Author + information
- Received June 2, 1999
- Revision received September 21, 1999
- Accepted October 27, 1999
- Published online February 1, 2000.
- Wayne Tworetzky, MDa,
- Phillip Moore, MDa,
- Janine M Bekker, MSa,
- James Bristow, MDa,
- Stephen M Black, PhDa and
- Jeffrey R Fineman, MDa,* ()
- ↵*Reprint requests and correspondence: Dr. Jeffrey R. Fineman, University of California, San Francisco, 505 Parnassus Avenue, Box 0106, M-680, San Francisco, California 94143-0106
To determine the effect of pulmonary blood flow (Qp) on nitric oxide (NO) production in patients with increased Qp due to an atrial septal defect (ASD).
Alterations in pulmonary vascular NO production have been implicated in the development of pulmonary hypertension secondary to increased Qp. In vitro, acute changes in flow or shear stress alter NO production. However, the effect of Qp on lung NO production in vivo is unclear.
Nineteen patients (2.4–61 years of age, median 17) with secundum ASD undergoing device closure were studied. Before, and 30 min after ASD closure, exhaled NO and plasma nitrate concentration were measured by chemiluminescence (NOA 280, Sievers, Boulder, Colorado).
Before ASD closure, all patients had increased Qp (Qp: systemic blood flow [Qs] of 2.0 ± 0.7) and normal mean pulmonary arterial pressure (13.4 ± 3.1 mm Hg). Atrial septal defect device closure decreased Qp from 6.0 ± 2.5 to 3.6 ± 1.3 L/min/m2(p < 0.05). Mean pulmonary arterial pressure was unchanged. Associated with the decrease in Qp, both exhaled NO (−22.1%, p < 0.05) and plasma nitrate concentrations (−17.9%, p < 0.05) decreased.
These data represent the first demonstration that acute changes in Qp alter pulmonary NO production in vivo in humans. Exhaled NO determinations may provide a noninvasive assessment of pulmonary vascular NO production in patients with congenital heart disease. Potential correlations between exhaled NO, pulmonary vascular reactivity and pulmonary hypertension warrant further study.
Nitric oxide (NO) is an endothelium-derived relaxing factor synthesized by the vascular endothelium from the oxidation of the guanidino nitrogen moiety of L-arginine (1). After certain stimuli, such as the receptor binding of specific vasodilators, NO is synthesized after activation of endothelial NO synthase (eNOS) (2,3). Once released from endothelial cells, NO diffuses into vascular smooth muscle cells, where it activates soluble guanylate cyclase, the enzyme that catalyzes the production of guanisone 3′,5′-cyclic monophosphate (cGMP) from guanosine 3′,5′-triphosphate. This results in increased smooth muscle cell concentrations of cGMP, thus initiating a cascade that results in smooth muscle relaxation (4). Recent evidence suggests that basal production of NO is an important mediator of pulmonary vascular tone, a modulator of pulmonary vascular reactivity and an inhibitor of platelet aggregation and smooth muscle mitogenesis (5–8). In addition, aberrations in NO activity have been demonstrated in patients with congenital heart disease and increased pulmonary blood flow (Qp), implicating a role for NO in the pathophysiology of pulmonary hypertension (9,10).
In vitro data suggest that acute changes in flow or shear stress regulate the production of endothelial NO production (11–13). However, the mechanisms by which NO production is regulated in vivo are unclear. The purpose of this study was to determine if acute changes in Qp alter endogenous NO production in patients with increased Qp due to congenital heart defects. To this end, we studied patients with increased Qp due to secundum atrial septal defects (ASDs) who were undergoing device closure of ASD in the cardiac catheterization laboratory. This experimental model provides a unique condition in which Qp is acutely changed without the effects of cardiopulmonary bypass. To determine changes in endogenous NO production, we measured both exhaled NO (an indirect determinant of pulmonary NO production) and plasma nitrate (an indirect determinant of whole body NO production) concentrations before and 30 minutes after ASD closure (14,15).
Children and adults with secundum ASDs were enrolled in the study. All patients were concurrently enrolled in a multicentered trial evaluating ASD closure with the CardioSEAL (Nitinol Medical Technologies, Boston, Massachusetts) device (16). Patients were excluded from the study if they had a history of reactive airway disease, renal insufficiency or were taking anti-inflammatory medication.
All patients were fasted for 12 h before the procedure. Patients were then brought to the cardiac catheterization laboratory where they were sedated, intubated and mechanically ventilated with an FiO2of 0.25. Normocarbic ventilation was maintained utilizing an Ohmeda Modulus 2 plus (Ohmeda Inc., Austell, Georgia). Throughout the procedure, FiO2, minute ventilation and ventilator flow were held constant. General anesthesia was maintained with intravenous or inhalational agents. Baseline (preclosure) measurements of pulmonary and systemic arterial pressures and heart rate were obtained (hemodynamic variables). Pulmonary and systemic arterial and venous blood samples were obtained for measurement of hemoglobin and hemoglobin oxygen saturation. Oxygen consumption was estimated based on heart rate and body surface area, and Qp and systemic blood flow (Qs) were estimated by the Fick equation. Before implantation of the device, after a minimum of 30 min of stable mechanical ventilation, breath-to-breath determinations of exhaled NO were obtained for 30 s. In addition, femoral venous blood was obtained for plasma nitrate (NOx) determinations. Thirty minutes after ASD device closure, repeat measurements of the hemodynamic variables, hemoglobin and hemoglobin oxygen saturation, Qp, Qs, exhaled NO and plasma NOxwere obtained. Each patient had exhaled NO determinations at least two times before the preclosure value was obtained to insure a steady-state value. To determine if cardiac catheterization alone, without changes in blood flows, altered plasma NOxconcentrations, plasma was obtained in six additional patients undergoing diagnostic catheterization. Samples were obtained before and 30 min after angiography in these patients. Exhaled NO was not determined in these patients because their trachea were not intubated.
The protocol was approved by the Committee on Human Research, University of California, San Francisco. Written, informed consent was obtained from all patients or their parents before cardiac catheterization.
Pulmonary arterial pressure was measured using a side-hole balloon catheter (Arrow Intl., Reading, Pennsylvania) connected via a fluid-filled system to a pressure transducer (Abbott Labs, North Chicago, Illinois). Mean pressure was obtained by electrical integration. Systemic arterial pressure was measured by an automated sphygmomanometer (Critikon Inc., Tampa, Florida) placed on the left arm. Heart rate was measured with a cardiotachometer. Systemic arterial blood gases and pH were measured using a 850 pH/Blood Gas Analyzer (Ciba-Corning Diagnostic, Tarrytown, New York). Hemoglobin and hemoglobin oxygen saturations were measured in whole blood using an OSM2 oximeter (Radiometer America Inc., Westlake, Ohio). Oxygen consumption was assumed based on heart rate and body surface area (17). Pulmonary and systemic blood flows were estimated by the Fick equation.
Exhaled NO determinations
Expiratory gas was sampled continuously form a side port attached to the ventilator circuit at a rate of 200 ml/min. Exhaled NO was measured using a chemiluminescence analyzer (NOA 280, Sievers, Boulder, Colorado) as previously described (18). The analog signal was digitalized and stored continuously on a computer using data acquisition software (Sievers). Utilizing an inline pressure transducer to determine the beginning and end of exhalation, NO was determined on a breath-by-breath basis. The average concentration of NO at end-expiration over a 30 s time period was used as the exhaled NO concentration. The preclosure values were taken immediately before delivery of the device, and the postclosure values were taken 30 minutes after device closure. Calibration was performed daily. Previous investigations have demonstrated linearity of the analyzer over the concentrations measured (18).
Blood samples (1 to 2 ml) were collected in iced vacutainer tubes. The samples were centrifuged (4000 g× 15 min) and the resulting plasma stored in polypropylene storage tubes at −70°C. Before analysis, plasma samples were deproteinized with 0.2M HCl. In solution, NO reacts with molecular oxygen to form nitrite and with oxyhemoglobin and superoxide anion to form NOx. The nitrite and NOxwere reduced using vanadium (III) and hydrochloric acid at 90°C. Nitric oxide was then purged from solution resulting in a peak of NO. Therefore, this value represents total NO, nitrite and NOx. This peak was then detected by chemiluminescence (NOA 280,Sievers). The detection limit is 1 nM/ml of NOx. Calibration was performed daily.
The mean ± standard deviation (SD) were calculated for the hemodynamic variables, Qp, Qs, exhaled NO and plasma NOxconcentrations. The hemodynamic variables, Qp, Qs and exhaled NO concentrations were compared before and after ASD closure by the paired ttest. Plasma NOxconcentrations were not normally distributed. Therefore, they were compared before and after ASD closure by the Wilcoxon signed-rank test. A p < 0.05 was considered significant.
Twenty-six patients were enrolled in the study. During cardiac catheterization, seven patients were excluded from the CardioSEAL ASD device closure protocol: five due to large ASD size (>20 mm) and two due to the presence of associated defects. Nineteen patients (age 2.4 to 61 years, median 12 years, 11 female and 8 male) completed the study; no additional patients were excluded. Two patients did not have exhaled NO determinations because of technical problems. These patients had plasma NOxdeterminations only. Two other patients did not have plasma NOxdeterminations because of error in sample handling. These patients had exhaled NO determinations only. All enrolled patients had both pre- and post-value determinations.
The median ASD size was 11.0 mm, with a range of 7 to 18 mm. Before ASD closure, all patients had increased Qp (Qp:Qs = 2.0 ± 0.7) and normal mean pulmonary arterial pressures (Table 1). Exhaled NO concentrations were unchanged over the 30 min period before ASD closure (7.89 ± 7.4 vs. 7.90 ± 7.2 ppb, n = 15).
Atrial septal defect device closure decreased Qp (Table 1, p < 0.05). Mean pulmonary and systemic arterial pressures were unchanged. Associated with this decrease in Qp, there was a significant decrease in both exhaled NO (from 7.6 ± 7.1 SD to 5.4 ± 6.9 ppb, p < 0.05) and plasma NOxconcentrations (from 43.4 ± 29.3 to 35.3 ± 26.6 μM, p < 0.05) (Figs. 1 and 2). ⇓⇓Systemic arterial blood gases and pH and hemoglobin were unchanged.
Plasma NOxconcentrations did not change in the six patients who underwent diagnostic catheterization without changes in blood flows (from 19.8 ± 11.3 to 21.0 ± 11.1 μM).
In vitro data suggest that acute changes in flow or shear stress regulate NOS activity. For example, in cultured endothelial cells, increases in flow produce an increase in NO production and eNOS gene expression (11–13,19). However, the acute effect of Qp on NO production in vivo in humans was unknown. This study represents the first demonstration that decreases in Qp decrease endogenous NO production in humans. To demonstrate the effects of Qp on NO production, we utilized a unique study design: we measured changes in exhaled NO and plasma NOxin patients undergoing nonsurgical ASD device closure in the cardiac catheterization laboratory. The majority of surgical procedures that change Qp utilize cardiopulmonary bypass. However, cardiopulmonary bypass changes pulmonary NO production independent of changes in Qp; it produces inflammatory lung injury and pulmonary vascular endothelial cell injury (20). During ASD device closure, patients undergo a significant decrease in Qp (∼50%) without cardiopulmonary bypass, making this an excellent model to assess the effect of Qp on NO production.
This unique study design also minimized the limitations of in vivo determinations of endogenous NO production. To determine changes in endogenous NO production, we measured changes in exhaled NO and plasma NOx. Exhaled NO is an indirect determination of pulmonary NO production (14). The major sources of exhaled NO are the nasopharynx, sinuses and respiratory tract (21). In this study, all patients were intubated for the procedure. Sampling in intubated patients excludes the nasopharynx and sinuses, isolating the source to the respiratory tract. Within the respiratory tract, all three isoforms of NOS have been found (22,23). The major constitutive isoform in the lung is eNOS, which is found predominantly in pulmonary vascular endothelial cells, with small amounts found in the bronchial epithelium. Small amounts of the other constitutive isoform, neuronal NOS, have been found in the nonadrenergic, noncholinergic nerves of the lung. The inducible isoform, iNOS, is found in the airway epithelium, inflammatory cells and alveolar pneumocytes. Cell culture studies suggest that several hours are required for the inducible NOS to produce NO following a stimuli (24). In this study, we determined changes within 30 min, suggesting that these changes are reflective of changes in constitutive NO production, the majority being eNOS-derived NO production. In addition to endogenous NO production, exhaled NO determinations may be altered by changes in minute ventilation, expiratory flow rate and inspired oxygen concentrations (25,26). However, during the study period all of these variables were held constant, isolating any change in exhaled NO to changes in endogenous production.
Plasma NOxis an indirect determination of total body NO production (15). Endogenous NO may be found in plasma as dissolved NO gas, nitrite anions and the majority as NOxanions (27). Utilizing vanadium and HCL reduction, both nitrite and nitrate are reduced to NO, which is then determined by chemiluminescence. Therefore, we determined all potential sources of endogenous NO production. We found a 17.9% decrease in plasma NOxconcentrations associated with a 40% decrease in Qp. Since plasma NOxconcentrations represent total body endogenous NO production, we were surprised by the magnitude of change demonstrated in the plasma concentrations in this study. In fact, since the half-life of plasma nitrate is 3.8 h, the changes demonstrated after 30 min may underestimate the total decrease (15). However, human and animal data suggest that endogenous pulmonary NO production is increased in subjects with increased Qp (28,29). Therefore, pulmonary NO production may be a significant proportion of total body NO production in this setting. In fact, one study demonstrated a similar 23% decrease in plasma NOxin children following surgical closure of ventricular septal defects (28). In addition to endogenous NO production, plasma NOxconcentrations are effected by diet, changes in renal function and changes in extracellular volume (15). During our study period, all patients were fasted for 12 h and had stable renal function, isolating any change in plasma NOxto changes in endogenous production (30).
In addition to the indirect measurements of endogenous NO production, one additional limitation of this study was noteworthy. Because the only patients intubated and mechanically ventilated during cardiac catheterization are those undergoing ASD device closure, identical control patients, who were undergoing catheterization without changes in blood flow, were not available. Therefore, we determined exhaled NO at least two times before obtaining preclosure values to insure that the values had reached a steady state. In addition, to determine if catheterization with angiography alone changed NO production, we measured NOxconcentrations in six additional patients undergoing catheterization without changes in blood flow. We found that cardiac catheterization alone did not change plasma NOx. Exhaled NO determinations were not made in these patients because they were spontaneously breathing.
In summary, this study is the first demonstration that changes in Qp mediate endogenous NO production in vivo in humans. We utilized a unique study population, patients undergoing device closure of ASD. This provided a study design which not only allowed the investigation of acute changes in Qp without the use of cardiopulmonary bypass, but also maximized the utility of both in vivo determinations of endogenous NO production. A limitation of the study was that the exact source NO production could not be determined by these noninvasive measurements. However, since the changes measured in this study occurred over a 30 min period, they most likely represent changes in the constitutive isoforms, the majority being changes in eNOS-derived pulmonary vascular NO production. It should also be noted that a reduction in the perfused surface area secondary to derecruitment of vessels, and not a reduction in the NO production of individual endothelial cells, cannot be excluded in this in vivo study design. Alterations in endogenous endothelial NO production have been implicated in the pathophysiology of pulmonary hypertension associated with increased Qp (6,9). Whether exhaled NO and plasma NOxwill provide clinically useful measures of NO production, and thereby be predictive of the progression of pulmonary hypertension and vascular reactivity, is unknown and warrants further study.
The authors thank Sarah Hudson, AN, Robert Powell, AN, Anne Marie Hagan, Patricia Macdonald and Clarke Cudd, BSN, for their expert technical assistance.
☆ This study was supported by grant 9640010N EIA from the American Heart Association (J.R.F.), grant FY97-0175 from the March of Dimes (J.R.F.) and grants HL60190 and HD 28825 from the NIH (S.M.B.).
- atrial septal defect
- guanisone 3′,5′-cyclic monophosphate
- endothelial NO synthase
- nitric oxide
- pulmonary blood flow
- systemic blood flow
- standard deviation
- Received June 2, 1999.
- Revision received September 21, 1999.
- Accepted October 27, 1999.
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