**7.1 Inhaled nitric oxide (iNO)**

64 Liver Transplantation – Basic Issues

Inhibition of NO was observed not to influence neutrophil migration to the injured sites. While this contradicts a number of other studies, based on their findings, the authors concluded that NO availability was unlikely to be involved in the post-ischemic oxidant stress and reperfusion injury (Jaeschke, Schini, & Farhood, 1992). Nevertheless, the majority of published literature has demonstrated the beneficial effects of NO during liver IRI. These conflicting results might be explained by the fact that the mechanism of NO-mediated protection varies depending on cell type, quantities supplied, laboratory methods applied, timing and duration of NO exposure. Here, we summarize some key studies studying

> 120 min NO derived from iNOS, antioxidant

45 min NO derived from eNOS,

blood flow after reperfusion, attenuated neutrophils infiltration.

liver blood flow after reperfusion, cytoprotective

antiproinflammatory cytokines, improves microcirculation by the cGMP pathway, Inhibit neutrophils infiltration and platelet aggregation.

60 min NO, improve peripheral

45 min NO, antioxidant,

microcirculation

60 min NO, antioxidant (Chattopadhy

30 min NO, antioxidant (Köken & Inal,

antioxidant, suppresses Kupffer cell function, regulated basal hepatic blood flow, but not affects

60 min NO, improve hepatic

NO or NOS Effects Reference

(M Isobe et al.,

(Aiba et al., 2001)

ay et al., 2008)

(Hines et al., 2005)

(Nilsson, Delbro, Wallin, & Friman, 2001)

(Cottart et al.,

2003)

2000)

1999)

Ischemic Time

endogenous NO and NOS in hepatic IRI Table 1.

min before ischemia

before ischemia and15 min before and to 45 min after reperfusion

before ischemia

chloride 24h before


nitroprusside or L-Name prior to ischemia

L-NAME or 8-bromo guanosine

monophosphate or rat atrial natriuretic peptide (ANP 1-28) 30 min before ischemia

Table 1. Effect of endogenous NO and NOS on liver IRI

3′5′-cyclic

Species Experimental

Dogs FK 409, 30 min

Rats L-arginine, 7 days before IRI

Rats L-NAME 60 min

ischemia

ischemia.

Rats - L-arginine or Sodium

Male Rats - Arginine or

Mouse - Gadolinium

Methods

Pigs Aminoguanidine, 5

Inhaled NO was approved by the U.S. Food and Drug Administration in December 1999, for the treatment of Persistent Hypertension of the newborn. Over the last decade, the primary advantage of iNO was seen to be its ability to selectively decrease pulmonary vascular resistance with minimal effects on systemic blood pressure; however, there is currently much interest in exploring its other benefits, including its antioxidant properties and its cytoprotective abilities (Zaky et al., 2009). In many animal studies, iNO decreased infarct size and left ventricular dysfunction after ischemia-reperfusion injury, increased coronary artery patency after thrombosis, increased blood flow in brain, kidney and peripheral vasculature, decreased leukocyte adhesion in bowel during ischemia reperfusion, and decreased platelet aggregation (McMahon & Doctor, 2006). Date *et al* reported the use of iNO in 15 out of 32 patients who suffered from immediate severe allograft dysfunction with iNO at 20 to 60 ppm. The mortality was significantly lower in the iNO group (7% and 24%, respectively). The gross benefits reported were that iNO improves oxygenation, decreases pulmonary artery pressure, shortens the period of postoperative mechanical ventilation, and reduces airway complications and mortality (Date et al., 1996). Likewise, a recent retrospective study also presented an improvement of overall respiratory functions. The authors encouraged the administration of iNO for the prevention and treatment of early graft failure in lung transplant recipients (Yerebakan, Ugurlucan, Bayraktar, Bethea, & Conte, 2009). Varadarajan *et al* were the first group to study the relationship between NO metabolism and IRI in human liver transplantation (Varadarajan et al., 2004). From their study, they concluded that reduced bioavailability of eNOS contributed to IRI one hour after portal reperfusion. On the other hand, iNOS did not contribute to early IRI after human liver transplantation. Clinical and mechanistic reports on therapeutic use of iNO demonstrating well beyond vascular relaxation, subsequently inactivated by oxyordeoxyhemoglobin in the red blood cells. iNO has various positive effects on extrapulmonary systems. However, how iNO mediates extrapulmonary effects remains unclear. Evidence supporting of stable forms of iNO is probably strongest for *S*-nitrosothiol (SNOs) and nitrite (McMahon & Doctor, 2006). In a prospective, blinded, placebo-controlled study, iNO at 80 ppm was administered to patients undergoing orthotropic liver transplantation (J. D. Lang et al., 2007). Many advantages were reported in the iNO group, including reduced platelet transfusion, an improvement in the rate at which liver function was restored post-transplantation on, and a decrease in the length of hospital stay. Most interesting was the finding of an approximated 75% reduction of hepatocellular apoptosis in patients treated with iNO (J. D. Lang et al., 2007). Possible biochemical intermediates of iNO including plasma and red blood cell nitrate, nitrite, S-nitrosothiols, C- or N-nitrosamines and red blood cell ferrous nitrosylhemoglobin. In this study, a detailed analysis indicated that the most likely candidate transducer of iNO on liver IRI was nitrite.

#### **7.2 iNO delivery systems**

An iNO delivery system should allow for constant and accurate measurements of NO and nitrogen dioxide [NO2] concentration in inspired gas as well as minimize the contact time between oxygen and NO in order to decrease the feasibility of producing high NO2

concentrations. The measurement of iNO and NO2 concentrations can be undertaken using chemiluminescence or electrochemical devices. There are some drawbacks of chemiluminescence devices such as cost, the need for a relatively high sample volume, noise and maintenance difficulties (Mupanemunda & Edwards, 1995). However, an electrochemical analyzer is relatively insensitive, and these measurements may be affected by pressure, humidity, temperature and the presence of other gases in the environment (Macrae et al., 2004). The delivery system should display the pressure of iNO in the cylinder and should have a backup power supply to avoid sudden discontinuation of iNO. Inhaled NO is usually supplied in nitrogen at various concentrations. The gas mixture concentration should be sampled downstream of the input port just proximal to the patient manifold. iNO also can be administered via nasal cannula, oxygen mask and oxygen hood (Ambalavanan, St John, Carlo, Bulger, & Philips, 2002). Finally, the exhausted gas should be scavenged by passing it through carbon and filters, soda lime or activated charcoal (Ambalavanan, El-Ferzli, Roane, Johnson, & Carlo, 2009).

#### **7.3 Potential toxicities during inhalation**

In the presence of high concentrations of O2, NO oxidizes to nitrogen dioxide (NO2). NO2 reacts with the alveolar lining fluid to form nitric acid. NO dissolves in the alveolar lining fluid reacts with O2 - yielding OONO, then decomposes into a hydroxyl anion (Pryor & Squadrito, 1995). Nitration of tyrosine residues of proteins is used as a marker of oxidative stress (Ischiropoulos, 1998). The rate at which NO is oxidized to NO2 depends on the square of NO concentration and fractional concentration of oxygen to which it is exposed. The Occupational and Health Administration recommend 5 ppm/8 hr/24 hour interval as the upper safe limit of human exposure (Fullerton & McIntyre, 1996). In order to protect against NO2 toxicity, iNO should be given with the least possible O2 concentration. Inhaled NO and NO2 concentrations should be monitored, exhaled gases should be scavenged and a soda lime canister should be placed in the inspiratory limb of the breathing circuit.

#### **7.4 Nitrite**

The simple molecule nitrite had been thought to be just an index of NO production for decades (Köken & Inal, 1999). Recently, a number of evidence suggests that nitrite is a promediator of NO homeostasis. Administration of nitrite at near physiological concentrations (<5 μg) leads to vasodilatation in animals and human studies (Fullerton & McIntyre, 1996). Gladwin *et al* observed that nitrite was metabolized across the peripheral circulation. In addition, nitrite caused an increase in peripheral forearm blood flow when 80 ppm iNO was administered (Shiva & Gladwin, 2009). Under distinct conditions such as hypoxia and acidosis, nitrite can be reduced to NO by a number of deoxyhemeproteins (hemoglobin, myoglobin, neuroglobin and cytoglobin), enzymes (cytochrome P450 and xanthine oxidoreductase), and components of the mitochondrial electron transport chain (Zaky et al., 2009). Since nitrite can be converted back to NO during hypoxia nitrite therefore is expected to be utilized during ischemia reperfusion injury. Furthermore, nitrite shows more potential benefits than NO in terms of safety and ease of administration. In other words, nitrite concentrations administered need only a small dose in order to increase plasma and tissue nitrite level several-fold. Routes of administration are oral, intravenous injection or infusion, intraperitoneal, nebulizer or topical (Duranski et al., 2005). Nitrite has now been demonstrated to have cytoprotective effects in animal models of ischemia reperfusion in organs. Duranski *et al* evaluated the effects of nitrite therapy in vivo murine models of hepatic and myocardial ischemia reperfusion injury and showed that nitrite was associated with cytoprotective effects. In the setting, nitrite reduced cardiac infarct size by 67% and limited elevations of liver enzymes in a dose-dependent manner. They also demonstrated that nitrite was reduced to NO regardless of eNOS and heme oxygenase-1 enzyme activities (Duranski et al., 2005). The exact mechanisms of how nitrite protects against the particular condition are being explored, but it appears that the benefit is mediated through the modulation of mitochondrial function by involving the posttranslational *S*-nitrosation of complex I to attenuate reperfusion oxygen radical generation and prevents cytochrome-C release (Shiva & Gladwin, 2009).
