Aerobic Metabolism

Oxygen (O ₂ ) is one of the most abundant elements in nature; the second most abundant component of breathing air, constituting 21% of its composition; and probably the most widely used drug in neonatology. The presence of oxygen will allow the complete combustion of glucose, amino acids, and free fatty acids in a highly efficient process that produces approximately 20 times more energy than anaerobic combustion. This process takes place within the mitochondria, which act as the energy factories of the cell ( Fig. 16-1 ). Substrates are metabolized into acetyl coenzyme A, which enters the tricarboxylic acid cycle (Krebs cycle) where energy in the form of highly energized electrons is liberated and transported by specific proteins (NADH, NADPH, FADH) to the electron transport chain (ETC) located in the inner mitochondrial membrane. Electrons, considered reducing equivalents, provide the energy necessary to maintain the electrochemical gradient that drives adenosine triphosphate (ATP) synthesis. Components of the ETC pump protons across the inner mitochondrial membrane against an electrochemical gradient, and the protons are taken in again by ATP synthase. In this process, energy is recovered and employed to transform adenosine diphosphate into ATP. Electrons are captured by oxygen thus permitting the formation of water and avoiding electron leakage and formation of free radicals; thus, each molecule of dioxygen will be completely reduced by four electrons. Metabolic substrates used to provide energy are highly organ specific; thus, the central nervous system and erythrocytes almost exclusively depend on glucose, whereas cardiac contraction uses energy provided from the combustion mainly of free fatty acids. Oxidative phosphorylation provides most of the ATP needed by the body and is especially relevant in aerobic-dependent tissues known as oxyregulators, such as brain. These organs cannot adapt for even short periods of time in the absence of oxygen without undergoing necrosis and/or apoptosis.

Nutrients are transformed into acetyl-CoA, which is metabolized in the inner mitochondrial space along the tricarboxylic acid cycle. During this process energized electrons are liberated and transported to the electron transport chain, creating a mitochondrial transmembrane potential (Ψ _m ). Energy is used to first extrude protons (H ⁺ ) that are thereafter taken in again by adenosine triphosphate (ATP) synthase, and liberated energy is employed to resynthesize ATP from adenosine diphosphate (ADP). Oxygen will be reduced with four electrons. This process is known as oxidative phosphorylation.

Reactive Oxygen Species, Redox Regulation, and Antioxidant Enzymes

The term reactive oxygen species (ROS) refers to a series of molecules derived from incomplete reduction of molecular dioxygen ( Fig. 16-2 ). Even under physiologic conditions, a small percentage of electrons will “leak,” causing a “partial” reduction of oxygen (oxygen with fewer than four electrons). The incomplete reduction of oxygen with one electron elicits the production of anion superoxide (•O ₂ ⁻ ), [•O ₂ ⁻ ] with the addition of two electrons will lead to the generation of hydrogen peroxide (H ₂ O ₂ ), and with a third electron, the highly reactive hydroxyl radical (OH) will be produced. ROS are also relevant in the regulation of nitric oxide (NO) metabolism and availability and therefore indirectly have an important influence on airway and vascular reactivity. Hence, when superoxide anion binds to NO, peroxynitrite (ONOO ⁻ ) will be produced. Peroxynitrite is a highly reactive nitrogen species but also influences vascular reactivity in such areas as the lung, where its production can lead to increased vasoconstriction, causing pulmonary hypertension. The production of ROS under physiologic and also pathologic conditions is highly dependent on the concentration of oxygen in the tissue. ROS are produced in hypoxic and hyperoxic situations and especially when the oxygen concentration fluctuates from hyperoxia to hypoxia and vice versa. ROS can be extremely aggressive when acting as free radicals, causing direct structural and/or functional damage and/or interfering with essential redox-regulatory elements. In other circumstances, ROS can be relatively stable and act as signaling molecules in physiologic processes.

Under normal conditions, oxygen undergoes a tetravalent reduction with four electrons. However, under certain circumstances, oxygen is reduced stepwise by one electron at a time. This leads to the formation of reactive oxygen species, some of which are free radicals (e.g., superoxide anion from monovalent reduction and hydroxyl radical from trivalent reduction), whereas others are not (hydrogen peroxide from divalent reduction). Hydrogen peroxide acts as a signaling molecule. Hydroxyl radical formation is enhanced in the presence of transition metals such as iron, copper, and manganese by the so-called Fenton chemistry. The antioxidant system neutralizes the chemical reactivity of free radicals. CAT , Catalase; GPx , glutathione peroxidase; TRx , thioredoxin; PRx , peroxiredoxin; SOD , superoxide dismutase.

The term free radical refers to any molecule capable of independent existence with one or more unpaired electrons in the outer shell (e.g., anion superoxide, hydroxyl radical). Free radicals form covalent bonds to share one electron with other molecules; however, the resulting molecule easily decomposes, leading to the formation of toxic products. Free radicals may also react with nonradical molecules in typical chain reactions, causing damage to DNA, proteins, and lipids or promoting the formation of adducts with DNA. Under very stressful conditions (ischemia–reperfusion, inflammation, or hyperoxia) damage caused by free radicals can lead to cell death by necrosis or apoptosis or marked cellular dysfunction. In addition to mitochondrial respiration, ROS are produced also by the cytochrome P450 monooxygenase system, xanthine oxidoreductase, nitric oxide synthases, heme oxygenases, and other enzymes involved in inflammatory processes. Moreover, in the presence of transition metals such as iron, copper, zinc, and manganese, the generation of ROS can be exponentially increased.

ROS also have a relevant role in cell physiology. At low/moderate concentrations, especially those ROS that are not free radicals (e.g., hydrogen peroxide) elicit an ample array of cellular responses acting as signaling molecules. Thus, ROS-mediated actions can be protective against ROS-induced oxidative stress and reestablish or maintain redox homeostasis. One of the most important roles of ROS is the regulation of NO production, the oxidative burst produced as a response to infectious agents by phagocytic NAD(P)H oxidase, or acting as sensing elements for regulating tissue oxygen needs, cell adhesion, immune responses, or induced apoptosis.

Redox Regulation

The concept of oxidative stress as a global imbalance affecting the entire economy no longer adequately explains redox biology. Central sulfur–disulfide couples, which include reduced and oxidized glutathione (GSH/GSSG), cysteine/cystine, and reduced and oxidized thioredoxin, function as reducing counterparts of H ₂ O ₂ and other oxidants in controlling the redox state of oxidizable thiols in proteins. These sulfur switches in which hydrogen peroxide has a relevant role are used for cell signaling, protein structure, protein trafficking, and regulation of enzyme, transporter, receptor, and transcription factor activity. Redox mechanisms control proinflammatory and profibrotic signaling, cell proliferation, apoptosis, and a range of other biologic processes by modifying the protein structure without involving oxidative structural or functional changes. This concept differs from previous thinking in which the importance of free radical mechanisms and macromolecular damage as an underlying mechanism was overemphasized.

Antioxidant Defenses

The redox balance at various sites in our organism requires the intervention of antioxidant defenses to counterbalance the generation of ROS. These mechanisms include both enzymatic and nonenzymatic processes. Antioxidant enzymes, through catalytic reactions, remove ROS and protect proteins through the use of chaperones, transition metal-containing proteins (transferrin, ferritin, ceruloplasmin), and low-molecular-weight compounds that function as oxidizing or reducing agents. Superoxide dismutases (SODs) constitute a family of enzymes located in the cytoplasm (Cu/Zn SOD), in the mitochondria (Mn/Cu SOD), or extracellularly (Zn/Cu SOD), which convert or dismutate superoxide anions to H ₂ O ₂ . Catalases and glutathione peroxidases (GPx) convert H ₂ O ₂ into H ₂ O and O ₂ . GPx couples H ₂ O ₂ reduction to water with the oxidation of GSH to GSSG. GSSG is again reduced to GSH by the activity of the pentose shunt. Other systems that 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 prooxidant, and generates biliverdin, an antioxidant-releasing iron, and carbon monoxide. Finally, nonenzymatic antioxidants such as GSH, vitamin C, vitamin E, and β-carotene also function to protect cells from the damaging effects of ROS. The ontogeny of enzymatic antioxidant expression progresses gradually during gestation. Compared to full-term infants, preterm infants have immature antioxidant defenses, which are more susceptible to ROS-associated conditions derived from hypoxia–reoxygenation, inflammation, or infection. In addition, transplacental passage of antioxidants also occurs late in gestation. In assays performed in human abortus materials, it has been shown that antioxidant enzyme activities in response to oxidant insults increase with advancing gestation. As a consequence, conditions occurring during pregnancy such as preeclampsia significantly alter placental antioxidant enzyme expression, causing a prooxidant burden for the fetus.

Biomarkers of Oxidative Stress

Oxidative stress can be assessed by various means. Direct damage to molecules such as proteins, lipids, and DNA can be measured, determining the results of the interaction with free radicals on their chemical structure. However, oxidative stress can also be indirectly measured by increased activity of antioxidant enzymes or increment in the concentration of oxidized antioxidants such as the GSH/GSSG ratio. In clinical research, determination of the concentration derived from free radical aggregation in biofluids such as blood, plasma, serum, urine, or cerebrospinal fluid has gained popularity. Table 16-1 summarizes the most widely employed biomarkers of oxidative stress. The GSH/GSSG ratio is a comprehensive indicator of redox status and can be determined in whole blood. Lipid peroxidation is determined by measuring malondialdehyde in blood or urine. However, isoprostanes, reflecting non-cyclooxygenase oxidation of arachidonic acid, are now widely employed because they are very stable and not influenced by diet, parenteral nutrition, or gestational age. The preferred method is liquid chromatography coupled to mass spectrometry. Isofurans, which reflect the oxidation of arachidonic acid under hyperoxic conditions, have been very satisfactorily employed in neonatal studies involving the use of oxygen. Other similar biomarkers are neuroprostanes and neurofurans. Neurofurans reflect the oxidation of docosahexaenoic acid specifically present in the brain and therefore may reflect damage caused by oxidative stress to brain tissue. Damage to proteins can be assessed by mass spectrometry determining ortho – or meta -tyrosine, which reflects the oxidation of circulating phenylalanine in a nonphysiologic metabolic pathway. Finally, determining the oxidation of the guanidine bases in urine or plasma assesses DNA damage, reflecting oxidation of the cell nucleus. There are many other biomarkers (RNA, glycoproteins, exhaled compounds, etc.) under evaluation, but these are still applicable only in more basic research.

Oxygen-Sensing Mechanisms and Physiologic Response

The following is a brief description of oxygen and hypoxia-inducible factor (HIF-1) interaction and erythropoietin (EPO) expression. Oxygen homeostasis is critical for the survival and function of cells and organisms. On one hand, oxygen in excess (hyperoxia) inevitably causes an excessive production of ROS, causing cell damage. On the other hand, the complete absence of oxygen (anoxia) is lethal. Physiologic systems are, therefore, adapted to achieve the proper equilibrium between oxygen demand and availability to ensure correct cellular functioning. A number of redox-sensitive transcription factors such as HIF-1, cAMP response element-binding protein, nuclear factor κB, activator protein 1, and p53 regulate gene expression in response to changes in the concentration of ROS.

Variations in oxygen concentration in the body elicit two types of responses. During acute hypoxia, pulmonary vasoconstriction and carotid body neurosecretion dependent on increased intracellular calcium levels mediated by ROS are essential for survival. If hypoxia is prolonged, activation of the HIF transcription factors will be triggered. The oxygen level in utero at the time of implantation is around 15 to 20 mm Hg and remains at this low level until the end of the first trimester, providing an adequate environment (reductive) for fetal and placental development. Angiogenesis is stimulated by low oxygen concentrations in tissue through transcriptional and posttranscriptional regulation of growth factors such as vascular endothelial growth factor (VEGF), EPO, placental growth factor, and angiopoietins 1 and 2. The master regulator of the cell’s adaptive response to hypoxia is HIF-1, a heterodimeric transcription factor comprising HIF-1α and HIF-1β subunits. HIF-1α protein is continuously synthesized; however, if the O ₂ concentration in tissue is normal or high, it will undergo proteasomal degradation. Under conditions below the specific critical oxygen threshold, HIF-1α protein will be stabilized and will bind to the hypoxia-responsive element of DNA and elicit the expression of numerous genes ( Fig. 16-3 ). HIF-1β is constitutively present in the cell nucleus. Activated genes, and especially VEGF and EPO, are meant to enhance O ₂ delivery to tissues.

HIF-1α protein is predominantly regulated posttranscriptionally by the O ₂ -dependent hydroxylation of two proline residues by the prolyl-hydroxylase enzymes, leading to ubiquitination and proteasomal degradation. HIF-1α is stabilized when the concentrations of oxygen are below the specific critical oxygen threshold, thus accumulating in the hypoxic cell. When oxygen overcomes metabolic needs, HIF-1α binds to the von Hippel-Lindau (pVHL) protein, which recruits a ubiquitin (Ub) ligase that targets HIF-1α for proteasomal degradation. HIF-1β is constitutively present in the cell nucleus. TGFα , Transforming growth factor α; EPO , erythropoietin; VEGF , vascular endothelial growth factor; PDGF-β , platelet-derived growth factor β.

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