Prevention of the Pulmonary Vasoconstrictor Effects of HBOC-201 in Awake Lambs by Continuously Breathing Nitric Oxide

This study was approved by the Subcommittee on Research Animal Care at the Massachusetts General Hospital, Boston, Massachusetts. Thirty-five Suffolk lambs (24.5 ± 2.7 kg, mean ± SD) were anesthetized with an intramuscular injection of ketamine hydrochloride (15 mg/kg; Hospira, Inc., Lake Forest, IL) as described previously.17After emergence from general anesthesia, all lambs were allowed to recover for at least 2 h in a large-animal mobile restraint unit (Lomir, Malone, NY) before starting the study. Mean arterial pressure (MAP), mean pulmonary arterial pressure (PAP), central venous pressure, pulmonary arterial occlusion pressure, heart rate, and cardiac output were measured as described previously.22Systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR) were calculated using standard equations.

Blood samples were collected before and after infusion of autologous whole blood or HBOC-201 as described previously.23Plasma thromboxane B²concentrations in plasma were determined with a Thromboxane B²ELISA kit (Neogen Corporation, Lexington, KY).

Nitrate and nitrite levels in plasma were determined in 15 lambs with a nitrate/nitrite fluorometric assay kit (Cayman Chemical Company, Ann Arbor, MI).24 

Hemoglobin and methemoglobin concentrations of whole blood and plasma were determined by the cyanomethemoglobin method measuring absorption at 540 nm and 630 nm with a spectrophotometer (Biomate 3; Thermoelectron Corporation, Waltham, MA).

HBOC-201 (12–14 g/dl, pH 7.6, methemoglobin ≤ 5%, containing 3% tetramer) is a preparation of glutaraldehyde-polymerized bovine hemoglobin in a buffered physiologic solution of electrolytes and was obtained from the Biopure Corporation (Cambridge, MA).

The tracheotomy tube was connected to a circuit consisting of a 3 l reservoir bag and a two-way nonrebreathing valve (Hans Rudolph, Kansas City, MO) to separate inspired gas from expired gas. Using volumetrically calibrated flowmeters (Cole-Parmer, Vernon Hills, IL), nitric oxide gas (900 ppm in nitrogen; Specialty Gases of America, Inc, Toledo, OH) was mixed with air and pure oxygen to obtain a final concentration of 80 ppm nitric oxide or 5 ppm nitric oxide both at Fio²= 0.3. Nitric oxide concentration was measured with a chemiluminescence nitric oxide analyzer (Sievers, Model 280 Nitric Oxide analyzer, Boulder, CO) connected to the inspiratory limb of the two-way valve. Nitrogen dioxide and oxygen levels were continuously monitored. Exhaled gases were scavenged via  a Venturi exhalation trap maintained at negative atmospheric pressure by the central vacuum system.

Forty-eight hours before the experiments, each lamb was anesthetized with an intramuscular injection of 15 mg/kg ketamine, and 12 ml blood/kg of body weight was withdrawn via  the jugular vein. The blood was collected in blood collection bags (Jorgensen Laboratories, Loveland, CO) containing sodium heparin (25 units/ml blood) and was stored at 4°C for 2 days before reinfusion for the control group. On the day of the experiment, either autologous whole blood (control group) or HBOC-201 (12 ml/kg) was administered. During the experiments, the lambs were awake and breathed spontaneously while receiving an intravenous infusion of lactated Ringer’s solution (15 ml · kg^-1· h^-1). All hemodynamic measurements and blood samples were obtained at baseline and every 15–30 min.

Blood gases were measured with groups receiving either whole blood or HBOC-201 with a blood gas analyzer (Rapidlab 840; Chiron Diagnostics, Medfield, MA). Oxygen delivery and oxygen consumption were calculated as previously described.25 

Four groups of lambs were studied. One group (n = 6) received an intravenous infusion of autologous whole blood (warmed to 37°C, 12 ml/kg over 20 min) while breathing at Fio²= 0.3. A second group (n = 5) received an intravenous infusion of HBOC-201 (12 ml/kg over 20 min) while breathing at Fio²= 0.3. A third group (n = 6) breathed 80 ppm nitric oxide at Fio²= 0.3 for 1 h followed by discontinuation of nitric oxide gas administration and infusion of HBOC-201 (12 ml/kg over 20 min) while breathing at Fio²= 0.3. A fourth group (n = 5) breathed 80 ppm nitric oxide at Fio²= 0.3 for 1 h followed by breathing 5 ppm nitric oxide at Fio²= 0.3 for 2 h both during and after the infusion of HBOC-201 (12 ml/kg over 20 min). After 2 h, nitric oxide breathing was discontinued and pulmonary and systemic hemodynamics were frequently monitored while lambs breathed at Fio²= 0.30. Nitrate and nitrite levels in plasma were determined before and after breathing nitric oxide.

Four additional lambs received an intravenous infusion of HBOC-201 (12 ml/kg over 20 min) followed by breathing sequential ascending concentrations of nitric oxide (0.5, 1, 2, 5, 10, 15, 30, 40, 60, and 80 ppm) for 15 min at each dose. Blood samples were taken for methemoglobin measurement in plasma and whole blood and for arterial blood gas tension and pH analysis (Rapidlab 840; Chiron Diagnostics, Medfield, MA) after breathing nitric oxide at each level.

Two additional lambs breathed nitric oxide (80 ppm for 1 h) followed by rapid discontinuation of nitric oxide gas and then breathed at Fio²= 0.3. MAP, PAP, pulmonary arterial occlusion pressure, central venous pressure, and heart rate were measured at baseline before nitric oxide breathing and every 5 min for 30 min after discontinuing nitric oxide breathing.

In 4 additional lambs, sodium nitrite (1 mg/kg dissolved in phosphate-buffered saline) was infused at a rate of 1 ml/min for 5 min before an intravenous infusion of HBOC-201 (12 ml/kg over 20 min) while breathing at Fio²= 0.3. Hemodynamic measurements were recorded for 3 h. Nitrate and nitrite levels in plasma were determined before and after infusion of sodium nitrite.

All data are expressed as mean ± SD. Statistical evaluations were performed with the Sigma Stat 3.0.1 program (Systat Software, Inc., San Jose, CA). A two-way repeated measures analysis of variance with interaction was used to compare data from different groups that were infused with HBOC-201 breathing air supplemented with or without nitric oxide, infused with HBOC-201 after a nitrite infusion, or infused with autologous whole blood while breathing air. Multiple group comparison analysis (plasma hemoglobin oxidation rate in sheep breathing increasing concentrations of nitric oxide) was performed by adding the Holm-Sidak procedure to the Student t  test after analysis of variance. When the parametric assumption was violated (i.e. , the normality test was failed), time and transfusion (whole blood, HBOC-201 supplemented with or without nitric oxide) were analyzed as variables with the Kruskall-Wallis test. A value of P < 0.05 was considered significant.

Infusion of autologous whole blood did not significantly alter MAP, PAP, SVR, or PVR (fig. 1A–D). Infusion of whole blood did not alter oxygen delivery or oxygen consumption (data not shown). In contrast, MAP, PAP, SVR, and PVR were increased immediately after HBOC-201 infusion (12 ml/kg) in lambs breathing at Fio²= 0.3 without added nitric oxide (P < 0.05; differs from autologous whole blood group). Administration of HBOC-201 did not significantly alter the oxygen delivery or consumption rate (data not shown). As we previously reported, pretreatment with inhaled nitric oxide (80 ppm for 1 h) blocked the systemic hypertensive effects of HBOC-201 challenge (P < 0.05; differs from HBOC-201 without inhaled nitric oxide; fig. 1A). However, pretreatment with inhaled nitric oxide was unable to prevent the HBOC-201–induced acute systemic vasoconstriction (P < 0.05, increased SVR differs from autologous whole blood group; fig. 1B) or pulmonary vasoconstriction (P < 0.05; increased PAP and PVR differ from autologous whole blood group; fig. 1, C and D). Cardiac output was lower in the group that received HBOC-201 infusion than in the group that received an autologous whole blood transfusion (P < 0.05). Pretreatment with nitric oxide inhalation (80 ppm) did not prevent the reduction of cardiac output (P < 0.05; differs from HBOC-201 group with or without inhaled nitric oxide; fig. 1E). The decrease in cardiac output after infusion of HBOC-201 with or without nitric oxide pretreatment resulted from a lower heart rate because stroke volume did not differ between experimental groups (data not shown).

Pretreatment with inhaled nitric oxide did not prevent the acute pulmonary hypertension induced in lambs by HBOC-201 infusion; we, therefore, hypothesized that concurrent breathing of low levels of nitric oxide might block the HBOC-induced pulmonary hypertension without causing significant plasma methemoglobinemia. However, it was known that breathing 80 ppm nitric oxide during HBOC administration induced marked plasma methemoglobinemia,21and we sought to identify a lower concentration of nitric oxide gas that would dilate the pulmonary vasculature when inhaled without inducing plasma methemoglobin formation. First, we studied lambs that received an infusion of HBOC-201, and plasma and whole blood methemoglobin levels were measured after breathing nitric oxide for 15 min at ascending concentrations from 0.5 to 80 ppm (fig. 2). We observed that the increase of plasma methemoglobin level was 1 ± 0% after breathing 5 ppm nitric oxide for 15 min, 3 ± 1% after breathing 30 ppm nitric oxide for 15 min, and 8 ± 1% after breathing 80 ppm nitric oxide for 15 min. However, the increase of methemoglobin levels within red blood cells was only 1 ± 0% after breathing at 80 ppm. Therefore, continuously breathing nitric oxide at less than 30 ppm after HBOC-201 administration did not cause marked oxidation of either plasma or intracellular hemoglobin.

To examine whether breathing a low concentration of nitric oxide during and after HBOC-201 infusion could block the acute pulmonary hypertension induced by HBOC-201 administration, we pretreated lambs with 80 ppm nitric oxide breathing for 1 h followed by infusion of HBOC-201 (12 ml/kg) while continuously breathing 5 ppm nitric oxide for 2 h. This two-level combination of nitric oxide breathing prevented the changes in systemic and pulmonary vascular resistance induced by HBOC-201 (P < 0.05; HBOC-201 differs from autologous whole blood and from HBOC-201 after inhaled nitric oxide; fig. 3, A–D). However, after 2 h, when nitric oxide breathing was acutely discontinued, the PAP and PVR immediately increased (fig. 3, B and D), but there were no effects on MAP or SVR. After infusion, cardiac output was lower in the group that received HBOC-201 than in the group that received autologous whole blood. After pretreatment with 80 ppm nitric oxide followed by continuously breathing 5 ppm nitric oxide, the HBOC-201–induced decrease in cardiac output was markedly attenuated (P < 0.05; differs from HBOC-201 group without inhaled nitric oxide; fig. 3E). After administration of HBOC-201, plasma methemoglobin levels in the group breathing at 80 ppm and then 5 ppm nitric oxide did not increase beyond the levels observed in the HBOC-201 group breathing at Fio²= 0.3 without nitric oxide (P = NS; fig. 3F).

To determine whether there was rebound pulmonary vasoconstriction after discontinuing nitric oxide breathing in normal lambs (that did not receive HBOC-201),26we obtained pulmonary and systemic hemodynamic measurements before and after the acute withdrawal of nitric oxide (80 ppm for 1 h). Our results demonstrate that there is no change of MAP, PAP, SVR, and PVR after discontinuing nitric oxide breathing in healthy lambs. (Supplemental Digital Content, fig. 1; MAP, PAP, SVR, and PVR of normal awake lambs before and after inhaling nitric oxide).

Thromboxane B²is the principal metabolite of the powerful endogenous vasoconstrictor thromboxane A².23It has been previously shown that severe acute pulmonary hypertension in lambs can be induced by infusion of thromboxane analogs as well as by the endogenous release of thromboxane A²after activation of complement.27To determine whether or not HBOC-201 infusion induced pulmonary hypertension via  synthesis and release of thromboxane metabolites by pulmonary intravascular macrophages,28we measured plasma thromboxane B²concentration before and after infusion of HBOC-201. Thromboxane B²levels did not differ before and after HBOC-201 infusion (data not shown).

Breathing nitric oxide leads to an accumulation of nitric oxide metabolites, including nitrate and nitrite.29,30Nitrite can be converted back to nitric oxide via  nitrite reductases, including deoxyhemoglobin.31To examine whether nitrite administration could prevent the pulmonary hypertension caused by HBOC-201 infusion, sodium nitrite (1 mg/kg) was administered intravenously as an infusion before HBOC-201 was infused (fig. 4). The nitrite infusion transiently lowered the MAP from baseline 99 ± 2 to 84 ± 3 mmHg (P = 0.008), but it had no effect on PAP or cardiac output (fig. 4, A, B, and E). Nitrite infusion attenuated the systemic hypertension induced by intravenous infusion of HBOC-201 (fig. 4A), but nitrite did not prevent the increases of PAP, PVR, or SVR or the decrease in cardiac output (P < 0.05; differs from autologous whole blood group; fig. 4, B and E). Plasma methemoglobin levels did not differ in lambs receiving HBOC-201 alone and lambs receiving HBOC-201 after a nitrite infusion (fig. 4F).

Nitrate and nitrite levels were measured in plasma samples taken from lambs pretreated with inhaled nitric oxide, from lambs treated with two levels of inhaled nitric oxide (80 ppm for 1 h before HBOC infusion and 5 ppm thereafter), and from lambs receiving a nitrite infusion (fig. 5). Plasma nitrate levels did not change in lambs receiving HBOC-201 alone, but they dramatically increased in lambs breathing nitric oxide and in lambs given a nitrite infusion (P < 0.05; groups differ from HBOC-201 alone group; fig. 5A). On the other hand, plasma nitrite levels did not change in sheep breathing nitric oxide, but levels were markedly increased in the lambs given intravenous nitrite before HBOC-201 administration. After the sodium nitrite infusion, the increase in plasma nitrite levels was transient, and nitrite concentrations returned to the baseline levels by 15 min after HBOC-201 infusion (fig. 5B).

In this study, we report that the intravenous administration of HBOC-201 caused marked pulmonary and systemic vasoconstriction and decreased cardiac output in awake lambs. Pretreatment with inhaled nitric oxide or intravenous nitrite did not prevent HBOC-induced pulmonary vasoconstriction or decreased cardiac output. We identified a low concentration of inhaled nitric oxide that prevented the increase in PVR and decrease in cardiac output when administered simultaneously with HBOC after pretreatment with 80 ppm nitric oxide for 1 h. The combination of pretreatment with 80 ppm nitric oxide and concurrent administration of a low concentration of inhaled nitric oxide (5 ppm) did not cause a significant increase of plasma methemoglobin levels.

Inhaled nitric oxide is a selective pulmonary vasodilator that has been used to treat pulmonary hypertension in babies and adults as well as to prevent chronic lung diseases associated with prematurity.32Accumulating evidence suggests that inhaled nitric oxide may have systemic effects on coagulation, inflammation, recovery from ischemia/reperfusion injury, and vascular tone, although the precise mechanisms responsible for these systemic effects are controversial.32–34In a recent study, we reported that pretreatment with 80 ppm inhaled nitric oxide for 1 h prevented the systemic hypertension induced by subsequent infusion of HBOC-201 in both mice and sheep without causing plasma methemoglobinemia.17Surprisingly, in the present study, despite pretreatment with inhaled nitric oxide, HBOC-201 infusion in the awake lamb caused acute pulmonary hypertension and a major reduction of cardiac output (fig. 1). Our studies with HBOC-201 infusion are consistent with the hemodynamic changes in various species reported by others.10,12,13,35The increase of systemic and pulmonary arterial pressure and elevation of systemic and pulmonary vascular resistance with a decreased cardiac output indicate a diffuse vasoconstrictor effect of HBOC-201 infusion. Although we found ample evidence of pulmonary vasoconstriction after HBOC infusions in animal studies,8–12,17we are unaware of studies measuring pulmonary hemodynamics while infusing HBOC-201 or other HBOCs in healthy human volunteers. It has been recently reported that the PAP significantly increased after HBOC-201 infusion in patients with an acute coronary syndrome undergoing percutaneous coronary interventions.36Therefore, we sought a method to prevent the pulmonary vasoconstriction associated with infusion of HBOC-201.

After infusing HBOC-201 into lambs, we studied the effects of inhaling increasing concentrations of nitric oxide. Surprisingly, we found that extracellular hemoglobin was resistant to oxidation by inhaled nitric oxide when the gas was breathed at levels below 30 ppm (fig. 2). We then evaluated whether nitric oxide if continuously inhaled at low levels would prevent the systemic and pulmonary vasoconstriction induced by HBOC-201 administration. We learned that pretreatment by breathing nitric oxide (80 ppm for 1 h) followed by breathing a low concentration of nitric oxide (5 ppm) during and after HBOC-201 challenge prevented the development of both systemic and pulmonary vasoconstriction in awake lambs (see fig. 3).

Our results suggest that continued breathing of low levels of nitric oxide will be necessary to reverse the persistent nitric oxide scavenging caused by HBOC-201 administration in the pulmonary circulation. Acute withdrawal of nitric oxide breathing in our lambs, even 2 h after HBOC-201 infusion, caused acute rebound pulmonary vasoconstriction (fig. 3). In contrast, sudden withdrawal of nitric oxide breathing in a normal sheep (without circulating HBOC-201) does not cause any hemodynamic alterations (see figure, Supplemental Digital Content 1, which shows MAP, PAP, SVR, and PVR of normal awake lambs before and after inhaling nitric oxide, https://links.lww.com/A601). The vasoconstriction observed after the sudden withdrawal of inhaled nitric oxide after HBOC-201 infusion is reminiscent of the rebound pulmonary hypertension occurring in adults with acute respiratory distress syndrome and children after cardiac surgery upon sudden withdrawal of nitric oxide inhalation. Slow reduction of the concentration of inhaled nitric oxide has been successful in preventing rebound pulmonary hypertension.37It is conceivable that slow weaning of inhaled nitric oxide may prevent rebound pulmonary hypertension in patients treated with HBOCs.

We considered the possibility that infusing HBOC-201, a foreign antigen, might induce pulmonary hypertension by activating complement with the elaboration of pulmonary thromboxane A², a potent endoperoxide vasoconstrictor.23We measured plasma thromboxane B²levels after HBOC-201 infusion. The levels remained low and were not different between animals receiving a control transfusion of autologous blood and those demonstrating HBOC-201–induced pulmonary hypertension (data not shown). These findings suggest that thromboxane generation does not contribute to the pulmonary hypertension associated with HBOC-201 administration.

In a comparison with inhaled nitric oxide, an intravenous infusion of a dose of sodium nitrite sufficient to induce systemic hypotension followed by HBOC-201 administration attenuated the development of systemic hypertension, but it did not prevent the development of acute pulmonary vasoconstriction (fig. 4). Serial measurements of nitrate and nitrite levels in plasma revealed that infusion of sodium nitrite increased levels of both metabolites (fig. 5). In comparison, breathing nitric oxide elevated only the plasma nitrate level but not the plasma nitrite level (fig. 5). These results suggest that the prevention of HBOC-induced vasoconstriction by nitric oxide inhalation is not attributable to the generation of nitrite.

The awake lamb is a commonly used model for pulmonary circulatory studies of the human newborn because it has a muscularized pulmonary arterial circulation. Thus, the lamb may be excessively susceptible to pulmonary vasoconstriction due to HBOC infusion. However, the pulmonary circulation of the human newborn is also muscularized as it is in adults with chronic pulmonary hypertension or other lung vascular diseases. It is conceivable that newborns and adults with pulmonary vascular diseases may be quite susceptible to vasoconstriction from HBOC infusion. We previously reported that very high levels of inhaled nitric oxide were required to partially reverse thromboxane-induced pulmonary hypertension.27In this study, the ability of low concentrations of nitric oxide breathing to completely prevent HBOC-201 pulmonary hypertension suggests that nitric oxide scavenging by HBOC causes this pulmonary hypertension (and not thromboxane A²release). High doses of inhaled nitric oxide can cause methemoglobinemia and may damage the lung and perhaps other organs.38On the other hand, thousands of infants with hypoxic respiratory failure and pulmonary hypertension are treated each year with inhaled nitric oxide, usually commencing at 20–80 ppm for at least several hours.39Most of these newborns survive without renal or respiratory failure; therefore, it is unlikely that short-term breathing of such doses of nitric oxide gas is highly toxic. However, the current study is not a study of a hemorrhagic shock model. We chose to study a top-load model without hypotension or shock in order to isolate and examine the hemodynamic effects of the agent that we infused (HBOC-201). Nonetheless, before clinical use of inhaled nitric oxide with HBOC to treat hemorrhage and shock, additional animal studies should be undertaken.

In conclusion, we report that pretreatment of lambs with inhaled nitric oxide followed by continuously breathing a low concentration of nitric oxide can prevent the pulmonary and systemic vasoconstriction induced by HBOC-201 administration without causing any significant elevation of plasma methemoglobin levels. This promising combination therapy merits further evaluation as a method to enable HBOC administration without vasoconstriction in patients with acute traumatic anemia.

The authors thank Hui Zheng, Ph.D., Assistant Professor in Medicine at Harvard Medical School, Massachusetts General Hospital Biostatistics Center, Boston, Massachusetts, for statistical assistance and help with data analysis.