Ann Thorac Surg 1997;63:414-418
© 1997 The Society of Thoracic Surgeons
Original Article: Cardiovascular
Nitric Oxide Dose Response During Moderate and Severe Hypoxia in Swine
Sherif G. S. Emil, MD,
Joel E. Berkeland, RRT,
James B. Atkinson, MD
Divisions of Pediatric Surgery and Respiratory Therapy, Children's Hospital Los Angeles, Los Angeles, California
Accepted for publication August 15, 1996.
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Abstract
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Background. Elucidation of factors that influence the dose response to inhaled nitric oxide is crucial to optimizing its therapeutic benefit. We investigated whether severity of hypoxia is one such factor.
Methods. Seven Yorkshire swine underwent 14 triphasic experiments: (1) control period of mechanical ventilation (fractional concentration of oxygen = 0.3); (2) induction of hypoxic pulmonary hypertension (fractional concentration of oxygen = 0.1 to 0.15); and (3) inhaled nitric oxide at 5, 10, 20, 40, and 80 parts per million (ppm). Hemodynamics and arterial blood gases were measured by pulmonary and systemic arterial catheters.
Results. Experiments were divided into two groups of seven based on hypoxia severity: severe (arterial oxygen tension, 25 to 40 mm Hg) and moderate (arterial oxygen tension, 41 to 60 mm Hg). The percent changes in mean pulmonary artery pressure after each dose were compared within each group by repeated measure analysis of variance and each dose was compared between the two groups by Student's t test. A statistically significant dose response existed in both groups (p < 0.02). Low doses resulted in significantly less vasodilation in the severe versus the moderate hypoxia group (5 ppm, 59% ± 6% versus 94% ± 7%, p = 0.003; 10 ppm, 69% ± 8% versus 99% ± 8%, p = 0.017).
Conclusions. Lower doses are significantly less effective in achieving maximal pulmonary vasodilation during severe hypoxia. Therefore, the degree of hypoxia is a determinant of the inhaled nitric oxide dose response.
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Introduction
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Nitric oxide (NO) was identified as endothelium-derived relaxing factor in 1987 by Palmer [1] and Ignarro [2] and their co-workers. Inhaled nitric oxide (INO) has been administered to diverse groups of adult and pediatric patients whose diseases included a major pulmonary hypertension component [3–13]. Choosing an optimal dose remains a somewhat controversial issue as initial doses were chosen somewhat arbitrarily and clinical experience has produced variable results. This controversy must be resolved to optimize therapeutic benefit and minimize toxic side effects. We used an animal model to investigate whether the degree of hypoxia influences the dose response to INO during pulmonary hypertension.
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Material and Methods
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Animal Preparation
Seven adolescent Yorkshire swine weighing 11 to 20 kg were used. The animals were first sedated with intramuscular atropine (0.04 mg/kg), xylazine (4 mg/kg), and ketamine (10 mg/kg) before halothane induction and endotracheal intubation. A 12F temperature probe (Hewlett-Packard 21090A; Hewlett-Packard, Andover, MA) was inserted in the rectum, a pulse oximeter probe (Nellcor Oxysensor II, Hayward, CA) was placed on the tongue, and electrocardiogram limb leads were affixed. Under continuous halothane anesthesia, femoral cut-downs were performed bilaterally. A femoral artery catheter was used for continuous systemic blood pressure monitoring, arterial blood gas analyses, and methemoglobin sampling. A 5 to 7F pulmonary artery catheter (Biosensors, Singapore) was introduced through the femoral vein and final position confirmed by chest roentgenogram. A cutdown on the contralateral femoral vein allowed for large caliber intravenous access and maintenance fluid infusion of normal saline solution at 4 mL • kg-1 • h-1. After the surgical procedure was completed, halothane anesthesia was discontinued and balanced continuous intravenous anesthesia was started with sodium pentobarbital (10 mg • kg-1 • h-1) and pancuronium bromide (0.2 mg • kg-1 • h-1). The animals then underwent the experiments described below. The protocol was approved by the Children's Hospital Los Angeles Animal Care Committee and adhered to the "Guide for the Care and Use of Laboratory Animals" (National Institutes of Health publication 86–23, revised 1985).
Experimental Design
The animals underwent 14 experiments, each consisting of three sequential phases. In phase I, the control phase, the animal was assigned randomly to either continuous pressure-controlled mandatory conventional ventilation (Servo 900 C; Siemens-Elema, Solna, Sweden) or high frequency oscillatory ventilation (Sensormedics 3100; Sensormedics, Anaheim, CA). The assigned ventilation mode was continued for the remainder of each experiment. Phase I lasted 30 minutes and consisted of normoxic ventilation (fractional concentration of oxygen, 0.30, and ventilator parameters set to maintain arterial carbon dioxide tension at 35 to 45 mm Hg). During phase II, the animal received a randomly assigned hypoxic gas mixture (fractional concentration of oxygen, 0.10 to 0.15). This hypoxic insult resulted in a wide range of arterial oxygen tension (PaO2) (25 to 60 mm Hg), but consistently established pulmonary hypertension, defined as a minimum of 25% increase in mean pulmonary artery pressure (MPAP) over baseline. Hypoxia lasted for 30 minutes before treatment with INO, and the fractional concentration of oxygen was not manipulated for the remainder of the experiment. Phase III consisted of treatment with INO gas (Gilmore Liquid Air, San Gabriel, CA), starting at 5 parts per million (ppm) and doubling the dose every 10 minutes to 80 ppm. Nitric oxide and nitrogen dioxide were continuously monitored on line by chemiluminescence (42H; Thermo Environmental Instruments, Franklin, MA). Each animal underwent a maximum of three independent consecutive experiments, in random order of hypoxia severity and ventilation mode, separated by 30-minute recovery periods before sacrifice with sodium pentobarbital overdose (120 mg/kg).
The magnitude of hypoxia was considered the independent variable and used to divide the 14 experiments into two groups (each n = 7) for analysis. Group A sustained severe hypoxia, PaO2, 25 to 40 mm Hg. Group B sustained moderate hypoxia, PaO2, 41 to 60 mm Hg.
Monitoring
During the course of the experiment, mean systemic arterial pressure, MPAP, central venous pressure, electrocardiogram, heart rate, and temperature were continuously monitored (Hewlett Packard 78534C) and recorded every 10 minutes (Hewlett Packard 78576A), coinciding with intermittent pulmonary capillary wedge pressure measurement. Duplicate thermodilution cardiac output measurements (9520A Cardiac Output Computer; American Edwards Laboratories, Irvine, CA) were obtained at baseline, during hypoxia, and after treatment with INO doses of 5, 20, and 80 ppm. Pulmonary and systemic vascular resistances (PVR and SVR) were calculated by standard formulas. During the treatment period (phase III), data were recorded 10 minutes after each new dose, just before the next one. Hemoglobin saturation with oxygen was monitored continuously (model N-100; Nellcor, Hayward, CA). Arterial blood gases were analyzed during each phase (model 170 pH/Blood Gas Analyzer; Corning, Mayfield, MA). Methemoglobin was measured by cooximeter at baseline and at the termination of each experiment.
Statistical Methods
The two primary outcome variables were MPAP and PVR. The secondary outcome variables were mean systemic arterial pressure, SVR, and cardiac output. Baseline (phase I) and hypoxia (phase II) values for the five hemodynamic parameters were compared between the two groups using two-tailed Student's t test. Paired t test was used to compare the two phases within each group. To quantify changes in MPAP and PVR during INO treatment, percent change was calculated as [(XHX - XNO)/(XHX - XBL) x 100], where X is MPAP or PVR, HX is the value during hypoxia, BL is the value at baseline, and NO is the value at the particular INO dose. Therefore, a transformed value of 0% would represent the hypoxic value before treatment and 100% would represent a complete return to the normoxic value with treatment. The raw and transformed values of MPAP and PVR at each dose of INO were then compared within each group by repeated measure analysis of variance and each dose was compared between the two groups by two-tailed Student's t test. A p value less than 0.05 was considered statistically significant. All analyses were performed using SAS statistical software (SAS Institute, Cary, NC).
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Results
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Table 1
compares groups A and B with respect to the five hemodynamic parameters at baseline and during hypoxia (phases I and II). There were no significant differences between the groups at baseline. The MPAP was higher during severe hypoxia (42.0 ± 0.85 mm Hg) versus moderate hypoxia (36.0 ± 2.57 mm Hg), closely approaching statistical significance (p = 0.061). Hypoxia resulted in a statistically significant elevation in MPAP and PVR in each group, and in a significant decrease in SVR in the severe group. There were no significant differences in PaO2, arterial carbon dioxide tension, or pH at baseline.
Figure 1 shows the MPAP at each intervention. A significant dose response existed for the five INO doses within each group. The MPAP was higher during severe hypoxia versus moderate hypoxia at each dose of INO, and this difference was statistically significant at doses of 5, 10, and 20 ppm.
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Fig 1. . Mean pulmonary artery pressure (MPAP; mean ± standard error of the mean) at each intervention in the severe and moderate hypoxia groups. A dose response exists in both groups, but the curve is much steeper in the severe hypoxia group, where the highest inhaled nitric oxide (NO) dose (80 ppm) is required to return the pulmonary artery pressure to baseline. (*p < 0.05 compared with the same intervention in the moderate hypoxia group.)
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Figure 2 shows the percent change in MPAP during each INO dose in the two groups. Again, there was a statistically significant dose response in both groups, and less vasodilation at each INO dose administered during severe hypoxia versus moderate hypoxia. At low doses (5 and 10 ppm), the difference between the groups was statistically significant.
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Fig 2. . Inhaled nitric oxide dose response displayed as percent change in mean pulmonary artery pressure (mean ± standard error of the mean) at each dose in the severe and moderate hypoxia groups. There is less dilation at each dose in the severe versus the moderate group. (*p < 0.05 compared with the same dose in the severe hypoxia group.)
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Figure 3 shows the percent change in PVR during low dose (5 ppm), medium dose (20 ppm), and high dose (80 ppm) NO. A statistically significant dose response existed in the severe hypoxia group and approached significance in the moderate hypoxia group (p = 0.09). There was less reduction in PVR at each dose during severe hypoxia versus moderate hypoxia; this difference was statistically significant at 5 ppm and closely approached significance (p = 0.059) at 20 ppm.
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Fig 3. . Inhaled nitric oxide dose response displayed as percent change in pulmonary vascular resistance (mean ± standard error of the mean) at doses of 5, 20, and 80 ppm in the severe and moderate hypoxia groups. There is less resolution in elevated pulmonary vascular resistance at each dose in the severe versus the moderate group. (*p < 0.05 compared with the same dose in the severe hypoxia group.)
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The INO at any dose did not have any further significant effect on mean systemic arterial pressure or SVR. Cardiac output did not differ significantly at 5, 20, or 80 ppm between the two groups. There was no significant change in PaO2, arterial carbon dioxide tension, or pH during NO treatment. There were no significant changes in central venous pressure or pulmonary capillary wedge pressure at any phase. Nitrogen dioxide and methemoglobin levels did not exceed more than 2.0 ppm and 1.40% of total hemoglobin, respectively. Group A experiments involved six separate animals and included two experiments on high frequency oscillatory ventilation. Group B experiments involved four separate animals and included three experiments on high frequency oscillatory ventilation. Analysis of covariance showed that neither this distribution nor order of experiments had any significant effect on outcome.
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Comment
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In this porcine model of hypoxic pulmonary hypertension, we found that INO effectively decreases pulmonary artery pressure in a dose-dependent manner during both severe and moderate hypoxia. However, this dose response was qualitatively and quantitatively different between the two conditions. During moderate hypoxia, the lowest INO dose (5 ppm) resulted in near maximal pulmonary artery dilation with doses more than 10 ppm lowering pulmonary artery pressure below normoxic baseline levels. During severe hypoxia, low doses (5 and 10 ppm) were significantly less effective in reversing pulmonary hypertension, when compared with the moderately hypoxic group. Maximal dilation during severe hypoxia was only obtained by high doses (40 and 80 ppm). A plateau in the vasodilator response was seen at more than 40 ppm in both groups.
The acute toxicity of INO is thought to be related to two major by-products of the administered gas, nitrogen dioxide and methemoglobin [11]. We used two different ventilation modes to familiarize our team with INO administration using the different modes, and to assess them with respect to production of nitrogen dioxide and methemoglobin. Neither continuous pressure-controlled mandatory conventional ventilation nor high-frequency oscillatory ventilation was associated with significant production of nitrogen dioxide or methemoglobin in our experiments. Although the statistical analysis did not point to ventilation mode as a major confounding variable, this cannot be completely ruled out due to the relatively small sample size.
Our study links the dose-response curve of INO therapy to the severity of hypoxia responsible for pulmonary hypertension. The efficacy, as well as the dose response, of INO has been investigated in several animal models of pulmonary hypertension [14–18]. Using a similar dose range to ours, a dose response was seen in U46619 (a thromboxane analogue) induced pulmonary vasoconstriction [14], normoxic and hypoxic isolated in situ neonatal pig lungs perfused by extracorporeal membrane oxygenation [15,] a newborn lamb persistent pulmonary hypertension of the newborn model created by intrauterine ductus arteriosus ligation [16], and a porcine adult respiratory distress syndrome model [17]. These studies also clearly showed a plateau in the dose response after 40 to 50 ppm INO, a phenomenon replicated in both our experimental groups. Etches and colleagues [18] did not find a statistically significant INO dose response in an acute hypoxic pulmonary hypertension model in the newborn piglet, but they attributed this finding to a small sample size. Therefore, the data from animal studies overwhelmingly support the existence of a dose-response pattern to INO therapy for pulmonary hypertension. Our study implicates the degree of hypoxia as one of the determinants of this dose response; a higher degree of hypoxia requires a higher INO dose.
Human case reports and small case series have produced more conflicting results. This is partly because of the different endpoints used in the various diseases and partly because of the arbitrary choice of one or two INO doses for most studies. However, several studies have reported dose-response data [5, 6, 9, 13]. A study of patients with adult respiratory distress syndrome showed no advantage of 36 ppm over 18 ppm of INO with respect to reduction in pulmonary artery pressure or improvement in oxygenation [5]. In an early trial of INO in neonates with persistent pulmonary hypertension, the investigators found 80 ppm to be superior to 20 and 40 ppm in improving postductal oxygen tension [6]. In a study of children with congenital heart disease where INO was used to reduce pulmonary vascular resistance during diagnostic catheterization, the researchers found a dose response in the 20 to 80 ppm range [9]. In a Canadian study of hypoxic neonates with or without echocardiographic evidence of pulmonary hypertension where the endpoint was improvement in oxygenation, the investigators found no significant differences in PaO2 or alveolar-to-arterial oxygen gradient for the 5 to 80 ppm dose range [13]. None of the human studies have shown significant acute toxicity with the spectrum of INO doses.
As expected, the human experience with INO is much more variable and diverse than the animal experience where the investigator can control many of the confounding factors that influence pulmonary vascular tone and oxygenation. In addition, Gerlach and colleagues [10] demonstrated that, although progressive increases in INO dose result in progressive pulmonary vasodilation in patients with adult respiratory distress syndrome, improvement in PaO2 appears to peak at 10 ppm with subsequent deterioration at higher doses. These researchers postulated that, whereas low INO doses improve ventilation-perfusion matching by preferentially vasodilating well ventilated alveoli, larger doses exacerbate mismatching by increasing blood flow to poorly ventilated lung units. Therefore, pulmonary vasodilation and changes in the ventilation-perfusion ratio are interrelated INO effects that both affect oxygenation. We used a model that did not induce parenchymal lung damage, specific ventilation-perfusion inequalities, or oxygenation fluctuations with INO treatment. This allowed us to delineate the INO dose response specifically on pulmonary vasodilation without confounding from oxygenation effects.
Previously, we have demonstrated that the degree and duration of hypoxia influence the overall response to INO during pulmonary hypertension, and that severe hypoxia predicts a stronger recurrence after discontinuing the drug [19, 20]. The present study extends our previous work, demonstrating that the degree of hypoxia strongly influences the INO dose response as well. This group of studies may have important clinical implications. It is conceivable that certain severely hypoxic patients who do not respond to low doses may respond to higher ones. Using INO early in the course of hypoxic pulmonary hypertension before severe deterioration in oxygenation has ensued may allow us to use smaller doses with the least likelihood of toxicity. These hypotheses remain to be tested in clinical trials.
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Acknowledgments
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We are grateful for the statistical analysis performed by Dr Linda Chan and Carla Rother of the Biostatistics Program, Children's Hospital Los Angeles.
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Footnotes
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Address reprint requests to Dr Atkinson, Division of Pediatric Surgery, UCLA Medical Center, 10833 LeConte Ave, Box 951749, Los Angeles, CA 90025-1749
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Abstract
Introduction
