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Liver Failure and Liver Disease
Article first published online: 20 APR 2006
Copyright © 2006 American Association for the Study of Liver Diseases
Volume 43, Issue 5, pages 1084–1091, May 2006
How to Cite
Gómez, F. P., Barberà, J. A., Roca, J., Burgos, F., Gistau, C. and Rodríguez-Roisin, R. (2006), Effects of nebulized NG-nitro-L-arginine methyl ester in patients with hepatopulmonary syndrome. Hepatology, 43: 1084–1091. doi: 10.1002/hep.21141
See Editorial on Page 912
Potential conflict of interest: Nothing to report.
- Issue published online: 20 APR 2006
- Article first published online: 20 APR 2006
- Manuscript Accepted: 28 JAN 2006
- Manuscript Received: 27 SEP 2005
- Red Respira-ISCIII–RTIC-03/11
- Comissionat per a Universitats i Recerca de la Generalitat de Catalunya. Grant Number: 2001 SGR00386
- Laboratorios Dr. Esteve, SA
Enhanced pulmonary production of nitric oxide (NO) has been implicated in the pathogenesis of hepatopulmonary syndrome (HPS). NO inhibition with NG-nitro-L-arginine methyl ester (L-NAME) in both animals and humans with HPS has improved arterial hypoxemia. We assessed the role of enhanced NO production in the pathobiology of arterial deoxygenation in HPS and the potential therapeutic efficacy of selective pulmonary NO inhibition. We investigated the effects of nebulized L-NAME (162.0 mg) at 30 and 120 minutes on all intrapulmonary and extrapulmonary factors governing pulmonary gas exchange in 10 patients with HPS (60 ± 7 [SD] yr; alveolar–arterial oxygen gradient, range 19–76 mm Hg; arterial oxygen tension, range 37–89 mm Hg). Nebulized L-NAME maximally decreased exhaled NO (by −55%; P < .001), mixed venous nitrite/nitrate (by −12%; P = .02), and cardiac output (by −11%; P = .002) while increased systemic vascular resistance (by 11%; P = .008) and pulmonary vascular resistance (by 25%; P = .03). In contrast, ventilation-perfusion mismatching, intrapulmonary shunt and, in turn, arterial deoxygenation remained unchanged. In conclusion, gas exchange disturbances in HPS may be related to pulmonary vascular remodeling rather than to an ongoing vasodilator effect of enhanced NO production. (HEPATOLOGY 2006;43:1084–1091.)
Hepatopulmonary syndrome (HPS) is defined as an arterial oxygenation defect induced by intrapulmonary vascular dilatations associated with hepatic disease.1 Arterial deoxygenation in HPS is caused by ventilation–perfusion mismatching and intrapulmonary shunt along with a diffusion–perfusion defect in most advanced cases.2 Few experimental and clinical evidences in HPS suggest that enhanced pulmonary production of nitric oxide (NO) plays a key role in the development of intrapulmonary vascular dilatations.3–10 Morphologically, the striking feature in HPS is widespread dilatation and increased number of pulmonary capillaries in alveolar regions11 that suggest a vasculogenic rather than a vasoactive pathobiological process.12
Studies with NO inhibitors in HPS, however, have highlighted that active NO-dependent pulmonary vasodilatation may contribute to the persistence of gas exchange abnormalities.10, 13, 14 In patients with HPS, there was a transient improvement in arterial oxygenation after intravenous methylene blue, an inhibitor of soluble guanylate cyclase and cyclic guanosine monophosphate production, a final molecular step for NO-mediated vasorelaxation.13, 14 Similarly, inhibition of NO synthase with nebulized NG-nitro-L-arginine methyl ester (L-NAME) in a patient with HPS improved arterial oxygen tension,15 suggesting that the selective inhibition of pulmonary NO through the inhaled route could be of potential therapeutic benefit. Nevertheless, the precise role of a NO-dependent pulmonary vasodilatation involved in the pathogenesis of abnormal pulmonary gas exchange in HPS remains far from clear.
To shed further light into the pathogenesis of HPS and to explore the potential therapeutic application of selective pulmonary NO inhibition, we designed the current study to assess thoroughly the effects of nebulized L-NAME on exhaled NO, mixed venous nitrite/nitrate, gas exchange, and hemodynamics in a series of patients with HPS encompassing the full spectrum of gas exchange severity.
Patients and Methods
Ten patients (3 women, 7 men; 60 ± 7 [SD] yr) with HPS were recruited over a period of 2 years. Liver disease was diagnosed according to clinical, biochemical, echographic, portal hemodynamic, and/or histopathological findings. Patients with hemorrhagic and/or infectious complications within the previous 6 months or with moderate to severe encephalopathy or renal failure were excluded. The degree of liver dysfunction was graded according to Child-Turcotte-Pugh classification. HPS was defined when, along with a diagnosis of liver disease, patients showed (1) increased alveolar–arterial oxygen gradient of 15 mm Hg or more while breathing room air in an upright position, with or without arterial hypoxemia (arterial oxygen tension <80 mm Hg), and (2) evidence of intrapulmonary vascular dilatations using transthoracic contrast-enhanced echocardiography.1 The study was approved by the ethical committee of our institution, and all patients gave written informed consent to participate.
This was an open, uncontrolled, prospective trial. All patients completed the study without side effects. Measurements were performed in a semirecumbent position breathing room air. A single dose of L-NAME (162.0 mg dissolved in 4.0 mL 0.9% saline; Sigma Chemical Co., St. Louis, MO) was administered via a continuous flow nebulizer (PARI LC Plus; PARI GmbH, Starnberg, Germany) over a 12-minute period. Dosage of L-NAME was based on previous studies in patients with asthma.16 Measurements were performed at baseline and 30 and 120 minutes after L-NAME nebulization. Maintenance of steady-state conditions was confirmed throughout the study as previously described.2, 16, 17
Pulmonary Gas Exchange.
Radial arterial (Seldicath; Laboratoire Pharmaceutique Saint-Leu-La-Foret, Cedex, France) and mixed venous samples were collected anaerobically, and hemoglobin, pH, and respiratory gases were analyzed in duplicate using standard electrodes (Model 860; CIBA-Corning, Medfield, MA). Minute ventilation and respiratory rate were recorded (Wright Respirometer MK8; BOC-Medical, Essex, UK). A non-rebreathing valve (Rudolph Valve #1500, Kansas City, MO) was used to collect the mixed expired gas through a heated-mixing box. Oxygen consumption and carbon dioxide production were calculated from mixed expired oxygen and carbon dioxide concentrations (CPX System; Medical Graphics, St. Paul, MN). The alveolar–arterial oxygen gradient was calculated using the measured respiratory exchange ratio. In addition, single-breath diffusing capacity for carbon monoxide (Jaeger; MasterSreen, Wüerzburg, Germany) after adjustment for hemoglobin concentration was measured before and also 3 hours after L-NAME inhalation.
Ventilation–perfusion (VA/Q) distributions were estimated via multiple inert gas elimination technique using mixed expired, arterial, and mixed venous inert gases in the customary manner.18 The dispersion of the two distributions (pulmonary perfusion and alveolar ventilation) on a log scale (Log SDQ and Log SDV, respectively) were used as indices of VA/Q mismatch (upper limits of normal, 0.60 and 0.65, respectively) (dimensionless).19 The difference among measured retentions and excretions of the inert gases corrected for the elimination of acetone, an overall descriptor of the combined dispersion of both blood flow and ventilation distributions, was calculated (normal values <3.0, dimensionless).
Systemic and Pulmonary Hemodynamics.
Pulmonary artery catheterization was performed using a transvenous balloon-tipped Swan-Ganz catheter (Baxter Healthcare, Irvine, CA). Intravascular pressures were continuously monitored (HP-7754 B; Hewlett-Packard, Waltham, MA). Two measurements were performed at each time point and the mean value was reported as the final result. Cardiac output was determined via thermodilution (HP-M1012 A; Hewlett-Packard). Derived hemodynamic variables—namely pulmonary and systemic vascular resistances—were calculated.
Exhaled NO and Serum Nitrite/Nitrate.
Exhaled NO measurements were performed with the offline method following standardized recommendations.20 Briefly, patients were asked to inhale orally NO-free air from a Douglas bag to total lung capacity and immediately perform a slow vital capacity maneuver against a calibrated resistor (Department of Biophysics, Universitat de Barcelona, Barcelona, Spain). Once dead space was removed, the exhaled air was directed to a Tedlar bag. Constant flow rate (≈50 mL · s−1) was ensured by online visual feedback of flow signal, integrated from mouth pressure signal (LabView; National Instruments, Austin, TX). The mean value of at least 3 measurements at each time point, analyzed within 30 minutes using a chemoluminiscence analyzer (CLD 700AL; EcoPhysics, Dürnten, Switzerland), was reported as the final result.21 Maintenance of comparable exhaled flow rate throughout the study was confirmed (baseline, 53 ± 5 mL · s−1; 30 min after L-NAME administration, 54 ± 5 mL · s−1; 120 min after L-NAME administration, 56 ± 8 mL · s−1). Ambient NO was recorded in all instances (range, 5–54 ppb). To measure mixed venous nitrite/nitrate, samples were ultrafiltered (PL-10 Ultrafree-MC centrifugal filter units; Millipore Corp, Bedford, MA) at 1,200g for 1 hour to remove proteins before analysis. Filtered serum was refluxed in glacial acetic acid containing sodium iodide. Under these conditions, nitrite/nitrate are reduced to NO which, after reacting with ozone, can be quantified by a chemoluminiscence detector (NOA 280; Sievers Instruments, Boulder, CO).22
The results of the study are expressed as the mean ± SD. Repeated-measures ANOVA was performed using version 10.0 of the SPSS statistical package (SPSS, Chicago, IL) to compare differences between baseline and each time point after L-NAME administration, including intersubject analysis for smoking habit. Post hoc pairwise comparisons were made using the Student t test; unpaired comparisons were made using the Mann-Whitney U test. Pearson's correlation was used to assess relationships between variables. A P value of less than .05 was considered significant.
Nine patients had cirrhosis (3 alcoholic, 2 hepatitis C, 2 alcoholic and hepatitis C, 1 cryptogenic, 1 hemochromatosis) and 1 had idiopathic portal hypertension (Table 1). Four patients were in Child-Turcotte-Pugh class A, 4 were in class B, and 2 were in class C. All patients had mild to severe dyspnea and only 1 had orthodeoxia.18 Five patients were smokers (range, 20–35 pack-years), 1 was an ex-smoker (15 pack-years), and 4 were nonsmokers. Lung function tests were normal in 8 patients (forced expiratory volume in 1 s, 95 ± 18% predicted; forced expiratory volume in 1 s/forced vital capacity ratio, 78 ± 5%; total lung capacity, 95 ± 11% predicted), whereas 2 smoking patients exhibited mild chronic obstructive pulmonary disease (forced expiratory volume in 1 s, 71 ± 0% predicted; forced expiratory volume in 1 s/forced vital capacity ratio ratio, 62 ± 2%; total lung capacity, 89 ± 6% predicted). As expected, all patients had reduced diffusing capacity (45 ± 17% predicted).
|Patient No.||Sex||Age (yr)||Diagnosis||Smoking||PaO2 (mm Hg)||AaPO2 (mm Hg)||Shunt (% QT)||Low VA/Q (% QT)||DISP R-E*||QT (L · min−1)||PVR (dyn · s · cm−5)||SVR (dyn · s · cm−5)||FENO, ppb|
|3||M||58||Ethol and HCV||Yes||83||20||2||1||8.5||5.4||37||1,230||17|
|5||M||65||Ethol and HCV||Yes||71||43||3||4||10.7||5.4||103||1,030||15|
Four patients had mild, 2 had moderate, 2 had severe, and 2 had very severe stages of HPS.1 Exhaled NO and mixed venous nitrite/nitrate were moderately increased (Tables 1, 2).21, 22 Six patients had moderate to severe hypoxemia (arterial oxygen tension, 55 ± 11 mm Hg; alveolar–arterial oxygen gradient, 58 ± 12 mm Hg), and 4 were normoxaemic (arterial oxygen tension, 85 ± 4 mm Hg) with an increased alveolar–arterial oxygen gradient (21 ± 3 mm Hg) only (Table 1). Arterial carbon dioxide tension was reduced (31 ± 3 mm Hg), minute ventilation was increased (8 ± 2 L/min−1), and oxygen consumption was within normal limits (221 ± 29 mL · min−1). VA/Q relationships were mildly to severely abnormal in all patients, as shown by increases in the dispersion of pulmonary blood flow (Log SDQ) and in the overall index of VA/Q heterogeneity (combined dispersion of pulmonary blood flow and ventilation distributions) (Tables 1, 2). In addition, mild to severe intrapulmonary shunt and mild low VA/Q regions were observed (Table 1). The mean distribution of pulmonary blood flow (mean Q) was close to normal (≈1.0) (0.88 ± 0.28). In contrast, the dispersion of alveolar ventilation (Log SDV) was normal or slightly increased, whereas the mean distribution of alveolar ventilation (mean V) was mildly to moderately increased (1.61 ± 0.47). Dead space was normal or slightly reduced (Table 2).
|Baseline||After L-NAME||P Value†|
|30 Minutes||120 Minutes|
|FENO (ppb)||22 ± 9||9 ± 4‡||10 ± 4§||.003|
|NO2/NO3 (μmol · L−1)||38 ± 24||35 ± 26||33 ± 20¶||.04|
|PaO2 (mm Hg)||67 ± 18||64 ± 17||63 ± 16||.06|
|AaPO2 (mm Hg)||43 ± 21||43 ± 21||44 ± 19||—|
|PvO2 (mm Hg)||38 ± 5||37 ± 5||37 ± 4||—|
|Shunt (% QT)||13 ± 13||13 ± 14||12 ± 14||—|
|Low VA/Q regions (% QT)||2.2 ± 2.7||3.1 ± 3.5||3.6 ± 3.3||—|
|Log SDQ||0.93 ± 0.33||0.99 ± 0.36||1.07 ± 0.34||—|
|Log SDV||0.67 ± 0.10||0.64 ± 0.08||0.62 ± 0.07||—|
|DISP R-E*||14.1 ± 7.1||14.0 ± 8.2||13.6 ± 7.7||—|
|Dead space (% of VE)||24 ± 15||28 ± 17¶||31 ± 11¶||.02|
Hemodynamically, there was a hyperdynamic circulatory state, characterized by high cardiac output and low systemic vascular resistance, mild reductions in both mean pulmonary artery pressure and pulmonary vascular resistance (<120 dynes · s · cm−5) and normal pulmonary artery occlusion pressure (Tables 1, 3).
|Baseline||After L-NAME||P Value†|
|30 Minutes||120 Minutes|
|QT (L · min−1)||6.4 ± 2.2||6.0 ± 2.4||5.7 ± 2.3†||.005|
|CI (L · min−1/m2)||3.5 ± 1.0||3.2 ± 1.1||3.1 ± 1.0†||.006|
|mPAP (mm Hg)||10.1 ± 4.2||10.2 ± 2.9||10.1 ± 2.9||—|
|PAOP (mm Hg)||4.4 ± 2.9||3.8 ± 2.4||4.3 ± 2.5||—|
|SVR (dyn · s · cm−5)||1098 ± 259||1158 ± 310||1219 ± 341‡||.03|
|PVR (dyn · s · cm−5)||81 ± 74||101 ± 81§||93 ± 74||.11|
|TPG (mm Hg)||5.8 ± 4.7||6.5 ± 4.1||5.8 ± 4.0||—|
|MAP (mm Hg)||84 ± 11||82 ± 13||82 ± 12||—|
|HR (beats · min−1)||69 ± 10||68 ± 11||67 ± 12¶||.04|
Arterial oxygen tension had an inverse relationship with both intrapulmonary shunt (r = −0.80; P = .006) and the combined dispersion of pulmonary blood flow and ventilation distributions (r = −0.82; P = .003), but no correlation was observed between any of the gas exchange descriptors and hemodynamic variables. Likewise, no correlation was shown between exhaled NO and gas exchange indices, hemodynamic variables, or serum nitrite/nitrate.
Exhaled NO was lower in smokers compared with both nonsmokers and ex-smokers (14 ± 2 ppb and 29 ± 6 ppb, respectively; P = .008), but no differences in nitrites/nitrates, gas exchange, or hemodynamic variables were observed between smokers and nonsmokers/ex-smokers.
Effects of L-NAME Nebulization.
Compared with baseline, exhaled NO substantially decreased both at 30 minutes (by −55%; P < .001) and 120 minutes (by −49%; P = .001) after L-NAME administration (Table 2; Fig. 1). In addition, mixed venous nitrite/nitrate maximally decreased at 120 minutes after L-NAME administration (by −12%; P = .02). Except for a trend to reduce arterial oxygen tension and an increased dead space over time (P = .02) after L-NAME administration, consistent with the simultaneous decreased cardiac output, no changes were observed in gas exchange descriptors (Tables 2, 3; Fig. 2).
Both cardiac output (by −11%; P = .002) and heart rate (by −4%; P = .01) maximally decreased at 120 minutes, whereas systemic vascular resistance increased (by 11%; P = .008). Furthermore, pulmonary vascular resistance maximally increased at 30 minutes after L-NAME administration (by 25%; P = .03) without changes in mean pulmonary artery and occlusion pressures and transpulmonary pressure gradient (Table 3; Fig. 3).
Intersubject analysis for smoking only showed that the reduction of exhaled NO was lower in smoking patients (P = .007) but no other differences in serum nitrite/nitrate, gas exchange and hemodynamic variables were observed after L-NAME administration.
No changes were observed in diffusing capacity for carbon monoxide after L-NAME administration (44 ± 16% predicted). No correlations were shown between changes in exhaled NO, hemoglobin levels, gas exchange indices, hemodynamic variables, and mixed venous nitrite/nitrate after L-NAME administration.
The most novel finding of our study was that inhibition of NO synthesis by nebulized L-NAME in patients with HPS did not modulate pulmonary gas exchange, despite reducing the hyperdynamic circulatory state and attenuating the pulmonary vasodilatation. Accordingly, these findings dispute the notion that in patients with HPS arterial deoxygenation is related primarily to an ongoing vasodilator effect of enhanced pulmonary NO production.
The concept that NO plays a key role in the development of HPS has been underlined experimentally.4, 6, 10, 23 In a rat HPS model, pulmonary vascular remodeling, gas exchange abnormalities, and blunted pulmonary vasopressor response were all linked to increased expression and activities of both pulmonary endothelial4, 23 and inducible NO synthase.6 Moreover, it has been shown that intravenous L-NAME acutely improved arterial hypoxemia,10 thereby suggesting that NO-dependent pulmonary vasodilatation actively contributes to VA/Q imbalance in HPS.
Unlike experimental studies, our findings showed that whereas systemic and pulmonary vasodilatation were considerably attenuated by inhaled L-NAME, VA/Q inequalities and, in turn, arterial blood gas disturbances remained essentially unchanged. From a gas exchange viewpoint, it is known that a decreased cardiac output improves intrapulmonary shunt and ameliorates arterial hypoxemia, all other things being equal.24 Instead, in our study, reduced cardiac output after L-NAME administration did not modulate VA/Q distributions while tending to reduce arterial oxygen tension. These findings are consistent with the concept that pulmonary circulation in patients with cirrhosis behaves paradoxically, combining a lower, or even absent, hypoxic vascular response with some degree of hypoxic vasoconstriction release.25 This in turn may reflect a more rigid and fixed pulmonary vasculature, less liable to adequately match alveolar ventilation to pulmonary blood flow balance.18
We did not investigate higher doses of L-NAME, and this can be a potential limitation of our study. However, the consistent reduction of exhaled NO along with moderate pulmonary vasoconstriction likely reflects the efficacy of L-NAME to inhibit pulmonary NO release at the current dosage. Exhaled NO in patients with HPS has been largely acknowledged as a key biomarker of pulmonary NO production before and after liver transplantation8 and also in comparison with patients who have cirrhosis without HPS.3 However, we used a higher dose of L-NAME than that administered to a patient with HPS in a recent case report that successfully reduced exhaled NO and improved arterial oxygen tension.15
Nevertheless, our results argue against an active functional effect of enhanced NO production on pulmonary circulation near the gas exchange zone, but not against the pathogenic role of NO, likely a key factor priming pulmonary vascular changes in HPS.1 In fact, on the basis of the favorable hemodynamic effects of inhaled L-NAME, prolonged modulation of NO production could improve pathological changes and its functional consequences.
Our findings are also at variance with those of a previous trial14 and a single case report13 involving patients with advanced HPS that improved arterial hypoxemia after intravenous methylene blue. Despite the fact that our patients encompassed the full spectrum of HPS severity,1 the gas exchange response to L-NAME was similarly ineffective, such that clinical characteristics cannot be considered to explain the current differences with those observed after methylene blue administration.14 It is noteworthy that intravenous methylene blue14 and inhaled L-NAME showed similar trends in pulmonary and systemic hemodynamic responses, suggesting that the different route of administration could not be invoked to explain the differences between agents. However, with inhaled L-NAME, the magnitude of such hemodynamic responses was lower on average, likely reflecting a reduced pharmacological potency or an insufficient NO inhibition at the dose given. It is possible, therefore, that the different results in methylene blue and L-NAME studies are related to different mechanisms of action—L-NAME through inhibition of NO synthase by competing with substrate, and methylene blue through inhibition of the soluble guanylate cyclase/cyclic guanosine monophosphate pathway.
The involvement of other vasodilatating molecules through NO-independent mechanisms in HPS, such as carbon monoxide26 or stimulated calcium-activated potassium channels by endothelial-derived hyperpolarizing factor,23 cannot be neglected. Enhanced pulmonary carbon monoxide production, enzimatically synthesized by heme oxygenase, is increasingly acknowledged in HPS. Inducible heme oxygenase is overexpressed in pulmonary macrophages in the rat HPS model, suggesting a key role in the development of intrapulmonary vascular dilatations.27, 28 Moreover, the potential contribution of increased carbon monoxide production to abnormal gas exchange has been highlighted recently in patients with HPS on the basis of a strong negative correlation between arterial oxygen tension and carboxyhemoglobin.29
Structural remodeling of the pulmonary vascular bed is thought to be the cause of the persistent low diffusing capacity for carbon monoxide along with a complete normalization of all the other gas exchange indices observed in patients with HPS after liver transplantation.30 Moreover, the diffusing capacity for carbon monoxide remained unchanged after L-NAME administration in our study, supporting the notion that this variable behaves in HPS as a marker of a structural derangement of the alveolar–capillary interface rather than as a functional descriptor of a diffusion-perfusion defect.31
Cigarette smoking was present in half of the HPS patients in our study. Tobacco smoke seems to play a role in inhibiting angiogenesis, which could influence the repair process and contribute to altered structural lung remodeling.32 Alternatively, cigarette smoking is associated with decreased NO production and may exert a negative feedback mechanism upon NO release in HPS.33 Although the biopathological sequelae of smoking in HPS patients have not been investigated, we previously reported that both VA/Q mismatching and abnormal hemodynamics in HPS conform a distinctive unique pattern barely influenced by coexisting chronic cardio-respiratory comorbidities.34 Likewise, no differences in gas exchange responses to inhaled L-NAME were shown according to smoking in the present study.
In conclusion, several therapeutic strategies other than liver transplantation have been investigated in patients with HPS in recent years, all with disappointing results.1 Even though earlier studies have highlighted the potential of NO synthesis inhibitors for the management of HPS, our findings, with one of the most robust techniques for the assessment of pulmonary gas exchange, detract from the use of such a therapeutic target. Future studies using higher doses of NO inhibitors in patients with HPS during more prolonged periods may be warranted.
We thank W. Jiménez Ph.D., for nitrite/nitrate analysis and J. Rigau, Ph.D., for helping with exhaled NO devices.
- 31Incomplete gas-exchange resolution after liver transplantation in hepatopulmonary syndrome [Abstract]. Eur Respir J 2003; 22: 19s., , , , , .