Oxygen Therapy in Neonatal Resuscitation

Oxidative Stress: Pathophysiologic Background

Perinatal asphyxia is a devastating disorder that affects roughly 2% of newborn babies in industrialized countries, but constitutes one of the leading causes of early neonatal death in nonindustrialized countries. Both ischemia and hypoxia reduce oxygen and glucose supply to neurons leading to ATP depletion and inactivation of ATP-depending ion pumps, causing intracellular accumulation of Na ⁺ (cell swelling), of ionic Ca ⁺⁺ (increased free radical formation and oxidative stress/damage), and inhibition of neurotransmitters’ recaptation at the synaptic cleft causing hyperexcitability. Altogether these pathophysiologic circumstances will not only lead to cell necrosis but will collectively predispose tissue to reoxygenation injury. During hypoxia, limited oxygen availability decreases oxidative phosphorylation, resulting in a failure to resynthesize energy-rich phosphates, including adenosine 5′-triphosphate (ATP) and phosphocreatine, resulting in an accumulation of purine derivatives, especially hypoxanthine. The tissue concentration of these metabolites is directly dependent on the intensity and prolongation of hypoxia ( Fig. 33.1 ). Upon resuscitation, restoration of oxygen and glucose supply may salvage neurons, but it will also activate the transformation of xanthine dehydrogenase in xanthine oxidase leading to the generation of a burst of oxygen and nitrogen free radicals that will trigger apoptotic and proinflammatory pathways, thus expanding the initial neuronal damage. Moreover, in the endothelium, ischemia promotes expression of certain proinflammatory gene products (e.g., leukocyte adhesion molecules, cytokines) and bioactive agents (e.g., endothelin, thromboxane A ₂ ) while repressing other “protective” gene products (e.g., prostacyclin, nitric oxide). Ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion.

Reperfusion and reoxygenation of ischemic tissues result in the formation of toxic reactive oxygen species, including superoxide anions (•O ₂ ⁻ ), hydrogen peroxide (H ₂ O ₂ ), hydroxyl radicals (•OH), and nitrogen reactive species, especially peroxynitrite (•ONOO-). Under physiologic conditions, hypoxanthine accumulated during the ischemia is further oxidized by xanthine dehydrogenase to uric acid in the cells containing this enzyme. However, during prolonged ischemia, xanthine dehydrogenase is converted to xanthine oxidase by specific proteases. Of note, xanthine oxidase uses oxygen as a substrate and during hypoxia-ischemia is unable to catalyze the conversion of hypoxanthine (resulting in the buildup of excess tissue levels of hypoxanthine). When oxygen is reintroduced during resuscitation, conversion of the excess hypoxanthine by xanthine oxidase results in the formation of reactive oxygen species, especially anion superoxide. Moreover, in the presence of nitric oxide, both superoxide and nitric oxide will combine in the formation of reactive nitrogen species, especially peroxynitrite. In tissues rich in ferrous iron such as the brain, Fenton chemistry will ensue, leading to the formation of the highly reactive hydroxyl radical. Interestingly, xanthine oxidase in humans is mainly restricted to the liver and intestine. It has been shown, however, that xanthine oxidase leaks out into the blood after hypoxia and hypotension, and the hypoxanthine-xanthine oxidase system may be detrimental in all parts of the body. Many other oxygen radical–generating systems are presently well described.

Reactive oxygen species and reactive nitrogen species are potent oxidizing and reducing agents that directly damage cellular structures. They are able to peroxidize membranes, structural proteins and enzymes, and nucleic acids. In addition, they are known to be extremely important regulators of intracellular signaling pathways that modulate DNA and RNA synthesis, protein synthesis, and enzyme activation, and directly influence the cell. Increasingly, the concept of redox code has acquired more relevance. The redox code defines the positioning of nicotinamide adenine dinucleotide (NAD, NADP), thiol/disulfide (GSH/GSSG; CysSH/CysS-S), and other redox systems as well as the thiol redox proteome as common elements for biologic processes. These control elements are functionally organized in redox circuits, which are controlled by central nodes constituted by sulfur/disulfide couples. These circuits function independently and are highly responsive for redox conditions, thus signaling and regulating biologic processes. The code is present in an oxygen-dependent life. Activation and deactivation cycles involving oxygen and hydrogen peroxide contribute to spatiotemporal organization for differentiation, development, and adaptation to the environment.

A vast array of enzymatic and nonenzymatic antioxidants has evolved in biologic systems to protect cellular structures against the deleterious action of free radicals. Antioxidant enzymes catalytically remove reactive oxygen species (ROS), thereby decreasing ROS reactivity, and protect proteins through the use of chaperones, transition metal-containing proteins (transferrin, ferritin, ceruloplasmin), and low molecular weight compounds that purposely function as oxidizing or reducing agents to maintain intracellular redox stability.

Clinically, antioxidant enzymes that have been most widely studied are the superoxide dismutases, catalases, and glutathione peroxidase. The most relevant nonenzymatic cytoplasmic antioxidant is reduced glutathione (GSH), a tripeptide (γ-glutamyl-cysteinyl-glycine). Hence, two molecules of GSH establishing a disulfide bond form oxidized glutathione and release one electron that is accepted by a free radical to stabilize the outer atomic shell. Thiol-disulfide strategy is extremely important in maintaining the reducing state in the cytoplasm and the cell redox status, essential for cell reproduction and maturation. Other systems to detoxify hydrogen peroxide in mitochondria and other organelles include glutaredoxin, thioredoxin, thioredoxin reductase, and the peroxiredoxins. Other enzymes with antioxidant and signaling functions are heme oxygenases (HO-1 and HO-2). HO-1 removes heme, a pro-oxidant, and generates biliverdin, an antioxidant, releasing iron and carbon monoxide. Finally, nonenzymatic antioxidants such as reduced glutathione, vitamin C, vitamin E, and β-carotene also function to protect cells from damaging effects of ROS.

Oxidative stress in a biologic system is defined as the imbalance of pro-oxidants and antioxidants in favor of pro-oxidants. Different biomarkers of oxidative stress have been used in biology and medicine. An indirect way of measuring oxidative stress is the detection of increased activity of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, or glutathione redox cycle enzymes. Another measure is to analyze the oxidized form of a nonenzymatic molecule such as oxidized glutathione. An increased concentration of oxidized glutathione or a decreased ratio of reduced to oxidized glutathione may indirectly reflect a pro-oxidant status.

For clinical purposes, the most widely employed markers of oxidative stress are those derived from the oxidant alteration of biologic molecules that convey the following characteristics: being chemically stable, reproducible, and easily measurable in biologic fluids. Urinary markers of oxidative stress are extremely valuable in neonatology, because urine sampling is readily available, allowing serial measurements without the need of supplementary blood sampling. In recent studies, reliable high performance liquid chromatography coupled to tandem mass spectrometry methods has been validated in the urine of newborn infants. Hence, protein oxidation can be readily measured, analyzing phenylalanine oxidation by the action of hydroxyl radicals that leads to the formation of ortho-tyrosine (O-tyr) and meta-tyrosine. In addition, the action of hypochlorous acid and peroxynitrite upon phenylalanine produces byproducts such as chlor-tyrosine and nitro-tyrosine that reflect inflammatory processes and nitrosative stress respectively. Hydroxyl radical aggression upon DNA can be assessed analyzing urinary concentration of oxidized guanidine bases. The byproducts 8-hydroxyguanine (8-oxo-Gua) and its 2′-deoxynucleoside equivalent 7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) are highly mutagenic. Urinary elimination of 8-oxo-Gua and 8-oxo-dG perfectly reflects nuclear attack by hydroxyl radicals. In experiments performed in a piglet model of hypoxia and reoxygenation, urinary elimination of metabolites O-tyr and 8-oxo-dG correlated significantly with the amount of oxygen used on reoxygenation. Lipid oxidation, especially arachidonic (isoprostanes, isofurans), docosahexanoic (neuroprostanes, neurofurans), and adrenic (di-homo isoprostanes) acids perfectly reflect damage to neuronal membranes and lipid components in gray and white matter respectively. Hence, F2-isoprostanes and isofurans, which are non-cyclooxygenase oxidative derivatives of arachidonic acid, are considered at present the most reliable markers of lipid peroxidation. These compounds are chemically stable, formed in vivo , present in all organic fluids and tissues, and are not affected by dietary content of lipids. Isofurans reflect oxidation in a high oxygen atmosphere and isoprostanes in a normoxic environment. In addition, byproducts derived from the oxidation of docosahexanoic acid such as neuroprostanes and neurofurans have also been considered very valuable biomarkers, especially related to oxidative damage of neuronal membranes, and adrenic acid reflects white matter damage by oxygen free radicals.

Oxidative Stress: Differences Between Resuscitation With 100% Oxygen and Room Air

In the absence of severe lung disease or cyanotic congenital heart disease, resuscitation with 100% oxygen has been shown to cause supra-physiologic arterial partial pressures of oxygen in the newly born infant. By contrast, resuscitation with room air increases the Pa o ₂ to physiologic levels only (i.e., approximately 70-80 mm Hg). Biomarkers of oxidative stress such as oxidized glutathione or antioxidant enzyme activity are significantly increased in patients receiving excessive oxygen. Thus, newborn infants resuscitated with pure oxygen exhibit higher oxidative stress after resuscitation than infants recovered with room air. Conspicuously, oxidative stress derived from resuscitation with pure oxygen may cause a long-lasting pro-oxidant status. Hence, in newborn babies resuscitated with 100% oxygen, oxidative stress was detected 4 weeks after birth. These infants had a decreased ratio of total blood reduced (GSH) to oxidized (GSSG) glutathione and oxidized DNA bases in urine at 1 month of life. No such effect has been observed in infants resuscitated with room air ( Fig. 33.2 ).

Hyperoxemia has also been associated with a series of negative side effects, including increased oxygen consumption and metabolic rate, increased activation of leukocytes and endothelial cells, and increased formation of reactive oxygen species and reactive nitrogen species. Prolonged vasoconstriction of cerebral arteries has also been shown. In a study involving premature infants younger than 33 weeks’ gestational age at 24 hours, cerebral blood flow was reduced by 20% in infants given 80% oxygen compared with infants for whom room air was used. This finding is in line with studies in newborn rats showing that the use of 100% oxygen for resuscitation reduces cerebral blood flow compared with room air resuscitation.