* Departments of Pediatrics-Neonatology and
Environmental and Community Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854; and
Department of Pharmacology and Toxicology, Rutgers University, Piscataway, New Jersey 08854
Received June 30, 2000; accepted September 19, 2000
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ABSTRACT |
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Introduction |
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Pulmonary Effects of Inhaled Nitric Oxide |
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In regard to the long-term adverse effects of inhaled nitric oxide, data from the medical and developmental follow-up of participants in the large trials are only now becoming available. The incidences of sensorineural hearing loss and moderate to severe cerebral palsy in surviving infants in the Neonatal Inhaled Nitric Oxide Study (2000) were reported to be increased in both the nitric oxide-treated and control groups relative to well infants, but were not correlated with the group assignment. Thus, inhaled nitric oxide was not associated with increased neurodevelopmental, behavioral, or medical abnormalities at 2 years of age. These findings are in accord with an earlier report of the 1- and 2-year follow-up of a smaller cohort of infants treated with nitric oxide for PPHN (Rosenberg et al., 1997). Long-term effects of inhaled nitric oxide on pulmonary function, bronchoreactivity, immune function, and hematologic parameters in infants who have recovered from PPHN have not been investigated.
In summary, it is clear that inhaled nitric oxide has found its place in the clinical arena as a pulmonary vasodilator, but that the place needs to be more clearly defined. Term and near-term infants with hypoxemic respiratory failure, as a group, appear to benefit from the treatment. These infants exhibit improved oxygenation, decreased need for extracorporeal membrane oxygenation (ECMO), and trends toward decreased mortality. In other patients, inhaled nitric oxide may be helpful in achieving specific therapeutic endpoints, such as pulmonary vasodilation following cardiac surgery (Fullerton et al., 1996). However, inhaled nitric oxide has not proven to be beneficial in other conditions, such as acute respiratory distress syndrome in adults (Dellinger et al., 1998
; Payen et al., 1999
) and respiratory distress syndrome in premature infants (Subhedar et al., 1997
; Franco-Belgium Collaborative NO Trial, 1999; Kinsella et al., 1999
; Hoehn et al., 2000
). For the most part, detectable, short-term toxicity has not been observed in clinical trials of inhaled nitric oxide in PPHN. However, based on the extensive literature on the biology and chemistry of nitric oxide in various systems, it is apparent that some collateral effects of inhaled nitric oxide are inevitable. The optimum indications and dosage for this therapy have not yet been determined, and vigilance must be maintained while short- and long-term data on the toxicology of inhaled nitric oxide are accumulating.
Formation of Nitrogen Dioxide
Nitric oxide is unstable in air and undergoes spontaneous oxidation to nitrogen dioxide (NO2; National Institute for Occupational Safety and Health, 1976). Using a second-order model, the rate constant for NO2 formation has been shown to be (1.2 ± 0.1) x 1011 ppm2 s1 (Sokol et al., 1999). Thus, the time to reach 5 ppm NO2 from 20 ppm nitric oxide in 100% oxygen is 12 min, while in air it is more than 1 h. At 80 ppm nitric oxide, formation of 5 ppm NO2 is expected after 3 min of contact with air (Foubert et al., 1992
). Continuous, in-line monitoring of NO2 concentrations is therefore the standard of care during the therapeutic administration of inhaled nitric oxide, which is almost exclusively delivered with high concentrations of supplemental oxygen.
NO2 is known to be directly toxic to the respiratory tract, and the Occupational Safety and Health Administration limits human peak exposure to 5 ppm (Centers for Disease Control, 1988). The acute pulmonary toxicity of NO2, thought to be several times greater than nitric oxide, has been studied more extensively, since it is a major component of urban air pollution (Frostell and Zapol, 1995
). Increased airway reactivity has been reported in humans at exposures as low as 1.5 ppm NO2 (Frampton et al., 1991
). Other toxic effects observed following inhalation of NO2 (
5 ppm) include altered surfactant chemistry and metabolism (Muller et al., 1994
), epithelial hyperplasia of the terminal bronchioles, increased cellularity of the alveoli in rats (Evans et al., 1972
), and diffuse inflammation (Muller et al., 1994
). At higher doses, the major toxicologic effect of NO2 is pulmonary edema (Centers for Disease Control, 1988
). In rats, histopathologic changes and increased lung weight have been reported following inhalation of 2550 ppm NO2 for 30 min (Stavert and Lehnert, 1990
). Interstitial fibrosis has also been observed in five different animal species following acute exposure to NO2 (Hine et al., 1970
). High doses of inhaled nitric oxide (> 100 ppm) appear to promote rather than protect against hyperoxic lung injury. This is most likely due to concurrent NO2 formation (Garat et al., 1997
). High-dose NO2 inhalation in humans presents clinically as irritation with mild dyspnea during exposure, followed by pulmonary edema after several hours of apparent recovery (NIOSH, 1976). This can culminate in death after the most severe exposures (Greenbaum et al., 1967
).
Given our understanding that even low levels of NO2 exert toxic effects on the lung, it is of concern that up to 5 ppm NO2 have been measured in systems delivering clinically relevant doses of inhaled nitric oxide (Schedin et al., 1999). Concentrations of nitric oxide and oxygen, minute volume ventilation, and residence time in the inspiratory part of the respiratory circuit all influence the rate of NO2 formation (Lindberg and Rydgren, 1999
). Moreover, since nitric oxide is added continuously to inhaled gas, even when the ventilatory gas flow is low or zero during the respiratory cycle, it is possible that tidal variations in NO2 concentrations result in brief but marked elevations in inhaled NO2. Current electrochemical or chemiluminescence analyzers have long response times and would not be likely to detect very brief fluctuations in NO2 levels. Nevertheless, at nitric-oxide doses less than 80 ppm, there were neither significant elevations in measured NO2 levels nor clinical evidence of NO2 toxicity in the major clinical trials of inhaled nitric oxide (Davidson et al., 1998
; Finer and Barrington, 2000
; Roberts et al., 1997
; Wessel et al., 1997
). It is apparent that inhaled nitric oxide can be delivered safely in well-designed, continuous-flow neonatal ventilatory circuits that minimize residence time in the respiratory circuit (Sokol et al., 1999
). Since standardized commercial apparatus for the delivery of inhaled nitric oxide has become available, earlier concerns regarding NO2 toxicity have largely subsided (Frostell and Zapol, 1995
). In addition, the Siemens Servo 900c and the Bear BP 2001 have been shown to safely deliver inhaled nitric oxide, without excessive accumulation of NO2, when the nitric oxide is delivered to the circuit close to the endotracheal tube (Breuer et al., 1997
). Most recently, concerns about NO2 toxicity have been raised when inhaled nitric oxide is administered during high-frequency oscillatory ventilation, which has been reported to provide better distribution of inhaled nitric oxide and to improve clinical outcome when compared to conventional ventilation (Hoehn and Krause, 1998
). As a result, this modality has become a primary (and, in some centers, the only) ventilatory strategy during nitric oxide delivery in neonates. However, during oscillatory ventilation, the tidal volume does not exceed the anatomic dead space in the airways. Although the flow dynamics during such ventilation are complex and not completely understood, it is clear that local gas stasis may occur under these conditions. In this regard, using a SensorMedics oscillator ventilator and an artificial test lung, Totapally et al. (1999) have shown that distal NO2 levels are sensitive to the frequency, amplitude, and inspired oxygen concentration. Thus, the "lung" NO2 concentrations increased dramatically to > 18 ppm when oxygen was increased to 93% or when the nitric oxide concentrations approached 80 ppm. Clearly, further studies using in vivo models are needed to determine whether NO2 generation is excessive, using current high-frequency ventilation strategies, and whether more stringent dosing limitations for inhaled nitric oxide should be recommended.
Pro- and Antioxidant Effects of Inhaled Nitric Oxide in the Lung
Nitric oxide can exert both pro- and antioxidant effects in the lung. Studies using nitric oxide donors or inhibitors of nitric oxide synthase in vitro have shown that nitric oxide increases airway microvascular leakage induced by substance P, tumor necrosis factor- (TNF-
, and leukotriene B4 (Johnson and Ferro, 1996
; Kageyama et al., 1997
; Miura et al., 1996
). In vivo, high-dose inhaled nitric oxide (100 ppm) also increases vascular injury during prolonged hyperoxia (Garat et al., 1997
). Conversely, nitric oxide has been reported to reduce injury in cultured alveolar epithelial cells following exposure to superoxide anion (Gutierrez et al., 1996
). Inhaled nitric oxide also increases survival in rats during prolonged hyperoxia (Nelin et al., 1998
), and decreases markers of injury to lung endothelial and epithelial cells (McElroy et al., 1997
).
The ultimate response of the lung most likely depends on the dose of nitric oxide administered and the presence of other reactive intermediates (Liaudet et al., 2000). In general, the antioxidant properties of nitric oxide are dominant under conditions of low-level or brief nitric oxide production. This is consistent with the signaling functions of nitric oxide under normal physiologic conditions. The antioxidant effects of nitric oxide appear to be due to scavenging of highly reactive oxygen intermediates such as superoxide anion. By reacting rapidly with superoxide anion (6.7 ± 0.9 x 109 M1sec1), nitric oxide effectively competes with Fe3+, thereby decreasing the generation of damaging hydroxyl radicals by the Fe3+-mediated Haber-Weiss reactions (Freeman, 1994
). Nitric oxide also has a direct scavenging effect on hydroperoxyl radicals and directly regulates oxygen consumption and oxidative phosphorylation in mitochondria by the reversible inhibition of cytochrome oxidase (Brown, 1999
). In addition, low levels of nitric oxide may exert antioxidant effects by increasing intracellular levels of reduced glutathione (Moellering et al., 1998
).
However, when nitric oxide generation is high or sustained, due to increased nitric-oxide synthase-II activity or administration of exogenous nitric oxide, cytotoxic reactive nitrogen intermediates can be formed. For instance, in aqueous solutions, nitric oxide decays to nitrate (NO2-) and dinitrogen trioxide (N2O3; Wink and Mitchell, 1998). N2O3 is a strong nitrosating agent that can form N-nitrosamines and S-nitrosothiols. S-nitrosothiols play a role in the regulation of intracellular metabolic pathways. Thus, inappropriate nitrosylation can interfere with cell surface-receptor functions (Broillet, 1999), ion channel activity (Xu et al., 1998
), and transcription factor activity (Peng et al., 1995
). S-nitrosothiols, such as the S-nitroso-adduct of reduced glutathione, can also inhibit glutathione metabolism (Liaudet et al., 2000
), leading to increased susceptibility to oxidant-mediated damage.
Nitric oxide toxicity may also be mediated by peroxynitrite (ONOO-), a highly reactive oxidant species formed by the rapid reaction of nitric oxide with superoxide anion (Koppenol et al., 1992). Peroxynitrite can induce lipid peroxidation (Grisham et al., 1999
) and inhibit mitochondrial respiration (Liaudet et al., 2000
). By reacting with superoxide anion, nitric oxide effectively competes with superoxide dismutase (SOD), which catalyzes the dismutation of superoxide anion to hydrogen peroxide. Since SOD is present intracellularly at high micromolar concentrations, the generation of peroxynitrite tends to occur mainly under conditions of high-nitric oxide flux or excessive superoxide-anion generation, such as during inflammation. In summary, it appears that nitric oxide may exert direct, beneficial, anti-oxidant effects, or toxic pro-oxidant effects resulting from the generation of toxic intermediates, including N2O3 and peroxynitrite. The relative importance of the beneficial and toxic effects is determined by the tissue concentration of nitric oxide, the concentrations of other reactive intermediates (including superoxide anion), and the presence of cellular antioxidants (including SOD and reduced glutathione).
Effects of Inhaled Nitric Oxide on Lung Leukocyte Recruitment and Activation
Recent studies have suggested that inhaled nitric oxide also exhibits both pro- and anti-inflammatory properties. At high concentrations, nitric oxide increases TNF- and interleukin-1 (IL-1) production by macrophages (Hill et al., 1996
; Wang et al., 1997
). In addition, alveolar macrophages isolated from animals exposed to inhaled nitric oxide (100 ppm) in vivo produce increased quantities of reactive oxygen and nitrogen intermediates (Weinberger et al., 1998
). In contrast, low-to-moderate doses of inhaled nitric oxide appear to decrease the number and activity of neutrophils in the lung (Kinsella et al., 1997
). Nitric oxide (50 ppm) has also been shown to reduce neutrophil migration from the vascular into the airway compartment (Guidot et al., 1996
), and to inhibit chemotaxis (Sato et al., 2000
). The mechanisms underlying these effects are unknown, but may be related either to the pro- or antioxidant activities of nitric oxide, or to direct effects of nitric oxide on lung-leukocyte adherence, activity or longevity. Inhaled nitric oxide can directly inhibit neutrophil adherence to endothelial cells (Fukatsu et al., 1998
), and this may contribute to reduced migration. Expression of the cell adhesion molecule, CD18, is also diminished in the presence of nitric oxide (Sato et al., 1999
). Neviere et al. (2000) recently demonstrated that inhaled nitric oxide reduces leukocyte adhesion and recruitment into the mesenteric vasculature. Thus, the actions of inhaled nitric oxide on circulating neutrophils may have implications beyond the lung.
The number of neutrophils in the lung following inhalation of nitric oxide may also be reduced by stimulating apoptosis. Neutrophils exposed to nitric oxide in vitro exhibit decreased viability (Daher et al., 1997). Nitric oxide also prevents LPS-induced attenuation of neutrophil apoptosis (Blaylock et al., 1998
). Fortenberry et al. (1998) reported that increased neutrophil apoptosis in vitro occurred at doses of inhaled nitric oxide greater than 5 ppm in the presence of 80% oxygen. The finding that superoxide dismutase blocked this effect suggests that peroxynitrite is a mediator of neutrophil apoptosis. The synergistic effect of inhaled nitric oxide and hyperoxia in promoting neutrophil apoptosis has recently been confirmed in vivo in piglets (Ekezekie et al., 2000).
Recent studies suggest that diminished lung neutrophil accumulation following inhalation of nitric oxide may be accompanied by reduced secretory and respiratory burst activity. Neutrophils exposed to nitric oxide (20 ppm) in vitro, in the presence or absence of supplemental oxygen, generate significantly decreased quantities of superoxide anion (Daher et al., 1997). Superoxide anion production by lung neutrophils is also reduced in experimentally induced Pseudomonas sepsis in the presence of inhaled nitric oxide (Bloomfield et al., 1997
). Increasing evidence suggests that decreased lung neutrophil numbers and activity during nitric oxide inhalation attenuates lung injury that is associated with severe pathogenic states. Nitric oxide has been reported to play a protective role in acute lung injury and mortality induced by platelet activating factor (Yoshikawa et al., 1997
) and to prevent capillary leakage caused by Pseudomonas sepsis (Bloomfield et al., 1997
) or by intratracheal administration of IL-1 (Guidot et al., 1996
). IL-2-induced lung damage, which is characterized by neutrophil sequestration, pulmonary congestion, and microvascular protein leakage is also significantly reduced by nitric oxide (Bouchier-Hayes et al., 1997
). Taken together, these results suggest that the ability of inhaled nitric oxide to promote or inhibit lung inflammation and tissue injury is determined by the cellular environment of the lung. Nitric oxide has a direct role in down-regulating leukocyte functional activity and longevity. However, under conditions that favor the production of pro-oxidant nitric oxide metabolites, including the sustained administration of high-dose inhaled nitric oxide, toxic oxidative stimuli can play a role in the activation and recruitment of leukocytes in the lung.
Antiproliferative Effects and DNA Alterations Induced by Inhaled Nitric Oxide
Nitric oxide is known to increase the activity of intracellular soluble guanylyl cyclase (Arnold et al., 1977), most likely by interacting with the heme moiety of the enzyme (Murad, 1994
). Guanylyl cyclase catalyzes the formation of cyclic GMP (cGMP), which acts as a second messenger. Regulation of cellular events by cGMP is accomplished by its interaction with specific classes of target proteins, including cGMP-regulated protein kinases or G-kinases. These soluble or membrane-bound enzymes are critical initiators of many intracellular signaling pathways (Lincoln and Cornwell, 1993). The vasodilatory actions of nitric oxide in smooth muscle are due, in part, to increased intracellular cGMP levels. However, increased cellular cGMP production can also lead to toxicity. By increasing guanylyl-cyclase activity and cytosolic cGMP, nitric oxide has the capacity to modulate DNA synthesis and decrease cellular proliferation. The importance of cGMP in mediating the antiproliferative effects of nitric oxide in vitro has been demonstrated in several systems, including vascular smooth muscle and human airway smooth muscle cells, using the cGMP analog 8-Br-cGMP, which activates cGMP-dependent protein kinase (PKG; Hamad et al., 1999; Yu et al., 1997). It has also been shown that vascular smooth muscle cells that overexpress PKG exhibit markedly diminished DNA synthesis and proliferation in response to low concentrations of nitric oxide or 8-Br-cGMP (Chiche et al., 1998
). Activation of PKG by nitric oxide leads to decreased mitogen-activated protein (MAP)-kinase activity. This is thought to be due to cGMP-mediated phosphorylation of the upstream kinases RAF and Raf-1, which couple extracellular growth factor-receptor tyrosine kinases to the MAP kinase cascade. Since MAP kinases are known to regulate DNA synthesis and cell proliferation, they represent a cGMP-dependent signal transduction pathway mediating the antiproliferative effects of nitric oxide (Yu et al., 1997
).
Nitric oxide has also been shown to inhibit the proliferation of cultured cells by mechanisms that are independent of cGMP (Heller et al., 1999; Kosonen et al., 1998
). Nitric oxide can alter the expression of cyclins (Guo et al., 1998
) and activate the cyclin inhibitor p21 (Ishida et al., 1997
). In addition, nitric oxide inhibits ribonucleotide reductase, which leads to cell-cycle arrest. In the lung, excessive proliferation of smooth muscle cells in response to hypoxia or injury can result in vascular obstruction and pulmonary hypertension. Inhaled nitric oxide at therapeutic doses has been shown to prevent neomuscularization and to attenuate vascular remodeling in the pulmonary arteries of hypertensive newborn rat pups following hypoxia (Roberts et al., 1995
) or monocrotaline administration (Roberts et al., 2000
). The mechanisms underlying the antiproliferative effects of inhaled nitric oxide in the lung remain unknown, although they appear to be independent of primary effects on pulmonary vascular tone, inflammation, or thrombosis (Roberts et al., 2000
). Further studies are required to determine whether the antiproliferative effects of inhaled nitric oxide interfere with desirable adaptive functions of the lung, or whether nitric oxide may, in fact, serve an additional therapeutic role in preventing some of the anatomic changes that are associated with severe pulmonary hypertension.
Nitric oxide is also known to induce structural alterations in DNA that are potentially genotoxic. Chromosomal aberrations in rat-lung cells in vivo and in TK6 human lymphoblastoid cells in vitro have been reported following nitric oxide treatment (Arroyo et al., 1992; Isomura et al., 1984
; Nguyen et al., 1992
; Wink et al., 1991
). Peroxynitrite can also initiate DNA base modifications. Incubation of purine nucleotides or of isolated DNA at physiologic pH with peroxynitrite in vitro leads to the generation of 8-nitroguanine. This compound is rapidly depurinated resulting in G:C
T:A transversions (Yermilov et al., 1995
). Other oxidized or deaminated base products that have been detected in DNA after exposure to peroxynitrite include 8-oxoguanine, hypoxanthine, and xanthine (Spencer et al., 1996
). These can cause specific transitions in the base sequence during DNA replication (Szabo and Ohshima, 1997
).
Nitric oxide and peroxynitrite can also induce DNA strand breaks (Salgo et al., 1995b), which can be prevented by superoxide dismutase or nitric oxide-trapping agents (Epe et al., 1996
). DNA single-strand breakage has been reported in intact cells exposed to peroxynitrite, indicating that peroxynitrite has the ability to enter cells and to induce nuclear changes (Salgo et al., 1995a
). Possible responses include initiation of DNA repair and/or cell death by necrosis or apoptosis. These findings elicit concern regarding reactive intermediates that may be formed in the extracellular milieu of the lungs of patients during the administration of inhaled nitric oxide.
Peroxynitrite-induced DNA strand breakage can also damage cells by activating the enzyme poly(ADP)ribosyltransferase (PARS; Szabo and Ohshima, 1997). PARS catalyzes the cleavage of NAD+ to ADP-ribose, the extension of ADP-ribose to poly(ADP-ribose) and nicotinamide, and the covalent attachment of poly(ADP-ribose) to nuclear proteins, including DNA polymerases and ligases. Generally, ADP-ribosylation results in a decrease in the activity of these enzymes (Lautier et al., 1993). It has been suggested that PARS functions to regulate gene expression and cellular differentiation, transformation, and division, and/or to slow cellular metabolism (Szabo and Ohshima, 1997
). Abnormal or sustained activation of PARS, as a result of exposure to oxidants like peroxynitrite, can rapidly deplete intracellular concentrations of its substrate, NAD+, resulting in cellular dysfunction and necrotic cell death. This sequence of events has been described in smooth muscle cells, macrophages, epithelial cells, and endothelial cells exposed to peroxynitrite, and can be reversed by inhibitors of PARS (Kennedy et al., 1997
; Szabo, 1996
; Szabo et al., 1997a
,b
). Peroxynitrite-induced PARS activation may also play a role in pulmonary epithelial hyperpermeability (Kennedy et al., 1997
; Szabo et al., 1997b
), and vascular smooth muscle hypocontractility and endothelial dysfunction in endotoxic shock (Szabo, 1996
; Szabo et al., 1997a
).
Effects of Nitric Oxide on Pulmonary Surfactant
Pulmonary surfactant, secreted by Type II pneumocytes, acts at alveolar air-fluid interfaces to lower surface tension, allowing for homogeneous expansion of the lung. Deficiency or dysfunction of surfactant plays a critical role in the pathogenesis of respiratory diseases, particularly in the newborn. Although surfactant is composed primarily of the phospholipids phosphatidylcholine and phosphatidylglycerol, surfactant proteins-A, B, and C (SP-A, SP-B, and SP-C) play important roles in the organization and function of surfactant complexes and regulate their recycling and secretion. Under physiologic conditions, nitric oxide can protect against oxidant injury and, in theory, protect the surfactant system against inactivation (Hallman et al., 1996a). In a small cohort of 12 infants with PPHN receiving
20 ppm of inhaled nitric oxide, the surface activity of airway specimens obtained by endotracheal suctioning was not detectably decreased relative to control infants with PPHN (Hallman et al., 1998
).
However, a number of studies have suggested that reactive nitrogen intermediates can interfere with surfactant activity by modifying surfactant proteins (Cifuentes et al., 1995; Haddad et al., 1993
; Robbins et al., 1995
). Peroxynitrite decreases the ability of SP-A to aggregate lipids, an effect associated with the appearance of nitrotyrosine residues (Haddad et al., 1994
). The surface activities of SP-B and SP-C are also diminished after exposure to peroxynitrite. In addition, brief stasis of nitric oxide and oxygen in the distal airways can lead to the generation of NO2, which has been shown to damage lung surfactant activity (Muller et al., 1994
). In this regard, surfactants retrieved from lambs or rats exposed to high doses of inhaled nitric oxide (80 or 100 ppm, respectively) exhibit reduced capacity to lower surface tension (Hallman et al., 1996b
; Matalon et al., 1996
).
The generation of methemoglobin (metHb) by inhaled nitric oxide in the alveoli may, in large part, determine the effects of nitric oxide inhalation on surfactant function in vivo (Hallman et al., 1996a). MetHb has been shown to directly inhibit surfactant activity, and the inhibitory effects of nitric oxide in vitro can be reproduced by metHb (Hallman et al., 1996a
,b
). Thus, under conditions characterized by high permeability lung edema (causing hemoglobin to be present in the airspaces), inhaled nitric oxide may inactivate surfactant by converting hemoglobin to metHb in the alveoli. Lung edema is most likely to be present under severe conditions, including NO2 toxicity as a result of excessively high doses of inhaled nitric oxide. However, even at therapeutic doses of inhaled nitric oxide, these data suggest that lung edema as a result of inflammation, infection, or ventilator-induced lung trauma places patients at risk for metHb-induced surfactant dysfunction. This may become clinically relevant under conditions of low surfactant content and high alveolar hemoglobin, which are often observed in severely affected infants with PPHN. Consistent with this possibility, estimates of alveolar hemoglobin concentrations in epithelial lining fluid in sick newborns suggest that inhibitory concentrations of metHb may be formed during inhalation of nitric oxide at therapeutic doses (Hallman et al., 1991
). Clearly, further studies are needed to better define the actions of inhaled nitric oxide on surfactant in PPHN. Adverse effects on surfactant function must be considered in the assessment of the toxicity and benefits of inhaled nitric oxide.
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Extrapulmonary Effects of Inhaled Nitric Oxide |
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Effects on Platelet Aggregation and Bleeding Time
In addition to regulating vascular tone, nitric oxide inhibits platelet adherence to endothelial cells as well as aggregation (Schini-Kerth, 1999). Bleeding times have been shown to be markedly decreased in mice lacking the gene for NOSIII, relative to wild-type control mice. This difference persisted even after controlling for endothelial nitric-oxide production, suggesting that platelet-derived nitric oxide plays a role in regulating hemostasis (Freedman et al., 1999
). Inhibition of endogenous nitric oxide production in humans by infusion of NG-monomethyl-L-arginine (L-NMMA) significantly shortened bleeding time (Albert et al., 1997
; Simon et al., 1995
). Furthermore, exposure of platelets to nitric oxide in vitro caused decreased ADP- and collagen-induced aggregation (Gries et al., 1998
). Therefore, a question arises as to whether exposure of platelets to inhaled nitric oxide by passage through the lungs alters their ability to regulate hemostasis. In rats, inhaled nitric oxide (15 ppm) increased bleeding time, reduced platelet aggregation, and increased platelet cGMP (Kermarrec et al., 1998
). Moreover, inhaled nitric oxide attenuated the procoagulant effect of acute endotoxin-induced pulmonary inflammation (possibly a beneficial effect) in this model (Kermarrec et al., 1998
). Several recent studies have examined the effects of inhaled nitric oxide on platelet function in healthy humans with conflicting results. In one study, inhalation of 30 ppm nitric oxide for 30 min had no effect on bleeding time or platelet P-selectin expression (Albert et al., 1999
). In contrast, Gries et al. (2000) reported that inhaled nitric oxide at 540 ppm for 2040 min inhibited both ADP- and collagen-induced human platelet aggregation and prolonged bleeding time. P-selectin expression and fibrinogen binding were also reduced. Reports on patients treated with inhaled nitric oxide support the idea that there may be systemic alterations in platelet function. For example, infants treated with inhaled nitric oxide (40 ppm) had bleeding times that were approximately twice as long as those observed 24 h after discontinuation of therapy (George et al., 1998
), although in vitro platelet aggregation was not affected. Clinical sequelae of increased bleeding tendency have not been reported in trials of inhaled nitric oxide in term or near-term infants with hypoxic respiratory failure/PPHN. However, in one study in premature infants with respiratory-distress syndrome, the incidence of intracranial hemorrhages (7 of 11 patients, mean gestational age 29.8 weeks) was higher in the nitric oxide-treated infants than in historical controls (Meurs et al., 1997
). Although this finding has not been reproduced in premature infants, it does constitute a biologically plausible concern regarding the toxicology of inhaled nitric oxide in a specific patient population. Similarly, in adults with ARDS, inhaled nitric oxide caused prolonged bleeding time, inhibition of platelet aggregation, and reduced P-selectin expression and fibrinogen binding (Gries et al., 1998
).
Methemoglobinemia
Inhaled nitric oxide can combine with hemoglobin to form nitrosylhemoglobin, which is rapidly oxidized to methemoglobin (metHb; United States Environmental Protection Agency, National Advisory Committee/AEGL, 1998). The affinity of nitric oxide for hemoglobin is about 1500 times greater than that of carbon monoxide (Gibson and Roughton, 1957), and the binding and formation of metHb is concentration- and time-dependent (Ripple et al., 1989
). Tissue hypoxia results when there is excessive metHb. Since the oxygen dissociation curve of metHb is shifted markedly to the left, it does not readily release oxygen. However, cyanosis does not appear until metHb levels are 1520%, and clinical symptoms of hypoxia (such as fatigue and dyspnea) do not generally become significant at levels below about 30% of hemoglobin. In both adults and children at doses below 100 ppm nitric oxide, MetHb formation is usually insignificant (Finer and Barrington, 2000
; Frostell et al., 1993
; Roberts et al., 1997
; Winberg et al., 1994
). Nevertheless, several occurrences of toxic metHb levels and clinical toxicity have been observed. MetHb levels rose to 9.4% after treatment of a lung transplant patient with 80 ppm nitric oxide for 8 h (Adatia et al., 1994
). In adults with pulmonary hypertension, peak metHb levels of 9.6% and 14% were reported after 108 and 18 h of 80 ppm nitric oxide inhalation (Wessel et al., 1994
). Inhalation of < 45 ppm nitric oxide resulted in a toxic level of 67% metHb in a patient with hydrochlorthiazide-induced pulmonary edema, which subsequently responded to treatment with methylene blue (Hovenga et al., 1996
).
The enzyme metHb reductase converts metHb back to Hb. Neonates exhibit reduced activity of this enzyme. Thus, they may be at greater risk than adults for developing significant methemoglobinemia after inhalation of nitric oxide. A level of 5% metHb has been reported in an infant receiving 20 ppm nitric oxide for 25 h (Frostell et al., 1993a). Wessel et al. (1994) reported metHb levels above 5% in 4 of 123 patients, mainly children, who had inhaled up to 100-ppm nitric oxide. Rapid and unexpected rises in metHb to toxic levels can occur in infants, leading to cyanosis and tissue hypoxia. In one case, metHb increased to 40% following treatment with 80 ppm nitric oxide for 26 h. Taken together with clinical efficacy data indicating that there is little or no marginal benefit of doses > 40 ppm, it seems prudent to recommend utilizing the lowest effective dose under 40 ppm.
Methemoglobinemia occurs when the kinetics of the reaction catalyzed by metHb reductase are such that its rate of formation exceeds its rate of elimination. In rats (Maeda et al., 1987) and rabbits (Sharrock et al., 1984
), nitric oxide binding to hemoglobin is rapidly reversible, with a half-life of 1520 min after the animals are returned to clean air. Young et al. (1994) reported that, in adults, maximal metHb levels were reached 35 h after nitric oxide inhalation began. The increase in metHb levels during nitric oxide inhalation, and its clearance following cessation of therapy displayed first-order kinetics. However, the favorable kinetics of elimination may differ in intensive-care infants and adults due to alterations in metHb reductase activity and/or to increased endogenous production of nitric oxide. In this regard, circulating metHb levels have been reported to be increased during septic shock in children, even in the absence of inhaled nitric oxide (Krafte-Jacobs et al., 1997
). Therefore, infusion of 12 mg/kg of methylene blue is advisable when metHb levels are toxic and there is a risk of tissue hypoxia. In a patient with 67% metHb, the level was reduced to 8% and cyanosis resolved within 1 h of treatment with methylene blue and the discontinuation of nitric oxide (Hovenga et al., 1996
). An infant with 40% metHb responded favorably within 20 min (Nakajima et al., 1997
). At standard doses, methemoglobinemia is a small and acceptable risk inherent in the use of inhaled nitric oxide. The complications and sequelae of methemoglobinemia can be averted effectively if the condition is diagnosed in a timely manner, using routine protocols for periodic surveillance of metHb levels as well as assessment of the appearance of cyanosis.
In summary, inhaled nitric oxide is a useful therapy for pulmonary hypertension in infants with hypoxic respiratory failure and possibly in some other groups of patients. However, pulmonary vasodilation is just one of many physiologic effects of inhaled nitric oxide that are dictated by its chemical and biological properties. Nitric oxide has oxidative effects in the lung, and can decrease neutrophil accumulation and surfactant function. Peroxynitrite and nitrogen dioxide generated from nitric oxide may also induce genotoxic alterations and/or tissue injury. Even outside of the lung, inhaled nitric oxide may alter vascular tone and platelet function, and methemoglobinemia can adversely affect tissue oxygenation. An understanding of the toxicology of inhaled nitric oxide is essential for defining the roles and utility of this therapy.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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