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Departments of Medicine and Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Simonson, Steven G., Karen E. Welty-Wolf, Yuh-Chin T. Huang,
David E. Taylor, Stephen P. Kantrow, Martha S. Carraway, James D. Crapo, and Claude A. Piantadosi. Aerosolized
manganese SOD decreases hyperoxic pulmonary injury in primates. I. Physiology and biochemistry. J. Appl.
Physiol. 83(2): 550-558, 1997.
Prolonged hyperoxia causes lung injury and
respiratory failure secondary to oxidative tissue damage mediated, in
part, by the superoxide anion. We hypothesized that aerosol treatment
with recombinant human manganese superoxide dismutase (rhMnSOD) would
attenuate hyperoxic lung damage in primates. Adult baboons were
anesthetized and ventilated with 100% oxygen for 96 h or until death.
Six animals were treated with aerosolized rhMnSOD (3 mg · kg
1 · day1
in divided doses), and six control animals did not receive enzyme therapy. Physiological variables were recorded every 12 h, and ventilation-perfusion ratio relationships were evaluated by using the
multiple inert-gas elimination technique. After the experiments, surfactant composition and lung edema were measured. We found that
rhMnSOD significantly decreased pulmonary shunt fraction (P < 0.01) and preserved arterial
oxygenation (P < 0.01) during hyperoxia. The rhMnSOD increased lung phospholipids,
phosphatidylcholine and disaturated phosphatidylcholine, and decreased
lung edema in this model. Testing of higher and lower doses of MnSOD (1 and 10 mg · kg1 · day1)
in two other groups of baboons produced variable physiological protection, suggesting a "window" of effective dosage. We
conclude that aerosolized MnSOD (3 mg · kg1 · day1)
affords significant preservation of pulmonary gas exchange during hyperoxic lung injury.
antioxidant enzymes; superoxide dismutase; oxygen; acute
respiratory distress syndrome; acute lung injury
INTRODUCTION PROLONGED EXPOSURE to 100% oxygen causes a diffuse
lung injury that leads to respiratory failure, similar to the acute
respiratory distress syndrome (ARDS). Oxygen toxicity and ARDS involve
oxidative stress in the lung. The lung injury is related in part to
production of superoxide anion
(O Among the primary antioxidant defenses in the lung are enzymes that
detoxify reactive oxygen species such as the superoxide dismutases
(SODs). Manganese SOD (MnSOD) is one of three SOD isoforms present in
mammalian tissues. MnSOD is normally found within mitochondria and
constitutes a smaller fraction of pulmonary SOD activity (~20% of
the total) than other isoforms of SOD, e.g.,
copper-zinc SOD (CuZnSOD) (27). The role of MnSOD in protection from
oxidative stress has been demonstrated in studies showing increases in
MnSOD mRNA and protein in response to sublethal hyperoxia and other forms of oxidative stress (5, 18, 19). Additionally, cytokines like
interleukin-1 and tumor necrosis factor increase MnSOD expression during the development of oxygen tolerance (22, 23).
Attempts to protect the lung from oxygen toxicity by using CuZnSOD have
had variable success. Most of the studies have required for
effectiveness a delivery system such as liposomes or conjugation of the
enzyme to polyethylene glycol (1, 3, 4, 24). MnSOD differs from CuZnSOD
because the protein carries a positive charge that may increase its
affinity for the plasma membrane when delivered to the cell surface.
This characteristic may affect its site of deposition and activity,
resulting in more efficacious antioxidant effects in the lungs.
Furthermore, the relative importance of different isoforms of SOD in
different oxidative injuries is not known.
We have established a primate model of pulmonary oxygen toxicity by
exposing baboons to 100% oxygen for 96 h (11, 12). The baboon is
appropriate for testing therapeutic interventions delivered by aerosol
because of the similarity of its lung to that of humans. This includes
a dichotomous airway-branching pattern so that localization of injury
and delivery of drugs should be similar. We hypothesized that
augmentation of pulmonary antioxidant defenses by administration of
aerosolized MnSOD would protect against pulmonary oxygen toxicity by
affording measurable physiological and biochemical protection from this
insult. A quantitative morphological study of the lung injury is
presented in the accompanying manuscript (28).
METHODS Adult male baboons (Papio
cynocephalus, Southwest Foundation, San Antonio, TX)
were used for the study. The animals were quarantined for at least 4 wk
before use. They were determined to be tuberculosis free and were
handled in accordance with American Association for the Accreditation
of Laboratory Animal Care guidelines. They were fasted overnight before
the experiment began and then sedated with intramuscular ketamine
(20-25 mg/kg). After orotracheal intubation with a no. 6 endotracheal tube, the animals were ventilated with a volume-cycled
ventilator (Puritan-Bennett, Kansas City, KS, model 7200a) at a tidal
volume of 12-15 ml/kg, positive end-expiratory pressure of 2.5 cmH2O, and a rate sufficient to
maintain an arterial PCO2
(PaCO2) of 35-45
Torr. Peak inspiratory flow was set at 15 l/min. The
animals were paralyzed with pancuronium (4-8 mg iv followed by 4 mg iv every 2 h), and sedation was maintained with ketamine (3-10
mg · kg Experimental protocol. The study was
designed to test the hypothesis that 3 mg · kg After the animals were prepared, they were monitored for ~2 h for
hemodynamic stability, and baseline cardiovascular, ventilator, and
ventilation-perfusion ratio
( MnSOD aerosol and delivery system.
Recombinant human MnSOD (rhMnSOD) was obtained from
Boehringer-Ingelheim Pharmaceuticals (Ridgefield, CT). Activity was
assessed by the cytochrome c reduction assay and found to be 3,100-3,580 U/mg. The drug was delivered via
ultrasonic nebulization (De Vilbiss Aerosonic Nebulizer, model 5000B)
in series with the inspiratory limb of the ventilator circuit. The
concentrations of protein in the aerosol were 1.0, 3.0, and 10 mg/ml,
respectively, for the three doses of rhMnSOD. Particle characteristics
were defined by using a cascade impactor and an endotracheal tube.
These studies revealed a mass median diameter of 2.5-3.0 µm and
a geometric SD of 2.5 µm for the aerosol. Typically, each dose of the
enzyme was delivered over a 20- to 30-min period. The nebulizer did not
alter the activity of the enzyme.
2·), a free radical that can
cause biological damage by itself or promote formation of other
reactive oxygen species (ROS), including hydrogen peroxide (H2O2),
peroxynitrite (ONOO), and
hydroxyl radical (· OH).
1 · h1)
and diazepam (0.4-0.8 mg/kg every 2 h) given through a peripheral venous catheter. Ampicillin (1 g) every 6 h intravenously and gentamicin (40 mg) and polymyxin (20,000 units) intratracheally every 4 h were given as prophylaxis against nosocomial pneumonia. A pulmonary
artery catheter (5-Fr) was inserted via a femoral incision to monitor
central venous, pulmonary arterial, and pulmonary arterial wedge
(occlusion) pressures; sample mixed venous blood; and determine cardiac
output. Cardiac output was measured by thermodilution (5-ml injectate)
performed in triplicate, with the average of three trials used for
calculations (Horizon 2000 monitor, Mennen Medical, Clarence, NY). A
femoral artery catheter was used to monitor blood pressure and obtain
blood samples for arterial blood-gas measurements. Arterial and mixed
venous blood PO2,
PCO2, and pH were measured by using a
pH/blood-gas analyzer (Instrumentation Laboratory, models 813 and 1304, Lexington, MA). Hemoglobin concentration, oxygen saturation, and oxygen
content were obtained by using a CO-oximeter (Instrumentation
Laboratory, model 482) programmed for baboon hemoglobin. Systemic
arterial, central venous, and pulmonary arterial pressures were
measured with Gould TDN-R pressure transducers and displayed on a
Horizon 2000 monitor.
1 · day1
of aerosolized MnSOD would improve physiological function during pulmonary oxygen toxicity in the baboon. The dose of enzyme was chosen
on the basis of preliminary studies in rodents. After completing the
untreated and 3 mg · kg1 · day1
treatment groups, we evaluated two other doses of enzyme for comparison. Thus four groups of six animals each were studied. One
group (control) of six animals was ventilated on 100% oxygen for
96 h or until death. These animals did not receive MnSOD. A
second group of animals was ventilated on 100% oxygen for 96 h and received aerosolized 3 mg · kg1 · day1
MnSOD in divided dose every 12 h. The third group of animals was
ventilated on 100% oxygen for 96 h and received aerosolized 1 mg · kg1 · day1
MnSOD in divided dose every 12 h. The final group of
animals received 100% oxygen for 96 h and aerosolized 10 mg · kg1 · day1
MnSOD in divided dose every 12 h. Treatment was initiated at the onset
of hyperoxia.
A/
)
measurements were obtained. Maintenance intravenous fluid was begun at
30 ml/h. Heart rate, temperature, arterial blood pressure, pulmonary
arterial pressures, fluid balance, and ventilator parameters were
recorded every hour. Additionally, every 12 h, arterial and mixed
venous blood gases, pulmonary arterial wedge pressure, and cardiac
output were recorded. Airway care consisted of endotracheal suctioning,
manual sigh breaths, position changes, and chest physiotherapy every 6 h. The animals were killed by KCl injection at 96 h, and autopsy followed immediately. An additional group of six animals was ventilated with air for 96 h under the same conditions. Selected data from this
group are included to provide normal reference values. The average
weight of the animals in the four experimental groups ranged from 14.4 to 16.2 kg.
A/
distribution.
A/
distributions were quantified by using the multiple inert-gas
elimination technique of Wagner et al. (25). These measurements were made at 0, 12, 36, 64, 84, and 96 h of hyperoxia. A
dilute solution of six inert gases (sulfur hexafluoride, ethane, cyclopropane, enflurane, diethyl ether, and acetone) in normal saline
was infused continuously via a peripheral vein at a rate of 2.5-3
ml/min. Duplicate samples of systemic arterial blood, mixed venous
blood, and mixed expired gas were collected at least 40 min after the
infusion was begun. Each blood sample was equilibrated with an equal
volume of nitrogen in a heated water bath (37°C) for at least 40 min. The equilibrated gas and expired samples were analyzed for sulfur
hexafluoride by an electron-capture detector and for the other five
gases by a flame-ionization detector (Varion Gas Chromatograph, model
3700 and Hewlett-Packard Chromatograph, model 5890, series II). The
data were directed to an analog-to-digital converter (Nelson Analytical
900 series interface) and stored on a computer for analysis. The
blood-gas partition coefficients of six inert gases also were
determined each day. Retention-partition coefficient and
excretion-partition coefficient curves were generated by using the
technique of enforced smoothing and least squares analysis. These
data were then transformed into
A/
distribution by using a 50compartment lung model. The shunt was
defined as A/
<0.005 and dead space as
A/
>100. The
log(A/)
SD values of perfusion and ventilation (SDq and SDv, respectively) were
used as indexes of dispersion of blood flow and ventilation, respectively (10).
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, Untreated animals; 
, animals
treated with recombinant human manganese superoxide dismutase (rhMnSOD)
at 3 mg · kg1 · day1;
, animals treated with 1 mg · kg1 · day1
of rhMnSOD; 
, animals treated with 10 mg · kg1 · day1
of rhMnSOD. Error bars are not shown for clarity; arterial
PO2 (PaO2) data are available in Table 2
(n = 6 in each group).
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Surfactant biochemistry. Total lipids were extracted from the bronchoalveolar lavage done in a lobe of the left lung at the end of the experiments (7) and separated into neutral and phospholipids (PLs) on silica gel columns and into PL classes by thin-layer chromotography (21). Disaturated phosphatidylcholine (DSPC) was isolated on neutral alumina columns after osmium tetroxide treatment of total PLs (15). Phosphorus was measured and assumed to be 4% of the PL by weight (2). Corrections for losses of lipid during processing were measured by adding a trace amount of [14C]dipalmitoyl phosphatidylcholine. Surfactant protein-A enzyme-linked immunosorbent assay was performed as previously described (11). These studies were performed only in animals receiving oxygen, air, and 3 mg · kg
1 · day1
MnSOD plus oxygen.
Statistical analysis. Data are
presented as means ± SE. Statistical analysis was performed by
using SAS statistical software (SAS Institute, Cary, NC).
Repeated-measures data from physiological measurements were analyzed by
using linear regression analysis allowing for both fixed and random
effects. Because there were missing values due to some animals dying
before 96 h, the standard repeated-measures model was employed (26).
Drug effects were compared at the 96 h time point, and slopes (rate of
change with time) were also compared among groups. The primary
comparison a priori was between the oxygen control
group and the 3 mg · kg1 · day1
rhMnSOD group by using a P value of
0.05 as significant. After two additional doses of rhMnSOD were added
to the experimental protocol, all enzyme doses were compared with the
oxygen control group and, in some cases, with each other. Because the
comparison of all three MnSOD groups with oxygen was post
hoc, we denoted as significant only the
P values that reached the 0.05 level
after a Bonferroni correction. Surfactant data and wet-to-dry weight ratios for the oxygen control group and the 3 mg · kg1 · day1
MnSOD group were compared by using the Student's
t-test or the Mann-Whitney test as
appropriate. Survival differences were compared by using Kaplan-Meier
analysis. Observations from animals not meeting physiological criteria
for stopping the experiment were considered censored observations.
RESULTS
1 · day1
of rhMnSOD. The sixth animal in the untreated group died at 84 h from
severe hypoxemia. One animal in the 1 mg · kg1 · day1
group died abruptly after 72 h without hypoxemia or acidosis. The sixth
animal in the 10 mg · kg1 · day1
group died after 72 h with severe acidosis (pH 7.08), but it was not
hypoxemic. Several animals in these three groups approached physiological criteria at 96 h for stopping the experiment
[PaO2 <60 Torr on
100% oxygen or mean arterial pressure (MAP) <60 mmHg]. This
included three animals in the untreated group, one in the 1 mg · kg1 · day1
group, and two in the 10 mg · kg1 · day1
group. All animals receiving 3 mg · kg1 · day1
rhMnSOD survived 96 h, showing no sign of impending physiological collapse. The survival difference between the untreated oxygen-exposed animals and those receiving 3 mg · kg1 · day1
of rhMnSOD during oxygen exposure approached statistical
significance (P = 0.06).
Hemodynamics. The cardiovascular
effects of 96 h of hyperoxia in control and MnSOD-treated animals are
reported in Table 1. In general, 3 mg · kg1 · day1
of rhMnSOD tended to minimize the changes in cardiovascular variables. Intravascular volume status and left ventricular end-diastolic pressure
as assessed by central venous pressure and pulmonary capillary wedge
pressure were maintained within the normal range in all
groups during the experiment. Central venous pressure remained between
6 and 8 mmHg and pulmonary capillary wedge pressure between 9 and 12 mmHg. MAP increased slightly in all groups during the first 48 h of hyperoxia. MAP then began to drop between 48 and 60 h in all
groups, except in animals receiving 3 mg · kg1 · day1
in which a slight decline began at 84 h. Systemic vascular resistance mirrored the MAP profile. Heart rate increased while cardiac output and
stroke volume decreased with time in all groups. The smallest change
occurred in the 3 mg · kg1 · day1
treatment group. Mean pulmonary arterial pressure increased 46% and
pulmonary vascular resistance increased 245% in untreated hyperoxia at
96 h but the increases were limited to 10 and 63%, respectively, in
the rhMnSOD group receiving 3 mg · kg1 · day1
of the enzyme. Hemoglobin concentration decreased by 3-4 g/dl in
all groups related to the effects of oxygen and blood withdrawn for
studies. The rate of decline in the hemoglobin concentration, however,
was significantly slower in all MnSOD-treated groups. Oxygen delivery
decreased in all groups, but this effect was significantly less in the
3 mg · kg1 · day1
rhMnSOD group. Systemic oxygen consumption remained constant. This
resulted in an increase in the oxygen extraction ratio and a decrease
in mixed venous PO2. The extent and
rate of decrease in the mixed venous
PO2 were significantly less in all
MnSOD-treated groups.
Respiratory variables,
A/
relationships, and gas exchange.
The effects of MnSOD on respiratory variables are reported in
Figs.1, 2, 3 and Tables 2 and 3.
PaO2 progressively decreased with
continuous exposure to 100% oxygen. Aerosolized rhMnSOD significantly attenuated the decline in PaO2 in all
treated groups, as shown in Fig. 1. Actual values and significance
levels are reported in Table 2. Figures 2 and 3 show the
changes in
A/
relationships during the hyperoxic exposure. The statistical analyses
of these changes compared with oxygen are provided in Table
3. Shunt fraction increased from 1% of
cardiac output at baseline to 40% at 84 h in the untreated control
group. We were unable to study three animals in this group using the
multiple inert-gas elimination technique at 96 h because the animals
died or exhibited non-steady-state conditions due to physiological
deterioration. The final
A/ data point represents only three stable surviving animals. Figure 2
shows that the increase in shunt fraction was limited to 10% of the
cardiac output at 96 h in the animals treated with 3 mg · kg1 · day1
of the enzyme (P = 0.001). A decrease
in shunt fraction was also seen in both of the other treated groups but
to a lesser extent. Dead space remained relatively constant in all
groups (Fig. 2). rhMnSOD treatment had inconsistent effects on
ventilation-perfusion matching, as seen in Fig.
3. In the group receiving 3 mg · kg1 · day1,
rhMnSOD treatment was associated with the least change in dispersion of
perfusion but the most change in dispersion of ventilation. Animals
treated with 1 or 10 mg · kg1 · day1
of enzyme had ventilation-perfusion relationships similar to untreated
animals. Total respiratory system compliance (including the ventilator
circuitry, chest wall, and lungs) decreased significantly over time in
all groups. Untreated animals showed a 42% decrease in compliance,
compared with 32% in animals receiving 3 mg · kg1 · day1
of enzyme (P = 0.12). Animals
receiving 1 or 10 mg · kg1 · day1
of enzyme had changes in compliance closer to these of the untreated group. Peak airway pressures were 16-17
cmH2O in all groups at baseline
and increased in the untreated oxygen-exposed animals to the 30-32
cmH2O range. Airway pressures in
animals receiving rhMnSOD at 3 mg · kg1 · day1
increased to ~25 cmH2O, whereas
animals in the other two treatment groups showed slightly greater
increases in peak pressures (Table 2). The minute ventilation required
to maintain a normal PaCO2 increased in
all groups but significantly less in animals treated with 3 mg · kg1 · day1
of enzyme (P = 0.01). Five of six
baboons treated with 10 mg · kg1 · day1
had a significant acidosis (pH <7.25) at 96 h or at death. This is in
contrast to one of six in the group receiving 1 mg · kg1 · day1,
none in animals receiving 3 mg · kg1 · day1,
and two in the untreated group. Differences between dosage groups did
not reach statistical significance after Bonferroni corrections were
applied to the data.
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1 · day1
attenuated many of the changes in lavage fluid caused by prolonged hyperoxia. Hyperoxia caused a decrease in lavage total PLs,
phosphatidylcholine (PC), and DSPC. Treatment with rhMnSOD
significantly increased PL, PC, and DSPC, even above values seen with
mechanical ventilation alone. Lavage protein increased in hyperoxic
lung injury, and this effect was decreased by rhMnSOD treatment,
despite nebulizing protein into the lung. The amount of surfactant
protein-A in the lavage fluid was not affected significantly by
rhMnSOD.
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1 · day1
rhMnSOD was 6.37 ± 0.53, and for those treated with 10 mg · kg1 · day1
the ratio was 6.48 ± 0.74.
DISCUSSION
This study demonstrated significant physiological
protection against pulmonary oxygen toxicity by aerosolized rhMnSOD (3 mg · kg1 · day1)
in baboons during prolonged exposure to hyperoxia. MnSOD preserved arterial oxygenation, decreased intrapulmonary shunt fraction, and
decreased pulmonary edema, compared with untreated oxygen-exposed animals. MAP was maintained, and trends in other cardiovascular variables suggested less physiological decline. Changes in surfactant composition induced by oxygen were attenuated by treatment with rhMnSOD. Lipid composition moved toward values seen in normal baboons
ventilated with air alone. We also found a dose-response effect with
lesser degrees of physiological protection at lower and higher doses of
MnSOD, including possible evidence of drug toxicity at high doses.
The improvement in arterial PO2
observed in the rhMnSOD-treated animals was related primarily to
decreased intrapulmonary shunt and, to a lesser degree, improved SDq.
The decrease in shunt fraction can be explained in two ways, by
improved ventilation or by diversion of blood flow from unventilated to
ventilated regions. Our data are most suggestive of improved (or
maintenance of) ventilation as an explanation for the lower
intrapulmonary shunt. This explanation is consistent with decreased
injury to the alveolar epithelium but not to the endothelium. Although
the changes in shunt and SDq also may be caused by effects of the treatment on the pulmonary vasculature, the lack of change in cardiac
output between treated and untreated animals, the smaller change in
pulmonary vascular resistance in the treated group, and similar
intravascular inflammatory changes by morphometry make this mechanism
less likely. SDv increased with hyperoxia and then
stabilized, an effect that occurs before the inflammatory phase of
hyperoxic injury in this species (8). The higher SDv in the 3 mg · kg1 · day1-treated
animals toward the end of the exposure may also reflect alveolar
epithelial protection in this group.
The mechanism whereby MnSOD produces its protection against hyperoxic
lung injury is presumably due to scavenging of
O2· by the enzyme. The enzyme
remains active after aerosolization, and blood levels of MnSOD activity
increase in rodents after aerosol therapy.
O2· production by lung tissue has
been shown to increase during exposure to hyperoxia (9). Moreover,
various stimuli have been shown to induce SOD activity in association
with tolerance to hyperoxia (20, 22, 23). These findings have supplied
the rationale for attempts to decrease lung injury from ROS by
exogenous SOD. Results have been varied, but several studies have shown
a positive effect when the drug is delivered to the air spaces (1, 3,
24). In the present study, MnSOD was administered in its native form
without the need for derivitization or a liposome-based delivery system
as has been required for other isoforms of SOD. A major difference
between MnSOD and CuZnSOD is that MnSOD carries a positive charge,
whereas CuZnSOD is negatively charged. This may significantly affect
the site and extent of deposition of the enzyme and the duration of activity. It also may be the reason that no special delivery strategy was needed for the drug.
Scavenging of O2· may be
beneficial in hyperoxic lung injury by decreasing tissue damage
directly caused by O2·, but
secondary effects of scavenging O2· may be equally or more
protective. The specific chemical species causing tissue injury during
most types of oxidative stress are not known.
O2·, H2O2
and nitric oxide NO · are relatively weak oxidants. Under appropriate conditions, O2· in the
presence of
H2O2
or NO · causes · OH and
ONOO formation, both of
which are strong oxidants and may damage lipids, proteins, or nucleic
acids during hyperoxia (18). Furthermore, if the
O2· reaction with NO · is a significant means for regulation of the biological effects of
NO ·, the effects on feedback mechanisms related to
NO · release may be altered, affecting multiple cell
functions, including oxidant production, vascular and airway tone, and
cytokine release. These important areas are being investigated in
studies at the cellular and organ level. Some of the physiological
findings in MnSOD-treated animals, such as decreased pulmonary artery
pressures and pulmonary vascular resistance, could be related in part
to increased NO · activity.
ROS are produced in hyperoxia intracellularly (e.g., by lung mitochondria) and extracellularly (e.g., by inflammatory cells). ROS usually react biologically at or near their site of production. This is important because attempts to decrease toxicity by scavenging ROS require the drug to be delivered to the sites of ROS production. Although our study did not directly assess the site of production of ROS, MnSOD was delivered to the airways and alveolar spaces. Other work (6) and the accompanying study (28) demonstrate MnSOD deposition along epithelial surfaces, in the airways and in alveolar macrophages, without evidence for significant epithelial cell uptake of the compound. This contrasts with studies that demonstrate oxygen tolerance being associated with increases in intracellular MnSOD activity (5, 19, 20, 22, 23). Additionally, hyperoxic lung injury can be attenuated in transgenic mice expressing increased intracellular MnSOD in epithelial cells (29).
Surfactant alterations in hyperoxic lung injury have been attributed to impairment of alveolar type II cells to synthesize surfactant of normal composition (13) as well as increased degradation of surfactant. MnSOD acting on the alveolar epithelial surface may have protected type II cell function in part, explaining the relatively minor surfactant changes seen in treated animals in this study.
Significant physiological deterioration including hypoxemia, decreased
pulmonary compliance, and ventilation-perfusion abnormalities occurred
with time despite treatment with MnSOD at all doses of the enzyme.
There are several possible explanations for the lack of complete
protection by the enzyme. First, it is possible that with continued
exposure to hyperoxia, O2· production by the lung and inflammatory cells exceeds the extracellular antioxidant defenses despite the presence of MnSOD. Second,
H2O2 production may exceed the ability of catalase and glutathione peroxidase to detoxify it. Third, sites of ROS production exist during
hyperoxia that are not accessible to aerosolized MnSOD (e.g., mitochondria). It is also possible that MnSOD
may affect cell regulatory mechanisms via macrophages or other cells,
altering, but not preventing, amplification of the inflammatory
cascade. In addition, some of the physiological derangement during
hyperoxia may be due to injury mechanisms not directly related to ROS,
such as release of proteases and elastase as well as other neutrophil products.
MnSOD at 1 mg · kg1 · day1
or 10 mg · kg1 · day1
was not as effective as 3 mg · kg1 · day1
in physiological protection against oxygen toxicity. It is notable that
the death of an animal receiving 10 mg · kg1 · day1
was associated with a severe metabolic acidosis (a group trend), whereas the early death of an animal receiving 1 mg · kg1 · day1
was not. Arterial oxygenation was significantly better than in untreated animals in all three treated groups, but there were no
differences among the three doses of rhMnSOD. In contrast to the 3 mg · kg1 · day1
group, MAP, mean pulmonary arterial pressure, and oxygen delivery were
not improved by treatment with rhMnSOD at 1 or 10 mg · kg1 · day1.
Thus a dosage "window" appears to provide significant protection without adverse effects.
This type of therapeutic window for dosing SOD has been observed by others in diverse models of oxidative stress and in different species (16, 17). Low-dose drug failure can be explained by a lack of a threshold quantity of drug required for efficacy. High-end dosage failure is more difficult to explain, however, several theories are tenable (16, 17). Excessive SOD may enhance lipid peroxidation by decreasing the free radical processes involved in terminating lipid peroxidation. Increased H2O2 production at a site not prepared for attack by H2O2 or a rapid burst of H2O2 production that exceeds regional peroxidase defenses may result from excessive SOD administration.
Toxicity related to manganese is a possible but less likely explanation
for the suboptimal outcome of the 10 mg · kg1 · day1
group. Manganese levels were not measured in this experiment, and the
avidity of this enzyme complex for manganese under in vivo conditions
is not known. Manganese can accumulate in mitochondria where it may
interfere with oxidative phosphorylation. This effect could explain the
acidosis and worse outcome at higher doses. The rhMnSOD, however, is
very stable in vitro, and it is difficult to inactivate the
enzyme chemically (T. Noonan, Boehringer-Ingelheim Pharmaceuticals, personal communication).
In summary, we have shown that aerosolized rhMnSOD provides
physiological protection against injury from continuous exposure to
100% oxygen in baboons. The protection was most prominent at a dose of
3 mg · kg1 · day1,
and possible toxicity was seen with the 10 mg · kg1 · day1
dose. Further studies are needed to determine whether the mechanism of
the protective effects is related to the catalytic activity of the
enzyme and its localization in the lung. The data suggest that
aerosolized MnSOD may be beneficial to patients with acute lung injury
who are exposed to high concentrations of inspired oxygen for a
prolonged period of time to prevent oxygen toxicity. The dose, however,
would have to be chosen carefully to avoid potential drug toxicity.
ACKNOWLEDGEMENTS
We acknowledge the technical expertise of John Patterson and Craig Marshall and the secretarial assistance of Louise Wilson.
FOOTNOTES
Address for reprint requests: C. A. Piantadosi, Box 3315, Duke Univ. Medical Center, Durham, NC 27710.
Received 19 August 1996; accepted in final form 31 March 1997.
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