Rationale: Decreased nitric oxide (NO) is considered an important pathogenetic mechanism in pulmonary arterial hypertension (PAH), but clear evidence is lacking. Objectives: We used multiple techniques to assess endogenous NO in 10 patients with untreated PAH (8 idiopathic and 2 anorexigen-associated PAH) and 12 control subjects. Methods: After a nitrite/nitrate-restricted diet, NO metabolites (NOx) were assayed in 24-hour urine collections and exhaled NO (FE^sub NO^) determined at multiple expiratory flows. Analysis of the relation between FE^sub NO^ and flow allowed derivation of three flowindependent parameters: airway wall concentration (C^sub W^), diffusing capacity (D^sub NO^), and alveolar concentration (C^sub A^). Seven patients underwent follow-up testing after 3 months of bosentan treatment. Results: At baseline, FE^sub NO^ was markedly decreased at the two lowest expiratory flows in PAH: 21 ± 4 versus 36 ± 4 ppb at 18 ml/second and 11 ± 2 versus 17 ± 2 ppb at 50 ml/second, for subjects with PAH and control subjects, respectively (p
Keywords: airway; endothelin; urine nitric oxide metabolites
Impaired nitric oxide (NO) generation has long been proposed as a potential mechanism in the pathogenesis of idiopathic pulmonary arterial hypertension (IPAH); however, assessment of endogenous NO in patients with IPAH has yielded conflicting results. Reduced expression of pulmonary vascular endothelial NO synthase (eNOS) was initially described (1), but this finding has not been confirmed by others (2). NO in exhaled breath has been investigated as an indicator of pulmonary NO production in IPAH, but decreased (3, 4), normal (5), and increased (6) values have been reported. These studies used variable methodology and included subjects receiving therapy that could affect exhaled NO (FE^sub NO^) concentrations (7). Circulating nitrite and nitrate metabolites (NOx) are largely derived from oxidation of NO (8) but are substantially affected by dietary factors (9) and glomerular filtration (10). Reports of plasma NOx levels in IPAH have been highly variable (4, 6, 11). Urinary NOx excretion is considered a valid indicator of whole-body NO production when dietary factors are controlled for (12).
This study used complementary techniques to assess endogenous NO in untreated patients with IPAH and anorexigen-associated PAH. FE^sub NO^ was measured at multiple expiratory flow rates to allow partitioning into airway and alveolar components (13). Nasal NO release was determined as a potential regulator of the pulmonary vasculature (14). Whole-body NO production was assessed by measuring NOx in 24-hour urine collections and plasma levels of NOx, L-arginine, L-citrulline, and the methylated arginines, asymmetric and symmetric dimethyl arginine (15), during a nitrate/nitrite-restricted diet. Moreover, serial testing was performed after therapy with the endothelin (ET) receptor antagonist bosentan. Some of the results of these studies have been previously reported in the form of an abstract (16).
This study was approved by the local institutional review board, and all participants provided written, informed consent. Ten adult patients with newly diagnosed IPAH or anorexigen-induced PAH (17) on no specific therapy were enrolled. Exclusion criteria included any factor that is known or suspected to alter respiratory or systemic NO and are listed in the online supplement. Twelve healthy nonsmoking volunteers served as control subjects. World Health Organization functional class (18) and 6-minute walk distance (19) were obtained at baseline and follow-up examinations.
Urine and Plasma Measurements
Subjects were asked to follow a nitrite/nitrate-restricted diet (20) for 48 hours. During the second day of the diet, a 24-hour urine collection was obtained. On the third morning, participants underwent FE^sub NO^ and nasal NO measurements (see below). Blood was collected into heparinized tubes and plasma separated immediately. Urine NOx was determined with a chemiluminescent analyzer (Sievers 280 NOA; Sievers Instruments, Boulder, CO) (21) and corrected for urine creatinine. Plasma NOx was similarly assayed after deproteinization with cold ethanol (21). L-arginine, L-citrulline, and asymmetric and symmetric dimethyl arginine were assayed by HPLC-mass spectrometry (22). One patient with IPAH did not provide urine or blood samples but underwent FE^sub NO^ measurements.
FE^sub NO^ Measurements
Online recording of FE^sub NO^ was performed according to the recommendations of the American Thoracic Society (23) at expiratory flow rates of 18, 50, 100, and 250 ml/minute. Using the method described by Silkoff and coworkers (13), the curvilinear relationship between FE^sub NO^ and flow rate was analyzed by nonlinear least squares regression to derive the airway wall NO concentration (C^sub W^), the diffusing capacity of NO from airway wall to lumen (D^sub NO^), and the alveolar NO concentration (C^sub A^) according to the following equation:
FE^sub NO^ = C^sub W^(1 - e^sup -Dno/V^) + C^sub A^ e^sup -Dno/V^,
where V = expiratory flow rate.
Ambient air (always
Of the 10 patients with PAH, 7 (including both of the anorexigen-associated cases) were restudied after 3 months of therapy with bosentan (Tracleer; Actelion Pharmaccuticals, South San Francisco, CA) administered according to the package insert.
Data are expressed as means ± SEM. Comparison of FE^sub NO^ values of the patients with PAH at baseline with the control group and between the patients at baseline with patients after bosentan was performed with analysis of variance. The mean FE^sub NO^ values at the different flow rates for each group were used to derive C^sub W^, D^sub NO^, and C^sub A^ by nonlinear regression as described above. Individual FE^sub NO^ data were also used to calculate these parameters, which were converted to natural logarithms (13) and compared between groups with a paired or unpaired t test, as appropriate. Nasal NO output and the urine and plasma variables were compared between the control and PAH baseline groups with an unpaired t test or the nonparametric Mann-Whitney test as appropriate. A Wilcoxon matched pairs lest was used to compare the seven bosentan-treated patients with PAH at baseline and at follow-up.
Table 1 shows the demographic and clinical characteristics of the subjects with PAH and control subjects. All had moderate-severe disease with a mean pulmonary arterial pressure of greater than 40 mm Hg, and all but one had World Health Organization class III-IV symptoms. Two had a history of fenfluramine use. Although anorexigen-associated PAH is classified separately from IPAH, there is no evidence that these groups differ clinically (24) or pathologically (25). Moreover, the various NO parameters obtained from these two individuals were comparable to the remainder of the IPAH group and excluding them from the analyses did not significantly alter the results. The control group was well matched in terms of sex, age, and height-factors that may affect FE^sub NO^ (26).
FE^sub NO^ concentrations were markedly reduced in the PAH group at baseline compared with control subjects (Figure 1). This was particularly evident at the lower expiratory flow rates (e.g., at 18 ml/second, mean FE^sub NO^ in the patients with PAH was 21 ppb compared with 36 ppb in control subjects). Application of the model described above to the mean group FE^sub NO^ data revealed that C^sub W^ was reduced to less than half the normal value in the patients at baseline (Figure 2A). When individual FE^sub NO^ data were used, the fit of two patients with PAH at baseline did not converge. The C^sub W^ of the other eight patients was 33.1 ± 11 ppb compared with 104 ± 34 in the control group (p = 0.04; Figure 2B). No significant differences in D^sub NO^ or C^sub A^ were observed.
Nasal NO output was similar in patients with PAH at baseline (512 ± 48 nl/minute) and control subjects (760 ± 125 nl/minute; p = 0.2).
Urine and Plasma NOx
Urinary excretion of NOx was significantly reduced in patients with PAH at baseline compared with control subjects (Table 2). In contrast, plasma NOx was similar in the two groups. Creatinine clearance, as expected, was mildly decreased in the PAH group.
Plasma L-arginine, L-citrulline, and Asymmetric and Symmetric Dimethyl Arginine
Table 2 summarizes the HPLC data for control subjects and patients with PAH at baseline. Although L-arginine levels were identical between the two groups, plasma L-citrulline was markedly decreased in the patients with PAH, resulting in a significantly lower L-citrulline/L-arginine ratio. The patients with PAH also demonstrated small but significant increases in asymmetric and symmetric dimethyl arginine plasma levels.
Changes in Response to Bosentan Therapy
All but one of the seven patients restudied after 3 months of bosentan therapy reported subjective improvement in symptoms. Six-minute walk distance increased from 296 ± 34 to 341 ± 41 m (p = 0.02). FE^sub NO^ at the lower expiratory flow rates increased dramatically, becoming similar to those observed in the control group. Figure 1 displays the comparison between all patients with PAH at baseline and the seven treated with bosentan at follow-up. Similar results were obtained when the comparison was limited to the seven bosentan-treated patients (data not shown).
Using mean group FE^sub NO^ data for the partitioning analysis, C^sub W^ similarly normalized, whereas D^sub NO^ and C^sub A^ did not appear to change (Figure 2A). We also performed this analysis for each individual patient at baseline and follow-up. For two of the bosentan-treated patients at baseline, the model fit did not converge. Comparison of individual C^sub W^ values among eight patients at baseline with seven patients after bosentan treatment revealed a marked increase from 33 ± 11 to 105 ± 44 ppb (p = 0.01; Figure 2B). Similar results were obtained when the comparison was limited to the five bosentan-treated patients whose data were available for partitioning analysis at both baseline and follow-up (p
Nasal NO output at follow-up (659 ± 146 nl/minute) was similar to baseline (498 ± 46 nl/minute; p = 0.7). Urinary excretion of NOx significantly increased after 3 months of bosentan therapy (Table 2). No significant changes were detected in any of the plasma variables, whereas a trend was noted for a reduction in creatinine clearance at follow-up.
The status of endogenous NO in PAH is unclear and highly controversial. This study demonstrated that patients with PAH have markedly reduced FE^sub NO^ at lower expiratory flow rates, which may be the result of decreased C^sub W^. We also show that whole-body NO production, as assessed by 24-hour urinary NOx excretion, is decreased. In response to treatment with the nonselective ET receptor antagonist bosentan, these abnormalities reverted to normal after 3 months.
Shortly after the discovery that NO was detectable in exhaled breath, investigators began to explore this critical vasoactive mediator in IPAH, yielding highly variable results (3,5,6). These studies were conducted before the realization that FE^sub NO^ varies widely with the expiratory flow rate and that the nasal passages need to be reliably excluded. Researchers at the Cleveland Clinic demonstrated significantly reduced lower respiratory tract NO concentrations in IPAH using a bronchoscopic technique (4). The current study uses the standardized procedure for online recording of FE^sub NO^ recommended by the American Thoracic Society (23), Also, unlike previous studies, only untreated patients were included. It is well recognized that epoprostenol administration can dramatically increase FE^sub NO^ (7, 27). Moreover, care was taken to exclude subjects with concomitant factors that could alter respiratory or systemic NO. The most important finding of our study is the profound reduction in FE^sub NO^ at lower expiratory flow rates in patients with PAH at baseline and the normalization of these abnormalities with bosentan therapy.
Partitioning and Anatomic Source of FE^sub NO^
Detailed analyses of pulmonary NO exchange dynamics have led to a two-compartment model (airways and alveolar region) describing three flow-independent NO exchange parameters during exhalation: mean NO concentration in the airway wall, total airway compartment NO diffusing capacity from the airway wall to lumen, and NO concentration in alveolar gas (28). Derivation of these parameters requires measurements of FE^sub NO^ at multiple expiratory flow rates, as described by Silkoff and coworkers (13). For all but two measurements (two patients with PAH at baseline), the data fit the model well, and the flow-independent parameters obtained in the control subjects were comparable to previously published results (28). On the basis of this model, we have shown that the reduced FE^sub NO^ concentrations observed in the PAH group were due to decreased C^sub W^. The normal nasal NO output in our patients with PAH indicates that this abnormality is localized to the lower respiratory tract.
Since the discovery of NO in exhaled breath, there has been considerable controversy regarding the extent to which the pulmonary vasculature contributes to FE^sub NO^. The term "airway wall" in the model used here encompasses all of the tissue between the lumens of the airways and blood vessels, including pulmonary and bronchial endothelium, vascular and bronchial smooth muscle, and airway epithelium. Because of its extremely high avidity for hemoglobin, most NO produced in the blood vessels is expected to be consumed within the vascular lumen. Although several lines of evidence support the contention that the majority of FE^sub NO^ originates from cells lining the airways and alveoli (29, 30), pulmonary vascular factors may also influence FE^sub NO^ (31). Thus, the precise origin of airway wall tissue NO remains to be fully elucidated.
Significance of Reduced FE^sub NO^ in PAH
Irrespective of the exact source of FE^sub NO^, our findings have potential clinical and mechanistic importance. The striking reductions at baseline and significant increases in FE^sub NO^ in response to therapy observed here and by others (4, 7), together with the recent suggestion that serial measurements may correlate with clinical outcomes (32), point to the potential utility of this noninvasive, easily obtained assessment as a clinical biomarker pending further validation. Although decreased FE^sub NO^ in IPAH may reflect reduced NO release from the pulmonary vasculature as a consequence of endothelial dysfunction, reduced lower airway NO may also have pathogenetic importance as a regulator of the adjacent pulmonary arteries (33).
Mechanism(s) for Decreased FE^sub NO^ in PAH
The main determinant of C^sub W^ is the production of NO in the airway wall tissue relative to its catabolism (13). Decreased pulmonary expression and/or activity of NOS could account for the low C^sub W^ observed in the patients with PAH. Recent studies in humans suggests that the type II, inducible (iNOS) isoform is the major determinant of FE^sub NO^ (34). Although the abundance of vascular endothelial NOS in PAH is debatable, there is no evidence for altered expression of any of the NOS isoforms in PAH airways (1, 35). However, available data are quite limited in this regard.
A major route of NO consumption is reaction with superoxide, forming peroxynitrite. There is considerable evidence for oxidative stress in PAH (36) and intense nitrotyrosine (a marker of peroxynitrite formation) expression in the lung (37). Thus, enhanced oxidative consumption of NO could explain reduced exhaled and airway wall concentrations. Oxidants can also directly suppress NOS activity (15). Enhanced binding of NO to metalloproteins is another potential mechanism for reduced airway NO (38).
NO is generated by the NOS-induced oxidation of L-arginine to L-citrulline. The reduction in plasma L-citrulline/L-arginine ratio observed in the patients with PAH suggests decreased NOS activity. We detected elevated asymmetric dimethyl arginine, a potent endogenous inhibitor of NOS (15), in PAH plasma. However, the magnitude of the observed increase is probably not biologically significant (39). Normal asymmetric dimethyl arginine levels in IPAH have been recently reported (35), whereas another, preliminary, study found significantly increased values (40). Similarly, the significance of our finding of high circulating symmetric dimethyl arginine in the patients with PAH is unclear. Although not a direct inhibitor of NOS, symmetric dimethyl arginine may interfere with L-arginine uptake into cells by competing with the cationic amino acid transporter (41), but considerably higher concentrations than detected here in the plasma would be required for this effect.
Twenty-four-hour urinary NOx excretion can serve as a qualitative indicator of whole-body NO production, once dietary intake and renal function are controlled for, and this has been shown to be reduced in systemic vascular diseases (42). A recent study in four patients with IPAH has shown results consistent with ours (43). Although reduced pulmonary NO production and/or consumption may be the predominant source of the lower urine NOx. a systemic or renal (12) contribution is not excluded. Plasma NOx has been assessed previously in IPAH with variable results (4, 11). Archer and coworkers (6) reported normal plasma NOx in IPAH, but increased values in fenfluramine-related PAH. The latter had a lower cardiac index and, consequently, lower glomerular filtration rate. There is evidence for diurnal variation in urinary NOx excretion (44). Hence, a 24-hour urine collection provides a more reliable global assessment of NO production than a single random plasma sample.
Changes in Endogenous NO in Response to Bosentan Therapy
We were surprised by the significant increase in FE^sub NO^ (and C^sub W^) and urinary NOx in response to bosentan. These changes suggest increased airway and possibly pulmonary and/or bronchial vascular NO production and/or reduced consumption. There is a well-recognized reciprocal relationship between ET and NO (45). Stimulation of ET-B receptors on vascular endothelial cells enhances NO release, whereas activation of ET-A receptors located predominantly on vascular smooth muscle inhibits NO production. Both receptors are abundantly expressed on airway epithelial and smooth muscle cells, mediating a host of biological effects (46). Analogous to the actions of ET in arteries, there is some evidence for the presence of bronchodilatory ET-B receptors on bronchial epithelial cells that release NO (47). ET-1 infusion acutely increased FE^sub NO^ in guinea pigs (48), and bosentan acutely reduced FE^sub NO^ in anesthetized dogs (49). Consistent with our results, however, is the normalization of a reduced FE^sub NO^ in chronically hypoxic pulmonary hypertensive piglets by chronic administration of an ET receptor-A selective antagonist (50). Both in vitro (51) and in vivo (52, 53) studies have demonstrated enhanced NOS activity in response to ET-A or combined ET-A+B receptor blockade. Several mechanisms may be involved, including enhanced transcription of eNOS (52), post-transcriptional regulation (53), and attenuation of the prooxidant effects of ET (54).
Given the small number of patients studied here, our results must be considered preliminary, and more research is required to further characterize the mechanisms accounting for our observations. An important limitation of our study is the lack of data regarding the level of NOx, oxidants, and nitroso-compounds within the epithelial lining fluid, which could be assessed in bronchoalveolar lavage fluid or exhaled breath condensate. Such information would help clarify the basis for our FE^sub NO^ findings. Previous studies have reported reduced NOx in bronchoalveolar lavage fluid (4) and exhaled breath condensate in patients with IPAH (32).
In summary, we have demonstrated that FE^sub NO^ at low expiratory flow rates is markedly reduced in untreated patients with PAH. Partitioning analysis reveals that this reduction is due to decreased airway wall tissue concentrations of NO. This is accompanied by a reduction in whole-body NO production as indicated by 24-hour urinary NOx excretion. Moreover, the ET receptor antagonist bosentan normalized these abnormalities after 3 months of treatment. Decreased production and/or increased consumption of pulmonary NO may contribute to the pathogenesis of PAH, and FE^sub NO^ may serve as a useful biomarker in this challenging disease.
Conflict of Interest Statement: R.E.G. received $3,000 in lecture fees per year for each of the past two years from Actelion Pharmaceutical, manufacturer of Tracleer (bosentan); H.C.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; G.B.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; R.A.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; S.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; J.T.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.
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Reda E. Girgis, Hunter C. Champion, Gregory B. Diette, Roger A. Johns, Solbert Permutt, and J. T. Sylvester
Divisions of Pulmonary and Critical Care Medicine and Cardiology, Departments of Medicine and Anesthesiology, Johns Hopkins University, School of Medicine, Baltimore, Maryland
(Received in original form December 15, 2004; accepted in final form May 3, 2005)
Supported by American Lung Association clinical research grant CG011N (R.E.G.).
Correspondence and reguests for reprints should be addressed to Reda E. Girgis, M.B., B.Ch., Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, School of Medicine, 1830 East Monument Street, 5th floor, Baltimore, MD 21205. E-mail: firstname.lastname@example.org
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Am J Respir Crit Care Med Vol 172. pp 352-357, 2005
Originally Published in Press as DOI: 10.1164/rccm.200412-1684OC on May 5, 2005
Internet address: www.atsjournals.org
Copyright American Thoracic Society Aug 1, 2005
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