Exhaled Nitric Oxide and Impaired Oxygenation in Cirrhotic Patients before and after Liver Transplantation

Methods

Patients

Twenty patients who underwent successful orthotopic liver transplantation at our hospital from August 1995 to February 1997 were recruited for our study. These patients came from a group of 45 patients who were evaluated at the Outpatient Clinic for Liver Cirrhosis during a scheduled visit [7].

All patients gave their informed consent to participate in the study, which was approved by the ethical committee. Included patients had to have been evaluated within 8 weeks before transplantation. Exclusion criteria were respiratory and cardiovascular disease, including clinically significant pleural effusion (larger than costophrenic angle on chest radiography); tense ascites; inability to perform lung function tests; current smoking habit or a smoking history of more than 10 packs per year (1 pack per year = 20 cigarettes per day for 1 year); forced vital capacity (FVC) and FEV (₁) less than 80% of predicted; FEV_1/FVC × 100 less than 70%; and an airway infection in the previous 4 weeks. All patients were reevaluated 3 to 12 months after transplantation.

Laboratory Testing

Patients underwent the following studies: pulmonary function tests, arterial blood gas analysis done while patients were breathing room air in a seated position, contrast-enhanced echocardiography, and measurement of nitric oxide in exhaled air. The hepatopulmonary syndrome was defined as an alveolar-arterial oxygen gradient greater than 15 mm Hg and echocardiographic evidence of intrapulmonary vasodilatations [4].

Lung volumes and carbon monoxide diffusing capacity were obtained according to standardized procedures [9, 10]. The reference values of Quanjer were used [11]. Arterial blood gas samples were obtained by percutaneous radial artery puncture while patients were seated and breathing room air. The alveolar oxygen tension was calculated by using the ideal air Equation and assuming a respiratory exchange ratio of 0.8. The alveolar-arterial oxygen difference was then derived. A gradient less than 15 mm Hg was considered normal.

Exhaled nitric oxide was measured on a chemiluminescence analyzer (Dasibi Environmental Corp., Glendale, California) that is sensitive to nitric oxide from 1 to 4000 parts per billion (ppb) by volume, adapted for on-line recording of nitric oxide concentration, at a sample gas flow of 250 mL/min according to European Respiratory Society recommendations [12]. The analyzer was calibrated daily against standard gas mixtures. While seated and wearing a noseclip, patients were asked to inhale nitric oxide-free air (<5 ppb) and to perform a slow expiratory vital capacity test over 20 to 30 seconds with a flow of 5 to 15 L/min against a low resistance (5 to 20 cm H₂O). Exhaled air was sampled for nitric oxide analysis by way of a Teflon tubing side arm attached to the mouthpiece. Nitric oxide concentration, flow, and pressure were simultaneously displayed against time on a computer screen. Three successive reproducible recordings were made at 2-minute intervals, and the mean values of the plateau (the last part of expiration) of nitric oxide concentration (expressed in ppb) were recorded. Because of flow dependence of exhaled nitric oxide concentration [13], flow rates at which nitric oxide measurements were performed in each patient before and after transplantation were compared and found to not be statistically significantly different (10.2 ± 2.37 L/min compared with 10.6 ± 2.28 L/min). Twenty nonsmoking healthy persons (mean age, 44.6 ± 11.2 years; 12 men) served as normal controls for exhaled nitric oxide concentrations.

Saline contrast-enhanced echocardiography was done by use of a peripheral intravenous line, as reported elsewhere [14]. A positive result on contrast-enhanced echocardiography (that is, indicating intrapulmonary right-to-left shunt) was defined as the delayed appearance (three to six beats after the initial appearance of contrast in the right side of the heart) of microbubbles in the left side of the heart.

Statistical Analysis

The variability of the outcomes (decrease in alveolar-arterial oxygen gradient and exhaled nitric oxide concentration after transplantation) was estimated from previous studies [3, 8]. The sample size was calculated on the basis of an expected 50% decrease in the outcomes after transplantation compared with pretransplantation values, for a two-tailed α value of 0.05 and a β value of 0.20. Means and SDs were calculated for each variable. The Student t-test for paired data was used to compare data before and after transplantation; the McNemar test was used when appropriate. Regression analysis was performed by using the least-squares method. A P value of 0.05 or less was considered statistically significant.

Role of the Funding Source

Our funding source had no role in the collection, analysis, or interpretation of the data or in the submission of the paper for publication.

Results

Eighteen patients (mean age, 48.3 ± 7 years; 14 men) completed the study. Two patients died after surgery (1 of pneumonia and 1 of stroke) 72 and 85 days after transplantation. Cirrhosis was alcoholic in 7 patients; viral in 8 patients; and autoimmune, cryptogenic, and associated with Wilson disease in 1 patient each. According to the Child-Pugh classification [15], 4 patients had class A cirrhosis, 4 had class B cirrhosis, and 10 had class C cirrhosis. Fifteen patients (83.3%) had esophageal varices.

Patients were reevaluated 8.5 ± 3.5 months (range, 3 to 12 months) after transplantation. Post-transplantation lung volumes and diffusion did not significantly differ from pretransplantation values: Total lung capacity increased from 95.4% ± 9.6% of predicted to 96.7% ± 10.4% of predicted, FEV₁ increased from 100.3% ± 13.3% of predicted to 103% ± 13.2% of predicted, FEV_1/FVC × 100 decreased from 80.5% ± 4.5% of predicted to 79.5% ± 4.2% of predicted, and the diffusing capacity of the lung for carbon monoxide divided by alveolar volume decreased from 76.5% ± 20.9% of predicted to 71.6% ± 17.2% of predicted.

Arterial blood gas data, exhaled nitric oxide concentrations, and findings on echocardiographic evaluation of intrapulmonary shunts before and after transplantation are given in Table 1. Before transplantation, the mean exhaled nitric oxide concentration was significantly higher in patients than in controls (13 ± 4.9 ppb compared with 5.75 ± 1.9 ppb; P < 0.001).

After transplantation, the alveolar-arterial oxygen gradient significantly decreased (from 17.3 ± 7.1 mm Hg before transplantation to 9 ± 5.2 mm Hg; P < 0.001), as did the exhaled nitric oxide concentration (from 13 ± 4.9 ppb to 6.2 ± 2.8 ppb; P < 0.001). The decrease in exhaled nitric oxide concentration after liver transplantation was significantly correlated with the decrease in alveolar-arterial oxygen gradient (r = 0.56; P = 0.014) (Figure 1).

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Figure 1. ppb = parts per billion. Correlation between the differences in alveolar-arterial oxygen gradient and exhaled nitric oxide concentrations before and after liver transplantation.

Before transplantation, intrapulmonary shunts were detected by contrast-enhanced echocardiography in five patients, all of whom met the criteria for the hepatopulmonary syndrome and had an alveolar-arterial oxygen gradient greater than 15 mm Hg. In these patients, the pretransplantation exhaled nitric oxide concentration was significantly higher than that in patients without the hepatopulmonary syndrome (18 ± 5.48 ppb compared with 11.07 ± 3.2 ppb; P < 0.005). After transplantation, the hepatopulmonary syndrome was no longer evident; the alveolar-arterial oxygen gradient returned to normal in all affected patients, even the two patients in whom contrast-enhanced echocardiography still showed intrapulmonary vasodilatations.

Discussion

We found a highly significant decrease in exhaled nitric oxide concentrations after liver transplantation that was correlated with the decrease in alveolar-arterial oxygen gradient. As in other investigations [1-3], respiratory function assessment done before liver transplantation revealed a high prevalence of widened alveolar-arterial oxygen gradient in our patients with advanced liver disease. The high concentration of exhaled nitric oxide in our patients before liver transplantation also confirms previous data [6, 7].

A decrease in exhaled nitric oxide concentrations accompanied by normalization of arterial oxygenation after liver transplantation was previously reported by Cremona and colleagues [8] in one patient with cirrhosis and the hepatopulmonary syndrome. Five of our patients met the minimal criteria for this syndrome; in all of these patients, transplantation corrected the syndrome. In our study, patients with the hepatopulmonary syndrome had a higher concentration of exhaled nitric oxide than patients without the syndrome, a finding consistent with previous observations [7, 8]. Liver transplantation seems to be the only effective cure for the hepatopulmonary syndrome, but other researchers have suggested that the procedure is not beneficial for hypoxemic patients whose PaO₂ does not increase to more than 150 mm Hg when they breathe 100% oxygen [4, 16]. As a result, an early diagnosis of the hepatopulmonary syndrome is important; the measurement of exhaled nitric oxide concentrations may be a relatively simple and noninvasive way to identify patients at risk for developing the syndrome. On the other hand, caution should be used in interpreting an increased exhaled nitric oxide concentration in unselected patients because increased nitric oxide concentrations have been reported in patients with common inflammatory airway disease, such as asthma or upper airway inflammation [12]. Nitric oxide is a powerful pulmonary vasodilator, and it seems to be the favored mediator of the widespread pulmonary vasodilation of cirrhosis, which is the key determinant of oxygen abnormalities [17]. In a rat model of chronic bile duct ligation, which reproduces the hepatopulmonary syndrome, Fallon and colleagues [18] showed that the endothelial nitric oxide synthase content of lung homogenates progressively increased. This increase was closely correlated with the development of alterations in gas exchange. This experimental finding agrees with the correlation between exhaled nitric oxide concentrations and alveolar-arterial oxygen gradient that we previously found in a larger series of patients with cirrhosis (25% of whom had the hepatopulmonary syndrome [7]) and with the present findings.

In conclusion, most cases of abnormal oxygenation in patients with cirrhosis are reversible after liver transplantation, and the improvement of oxygenation is correlated with the decrease in exhaled nitric oxide concentrations. These observations reinforce the hypothesis that nitric oxide is an important mediator of impaired oxygenation in cirrhosis. The measurement of exhaled nitric oxide may be useful for identifying patients with abnormal oxygenation who are at risk for developing the hepatopulmonary syndrome.

Dr. Scappaticci: Fisiopatologia Respiratoria, Ospedale Molinette, c.so Bramante 88, 10126 Torino, Italy.

Dr. Polizzi: USL 8 Carmagnola, via Ospedale 13, 10022 Carmagnola, Torino, Italy.

Drs. Ottobrelli and Cerutti: Centro Trapianto Epatico, Ospedale Molinette, c.so Bramante 88, 10126 Torino, Italy.

Drs. Morello and Bergerone: Divisione di Cardiologia dell'Universita, Ospedale Molinette, c.so Bramante 88, 10126 Torino, Italy.