Free Radical Biology & Medicine, Vol. 37, No. 7, pp. 1010–1017, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter
doi:10.1016/j.freeradbiomed.2004.06.039
Original Contribution NITRIC OXIDE AND PULMONARY ARTERIAL PRESSURES IN PULMONARY HYPERTENSION ROBERTO F. MACHADO,* MEDHA-VINI LONDHE NERKAR,*,y RAED A. DWEIK,* JEFFREY HAMMEL,z ALLISON JANOCHA,y JACQUELINE PYLE,* DANIEL LASKOWSKI,*,y CONSTANCE JENNINGS,* ALEJANDRO C. ARROLIGA,* and SERPIL C. ERZURUM*,y *Department of Pulmonary and Critical Care Medicine; yDepartment of Cancer Biology; and Department of Biostatistics and Epidemiology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195, USA
z
(Received 29 January 2004; Revised 10 May 2004; Accepted 24 June 2004) Available online 23 July 2004
Abstract—Decreased production of vasodilator substances such as nitric oxide (NO) has been proposed as important in development of pulmonary arterial hypertension (PAH). We hypothesize that NO measured over time serves as a non invasive marker of severity of PAH and response to therapy. We prospectively and serially measured exhaled NO and carbon monoxide (CO), a vasodilator and anti-inflammatory product of heme oxygenases, in 17 PAH patients in conjunction with hemodynamic parameters over 2 years. Although pulmonary artery pressures and NO were similar in all patients at entry to the study, NO increased in the 12 individuals who survived to complete the study, and correlated with change in pulmonary artery pressures. In contrast, CO did not change or correlate with hemodynamic parameters. Investigation of NO–oxidant reaction products in PAH in comparison to controls suggests that NO synthesis is impaired in the lung and that reactive oxygen species may be involved in the pathophysiology of pulmonary hypertension. Endogenous NO is inversely related to pulmonary artery pressure in PAH, with successful therapy of PAH associated with increase in NO. D 2004 Elsevier Inc. All rights reserved. Keywords—Pulmonary hypertension, Nitric oxide, Vasodilation, Carbon monoxide, Reactive oxygen species, Free radicals
survival in these patients [4]. Other experimental data suggest that carbon monoxide (CO), endogenously produced through heme oxygenases and having significant antiproliferative and vasodilatory effects on the pulmonary circulation, may also be involved in the pathogenesis of PAH [5–10]. Current clinical markers of prognosis include hemodynamic variables, including degree of pulmonary hypertension and the function of the right ventricle [11]; however, noninvasive quantitative tests for pulmonary hypertension, monitoring disease progression and evaluation of response to therapy, are not available. We and others have previously shown that individuals with PPH have lower than normal exhaled NO and that NO production in the lung is inversely related to the degree of pulmonary hypertension [12]. Further, individuals with PAH have an increase in exhaled NO concomitant with a decrease in pulmonary artery pressure after initiation of vasodilator therapy with the prostacyclin
INTRODUCTION
Pulmonary arterial hypertension (PAH) is a rare disease of unknown etiology leading to the development of severe precapillary pulmonary hypertension characterized by impaired regulation of both pulmonary hemodynamics and vascular growth [1]. PAH may be associated with known diseases, such as collagen vascular diseases and portal hypertension, but, in the absence of an identifiable etiology, is classified as primary pulmonary hypertension (PPH, PAH class 1.1) [2]. Abnormalities in vasodilator substances such as nitric oxide (NO) have been proposed as important in the development of PAH [3], and vasodilator therapy has been shown to prolong Address correspondence to: Serpil C. Erzurum, M.D., Director, Lung Biology Program, Cleveland Clinic Foundation, Lerner Research Institute 9500 Euclid Avenue/NB40, Cleveland, OH 44195, USA; Fax: (216) 445 6269; E-mail:
[email protected]. 1010
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epoprostenol [13]. We hypothesized that serial measurement of NO over time may serve as a noninvasive marker of severity of PAH and response to therapy. To evaluate the relative roles of NO and CO in PAH, we prospectively and serially determined exhaled NO and CO in 17 individuals with pulmonary hypertension in conjunction with hemodynamic parameters over a 2year period. To investigate the role of oxidant species in the mechanisms that decrease NO, NO–oxidant reaction products were assessed noninvasively by collection of serum and exhaled breath condensate from PAH patients and healthy control individuals. Exhaled breath condensate has been increasingly used as a noninvasive method to sample the lower airway fluid with measurable characteristics that differ in health and disease [14]. Specifically, alterations in exhaled breath condensate levels of several NO reaction products have been demonstrated in tobacco use [15] and inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, and pneumonia [16–19]. In the context of a pro-inflammatory environment in PAH lungs [12,20], we hypothesized that NO produced in the lungs of PAH patients is increasingly consumed by reactive oxygen species, such as superoxide.
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months later at the end of the study (time 3). As part of standard care, follow up right heart catheterization was performed approximately 12 months after initiation of therapy. Both the baseline screening pulmonary artery pressures (PAPs) and subsequent PAP at 12 months (time 2) were available for use in the study. NO and CO were measured in the exhaled breath of patients at entry into the study (time 1) and subsequently at 12 months (time 2) and end of study (time 3). For the convenience of volunteers in the study, measurements were performed at times before or after the prescribed plan of 12 and 24 months. Thus, the average time between measurements is given in the results. In addition, eight nonsmoking individuals fulfilling the NIH and WHO criteria for pulmonary arterial hypertension (PPH, PAH class 1.1) and 46 nonsmoking healthy volunteers underwent study of exhaled breath condensate for evaluation of NO reaction products at a single time. Pulmonary function studies performed according to American Thoracic Society recommendations were available for all volunteers [23]. The study was approved by the Cleveland Clinic Foundation institutional review board and written informed consent was obtained from all individuals. An overview of the study design is shown schematically in Fig. 1.
METHODS
Individuals with PAH were identified for enrollment into the prospective study if they met the NIH registry diagnostic criteria for pulmonary hypertension [21] and were classified according to the World Health Organization criteria as PAH class 1 [22]. Specifically, participation was restricted to patients who had pulmonary hypertension determined by right heart catheterization as part of their standard care; i.e., right heart catheterization was not done for research purposes. All individuals underwent a trial of calcium channel blockers. Parenteral prostacyclin-based vasodilator therapy with epoprostenol or teprostinil (UT-15) was started on those individuals who did not respond to calcium channel blockers, according to the discretion of the patients’ physicians. Noninvasive measure of pulmonary artery hypertension was estimated by right ventricular systolic pressure (RVSP) at echocardiography performed at enrollment to the study (time 1) and approximately 24
Measurement of exhaled gases As previously described [24], exhaled gases were obtained by an off-line method in agreement with American Thoracic Society standards for exhaled NO determination. Briefly, individuals inhaled NO-free air to total lung capacity and exhaled against 10 cm water pressure, to meet the American Thoracic Society recommended flow rate of 0.35 L/s, into a Mylar collection bag (Physiological Measurement Systems, Bay Village, OH, USA). All individuals were seated at rest for at least 15 min before gases were collected. Exhaled CO was measured in the exhaled gases with a Siemens Ultramat 6 infrared analyzer (Karlsruhe, Germany) that was adapted for use in this study. The analyzer was sensitive to a concentration of 100 ppb for CO. NO concentrations were determined by using a chemiluminescence analyzer (Sievers Instruments, Boulder, CO, USA).
Fig. 1. Prospective study design and timeline. Enrollment into the study occurred shortly after baseline evaluation at time one.
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Collection of exhaled breath condensate
Statistical analysis
Exhaled breath condensate (EBC) was collected using a condenser, which allowed the noninvasive collection of nongaseous components of the expiratory air (EcoScreen, Jaeger, Wqrzburg, Germany). Subjects breathed through a mouthpiece with a two way non-rebreathing valve, which also served as a saliva trap. They were asked to breathe at a normal frequency and tidal volume, for a period of 15 min. The condensate (at least 0.5 ml) was collected as ice at 208C and immediately stored at 708C.
Quantitative data are summarized as means F SE; categorical data are summarized by frequencies. Associations between pairs of variables are described by Pearson’s correlation coefficient and a test for nonzero correlation. Two-tailed t-test statistics and analysis of variance were used where appropriate. All tests were performed at individual significance levels of a .05.
NO reaction products and protein in EBC Nitrite and nitrate concentrations were determined using the ISO-NOP Nitric Oxide Sensor (World Precision Instruments, Sarasota, FL, USA), an amperometric sensor specific for nitric oxide [25,26]. The sensor was immersed in 10 ml of 0.1 M sulfuric acid and 0.1 M potassium iodide, a solution in which NO is liberated from nitrite. ZnSO4 and NaOH were used to remove fatty/ particulate materials and proteins from serum (Nitralyzer II, World Precision Instruments, Sarasota, FL, USA). Sample nitrite concentration was determined by adding 50 Al of exhaled breath condensate sample, or 150 ul of serum, to the reaction vessel. Current was recorded using DUO 18 data acquisition software (World Precision Instruments). The difference in average current between the peak due to the sample and region immediately preceding the peak was measured, and the nitrite concentration was calculated based on authentic standards of nitrite. Total nitrite and nitrate (NOx ) concentration was determined in replicate samples by reducing nitrate to nitrite using the Nitralyzer II (World Precision Instruments), which uses a cadmium–copper pellet to reduce nitrate to nitrite according to the following reaction: NO 3 + H2O + Cd Y NO2 + Cd(OH)2. Conversion using this protocol with authentic nitrate was determined to be 100%. After reduction, nitrite was measured by the ISO-NOP. Nitrate was determined by subtracting the nitrite from the total NOx for each sample. Saline controls were also evaluated as negative control for nitrite and nitrate. EBC protein concentration was determined using the CBQCA Protein Quantitation Kit (Molecular Probes, Eugene, OR, USA). Briefly, samples were prepared by mixing 100 Al of EBC with 35 Al of 0.1 M sodium borate buffer, pH 9.3, in a 96-well microplate. Five microliters of 20 mM KCN was added to each sample. Ten microliters of a 5 mM working solution of the ATTO-TAG CBQCA solution was added to each sample. The plate was protected from light and incubated for 1 h, then read on a Victor 2 Fluorescence microplate reader (PerkinElmer Life Sciences, Boston, MA, USA) with excitation at 485 nm and emission at 535 nm.
RESULTS
Clinical characteristics of patients in the prospective study Seventeen PAH patients were enrolled in the study; 5 died during the study and 12 completed the study. Tables 1 and 2 describe the characteristics of the study population and hemodynamic measures determined at screening and during times 1 to 3 of the study. Associated causes of PAH were scleroderma (n = 3), portopulmonary hypertension (n = 4), Eisenmenger’s syndrome (n = 1), and sarcoid vasculitis (n = 1). Mean time from initial diagnostic cardiac catheterization/initiation of therapy to entry into the study with time 1 exhaled NO measurement and echocardiographic estimation of PAP by determination of right ventricular systolic pressure (RVSP) was 4 F
Table 1. Clinical Characteristics of PAH Patients in Longitudinal Study
Survivors (n = 12)
Early mortality (n = 5)
51 F 4 12
54 F 2 5
Ethnicity Caucasian African-American Hispanic Asian
6 3 2 1
5 0 0 0
PPH
7
1
Smoking history
1
0
Age Female sex
Lung function (% predicted) Forced vital capacity FEV1a Total lung capacity DLCO
80 F 71F 92 F 65 F
Mean PAP (mm Hg)
54 F 4
6-min walking distance (ft) Vasodilator therapy Epoprostenol/teprostinil/nifedipine
4 5 4 5
1178 F 82 8/3/1
81 84 82 51
F F F F
4 4 4 6
52 F 8 1342 F 72 4/1/0
a FEV1, forced expiratory volume in 1 s; DLCO, carbon monoxide diffusing capacity.
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Table 2. Hemodynamic Parameters and Exhaled NO and CO Survivors (n = 12)
Early mortality (n = 5)
Systolic PAPa (mm Hg)
Cardiac index (l/min/m2)
NO (ppb)
CO (ppm)
Baseline screening 1 2
87 F 7 83 F 8 81 F 7
2.7 F 0.3 — 3 F 0.3
— 8F1 9.2 F 1
— 1.3 F 0.3 1.5 F 0.4
3 p-valuec
74 F 4 .470
— .128
15 F 1 .001
1.3 F 0.4 .880
Time
a b c
Systolic PAPa (mm Hg) 74 F 11 87 F 9 81 F 7 97 F 9b — .182
Cardiac index (l/min/m2)
NO (ppb)
CO (ppm)
3F1 — 3F2 — — .334
— 9F1 9F1 — — .587
— 1.4 F 0.5 1.7 F 0.4 — — .580
PAP determined by right heart catheterization at baseline and time 2, and estimated at times 1 and 3 by RVSP measured by echocardiogram. PAP estimated by RVSP at echocardiography prior to death. p value of ANOVA over time for more than two values, or Student’s t test for paired comparisons.
2 months. Second measurement of NO and right heart catheterization determination of PAP (time 2) occurred 9.3 F 1.3 months from time 1, and measurement at time 3 of NO and echocardiographic determination of PAP occurred 7.0 F 0.9 months from time 2. Echocardiographic measurement of RVSP accurately estimates pulmonary artery pressures, with mean differences of 11 F 2 mm Hg between echocardiogram and invasive measures [27]. Here, RVSP at echocardiography also accurately predicted systolic PAP as determined by right heart catheterization at baseline screening (correlation of systolic PAP to RVSP, R = .794, p b .001). Thus, RVSP was used as a good noninvasive estimate of systolic PAP. In the 12 patients who completed the study, systolic PAP appeared to decrease in survivors (PAP time 1 vs. time 3, p = .06) and increase in those patients who died ( p = .182) (Table 2). Cardiac index increased in some individuals, but did not change significantly in the survivors ( p = .128) (Table 2) or in the 5 patients who expired prior to completion of the study ( p = .334) (Table 2). NO at entry into the study did not differ between survivors and patients who died ( p = .53) (Table 2). Interestingly, NO increased 2-fold from time 1 to time 3 in the surviving patients ( p b .001), although no significant change in NO was detected from time 1 to time 2 of the study (Fig. 2). In contrast, CO did not change throughout the study in survivors or in the patients who died (Table 2). Notably, NO at entry into the study (time 1) was correlated with change in PAP of survivors (NO vs. DPAP(TIME 1–3), r = .56, p = .054). These findings suggest that NO values may predict PAPs and changes in pulmonary hypertension, such that the lower NO values are associated with higher pulmonary artery pressures and smaller decrease of pulmonary hypertension over time. Finally, NO levels at entry (time 1) were inversely correlated with total months since diagnosis (r = .69, p = .011). In other words, a longer time with a diagnosis of
Fig. 2. Exhaled NO in patients with pulmonary hypertension [PAH 1.1 (PPH), open circles; PAH 1.2, closed circles]. In contrast to those patients who expired, individuals who completed the study had a progressive increase in exhaled NO. Successful vasodilator therapy, defined by survival of patients, appears to restore NO and slow the progressive increase in pulmonary arterial pressures.
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pulmonary hypertension was associated with a lower NO value at entry into the study, perhaps suggesting a global decrease in NO over long periods with the disease [12]. Nevertheless, vasodilator therapy restored NO production in the majority of patients evidenced by increase in exhaled NO in the patients who responded to therapy and survived (Fig. 3). NO reaction products in airways of PPH patients The effects of NO are dependent on its biologic halflife, which is largely influenced by its reactions with reactive oxygen species in pro-inflammatory environments [28]. To investigate increased oxidative consumption of NO and its role in PAH, exhaled breath condensate was collected from patients with PPH (n = 8, age 47 F 5 years, female 7, FVC 84 F 4%, FEV1 81 F 4%, mean PAP 71 F 9 mm Hg, prostacyclin use in 4) and healthy volunteers (n = 46, age 25 F 1 years, female 22, FVC 96 F 2%, FEV1 94 F 2%). Total protein in EBC was similar among PPH and controls (Ag/ml: PPH 11 F 7, control 3 F 0.8; p = .24). Variation in measures was determined by collection of EBC from a subgroup of individuals on 2 separate days (n = 7; 5 controls, 2 PPH). Coefficient of variation for total protein in EBC was 27 F 13%, and coefficient of variation for total NO reaction products in EBC was 30 F 7%. Total NO reaction products in EBC from PPH patients were lower than in healthy controls (NOx AM: PPH 7.2 F 1.5, control 18.1 F 1.5; p b .001). Nitrate was the predominant NO product detected in breath condensate in controls (Fig. 4). When compared with control individuals, PPH patients had decreased nitrate levels (NO3 AM: PPH 3.2 F 1.0, control 15.7 F 1.3; p b .001) but similar nitrite levels (NO2 AM: PPH 4.0 F 1.3, control 2.4 F 0.4; p = .2) (Fig. 4). Correction for NO reaction products relative to total protein revealed similar results [(NOx Amol/mg: PPH
4.3 F 2.2, control 20.3 F 2.9; p b .001: NO3 Amol/ mg: PPH 1.1 F 0.5, control 17.6 F 2.7; p b .001) (NO2 Amol/mg: PPH 2.8 F 1.9, control 2.4 F 0.3; p = .8)]. Nitrate levels were directly correlated with PAP (r = .82, p = .01). On the other hand, nitrite, similar to NO, was inversely correlated with length of time with diagnosis (r = .75, p = .03). Systemic alterations in NOx were not detected, as serum NOx of PPH patients was similar to that of healthy controls (NOx AM: PPH 28 F 4, control 26 F 5; p = .716). DISCUSSION
Ever since endothelium-derived relaxing factor was pharmacologically defined as identical to NO activity, NO has been proposed as the major physiologic regulator of blood vessel tone [29–31]. Here, we provide evidence that endogenous NO production is closely related to pulmonary artery pressures in the pathologic state of pulmonary hypertension in the human lung. It is also important to note that the majority of patients in this study were on prostacyclin vasodilator therapy. The prostacyclin epoprostenol has been shown to improve survival in patients with PAH through direct relaxation of vascular smooth muscle cells and other effects, e.g., inhibition of production and secretion of endothelin [32]. Prostacyclin may mediate some of these effects through mechanisms involving NO. For example, inhibition of the production and secretion of endothelin 1 is via a cGMP-dependent mechanism, which may involve NO as an intermediary signaling molecule [33]. In support of this, the use of prostacyclin in patients with pulmonary hypertension increases exhaled NO within 18 to 24 h of administration of the drug [13,34]. It has also been speculated that prostacyclin may arrest or reverse factors involved in proliferative or occlusive pulmonary vascular disease; i.e., prostacyclin may remodel the pulmonary vasculature [32]. In support of the concept of remodel-
Fig. 3. Comparison of the behavior of NO (open circles) and pulmonary artery pressures (asterisks) determined by right heart catheterization or estimated by RVSP at echocardiography in PAH patients. Paired NO value for the final PAP estimated by RVSP at echocardiography in individuals prior to death was not obtainable.
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Fig. 4. (A) Decreased NO reaction products in the airways of patients with PPH. Total NOx and nitrate levels are decreased, whereas nitrite levels are similar to those of healthy controls. Total protein in EBC does not significantly differ between PPH patients and controls. (B) NO reaction products, specifically NOx and nitrate, are also significantly decreased, when EBC is normalized to total protein. (C) Correlation of nitrate in EBC and systolic PAP estimated by echocardiography. Plots of values in graphs (A) and (B) represent medians F 25–75% interquartile range, with individuals outside these ranges represented by single dots.
ing, long-term treatment with epoprostenol for a mean time of 18 months reverts an initial lack of response to vasodilators in PAH patients [35]. The absence of complete long term data from patients who expired
precludes us from concluding that exhaled NO is a marker of remodeling or a better prognosis. However, our finding of a substantial increase in NO in surviving patients at greater than 9 months after initiation of
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prostacyclin could suggest a remodeling effect on the pulmonary vasculature. For example, regression of pulmonary vascular lesions over time may allow increased endothelial surface area for NO synthesis, improve endothelial cell functions including NO synthesis, or enhance NO diffusion. Subsequently, increased NO production may further enhance regression of vascular structural abnormalities through inhibition of cell proliferation and migration. Inflammation and consequent oxidative stress may also play a role in the pathophysiology of PAH. Inflammatory necrotizing arteritis and increased infiltration of macrophages and lymphocytes are seen in the plexogenic lesions characteristic of pulmonary hypertension [20]. Patients with PAH have higher serum levels of the pro-inflammatory cytokines interleukin-1h and interleukin-6 than healthy controls or patients with pulmonary hypertension associated with hypoxemia [36]. Urinary levels of isoprostaglandin F2a, a marker of lipid peroxidation, are increased in PAH patients and inversely correlated to vasoreactivity to inhaled NO [37]. Further, increased nitrotyrosine expression and elevated levels of 5-oxo-eicosatetraenoic acid and leukotriene B4 5-hydroxyeicosatetraenoic acid, along with lower superoxide dismutase activity, have recently been demonstrated in lungs of PAH patients [38]. In this context, although decreased production in the lung may be a primary determinant of the low levels of NO in PAH, increased NO consumption by reactive oxygen species may also limit NO bioavailability [12,38]. Consequently, reactive oxygen species may contribute to the generation of pulmonary hypertension, in part, by decreasing the half-life of NO. Here, patients with PAH have decreased levels of total NO reaction products in EBC but not in serum, supporting the concept that synthesis of NO is impaired specifically in the lung. Nitrite and nitrate are the primary NO reaction products in the lung epithelial lining fluid [28]. Formation of nitrate occurs in part through decomposition of peroxynitrite (ONOO-), a reactive nitrogen species generated by reaction of NO and superoxide [28]. The direct correlation between EBC nitrate and PAP suggests that reactive oxygen and nitrogen species are associated with the pathophysiology of PAH. This preliminary study provides evidence for the concept that endogenous lung derived NO is a determinant of human pulmonary artery pressure, and demonstrates a role for monitoring exhaled NO in patients in the pathologic state of pulmonary hypertension. Successful long term outcome is associated with increase in NO after many months of therapy. However, it is unclear whether increase in NO is specific only to survivors with PAH. Further, it is not apparent whether increase in NO is a mechanism of
improvement or a specific marker of improvement, e.g., greater vascular bed with regression of pulmonary vascular lesions. Future longer studies may define whether increase in NO is sustainable and if it indicates regression of disease. Acknowledgments—We thank K. Stelmach for help with patient recruitment. This work was supported by NIH Grants HL60917, HL04265, and HL68863 and, in part, by Public Health Service Research Grant MO1-RR018390.
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[29] Ignarro, L. J.; Buga, G. M.; Wood, K. S.; Byrns, R. E.; Chaudhuri, G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci. USA 84:9265 – 9269; 1987. [30] Palmer, R. M.; Ferrige, A. G.; Moncada, S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524 – 526; 1987. [31] Cooper, C. J.; Landzberg, M. J.; Anderson, T. J.; et al. Role of nitric oxide in the local regulation of pulmonary vascular resistance in humans. Circulation 93:266 – 271; 1996. [32] Fishman, A. P. Epoprostenol (prostacyclin) and pulmonary hypertension. Ann. Intern. Med. 132:500 – 502; 2000. [33] Prins, B. A.; Hu, R. M.; Nazario, B.; et al. Prostaglandin E2 and prostacyclin inhibit the production and secretion of endothelin from cultured endothelial cells. J. Biol. Chem. 269:11938 – 11944; 1994. [34] Forrest, I. A.; Small, T.; Corris, P. A. Effect of nebulized epoprostenol (prostacyclin) on exhaled nitric oxide in patients with pulmonary hypertension due to congenital heart disease and in normal controls. Clin. Sci. (Lond.) 97:99 – 102; 1999. [35] Ziesche, R.; Petkov, V.; Wittmann, K.; et al. Treatment with epoprostenol reverts nitric oxide non-responsiveness in patients with primary pulmonary hypertension. Heart 83:406 – 409; 2000. [36] Humbert, M.; Monti, G.; Brenot, F.; et al. Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am. J. Respir. Crit. Care. Med. 151:1628 – 1631; 1995. [37] Cracowski, J.-L.; Cracowski, C.; Bessard, G.; et al. Increased lipid peroxidation in patients with pulmonary hypertension. Am. J. Respir. Crit. Care. Med. 164:1038 – 1042; 2001. [38] Bowers, R.; Cool, C.; Murphy, R. C., et al. Oxidative stress in severe pulmonary hypertension. Am. J. Respir. Crit. Care. Med. 169:764 – 769; 2004 ABBREVIATIONS
EBC — exhaled breath condensate FEV1 — forced expiratory volume in 1 s FVC — forced vital capacity PAH — pulmonary arterial hypertension PAP — pulmonary arterial pressure PPH — primary pulmonary hypertension RVSP — right ventricular systolic pressure
