Inhaled Nitric Oxide Reduces Pulmonary Transvascular Albumin Flux in Patients with Acute Lung Injury

Methods: Transvascular albumin flux was measured by a double radioisotope method using^99mTc-labeled albumin and⁵¹Cr-labeled autologous red blood cells. Radioactivity of both isotopes was externally measured over the right lung by a gamma scanner and simultaneously in arterial blood. The normalized ratio of^99mTc/sup 51 Cr lung to^99mTc/sup 51 Cr blood (normalized index) was calculated. The normalized slope index which is the slope of the regression line of the normalized index versus time represents the accumulation rate of albumin in the interstitial space of the lungs. Normalized slope index and pulmonary capillary pressure were determined before, during, and after inhalation of 40 ppm nitric oxide. Pulmonary capillary pressure was estimated using the visual analysis of the pressure decay curve after pulmonary artery occlusion.

Results: Normalized slope index decreased from 0.0077 plus/minus 0.0054 min sup -1 (SD) off nitric oxide to -0.0055 plus/minus 0.0049 min sup -1 (P < 0.01) during nitric oxide and increased to 0.0041 plus/minus 0.0135 min sup -1 after nitric oxide. Pulmonary capillary pressure declined from 24 plus/minus 4 mmHg off nitric oxide to 21 plus/minus 4 mmHg during nitric oxide (P < 0.01), whereas pulmonary artery wedge pressure and cardiac output did not change.

Conclusions: It is concluded that 40 ppm inhaled nitric oxide decreases pulmonary transvascular albumin flux in patients with acute lung injury. This effect may be the result of the decrease in pulmonary capillary pressure. (Key words: Gases, nitric oxide: pulmonary capillary pressure; pulmonary edema; transvascular fluid filtration. Lung: acute injury; increased pulmonary permeability. Measurement techniques: double isotope technique.)

PULMONARY edema as a result of enhanced microvascular permeability is one of the pathologic hallmarks of acute lung injury (ALI). The presence of enhanced microvascular permeability in ALI has been demonstrated by various measurement techniques. Among others, radioisotope techniques have been used to demonstrate the capillary leak with an increased transcapillary protein flux in animal models of ALI and in patients with ALI and acute respiratory distress syndrome (ARDS). In the presence of a pulmonary capillary leak, the hydrostatic pressure in the capillaries becomes the decisive factor for the net filtration of fluid entering the interstitial space of the lungs. Therefore, one of the therapeutic goals in the treatment of ALI is the reduction of the pulmonary capillary pressure (PCP) to reduce edema formation. In the past, the effects of various vasodilators on pulmonary artery pressure and PCP have been studied. Nitroprusside lowers pulmonary artery pressure in endotoxin-challenged sheep and in dogs with oleic acid lung injury. Prostaglandin E¹decreases PCP in sheep lungs preconstricted with a thromboxane analog and reduces pulmonary vascular outflow pressure in oleic acid-injured dog lungs whereas hydralazine does not affect PCP. In patients with ARDS, PCP is decreased by prostaglandin E¹, prostacyclin, and nitroglycerin. .

In spite of reduction of PAP and PCP, prostaglandin E¹failed to attenuate edema formation in an animal model of lung injury. Prostacyclin increased pulmonary edema formation in thromboxane-induced pulmonary hypertension probably by increasing vascular surface area. The only vasodilator that has been shown to reduce edema formation is nitroprusside. In patients with ALI or ARDS, however, systemic administration of vasodilators is hampered by severe side effects such as systemic hypotension and deterioration of pulmonary gas exchange. .

Inhaled nitric oxide is a selective pulmonary vasodilator that decreases pulmonary artery pressure in a variety of pathologic conditions associated with pulmonary hypertension. Intrapulmonary right-to-left shunt decreases and oxygenation improves in patients with ARDS during inhalation of nitric oxide. We recently reported that in patients with ALI inhalation of 40 ppm nitric oxide causes predominantly vasodilation of the pulmonary venous vasculature thereby decreasing PCP. We therefore hypothesized that the nitric oxide-induced decrease in PCP is accompanied by a decrease in transvascular fluid filtration in patients with ALI.

After approval by the local ethics committee and obtaining informed consent of the patients' families, nine consecutive patients without a history of previous lung disease who fulfilled the clinical and radiologic criteria of ALI were included in this study. The clinical characteristics of these patients are summarized in Table 1. The severity of acute lung disease was assessed by the lung injury score according to Murray et al. This scoring system includes a chest roentgenogram score, a hypoxemia score, a positive end-expiratory pressure score and a respiratory system compliance score. A score of 0 indicates no lung injury, a score of 0.1–2.5 indicates mild-to-moderate lung injury, and a score of 2.6–4 indicates severe lung injury. Median lung injury score was 2.75 ranging from 1.75 to 3.5. The patients' lungs were ventilated with a pressure-controlled ventilator (Servo 900 C, Siemens Elema, Lund, Sweden) with tidal volumes of 5–14 ml/kg body weight, respiratory rates of 10–20/min and 10–16 cm H²O of positive end-expiratory pressure. The fraction of inspired oxygen (FIO²) was maintained at 1.0 throughout the investigation. Nitric oxide was administered as described earlier. Nitric oxide and nitric dioxide concentrations were monitored continuously by electrochemical sensors (GS 8641 nitric oxide and GS 8650 NO², Bieler & Lang, Achern, Germany). Methemoglobin was measured photometrically. The patients were sedated with flunitrazepam (2 mg/h) and paralyzed with pancuronium (6 mg/h).

All patients had a pulmonary artery flow-directed thermodilution catheter (model SP 1507, Spectramed, Dusseldorf, Germany) and a radial arterial catheter in place. Systolic, diastolic and mean pulmonary artery pressure (MPAP), central venous pressure, mean arterial pressure, and heart rate were monitored continuously. Cardiac output was determined by averaging three thermodilution measurements using 10 ml room temperature saline and a cardiac output computer (Sirecust 1281, Siemens, Erlangen, Germany). All pressure measurements were performed at end-expiration with the patient supine and the calibrated pressure transducers (Medex Novotrans II MX 860, Hilliard, OH) zeroed to atmospheric pressure. The zero reference level was two thirds of the sagittal thoracic diameter ventral of the vertebral column. Pulmonary capillary pressure was estimated by visual analysis of the pressure decay curve after pulmonary artery occlusion. When MPAP was constant, the balloon of the pulmonary artery catheter was inflated and the pressure profile recorded at a chart speed of 6.25 mm/s on a precalibrated recorder (Siredoc 220, Siemens, Erlengen, Germany) until the wedge pressure was obtained. The inflection point, i.e., PCP, was determined by placing a ruler on the rapid component of the pressure decline adjusted for the best fit and marking the point at which the slow component of the pressure profile deviated from the rapid component (Figure 1). Three pressure profiles were obtained with each set of measurements. The coefficient of variation of the PCP estimation was < 3%. Pulmonary artery wedge pressure was determined when the pressure decay curve had reached a stable level. After PCP determination, pulmonary vascular resistance (PVR) was calculated using standard formulas and divided into arterial and venous resistance. Pulmonary arterial resistance (PVR^art) was calculated as the pulmonary arterial pressure gradient (MPAP-PCP) divided by cardiac output, and pulmonary venous resistance (PVR^ven) as the pulmonary venous pressure gradient (PCP-pulmonary artery wedge pressure) divided by cardiac output. Arterial and mixed-venous blood gas tensions, hemoglobin oxygen saturation, total hemoglobin concentration, and hematocrit were determined with an ABL 510 radiometer (Copenhagen, Denmark). Intrapulmonary venous admixture was calculated by standard formulas.

Pulmonary transvascular albumin flux was measured by a double radioisotope technique. One hour before the trial, 40 ml of blood was withdrawn from the patient. Red blood cells were labeled with 10 Megabecquerel (MBq)⁵¹Chromium as intravascular tracer, and 10 mg human albumin (TCK-2, CIS Bio International, Gif sur Yvette, France) were labeled with 10 Mbq^99mTechnetium as diffusible tracer. The labeling efficiency was > 97% as determined by chromatography. Ten minutes before data acquisition, labeled erythrocytes and labeled albumin were injected intravenously. External gamma counting was performed with a 2-inch probe detector (Ortec, Berthold, Bad Wildbad, Germany) that was placed over the upper part of the right hemithorax after measuring background activity. Radioactivity of each of the two tracers was measured one after the other, with each of the measurements lasting 1 min. This sequence was repeated once. The mean of data pairs was used for further calculations. Measurements were repeated every 10 min. Simultaneously, radioactivity of both tracers was measured in arterial blood samples.

To assess the transvascular pulmonary albumin flux, the normalized index (NI) described by Roselli et al. was used. Briefly, for calculation of the normalized index the measured gamma counts were corrected for background activity and radioisotope overlap.^99mTechnetium activity was corrected for radioactive decay. Values were normalized by expressing them as a percentage of initial values. The normalized index is the ratio of normalized^99mTechnetium/sup 51 Chromium over the lung to^99mTechnetium/sup 51 Chromium of blood. The normalized slope index (NSI), which is the slope of the regression line of the normalized index versus time, represents the accumulation rate of albumin in the interstitial space of the lungs. Systemic and pulmonary hemodynamic variables, gas exchange values, and transvascular albumin flux were determined before, during, and after nitric oxide inhalation at a concentration of 40 ppm. A concentration of 40 ppm was chosen because 30–40 ppm seems to produce maximum pulmonary vasodilation. Each study period lasted approximately 40 min. Measurements were made when hemodynamics were stable.

Data are expressed as mean plus/minus standard deviation. Values were compared before and during nitric oxide inhalation using the Wilcoxon test for paired data. A P value of less than 0.05 was considered significant.

The effects of inhaled nitric oxide on hemodynamics and gas exchange are listed in Table 2. Inhalation of 40 ppm nitric oxide decreased MPAP, PCP, and PVR. PVR^vendecreased, PVR^artdid not change. Intrapulmonary venous admixture also decreased. The count rates of^99mTechnetium and⁵¹Chromium over the lung and in blood are summarized in Table 3. The values for the NSI and the regression coefficients are listed in Table 4. Mean NSI decreased during nitric oxide. Typically, this decrease occurred with a delay of 5–15 min. Figure 2is a representative example of the time course of the normalized index during nitric oxide inhalation. In all but one patient (patient 4) NSI increased again after discontinuation of nitric oxide. In patient 4, PCP remained low after discontinuation of nitric oxide. The change of NSI during nitric oxide inhalation correlated with the change of PVR^ven(r = 0.83;Figure 3). The more pronounced the reduction in PVR^venduring nitric oxide inhalation the more marked was the change of the NSI. The NO sub 2 concentration did not exceed 2 ppm, and the methemoglobin concentration remained constant throughout the study (1.3 plus/minus 0.2 vs. 1.4 plus/minus 0.2%, NS).

The main findings of this clinical study are that inhalation of nitric oxide at a concentration of 40 ppm decreases PCP and reduces transvascular albumin flux in patients with acute lung injury. Such a response has not previously been reported in a clinical investigation of nitric oxide inhalation.

According to the Starling equation for transcapillary fluid filtration, the net filtration of fluid entering the interstitial space depends on the filtration coefficient, the hydrostatic and oncotic pressure gradients and the reflection coefficient, the latter being dependent on microvascular permeability. Roselli et al. have shown that pulmonary microvascular permeability can be described by a two-pore model. Validity of this model has been demonstrated in bovine endotoxemia-induced ALI. After administration of endotoxin, the radius of the large pores in pulmonary microvessels as well as the numeric proportion of small to large pores increase. During the early phase of endotoxemia, microvascular pressure increases and membrane surface decreases. During the late phase of endotoxemia, large pore radius further increases. When microvascular permeability is enhanced, the hydrostatic pressure in the capillaries becomes the decisive factor of net fluid filtration into the interstitial space. Pulmonary venoconstriction as in ALI leads to a rise in pulmonary capillary pressure, promoting edema formation. Grimbert et al. have demonstrated that an increase in PCP by 3 mmHg results in an eightfold increase in transvascular fluid filtration in dog lungs with increased vascular permeability after acid aspiration. Reduction of PCP has, therefore, become one of the therapeutic goals in the treatment of ALI.

In the past, various vasodilators have been shown to reduce PCP and/or pulmonary artery pressure. Nitroprusside decreases pulmonary artery pressure in sheep after infusion of endotoxin, and in dogs with oleic acid lung injury. Prostaglandin E¹decreases PCP in sheep lungs preconstricted with a thromboxane analog, and pulmonary vascular outflow pressure in oleic acid-injured dog lungs. In contrast, hydralazine does not affect PCP. In patients with ARDS, prostaglandin E¹, prostacyclin, and nitroglycerin decrease PCP. Pulmonary vasodilation by itself, however, will not decrease fluid filtration. In fact, if cardiac output increases as a result of systemic vasodilation, pulmonary edema formation may ensue. Prostacyclin has been shown to increase cardiac output and to promote lung edema formation induced by a stable thromboxane analog probably by increasing pulmonary vascular surface area. Prostaglandin E¹failed to reduce edema formation in an animal model of ALI. Nitroprusside is known to reduce edema formation in animal models of ALI. In patients with ALI or ARDS, however, systemic administration of vasodilators is hampered by severe side effects such as systemic hypotension or deterioration of pulmonary gas exchange. Inhaled nitric oxide, in contrast, reduces pulmonary artery pressure without affecting systemic arterial pressure, improves gas exchange in patients with ARDS, and reduces PCP in isolated lung preparations and in humans. .

In the current study, mean PCP decreased during nitric oxide inhalation. This decrease was accompanied by a decrease in mean NSI. NSI varies between 0.001 and 0.002 min sup -1 in noninjured isolated in situ lungs and in healthy sheep lungs. When pulmonary microvascular permeability was increased by perilla ketone, NSI increased fourfold to fivefold to 0.01 min sup -1. The increase of NSI was paralleled by an increase in wet-to-dry ratio of the lungs. Gorin et al. demonstrated in sheep that transvascular^113mIn-transferrin flux correlated well with lung lymph accumulation of the tracer protein after Pseudomonas aeruginosa infusion. In dog lungs, the protein leak index, which is identical to the NSI, increased from 0.0016 min sup -1 at baseline to 0.0041 min sup -1 after infusion of endotoxin. Dauber et al. measured the albumin leak index, which corresponds to NSI, in dog lungs after thiourea injury. The albumin leak index increased nearly fourfold from 0.0008 to 0.0027 min sup -1. In humans with ARDS, the protein flux units, which are 1,000-fold NSI, were 3.2 compared to 0.2 in control subjects. Hunter et al. observed in ARDS patients a plasma protein accumulation index of 0.0029 min sup -1 versus 0.0002 min sup -1 in healthy subjects. An NSI of 0.0035–0.0207 min sup -1 (mean 0.0077) in our patients without nitric oxide indicates an abnormal pulmonary microvascular permeability favoring transvascular fluid filtration. The decrease of NSI during inhalation of 40 ppm nitric oxide suggests a reduction in fluid efflux into the interstitial space of the lungs (T4-4). This effect occurred 5–15 min after nitric oxide inhalation in all patients. Moreover, NSI was slightly negative during nitric oxide administration suggesting a resorption of fluid from the lung tissue into the vascular bed. The effect of fluid resorption, however, may be transient. During edema formation before nitric oxide inhalation, interstitial pressure will be elevated. When PCP is decreased by nitric oxide inhalation, interstitial pressure may be transiently higher than PCP. However, after some fluid resorption, interstitial pressure should drop, and fluid resorption across the alveolar capillaries should cease. A longer period of nitric oxide inhalation would be needed to demonstrate this. Blomqvist et al. observed an unexpected rapid and complete resolution of bilateral pulmonary infiltrates during the first 120 h of nitric oxide inhalation in a 58-year-old patient with ARDS secondary to a pneumococcal pneumonia. The authors hypothesized that the reduction of microvascular pressure during nitric oxide inhalation might have contributed to the rapid disappearance of the pulmonary infiltrates.

Rossaint et al. measured extravascular lung water in seven patients with ARDS by a double-indicator dilution method. They observed no significant change in extravascular lung water during prolonged exposure to nitric oxide gas. The difference may be explained by different disease entities. The lungs of their patients were ventilated for a longer period than ours before lung water measurements were made. At that time, microvascular permeability may have returned to normal. Measurement of extravascular lung water by a double-indicator dilution method reveals only large changes in extravascular lung water. Moreover, extravascular lung water determination by double-indicator technique may be affected by changes in cardiac output and regional lung perfusion. .

There was a good correlation between the change in PVR^venand the change in NSI during inhalation of nitric oxide. The more pronounced the reduction in PVR^ven, the more marked was the reduction in NSI (F3-4). In the current study, PVR^vendecreased by 30%, whereas PVR^artdid not change significantly (T2-4). In a larger series of patients, PVR^artdecreased as well but to a lesser extent than PVR^ven. This is in contrast to studies in isolated lung preparations. In endothelin-preconstricted rat lungs perfused with Krebs-Henseleit solution, Roos et al. demonstrated a more pronounced postcapillary vasodilation during inhalation of 170 ppm nitric oxide. In blood-perfused lungs, however, the extent of precapillary and postcapillary vasodilation was similar. In Krebs-perfused rabbit lungs preconstricted with the thromboxane analog U-46619, Lindeborg et al. found no change in the longitudinal distribution of pulmonary vascular resistance during inhalation of nitric oxide. The differences in results between our study and previous experimental work may be explained by the models of pulmonary hypertension used in the isolated lung preparations. In both experimental studies, the vasoconstrictors produced precapillary as well as postcapillary vasoconstriction. Nitric oxide dilates preconstricted pulmonary vessels and has no effect on the unconstricted pulmonary circulation. In animal studies, a number of mediators involved in acute lung injury such as thromboxane, endotoxin, platelet activating factor, and leukotrienes have been shown to constrict predominantly the pulmonary venous vasculature. In patients with ARDS venous vascular resistance was higher than in patients with healthy lungs. Rimar et al. observed no effect of inhaled nitric oxide on pulmonary venous resistance in preconstricted isolated rabbit lungs during orthograde perfusion. When reversing the direction of perfusion thereby producing a marked venoconstriction, inhaled nitric oxide reduced venous vascular resistance. When venoconstriction is predominant as in ALI, nitric oxide dilates postcapillary vessels and facilitates drainage of blood from pulmonary capillaries thereby lowering PCP. In that instance, transvascular fluid efflux is reduced particularly in the presence of enhanced pulmonary microvascular permeability.

We did not examine the effects of nitric oxide concentrations less than 40 ppm. The dose-response of inhaled nitric oxide on PCP in humans with ALl has not yet been studied. The results of dose-response studies on pulmonary artery pressure in humans with ARDS are conflicting. Rossaint et al. examined the effects of 18 and 36 ppm nitric oxide on MPAP. A concentration of 18 ppm was as effective as 36 ppm. Young et al. administered 8, 32, and 128 ppm nitric oxide to patients with respiratory failure. During inhalation of 32 ppm, MPAP decreased by 3.2 mmHg compared to 1.7 mmHg during inhalation of 8 ppm nitric oxide. A concentration of 128 ppm nitric oxide was not more effective than 32 ppm. Bigatello et al. observed a dose-related decrease in pulmonary artery pressure. In 7 of 11 patients studied, the maximum reduction in MPAP was reached at a concentration of 20 ppm or less whereas in the remaining four patients 40 ppm nitric oxide were more effective than 20 ppm. In the current study, we chose a concentration of 40 ppm because it seems to produce maximum pulmonary vasodilation. However, a dose-response regarding albumin flux has yet to be established.

Reduction of PCP may not be the only mechanism by which inhaled nitric oxide reduces transvascular fluid filtration. Kavanagh et al. found that inhaled nitric oxide attenuated the increase in microvascular permeability after oxidant-induced lung injury in an isolated rabbit lung preparation.

This study demonstrates that inhalation of nitric oxide at a concentration of 40 ppm reduces pulmonary transvascular albumin flux in patients with increased pulmonary microvascular permeability and lowers PCP. The long-term effect and dose-response of nitric oxide have yet to be established. If no adverse effects of nitric oxide will be found after long-term use in clinically relevant doses, nitric oxide may become the preferred agent in the treatment of disease states where pulmonary capillary hypertension and increased pulmonary microvascular permeability are the leading pathophysiologic determinants.