Exogenous inhaled nitric oxide as a selective pulmonary vasodilator

Exogenous Inhaled Nitric Oxide as a Selective Pulmonary Vasodilator Geoffrey N.

Morris, George F. Rich, and Roger A. Johns

ENDOGENOUS NITRIC OXIDE

N 1980, AN UNIDENTIFIED vasodilating factor released from endothelium was reported as the mediator of acetylcholine-induced vasodilation and named endothelium-derived relaxant factor.~ Endothelium derived relaxant factor diffuses from the endothelium to the vascular smooth muscle where it activates soluble gaunylyl cyclase producing an increase in cyclic 3',5' guanosine monophosphate (cGMP) resulting in vascular relaxation. Endothelium derived relaxant factor was subsequently found to be the common mediator of a wide array of endogenous and exogenous vasodilators.2'3 In 1986, endothelium derived relaxant factor was recognized as nitric oxide (NO). 4'5 Nitric oxide is now known to be a novel signaling molecule with wide ranging physiological and pathophysiological actions in the cardiovascular, immune, and nervous systems. In blood vessels, NO is produced by the endothelium where it is a primary determinant of resting vascular tone through basal release, and causes vasodilation when synthesized in response to a wide range of vasodilator agents.2"6 It also inhibits platelet aggregation and adhesion. It plays a major role in disease states such as atherosclerosis and hypertension, cerebral and coronary vasospasm, and ischemia-reperfusion injury. In the immune system, it is an effector mechanism for macrophage-induced cytotoxicity7 and its overproduction is an important mediator of the septic shock syndrome. In neuronal tissue, NO seems to subserve multiple functions,s It is present in several specific neuronal pathways and is known to mediate the N-methyl D-aspartate (NMDA) and acetylcholine receptor stimulated increases in neuronal cGMP. Nitric oxide has been implicated in long-term potentiation in the CA I region of the hippocampus8-1~thus mediating an important step in learning and memory. It is the potential agent responsible for N-methyl D-aspartate-mediated cytotoxicityI~ and is the mediator of nonadrenergic, noncholinergic neurotransmission.~2~4 Recent studies have suggested

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Seminars in Anesthesia, Vo115, No 1 (March), 1996: pp 47-60

a role for NO in mediating central nociceptive pathways ~5''6 and a possible involvement in mechanisms of anesthesia.IV't8 The L-arginine to NO pathway is being explored as a physiological mediator in other cells and tissue types where it is present, including bronchial epithelium, renal tubular and juxtaglomerular cells, gastrointestinal epithelium, hepatocytes and Kuppfer cells, the pituitary, the thyroid, the ovary, the uterus, and the adrenal medulla) 9"2~ Three major NO synthase (NOS) isoforms have been described. ~9"2t They have been sequenced and cloned, and are biochemically similar while retaining important differences. Two require calcium and calmodulin binding for activation and are constitutively expressed (ie, normally present) in neurons and in endothelium. These constitutive enzymes are activated by an increase in cytosolic free Ca 2+ levels and subsequent binding of Ca 2§ to calmodulin. In endothelium, the enzyme (eNOS) is bound to the membrane by myristoylation providing a lipophilic anchor in the bilayer. 22 In neural tissue the enzyme (nNOS) is in a soluble form in the cytosol. The third isoform is only expressed after induction by cytokines or microbial products such as endotoxin and participates in host defense, mediating many of the cytotoxic actions of macrophages. This inducible isoform (iNOS) has calmodulin tightly bound as a subunit and produces NO continuously and in large amounts without a calcium requirement. Whereas iNOS is present in the macrophage under basal conditions, it is not normally found in endothelial cells or vascular smooth muscle. Many cell types seem capable of expressing iNOS after exposure to the appropriate cytokines. Unlike the constitutive form, the induced form is present in vasFrom the Department of Anesthesiology, Universityof Virginia Health Sciences Center, Charlottesville, VA. Address reprint requests to George Rich, MD, Department ofAnesthesiology, Box 238, Universityof Virginia Health Sciences Center, Charlottesville, VA 29908. Copyright 9 1996 by W.B. Saunders Company 0277-0326/96/1501-000755.00/0

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cular tissues only after induction by cytokines23-25 (eg, endotoxin-induced sepsis). Both inducible and constitutive NO synthases contain a heme moiety and are members of the cytochrome P450 family. These NOS synthases oxidize L-arginine in a stepwise manner to form NO and citrulline as primary products. 26-2s One molecule of 02 is incorporated into NO and one into L-citrulline. Limiting the availability of substrate molecular 02 is the likely mechanism by which hypoxia inhibits NO synthase activation and NO-dependent vasodilation.29 All forms of the enzyme can be specifically and competitively inhibited by analogs of L-arginine. These inhibitors are proving beneficial in elucidating the physiological and pathophysiological roles of the NO pathway. Nitric oxide itself can be inactivated after its production. It binds avidly to hemoglobin and other hemoproteins and also interacts with superoxide to form peroxynitrite.3~The regulation of cellular superoxide dismutase activity and subsequent superoxide levels has been suggested as a potential physiological regulatory mechanism.31 Of clinical significance, the rapid binding and inactivation of NO by hemoglobin in the pulmonary vessels prevents it from reaching the systemic circulation and is thereby responsible for its selective pulmonary vasodilator effect when used as an inhalational agent. 32 Interference with NO activation of guanylyl cyclase is another means of blocking NO action. Methylene blue has been commonly used for this purpose, but its avid role as an electron acceptor makes it very nonspecific and potentially toxic. 33 Indeed, it has been recently reported that methylene blue exerts much of its action by inhibiting NO synthase in addition to guanylyl cyclase. 34 The primary biological function of NO is the activation of soluble guanylyl cyclase to increase the cGMP content of several tissues. Nitric oxide binds with the heine moiety of guanylyl cyclase causing a conformational change and leading to increased catalytic activity.35 Cyclic GMP then serves as a "second messenger" carrying out many of the actions of NO. There are two major forms of guanylyl cyclase: (1) a particulate form, which is membrane bound and activated only by atrial natriuretic peptides, and whose function is still being defined; and (2) a soluble form, which is present in the cytosol and is activated by NO. Nitric oxide is the most potent and effective ac-

MORRIS, RICH, AND JOHNS

tivator ofguanylyl cyclase but both carbon monoxide and the hydroxyl radical have been proposed as additional physiological activators of soluble guanylyl cyclase. However, they seem to be less important than NO and their role remains undefined.35 There are three known classes of receptor proteins through which the diverse physiological functions of cGMP are mediated. These include cGMP-dependent protein kinases, cyclic nucleotide-binding phosphodiesterases, and ion channel proteins which directly bind cGMP and are regulated by such action. The ability of cGMP to directly gate ion channels is particularly important to the role of the NO-guanylyl cyclase pathway in the nervous system. Nitric oxide, a reactive free radical has also been reported to have other, non-cGMP mediated actions. It has been shown to activate adenosine diphosphate ribosyltransferase36 and to inhibit a number of other enzymes.37"4~These protein/enzyme interactions of NO have been suggested to be a result of its ability to form complexes with both heme and non-heme iron proreins. 41 The feedback inhibition of NO synthase has been reported recently and is a potentially important mechanism for regulation of this signaling pathway.42'43 The binding of NO to the heme of NO synthase would be a likely mechanism of this observed inhibition. Nitric oxide also interacts avidly with reactive oxygen species resulting in its rapid inactivation. It combines with superoxide radical to form peroxynitrite and with oxygen to form nitrite, nitrate, and nitric acid. 3~ This explains the ability of high oxygen concentrations to inhibit NO and of superoxide dismutase (SOD) to markedly prolong its biological half-life.29'44Nitric oxide has recently been shown to play an important role in gene regulation. It is capable of activating several DNA and RNA binding factors and has also been shown capable of inducing DNA mutation through deamination, oxidation, and crosslinking.23'45'46 INHALED NITRIC OXIDE/EXOGENOUS NITRIC OXIDE

Inhaled NO theoretically produces pulmonary vasodilation by the same mechanism as endogenous NO. Nitric oxide does not dilate the systemic circulation because NO binds rapidly to hemoglobin with a high affinity and is inactivated

NITRIC OXIDE

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as it is exposed to the pulmonary circulation. Because it is delivered via the respiratory system, inhaled NO is distributed only to those areas of lung that are ventilated and dilates only those vessels directly adjacent to the ventilated alveoli. Areas of the lung which are collapsed do not receive NO and therefore do not vasodilate. Ventilation perfusion matching is thus preserved or improved by inhaled NO therapy. Inhaled NO may also improve oxygenation by increasing pulmonary blood flow. Anesthesiologists and critical care physicians may be interested in administering inhaled NO in several situations: (l) adults with primary pulmonary hypertension; (2) infants with persistent pulmonary hypertension of the newborn; (3) newborns with respiratory distress syndrome; (4) adults with acute respiratory distress syndrome (ARDS); and (5) patients with pulmonary hypertension associated with heart and lung surgery. Inhaled NO may also have a diagnostic role in the preoperative assessment of patients for heart and/or lung transplants.

via a face mask and later via a transtracheal catheter. Administration continued for 68 days until a heart lung transplant was performed. The explanted lungs showed no evidence of significant NO toxicity. Kinsella5~described a case in which a 5-month old child with severe PHTN in whom standard pharmacological agents (isoproterenol, prostaglandin El, tolazoline, and nifedipine) caused systemic hypotension. In contrast, inhaled NO (20 ppm) improved oxygenation without a change in systemic arterial pressure. Nitric oxide was continued at 20 ppm for 20 hours followed by 6 ppm for a further 45 hours resulting in sustained pulmonary vasodilation. It is postulated that chronic PHTN involves pulmonary vascular endothelial dysfunction with reduced endogenous NO synthesis. In these patients inhaled NO may function as replacement therapy. Studies have indicated a role for inhaled NO in the treatment of PHTN in cases that are potentially reversible. It is unlikely to be helpful when there is no underlying reversible pulmonary pathology.

Treatment for Pulmonary Hypertension

Treatment for Persistent Pulmonary Hypertension of the Newborn

In 1991 Pepke-Zaba47 showed that when inhaled NO (40 ppm) was administered to patients with severe pulmonary hypertension (PHTN), the pulmonary vascular resistance (PVR) decreased by 5% to 68% but was not associated with a decrease in systemic vascular resistance. The onset and offset times of the vasodilatory effects of inhaled NO were less than 3 minutes. Benzing48 studied the effects of 40 ppm inhaled NO for 30 minutes on 18 patients with PHTN after acute lung injury and found it resulted in an abrupt decrease in mean pulmonary artery pressure (PAP) while pulmonary artery wedge pressure (PAWP) and cardiac output remained constant. Rich49 administered 20 ppm inhaled NO for 6 minutes to 20 patients while undergoing one lung ventilation during cardiac surgery. Inhaled NO decreased PAP and PVR in the patients with moderate PHTN but had no significant effect on these variables in the patients without PHTN. Sustained inhalational NO therapy is also being investigated. Snell 5~ reported the case of a women who after 10 years of increasing disability had end-stage primary pulmonary hypertension and was awaiting transplantation. Inhaled NO at 40 ppm was successfully administered initially

Persistent pulmonary hypertension of the newborn (PPHN) is a life-threatening condition characterized by an elevated PAP, and augmented right to left extra pulmonary shunting through the patent ductus arteriosus and foramen ovale. This results in systemic arterial hypoxemia and acidosis. Persistent pulmonary hypertension of the newborn results from a variety of insults such as sepsis and meconium inhalation or it may be idiopathic. The histopathological findings include smooth muscle hyperplasia and hypertrophy of the pulmonary arteries. Persistent pulmonary hypertension of the newborn has a good prognosis if the infant is supported through the acute phase of the illness. Current treatment of this condition comprises of hyperventilation, alkalosis, hyperoxia, inotropic support, and intravenous vasodilators, which may add to the morbidity and mortality. Extracorporeal membrane oxygenation (ECMO) is often required but is invasive, expensive, and may predispose to intraventricular hemorrhages. Roberts 52 studied 6 infants with PPHN. Inhaled NO at 80 ppm was administered for 30 minutes with a rapid improvement in the pre-

50 ductal oxygenation in all patients (from 88% to 97% with FIO2 = 0.9). Postductal oxygenation also improved in 4 patients, without a change in PaCO2 or systemic blood pressure. Hypoxemia returned within 5 minutes on discontinuation of NO in 5 of the infants who subsequently required ECMO. In one infant, improved oxygenation was maintained after withdrawing inhaled NO and ECMO was not required. K_insella53 administered inhaled NO for 24 hours in 9 newborns with severe PPHN who were candidates for ECMO. Arterial oxygenation improved progressively with inhaled NO (20 ppm) over the first 4 hours. Despite decreasing the inhaled NO concentration to 6 ppm, the improvement in oxygenation was sustained in 8 patients. Finer54 studied 23 critically ill, hypoxic near-term infants referred for ECMO. Thirteen of these had echocardiographic evidence of PPHN. Eleven of the infants with PPHN responded to inhaled NO (5 to 80 ppm for 15 minutes) with an increase in oxygen index whereas only 3 of the 10 without PPHN responded. Infants who had a successful response continued to receive the lowest dose of NO associated with improvement (maximum dose = 20 ppm). An attempt was made to wean the infant from NO every 24 hours by decreasing by 5 ppm every 15 minutes. The mean duration of therapy was 40 hours ( + / - 3 6 hours). Overall 11 infants required ECMO, 7 of whom had initially responded to inhaled NO. Varnhold 55 has shown that about 50% of neonates with PPHN who fail to respond to conventional ventilatory support can be treated successfully with high frequency oscillatory ventilation (HFOV), thus avoiding ECMO. Kinsella noted that in some cases NO during HFOV improved oxygenation greater than HFOV alone, or NO and intermittent positive pressure ventilation. In patients with severe PPHN, optimal management may include HFOV to recruit lung volume, thus promoting effective delivery of inhaled NO. Endogenous NO modulates vascular tone in the fetal and postnatal lung, and contributes to the normal decline in PVR at birth. It has been hypothesized that decreased release of endogenous NO and other abnormalities in endothelial function may play a critical role in the development of PPHN. Inhaled NO may provide an effective adjuvant therapy for treating severe

MORRIS, RICH, AND JOHNS PPHN, but responsiveness may depend on specific diseases associated with PPHN and optimized ventilator strategies. In some cases inhalational NO therapy may obviate the need for ECMO with its accompanying side-effects and may be sufficient to resolve PPHN. There are still many unresolved issues concerning the use of inhaled NO to treat PPHN. These questions include the optimal concentration of inhaled NO and the duration of administration which may be related to patient variables including severity of disease. It is uncertain what are the optimal adjunct supportive therapies in addition to NO. Perhaps the most serious question is the potential toxicity of prolonged inhaled NO on immature lungs (see toxicity).

Treatment for Newborns With Severe Hypoxemia Karamanoukian 56 studied 9 infants with hypoplastic lungs administered inhaled NO (80 ppm) for 20 minutes. All patients required ECMO support, which lasted from 5 to 17 days (mean 9). Nitric oxide inhalation before ECMO did not change postductal PaO2, arterial oxygen saturation, or oxygenation index. After decannulation from ECMO, NO inhalation increased postductal PaOz and arterial oxygen saturation. This could be caused by maturational changes occurring in the endothelium while on ECMO. In neonates with respiratory distress syndrome, hypoxemia may result from fight to left extra pulmonary shunt (EPS) or intrapulmonary shunt. Extra pulmonary shunt through the ductus arteriosus and/or the foramen ovale is the result of increased PVR. Inhaled NO reduces intrapulmonary shunt by improving ventilation-perfusion matching, and reduces EPS by decreasing PVR. Roze 57 administered 20 to 30 ppm NO to 17 neonates with severe hypoxemia. The infants were divided into EPS (n = 9) and non-EPS (n = 8) groups. They observed improved oxygenation in both groups but the NO was more effective in the infants with EPS. Recently Kinsella58 reported on the use of inhaled NO during transport of six infants. Each infant had severe hypoxemia with marked lability despite receiving aggressive treatment with mechanical ventilation and 100% inspired O2. Twenty ppm inhaled NO acutely improved oxygenation, decreased the cardiopulmonary la-

NITRIC OXIDE bility associated with severe hypoxemic respiratory failure, and sustained improvement during transport.

Bronchodilation Nitric oxide should produce bronchial smoothmuscle relaxation by the same mechanism that produces pulmonary vascular smooth muscle vasodilation. H6gman et a159 studied the effects of 80 ppm of inhaled NO for 10 minutes on 6 healthy volunteers, 6 subjects with hyperreactive airways constricted with inhaled methacholine, 13 patients with asthma, and 4 patients with chronic obstructive lung disease. There was no effect in the normal and chronic obstructive lung disease groups. Inhaled NO attenuated bronchconstriction in the methacholine group and had a weak bronchodilatory effect in the asthmatic group. The response in the asthmatic group was much less and of shorter duration than observed in asthmatic patients after the inhalation of/3sympathomimetic drugs. The same conclusions were reached by Sanna6~ after observing the effects of 80 ppm NO for 10 minutes on 7 men after methacholine-induced bronchospasm.

Adult Respiratory Distress Syndrome and Chronic Obstructive Lung Disease Acute pulmonary hypertension and fight to left shunting of venous blood consistently occurs in severe ARDS. Supportive treatment has included intermittent positive pressure ventilation with elevated inspired oxygen concentrations. High levels of inspired oxygen may damage the diseased lung and conventional ventilation has been associated with barotrauma and volutrauma. More recently, permissive hypercapneic techniques, placing patients in the prone position, dehydration, and HFOV have been used. Severe hypoxemia may also be treated by ECMO or intravenous oxygenation systems. Intravenous vasodilators inhibit hypoxic pulmonary vasoconstriction and decrease PVR in all areas of the lung. The net effect is a decrease in oxygenation secondary to increased venous admixture and/ or pulmonary shunt. In marked contrast inhaled NO, in addition to reducing PVR, should cause steal of blood flow toward ventilated areas increasing arterial oxygenation by reducing intrapulmonary shunting and improving the matching of ventilation to perfusion.

51 Rossaint6~studied 9 patients with severe ARDS administered 18 ppm and 36 ppm inhaled NO for 40 minutes. The PAP decreased and the ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen (PaO2/FIO2) increased. Inert gas analysis revealed that the beneficial effects of inhaled NO were caused by improved matching of ventilation to perfusion. In the same study, seven patients continued with 5 to 20 ppm of inhaled NO for 3 to 53 days with continued benefit. During brief daily discontinuation of inhaled NO, the PAP consistently increased and the PaO2/FIO2 decreased. Gerlach62 suggested the mean effective dose (EDs0) for the inhaled NO concentration which improved oxygenation was slightly lower than the concentration which reduced PVR. They postulated that higher concentrations of NO worsened oxygenation by diffusing into nonventilated areas. However, more recent work by Lowson63 investigating the response to varying concentrations of inhaled NO on patients with ARDS found that the maximum decrease in PVR occurred at lower concentrations (0. l ppm) than the maximum improvement in oxygenation (l to l 0 ppm). Puybasset64 also determined that in ARDS patients that the therapeutic concentration of inhaled NO was the same for oxygenation and PVR (0. l to 2 ppm). Bigatello 65 and others have shown that the decrease in PVR in response to inhaled NO, is proportional to the baseline PVR. A retrospective study of 87 patients with severe ARDS 66 showed that beneficial effects of NO inhalation can be observed in most patients but that NO may fail to improve oxygenation or to reduce PVR without obvious explanation. It is generally observed that the response to NO varies widely among ARDS patients and in the same patient at different times. It is possible that preexisting pulmonary disease and concomitant administration of other drugs may contribute to the variability of NO responses. Fierobe67 evaluated the effects of 5 ppm inhaled NO on right ventricular (RV) function in 13 patients with ARDS. Although the stroke volume did not change, the resulting decrease in PVR was associated with an increase in RV ejection fraction, a trend toward decreased RV endsystolic and end-diastolic volumes and a decrease in right atrial pressures. They concluded that in patients with ARDS who did not have RV failure,

52 inhaled NO reduces the loading conditions of the RV without changing RV output. The effects of combining inhaled NO with other therapies have also been studied, Puybasset68 administered 2 ppm inhaled NO to 11 patients with ARDS mechanically ventilated during both normocapnic and hypercapnic conditions and a significant increase in oxygenation was observed in both states. Inhaled NO completely reversed the increase in PVR induced by permissive hypercapnia. Wysocki69 studied the effect of 5 to 10 ppm inhaled NO and intravenous almitrine bismesylate on gas exchange in 17 patients with ARDS. Intravenous almitrine bismesylate may improve arterial oxygenation in patients with ARDS by enhancing hypoxic pulmonary vasoconstriction. Inhaled NO and almitrine bisrnesylate had additive effects on gas exchange while decreasing mean PAP. In animal studies, 7~ inhaled NO has been shown to have synergistic effects when administered in combination with exogenous surfactant or with the phosphodiesterase inhibitor, Zaprinast (Mand B-22948; RhonePoulenc Rorer, Dagenham, Essex, United Kingdom). Pulmonary hypertension frequently accompanies chronic obstructive lung disease. Adnot72 studied 13 patients with chronic obstructive lung disease and PHTN who breathed NO mixtures of 5, 10, 20, and 40 ppm during four sequential 10 minute periods. These were compared with the responses to three sequential 10 minute periods of intravenous infusions of acetylcholine at 5, 10, and 15 mg/min. The decreases in PVR were similar in response to 40 ppm NO or 15rag/ kg acetylcholine. Acetylcholine produced both pulmonary and systemic vasodilation and impaired arterial oxygenation (PaO2 reduced from 57 + / - 3 to 48 § 2 mm Hg) whereas inhaled NO induced selective pulmonary vasodilation and improved arterial oxygenation (PaO2 increased from 57 + / - 3 to 60 + / - 3). Low doses of NO (<2 ppm) can improve gas exchange. The risks of toxicity related to inhalational NO (see Toxicity) are likely to be reduced if the lowest effective concentration is used. The ability of NO to divert pulmonary blood flow to ventilated lung regions and thereby increase PaO2 is dependent on an element of pulmonary vasoconstriction being present. Those with the greatest degree of PHTN seem to respond the

MORRIS, RICH, AND JOHNS most to NO inhalation. So far decreased ventilatory time or mortality has not been shown, however larger randomized prospective trials are ongoing.

Adult Cardiac Surgery A variety of cardiac surgical procedures may lead to elevated PVR postoperatively. It has been suggested that during cardiopulmonary bypass (CPB) the endothelium is damaged and postoperative endothelial dysfunction may contribute to the elevated PVR. This may in turn result in RV dysfunction and RV failure. Intravenous vasodilators reduce PAP but the accompanying reduction in mean arterial pressure can decrease right coronary perfusion pressure, which further exacerbates RV dysfunction. The selective pulmonary vasodilator action of inhaled NO thus holds great potential. Patients with mitral valve disease frequently have accompanying pulmonary hypertension. Six patients who had chronic mitral stenosis and pulmonary hypertension, were administered 40 ppm of inhaled NO after mitral valve replacement. 73 Inhaled NO selectively reduced PVR and PAP. Snow TM studied 12 patients undergoing elective mitral valve repair or replacement and 10 patients undergoing elective coronary artery bypass grafting. Forty ppm of inhaled NO for 20 minutes during the first 2 hours after the operation caused a significant reduction in PVR in patients who had preexisting pulmonary hypertension, but no change in hemodynamic state in the coronary artery bypass grafting group of patients who had normal PAP levels. Rich 75 studied 20 patients undergoing cardiac surgery and showed that 20 ppm inhaled NO was a selective pulmonary vasodilator before and after CPB. Further the NO-induced pulmonary vasodilatation was proportional to the baseline PVR and was not altered by CPB, the presence of a ventricular assist device, or the infusion of nitrovasodilators. It would seem that whatever effects CPB has on endothelial function the response to exogenous NO is unchanged.

Pediatric Cardiac Surgery Congenital heart disease (CHD) may be complicated by pulmonary arterial smooth muscle hyperplasia, hypertrophy, and hypertension. Increases in PVR are common in these patients

NITRIC OXIDE and seem to be exacerbated after CPB. Wessel TM used inhaled NO and endothelium-dependent vasodilator acetylcholine to evaluate endothelial function after CPB. Pulmonary vasodilation with acetylcholine was seen in all preoperative patients (children with CHD and PHTN) but was attenuated in postoperative patients. However, inhalation of 80 ppm NO in postoperative patients lowered PVR. These findings suggest that CPB may be responsible for postoperative dysfunction of the pulmonary endothelium and may contribute to postoperative pulmonary hypertension. Winberg77 studied the short-term effects of inhaled NO in 22 infants and children aged 3 to 32 months with CHD undergoing preoperative cardiac catheterization. All but one had intracardiac shunt lesions and 13 had increased PVR. During catheterization the patients inhaled 40 ppm NO. Inhaled NO did not affect the systemic circulation but there was a significant reduction in PVR in the 13 infants with PHTN. Beghetti 78 administered inhaled NO to seven children with severe PHTN after cardiac surgery. In 6 of the 7 children, NO inhalation at 15 ppm selectively decreased mean PAP and concomitantly increased the PaO2. In responders, attempts at early weaning from NO were unsuccessful and concentrations of less than l0 ppm were continuously administered for a median of 9.5 days (range 4 to 16 days). One child later died and five were discharged. Months after surgery, Doppler echocardiography showed evidence of regression of PHTN in all five children. There are case reports documenting the successful treatment of PHTN with inhaled NO after VSD repair, 79'8~ after arterial switch procedures, 81"82after Fontan procedures, 83'84 and after surgery to correct total anomalous venous return. s5'86 In all instances, inhaled NO reduced pathologically increased PVR without affecting systemic circulation and without apparent side effects.

Reversibility-Diagnostic Studies The decision to operate on a patient may depend on the reversibility of the pulmonary vasoconstriction. In children with CHD, it is often desirable to determine the vasodilatory capacity of the pulmonary circulation during preoperative cardiac catheterization to predict the postoperative PVR. Hyperoxic breathing has been used

53 to selectively dilate the pulmonary vasculature; so have intravenous vasodilators (prostacyclin, tolazoline prostaglandin Em, sodium nitroprusside) which are nonselective and dilate the systemic and pulmonary circulations. Roberts 87 compared the pulmonary vasodilator potency of inhaled NO with oxygen. Ten pediatric patients with CHD and PHTN inhaled 80 ppm NO at FIO2 = 0.21 and FIO2 = 0.9. The study showed that inhaled NO showed a greater selective pulmonary vasodilatory effect than hyperoxic breathing.

Cardiac Transplant Preoperative PHTN in heart transplant candidates is associated with increased risk of intraoperative and early postoperative death as a result of acute RV failure of the donor heart after orthoptic heart transplantation. The RV of the donor heart is vulnerable to acute dysfunction when confronted with an increase in afterload. Patients with reversible PVR can be considered suitable for orthoptic heart transplantation and an effective vasodilator can be used after surgery to prevent RV failure. Keiler-Jensen88 used a brief period of inhaled NO 20 ppm to evaluate the reversibility of the PHTN in 12 candidates for orthoptic heart transplantation compared with sodium nitroprusside and prostacyclin in doses that lowered the mean arterial pressure by 15%. They concluded that NO provided a simple, selective, and standardized test of the pulmonary vasodilator potential. Girard 89documented a case when inhaled NO was used successfully in the management of a man with fight ventricular failure after orthoptic heart transplantation. Bocchi 9~described 3 cases which suggested that inhaled NO may lead to the development of pulmonary edema in patients with heart failure and severe PHTN. Two patients had ischemic cardiomyopathy and the other idiopathic dilated cardiomyopathy. After the inhalation of NO (40 to 80 ppm), cardiac output increased and PVR decreased in all patients. Right atrial pressure increased or remained the same. Pulmonary artery wedge pressure (PAWP) increased in 2 patients but could not be measured in the third. After a few minutes of NO inhalation, all 3 patients developed pulmonary edema. Despite stopping NO inhalation the elevated PAWP persisted. The investigators suggested that the acute reduction in

54 PVR caused an acute increase of RV output. The resulting increase of blood return to the impaired left ventricle may have caused the increase in PAWP. However, Semigran9~ also noted that inhaled NO caused an increase in PAWP in 16 patients with severe chronic heart failure but none developed pulmonary edema. Further studies are needed to ascertain the consequences of inhaled NO therapy in patients with severe heart failure.

Lung Transplantation After lung transplantation, transient graft dysfunction may occur and may be complicated by a reperfusion syndrome manifested by PHTN and edema, RV failure and respiratory failure. Five patients with PHTN after lung transplantation were administered 80 ppm inhaled NO which lowered mean PAP, PVR, and shunt fraction. 92 The trial of NO markedly improved oxygenation in two patients who continued to receive NO at 10 ppm while their PVR declined. A decrease in the endogenous production of NO by damaged endothelium, faster inactivation of NO, or an increase in circulating vasoconstrictors may be implicated in graft dysfunction. Selective pulmonary vasodilation with the associated improvement in intrapulmonary shunting and oxygenation suggest inhaled NO may be a very suitable agent to treat graft dysfunction after human lung transplantation.

Toxicity of Inhaled Nitric Oxide and Nitrogen Dioxide The potential for the clinical use of NO has to be balanced by the potential toxic effects of this therapy. Nitric oxide is an oxide of nitrogen formed during high temperature combustion processes and is a common atmospheric pollutant at levels near 10 ppm. It is present in cigarette smoke (400 to 1000 ppm) and in the exhaust from motor vehicles. Inhalation of NO and nitrogen dioxide (NO2) in high concentrations has caused death in humans. Nitrogen dioxide is readily solubilized in the moisture of the respiratory epithelium forming nitric and nitrous acid. In sufficient concentration these acids cause severe burns throughout the entire respiratory system and acute interstitial pneumonitis. This is the mechanism of Silo Filler's disease. 93 CluttonBrock94 reported two case of poisoning with NO, NO2, and other higher oxides of nitrogen after

MORRIS, RICH, AND JOHNS contamination of nitrous oxide cylinders, resulting in acute pulmonary injury, methemoglobinemia, asphyxia, and in one case death. Nitric oxide is converted to NO2 in the presence of oxygen. This is a second order reaction and depends on the concentrations of NO and oxygen and the time of contact. At low NO concentrations the reaction is minimal. However low concentrations of NO2 are extremely toxic. Patel and Block95 evaluated the effect of exposure of pig endothelial cells to 5 ppm NO2. They found that it changed the physical state of the membrane lipids, decreasing plasma membrane fluidity, and impaired the membrane function. Devalia et a196 cultured human bronchial epithelial cells and studied the effects on the synthesis of inflammatory cytokines after exposure to 400 or 800 ppb for 6 hours. Their findings suggest that exposure to NO2 at these concentrations may induce the synthesis ofproinflammatory cytokines from airway epithelial cells and consequently play an important role in the cause of airway disease. Frampton 97 found in healthy subjects that 1.5 ppm NO2 for 3 hours increased airway reactivity whereas repeated 15-minute exposures to 2 ppm did not. Carson 98 studied seven healthy adults breathing 2 ppm NO2 for 4 hours. In six of seven epithelial samples, the luminal border membranes of ciliated cells were ultrastucturally altered. They conclude that although the adverse effects on mucociliary cells caused by acute exposure are most likely minimal, injury patterns might seem more prominently with higher NO2 levels and/or more extended exposure. Rasmussen 99 administered 14 healthy subjects inhaled NO2 at 2 to 3 ppm for 5 hours. Physiological and biochemical measurements were obtained during the exposures and until 24 hours after. A 14% decrease in serum glutathione peroxidase activity (involved in antioxidant defense) was observed 24 hours after the start of NOe exposure, and a 22% decrease in alveolar permeability 11 hours after the start. These results suggest that a delayed response is a feature of the human reaction to NO2. In 1979, Hugod l~176 reported that the continuous exposure of 10 rabbits to 43 ppm NO (and 3.6 ppm NO2) did not cause morphological changes in the lungs. Stavert and Lehnert 1~ gave rats high concentrations of inhaled NO up to 1,500 ppm NO for 15 minutes, or 1000 ppm NO

NITRIC OXIDE for 30 minutes and produced no demonstrable increases in lung wet weights, nor resulted in any detectable histopathological changes in the lungs. In a study on 191 healthy human subjects von Nieding ~~ looked at the effects of inhaled NO, at various concentrations from 10 to 30 ppm, on lung function. They found that airway resistance as measured before and after a 15 minute exposure to NO at concentrations above 20 ppm showed a statistically significant increase. Comparing NO2 and NO, NO was markedly less toxic.

Free Radical Formation During normal cellular aerobic metabolism 98% of oxygen is fully reduced to water. About 2% of molecular oxygen is metabolized to superoxide or hydrogen peroxide.~~ These partially reduced oxygen species can directly oxidize biomolecules or be converted into even more reactive species such as the hydroxyl radical. Normally, endogenous tissue antioxidant defenses such as the SOD maintain intracellular concentrations of reactive oxygen species in the nanomolar range or less. In aqueous solutions, NO reacts rapidly with superoxide to form peroxynitrite which is highly toxic. Peroxynitrite is a strong oxidant and it readily catalyzes membrane lipid peroxidation and oxidizes DNA bases. It will react with metal or metalloproteins such as SOD to form potent and toxic subspecies. It inhibits epithelial cell ion channels. It is a major macrophage-derived oxidant contributing to the cytotoxic action of these cells. It is batericidal and is a likely mediator of host defense mechanisms) ~ The reaction of NO with superoxide may be particularly disconcerting because tissue superoxide formation is increased in the presence of hyperoxia, ischemia-reperfusion, sepsis and in both acute and chronic inflammatory disease. In these conditions, pulmonary cells are both key sources and targets of reactive oxygen species. Superoxide dismutases has been shown to protect ischemic tissue in experimental models when injected into the circulation just before reperfusion. Injury to the endothelium is a major consequence of ischemia/reperfusion injury, causing edema formation due to the loss of barrier function and favoring platelet adhesion to the endothelium. The protective action of SOD may in part be caused by preventing the decomposition of NO

55 by scavenging superoxide, which would help maintain normal vasodilatation and inhibit thrombosis. Beckman 3~ showed that SOD also protects endothelium by preventing the formation of peroxynitrite.

Formation of MetHemoglobin Nitric oxide combines rapidly with hemoglobin to form nitrosyl Fe (II) hemoglobin with an affinity 1,500 times higher than that measured for carbon monoxide. When oxygen is present nitrosyl hemoglobin is oxidized to Fe (III) Methemoglobin (MetHb). Methemoglobin is thereafter converted to hemoglobin by the enzyme MetHb reductase. When breathing low concentrations of inhaled NO most reports indicate clinically insignificant increased levels of MetHb. 85:~ However high levels have been reported in certain patients and frequent monitoring (every 4 hours for 24 hours, then daily) is essential. 66's5"1~ Infants are at increased risk because fetal hemoglobin is more prone to oxidation to MetHb than adult type (twice the rate) and there is reduced activity of MetHb reductase in the newborn. Certain ethnic groups including Native Americans and Eskimos have a relative deficiency of MetHb reductase placing them at a higher risk of developing methemoglobinemia.

Inhibition of Platelet Aggregation Endogenous NO causes an inhibitory effect on platelet aggregation and adhesion at the endothelial cell-blood interface by increasing cGMP. Hrgman t~ studying rabbits, noted a 14% increase in bleeding time while breathing 30 ppm NO for 15 minutes, and a 20% increase while breathing 300 ppm. Subsequently they studied six healthy human volunteers and showed that the bleeding time ratio increased to 1.33 after NO inhalation for 15 minutes (30 ppm) but decreased to I. 14 and 1.04, 15 and 30 minutes after discontinuation, respectively. Samama 1~ studied 6 patients with ARDS, and without preexisting coagulation disorders. The lungs were mechanically ventilated with inhaled NO at various concentrations (1, 3, 10, 30, and 100 ppm) randomly administered. After NO there was a statistically significant decrease in ex vivo platelet aggregation. Ivy bleeding time remained within normal values, and variations after NO did not correlate with changes in platelet aggregation. There have

56 been no reports thus far of bleeding complications occurring as a result of inhaled NO therapy.

Mutagenicity Nitric oxide may interfere with DNA synthesis and may induce DNA strand breakage or point mutation. The small molecular size and lipophilicity of NO make it readily accessible to cells. The combination of NO and NO2 yields a potent nitrosylating agent which has been shown to react with secondary amines to yield carcinogenic Nnitrosamines. Nitrosylation of primary amines, as found on DNA bases, results in immediate deamination with the possibility of a point mutation occurring at this site in the absence of repair. Macrophage-derived N O is cytostatic to tumor cells and microbial pathogens. Kwon 1~ determined that one of the molecular targets for the cytostatic action of NO is ribonucleotide reductase, a rate-limiting enzyme in DNA synthesis. Arroyo 1~ observed significantly enhanced mutation in Salmonella typhimurium when exposed to low concentrations (up to 20 ppm) of NO in the presence of oxygen. Nguyen 110 added NO to intact human lymphoblastic cell lines in culture and to aerobic solutions o f D N A , RNA, guanine, or adenine. Nitric oxide induced dose-dependent DNA strand breakage.

Metabolic Fate of Nitric Oxide Nitric oxide reacts with hemoglobin forming nitrosyl-hemoglobin, and from this, nitrate and nitrate are generated.l 11 Large amounts of nitrate are excreted into the urine and a certain amount is discharged through the salivary glands and transformed to nitrite. Some of this nitrite is converted to nitrogen gas in the stomach. Nitrate in the intestine is partly reduced to ammonia, is reabsorbed into the body, and converted to urea.

Suppression of Endogenous Nitric Oxide Sudden discontinuation of inhaled NO has been reported to result in severe pulmonary vasoconstriction and marked deterioration in oxygenation. 86 Several recent studies have shown that NO itself acts to regulate its own release by feedback inhibition o n N O N . l ! 2 Studies by ROOS 113 indicate that chronic inhaled NO (20 ppm for 3 weeks) reduces endogenous NO production, and the smooth muscle response to NO

MORRIS, RICH, AND JOHNS in hypoxic rats. This is not surprising because it is known that NO donors inhibit NOS in cell cultures. TM These studies which have shown downregulation of endogenous NO may explain rebound vasoconstriction and desaturation on discontinuing inhaled NO. As a result it may be necessary to slowly wean inhaled NO.

Nitric Oxide Administration~Delivery Inhalation circuits should insure accurate delivery of NO while maintaining low levels of NO2 (<5 ppm). Stock tanks of NO should not exceed 1,000 ppm and should ideally be mixed with the carrier gas (usually 02 and/or air) immediately before inhalation to decrease contact time. Foubert 114 calculated the time to reach 5 ppm NO2 from a mixture o f 20 ppm NO in 100% oxygen is 12 minutes and in air is more than 1 hour. In ventilated patients maintaining a constant inspired NO concentration despite changes in minute and tidal volumes may be problematic. Most pediatric ventilators pass a continuous gas stream around the ventilator system. Ventilation is achieved by altering the pressure and is independent of the flow rate of the gas stream. Thus, when a fixed concentration of NO is added to the inspiratory limb the delivered concentration of NO will be constant and independent of changes in minute ventilation. In adult ventilators flow occurs only during inspiration and changes with alterations in minute ventilation and tidal volume. Moors, 115 using a Siemens Servo 900 ventilator (Siemens-Elema, Sweden), compared the effects of adding NO 40 ppm at the low pressure gas supply, or into the inspiratory limb. They found that when NO was continuously added to the inspiratory tubing in an intermittent flow ventilator there was poor mixing of gas and potentially high NO and NO2 levels. Stenqvist 116 describes a system using a Siemens 900C ventilator, two mass flow regulators, and a soda-lime absorber. He mixed the NO and the breathing gas immediately before the ventilator gas intake to avoid variations in the final NO concentration. A soda-lime CO2 absorber which absorbs about 75% of the NO2 and also some of the NO passing through it, was placed in the inspiratory limb between the ventilator and the Ypiece. This system allows adequate mixing of the NO with inspiratory gases but keeps the concen-

NITRIC OXIDE

57

tration o f NO2 low. Y o u n g 117 describes a system using two p n e u m o t a c h o g r a p h s a n d a mass flow controller, designed to m a t c h the nitric oxidenitrogen m i x t u r e flow rate to the gas flow in the inspiratory l i m b of the ventilator. T h e injected dose o f N O is calculated a s s u m i n g there is n o N O in the inspired gas stream. It should n o t therefore be used in rebreathing systems. However it can be used with a n y ventilator i n c l u d i n g the c o n t i n u o u s flow pediatric systems. The concentration o f N O a n d NO2 in a sample o f gas m a y be measured by c h e m i l u m i n e s c e n c e , infrared analysis, R a m a n spectroscopy, a n d electrochemical methods. Chemiluminescence m a y b e the most accurate m e t h o d o f m e a s u r i n g N O c o n c e n t r a t i o n , b u t is expensive a n d c u m bersome. Electrochemical analyzers are i n e x p e n sive a n d m o r e suitable for r o u t i n e clinical use in most instances.

CONCLUSION Nitric oxide is a selective p u l m o n a r y vasodilator in adult a n d pediatric patients a n d m a y be a useful therapeutic agent in the t r e a t m e n t o f P H T N from a variety o f causes. I n h a l e d N O is also effective in treating hypoxemia as it improves ventilation to perfusion m a t c h i n g a n d decreases p u l m o n a r y shunt. Large clinical trials will determ i n e if inhaled N O decreases mortality or i m proves ventilator weaning. T h e m o s t i m p o r t a n t questions regarding inhaled N O therapy c o n c e r n its potential toxicity.

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