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European Journal of Heart Failure 2001 3(1):27-32; doi:10.1016/S1388-9842(00)00116-1
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© 2001 European Society of Cardiology

Increased nitric oxide in exhaled air in patients with rheumatic heart disease

Zehra Gölbasla,*, Sibel Dinçerb, Hakan Bayola, Belgizar Ugurlub, Dilek Çiçeka, Telat Kelesa, Sinan Aydogdua and Deniz Erbasb

a Department of Cardiology, Ankara Numune Education and Research Hospital Ankara, Turkey
b Department of Physiology, Gazi University Medical Faculty Ankara, Turkey

* Corresponding author. Tel.: +90-31-2310-3030/+90-31-2310-3635; fax: +90-31-2310-3460. E-mail address: golbasiz{at}anh.gov.tr (Z. Gölbas1).


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Endogenous production of nitric oxide and its presence in exhaled air was observed in humans. Prior studies have yielded contrasting information about the production of nitric oxide in patients with heart failure.

Aims: The aim of this study was to measure nitric oxide in the exhaled air of patients with chronic rheumatic heart disease with and without pulmonary hypertension.

Methods: Seventy-four patients (6 patients had isolated mitral stenosis; 13 patients had combined mitral stenosis and mitral regurgitation; 1 patient had isolated mitral regurgitation; 54 patients had combined mitral and aortic valve disease) and 27 healthy subjects were entered in the study. The nitric oxide concentration in exhaled air was determined with a chemiluminescence analyser. Echocardiography was performed in all patients to assess the severity of the valve disease and for the measurement of pulmonary artery pressure.

Results: The level of exhaled nitric oxide was significantly greater in patients with rheumatic heart disease than in controls. The value of nitric oxide concentration in exhaled air was significantly increased in patients with pulmonary hypertension, as compared with patients who had normal pulmonary artery systolic pressure.

Conclusion: We found increased nitric oxide in the exhaled air in patients with rheumatic heart disease, especially in those with pulmonary hypertension, compared with healthy patients.

Key Words: Nitric oxide • Heart failure • Rheumatic heart disease • Pulmonary hypertension

Received January 4, 2000; Revised April 20, 2000; Accepted June 20, 2000


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Nitric oxide (NO) is known to be a potent vasodilator and it is produced in all blood vessels, including the pulmonary circulation [1,2]. Nitric oxide is synthesized in pulmonary endothelial cells from L-arginine by endothelial NO synthase. It activates soluble guanylate cyclase, increasing the concentration of cyclic guanosine monophosphate in smooth muscle cell, resulting in vasodilation. Prior studies have yielded contrasting information about the production of NO in patients with heart failure. In addition, there is conflicting information about the role of NO in the regulation and maintainence of the basal pulmonary vascular tone [2–4]. Increased resistance to pulmonary blood flow leads to a condition known as passive pulmonary hypertension. The resistance to pulmonary venous blood flow may arise from various disorders but is most commonly seen in patients with left-sided valvular disease. The aim of this study was to measure NO in the exhaled air of patients with chronic rheumatic heart disease with and without pulmonary hypertension.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The investigation conforms with the principles outlined in the Declaration of Helsinki.

2.1. Patients
A total of 81 patients with rheumatic heart disease were entered into the study. Seven patients, who had no measurable tricuspid regurgitant signals, were excluded from the study. Seventy-four patients (56 women and 18 men; mean age: 42.0±13.8 years; range: 21–81) were assessed. Six patients had isolated mitral stenosis, 13 had combined mitral stenosis and mitral regurgitation, 1 had isolated mitral regurgitation, and 54 had combined mitral and aortic valve disease. Tricuspid stenosis was only detected in four of the patients who had combined mitral and aortic valve disease. Fractional shortening was measured below 25% only in two of the patients who had combined mitral and aortic regurgitation. Atrial fibrillation was detected in 16 patients. Patients were stratified according to baseline functional class: 27 patients were NYHA class I, 37 patients were NYHA class II, and 10 patients were NYHA class III. In all patients, vital capacity (VC) and forced expiratory volume at first second (FEV1) were measured. Patients with obstructive and/or restrictive patterns were excluded from the study. Patients with a previous diagnosis of bronchial asthma and patients with any upper or lower respiratory tract infection during the period of at least 4 weeks preceding the study were excluded from the study. We also excluded patients with previous history of myocardial infarction and/or patients who had electrocardiographic findings of myocardial infarction. The following drugs were administered: benzatine penicilline G (36), digoxin (17), aspirin (24), diuretic (13), angiotensin converting enzyme inhitor (4), coumadin (2), and calcium antagonist (1). All medications were discontinued 24 h before each study began. Alcohol, caffeine, and cigarettes were prohibited in the 12-h period before the study.

Twenty-seven healthy non-smoking age- and gender-matched subjects (18 women and 9 men; mean age 40.5±12.6 year; range 20–65) were studied as a control group.

2.2. Nitric oxide measurement
The NO concentration in exhaled air was determined with a chemiluminescence analyser (Sievers NOA 280, Boulder, CO, USA). The sensitivity of the analyser to NO ranged from less than 1 ppb–500 000 ppb. The system was calibrated before each study using a certified NO gas and NO free air. During the study period, the room air levels of NO concentration were less than 10 ppb. Experiments were performed with subjects in a sitting position and breathing normally for 10 min. Each subject was asked to inhale synthetic air free of NO by filtering through a large charcoal filter. The subjects, with nose occluded, were asked to produce a slow expired vital capacity maneuver over 20–30 s through a mouth breathing face mask connected to a Y-shaped valve. Three recordings were made at 2-min intervals and the average was recorded for each subject.

2.3. Echocardiographic measurement
The echocardiographic examination was performed with a GE Vingmed System FiVe ultrasound machine. M-mode, two-dimensional, and Doppler echocardiograms were obtained with the subjects in the left lateral decubitis position. The left ventricular dimension was measured in the long axis view of the left ventricle. The left ventricular fractional shortening was obtained using the Teichholz equation. Mitral stenosis was quantified by planimetry of 2-D images, Doppler measurement of transvalvular gradients, and estimation of valve area by the pressure half-time method [5–7]. Doppler methods, including assessment of regurgitant jet characteristics (length, height, area, and width at the vena contracta), were used to assess the severity of valvular regurgitation [8]. Tricuspid stenosis was quantified by Doppler measurement of transvalvular gradients, and the valve area was estimated using the pressure half-time method [9]. Aortic stenosis was quantified using Doppler measurements of transvalvular gradients, and the valve area was estimated using the continuity method [10]. The maximal velocity of tricuspid regurgitant jet was assessed by continuous wave Doppler echocardiography from a low parasternal, long-axis view of the right ventricular inflow or apical and subcostal views. The pressure gradient between the right ventricle and right atrium was calculated by applying the Bernoulli equation [11]. An estimate of right atrial pressure was added to the gradient. A calibrated estimate of right atrial pressure by use of phasic respiratory inferior vena caval dimensions was used [12]. Pulmonary hypertension was diagnosed if pulmonary artery systolic pressure was over 35 mmHg. Measurements represent an average of three beats for patients in sinus rhythm and 10 beats for patients in atrial fibrillation.

2.4. Statistics
Data were analysed with the SPSS for Windows statistical package and are presented as mean±S.D. Differences between mean values were calculated using the Student's unpaired t-test. Univariate comparisons between groups were made with non-parametric tests: (Kruskal–Wallis tests for multigroup comparisons and Mann–Whitney tests for two-group comparisons). The chi-square test and Fischer's probability test were used to compare proportions. Differences were considered significant when P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
Patient characteristics including severity of valvular disease are given in Table 1. The level of exhaled NO was significantly greater in patients with rheumatic heart disease than in controls (18.2±16.7 ppb vs. 12.3±7.9 ppb, respectively; P<0.05). The value of NO concentration in exhaled air was significantly increased in patients with pulmonary hypertension, as compared with patients who had normal pulmonary artery systolic pressure (21.4±20.1 ppb vs. 14.1±9.6 ppb, respectively; P<0.05) (Table 2 and Fig. 1). Pulmonary artery pressure was significantly higher in the study group than in the controls (44.6±18.1 mmHg vs. 15.0±4.2 mmHg; P<0.0001). Fourty-two patients had pulmonary hypertension, and 37 of them had mitral stenosis. Pulmonary artery pressure of patients with pure and/or predominant mitral stenosis was significantly higher than that of the patients without predominant mitral stenosis (61.2±22.4 mmHg vs. 35.0±10.0 mmHg, respectively; P<0.0001). The NO concentration in the exhaled air of patients with pure and/or predominant mitral stenosis was also significantly higher than that of the patients without predominant or pure mitral stenosis (28.9 ±22.7 ppb vs. 15.1±10.6 ppb; P<0.05). No correlation was found between the severity of the pulmonary hypertension and the levels of exhaled NO. In addition, no correlation was observed between NO concentrations and age, sex, body surface area, smoking habits, functional capacity, mitral valve area, presence of atrial fibrillation, blood pressure, or cholesterol levels.


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  		Table 1 Patient characteristics

 


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  		Table 2 Mean values of exhaled nitric oxide and pulmonary artery systolic pressure in patients with rheumatic heart disease and normal individuals

 


Figure 1
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  		Fig. 1 Individual values of concentration of NO (ppb) in patients and in healthy controls. PH, pulmonary hypertension.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The findings of the present study demonstrate that NO concentration in exhaled air was increased in patients with rheumatic heart disease compared with normal subjects. NO concentration in exhaled air was significantly greater in patients with rheumatic heart disease with pulmonary hypertension than in patients with normal pulmonary artery systolic pressure.

In the pulmonary circulation, NO mediates vasodilation to some stimuli and moderates vasoconstriction to others, but there is conflicting information about its role in maintaining the basal pulmonary vascular tone [2–4]. NO has a potent pulmonary vasodilatory effect that could potentially modulate the development of chronic hypoxia-induced pulmonary arterial hypertension. Endothelium-dependent pulmonary artery relaxation in vitro is impaired in patients with chronic obstructive lung disease [13]. It has been suggested that such impairment could contribute to the development of pulmonary hypertension in these patients. In addition, decreased NO production and decreased endothelial nitric oxide synthase were found in the lungs of newborn pigs subjected to chronic hypoxia [14]. Reduced expression of endothelial nitric oxide synthase was found in the lungs of patients with pulmonary hypertension [15]. However, not all evidence supports the idea that chronic hypoxia decreases NO production in the lung. Some investigators have found increased expression of endothelial nitric oxide synthase mRNA and protein in the hypoxic rat lung [16]. Also, it was shown that NO production and NO-dependent vasodilation were enhanced in the lungs of rats subjected to chronic hypoxia [17,18].

Increased resistance to pulmonary blood flow leads to a condition known as passive pulmonary hypertension, where there is an elevation in pulmonary artery pressure but no significant elevation in pulmonary vascular resistance. The resistance to pulmonary venous blood flow may arise from various disorders but is most commonly seen in patients with left-sided valvular disease. Could the resistance due to increased left atrial pressure against pulmonary flow be a mechanical stress for pulmonary arterial endothelium in our patients? If this is the case, high levels of NO in our patients with pulmonary hypertension could be due to this mechanical stress. Although endothelial nitric oxide synthase in the lung and pulmonary artery is upregulated in hypoxic rats, it is unclear whether the upregulation is caused by the hypertension or some other effect of the hypoxic exposure [19]. Also, it was shown that endothelial nitric oxide synthase gene expression of cultured endothelial cells is increased by shear stress [20,21].

Prior studies have yielded contrasting information about the production of NO in patients with heart failure. In one study, it was shown that the levels of plasma nitrate, a stable end product of NO production, were increased in patients with congestive heart failure [22]. However, another study showed that the concentration of NO in exhaled air was reduced in patients with low cardiac output [23]. In addition, it was shown that NG-monomethyl-L-arginine, a specific inhibitor of NO synthesis, increases the pulmonary vascular resistance in patients with heart failure, suggesting an enhanced basal production of NO [24]. These data suggested that vascular NO might be another example of a failed counter-regulatory vasodilator system in heart failure [24].

Data from pulmonary hypertension associated with connective tissue disease has suggested low exhaled NO levels. In one study, it was shown that exhaled NO is decreased in patients with systemic sclerosis and pulmonary hypertension when compared with both normal individuals and patients with systemic sclerosis without pulmonary hypertension [25]. There is evidence that the endothelium is damaged early in the course of systemic sclerosis. These data suggest that patients with systemic sclerosis and pulmonary hypertension have diminished endothelial production of nitric oxide [25].

In vitro studies have shown that cytokines induce NO synthase in tissue, including vascular endothelium, smooth muscle, and bronchial epithelium [26,27]. There are many reports showing increased plasma concentration of inflammatory cytokines in patients with acute rheumatic fever [28,29], but there are no data showing increased plasma levels of cytokines in patients with chronic rheumatic heart disease. Therefore, the increased level of NO in our patients as a result of stimulation by the cytokines remains unlikely.

NO in the exhaled air could be derived from sources other than the lower respiratory tract, e.g. the upper respiratory tract and esophagus. However, such possibilities remain unlikely in our patients. The epithelium of the upper respiratory tract is the source of exhaled NO in patients with airway inflammatory disease such as asthma [30]. There are no data showing that inflammation of the airway is a feature of patients with chronic rheumatic heart disease. We used the nose clip during the expiratory maneuver, which minimized the nasal contribution to the concentration of exhaled NO. In addition, in a study that measured NO via a bronchoscope, it was shown that NO levels measured at the mouth reflect levels in the lower respiratory tract [31].

In conclusion, we found increased NO in the exhaled air in patients with rheumatic heart disease, especially in those with pulmonary hypertension, compared with the healthy subjects. Therefore, it may be suggested that increased NO levels in our patients may be a regulatory mechanism against increased pulmonary artery pressure. Additional studies are required to determine whether altered pulmonary vascular endothelial NO production plays a pathogenetic role in the development of pulmonary hypertension in patients with rheumatic heart disease.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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