynes.s.cm-5)
0
20
40
60
80
100
120
0
20
40
60
80
ADMA
infusion
*
120
time (min)
100
140
160
180
200
220
Figure I A
stroke volume (mL)
70
75
80
85
90
95
100
105
110
115
120
0
20
40
60
80
ADMA
infusion
*
120
time (min)
100
140
160
180
200
220
Figure I B
1
ONLINE DATA
Figure I. Effect of 0.10 mg ADMA/kg/min on pulmonary vascular resistance (Figure I A) and
stroke volume (Figure I B) in 7 healthy volunteers. * = p<0.05 - comparison between
pre-infusion (baseline) and post-infusion data. Data are presented as mean ±SEM. Please
see www.ahajournals.org
-1-
Table I. Cardiovascular risk factors and characteristics of patients with
IPAH and controls.
Controls
IPAH
n
22
57
Age (years)
48 ± 14
49 ± 14
Male/female
9/13
19/38
cholesterol (mg/dL)
186 ± 24
163 ± 36
triglyceride (mg/dL)
110 ± 61
122 ± 47
blood glucose (mmol/L)
4.6 ± 0.8
4.7 ± 0.6
Homocysteine (µmol/L)
10.5 ± 3.4
13.2 ± 4.0*
patients
* - p<0.05 – comparison between controls and IPAH patients