Oxidative stress (Proceedings)

Oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. All forms of life maintain a reducing environment within their cells. This reducing environment is preserved by enzymes that maintain the reduced state through a constant input of metabolic energy. Disturbances in this normal redox state can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA.

Oxidative injury is important is important in many disease processes in veterinary medicine. Oxidative injury occurs in GDV, mesenteric torsion, systemic inflammatory response syndrome, sepsis, and any disease process that results in ischemia and reperfusion injury. A clear understanding of the pathophysiology of oxidative injury will allow possible avenues for treatment options.

Oxidant Injury

Because O2 removes electrons from other atoms and molecules, oxidation is also described as the loss of electrons by an atom or molecule. The chemical species that removes the electron is termed the oxidizing agent or oxidant. The companion process (i.e., the gain of electrons by an atom or molecule) is called a reduction reaction, and the chemical species that donates the electrons is referred to as a reducing agent.

As O2 reacts with a carbon skeleton (organic molecule) electrons are removed from the carbon molecule. This disrupts covalent bonds and energy in the form of heat, light, or sound is generated. The organic molecule then breaks down into smaller fragments. When the reaction is over the smaller compounds are broken down into simple carbon and hydrogen, which is CO2 and H2O.

Oxygen itself is a weak oxidizing agent, but some of its metabolites are potent oxidizing agents. Oxygen in its natural state is a diatomic molecule (molecules made only of two atoms, of either the same or different chemical elements). An atom or molecule that has one or more unpaired electrons in its outer orbital is termed a free radical (capable of independent living). The complete reduction of oxygen to water requires the addition of four electrons and four protons.

Figure 1

Superoxide Radical

The first reaction adds one electron to O2 and produces the superoxide radical. Superoxide has one unpaired electron and is less of a free radical than O2. Superoxide is neither a very reactive radical nor a potent oxidant. Although it is implicated in tissue damage such as in reperfusion injury. The toxicity of superoxide radical is most likely due to the high production of the radical on a daily basis.

Hydrogen Peroxide

The addition of one electron to superoxide radical creates hydrogen peroxide. Although not a free radical, it is a strong oxidizing agent. It is a powerful cytotoxin and crosses cell membranes easily. Well known to damage endothelial cells. This bond ruptures easily and forms two hydroxyl radicals, and iron donates an electron to one of the hydroxyl radicals and forms hydroxyl ion. The electron is donated by iron in its reduced form, which serves as a catalyst in the reaction. Iron is considered a powerful pro-oxidant.

Hydroxyl Radical

The hydroxyl radical is the king of free radicals, it is one of the most reactive molecules in biochemistry. It usually reacts with other molecules with in five molecular diameters of its origin.

Hypochlorous Acid

The metabolism of oxygen in neutrophils has an additional pathway that uses myeloperoxidase enzyme to chlorinate H2O2 producing hypochlorous acid (hypochlorite).

H2O2 + 2Cl- → 2 HOCl

When neutrophils are activated the conversion of superoxide increases 20 fold. This is the respiratory burst. When it reaches H2O2, about 40% is diverted to hypochlorite (active ingredient in bleach) production and the remainder forms the hydroxyl radical. It is a powerful germicide.

Only about 2% of the intermediate compounds escape into the cytoplasm. This is due to the fact that cytochrome oxidase carries on the reactions in a deep recess that blocks the escape of the intermediates. Once they escape their mobility and toxicity differ. The superoxide radical and H2O2 are mobile but not very toxic, but the hydroxyl radical is very toxic but not very mobile.

Lipid Peroxidation

Lipid peroxidation is responsible for the oxidative damage to cell membranes, due to the high content of Polyunsaturated fatty acids. The lipophilic interior of cell membranes is rich in polyunsaturated fatty acids. Oxidation increases the melting point and makes them less fluid. They then lose their selective permeability and are effected by osmotic disruption.

Figure 2

Nitric Oxide Transformation

In the presence of superoxide radical, nitric oxide radical generates a powerful oxidant called peroxinitrite, which is 2000 more potent than H2O2 as an oxidizing agent.

NO + O2 → ONOO –

It can cause tissue damage directly or breakdown into hydroxyl radicals and nitrogen dioxide, and then hydroxyl radical causes the damage.

Antioxidant Protection (see figure 1 above)

Any substance that can reduce or delay the oxidation of a substance is called an antioxidant.

Enzyme Antioxidants

Superoxide dismutase

It causes the dismutation of superoxide radical to form H2O2, if the catalase and peroxidase are unable to reduce H2O2 to H2O, then it is said to be a pro-oxidant.

Catalase

Catalse is an iron containing heme that is able to reduce H2O2 to H2O. It is lowest concentration in cardiac and neurons. Inhibition of catalase does not enhance the toxicity of H2O2 to endothelial cells so its role as an antioxidant is questionable.

Glutathione Peroxidase

The glutathione peroxidase reduces H2O2 to H2O by removing electrons from glutathione in its reduced form and then donating the electrons to H2O2.

H2O2 + 2 GSH → 2 H2O + GSSG

Selenium

The activity of glutathione peroxidase enzyme is dependant on the trace element selenium.

Nonenzymatic oxidants

Glutathione

One of the major antioxidants in the body. Glutathione acts a reducing agent by virtue of the sulfhydryl group in its cysteine residue. It reduces H2O2 to water. It is found in the lung, liver, endothelium, and intestine. It is primarily an intracellular antioxidant. It does not cross the membrane and if administered IV has little effect.

N-Acetylcysteine

It is a glutathione analogue that is used as a mucolytic agent. It is able to cross cell membranes, unlike glutathione.

Vitamin E (α-tocopherol)

Is a lipid soluble vitamin that antagonizes the peroxidative injury of membrane lipids. Vitamin E is the only antioxidant capable of halting the propagation of lipid peroxidation. When a propagation wave hits Vitamin E, Vit E is transformed into a free radical (poorly reactive), and then vitamin C will then donate an electron to convert vitamin E back. Therefore vitamin C deficiency can hinder vitamin E's ability act as an antioxidant.

Vitamin C

Vitamin C is a reducing agent that can donate electrons to free radicals and fill there electron orbitals. It is water soluble and operates in the extracellular space. It operates mainly in the lungs. The problem with vitamin C is its ability to promote rather than retard the formation of oxidants in the presence of copper and iron. Vitamin C reduces iron to it Fe II state, aiding in the absorption from the GIT. However Fe II can promote the production of hydroxyl radicals from H2O2. Thus Vitamin C can function as an oxidant by maintaing iron in its reduced form. Increases of free iron are associated with inflammation, blood transfusions, reductions in iron binding proteins. The presence of these conditions raise serious doubt for the use of Vitamin C in ICU patients.

Plasma Antioxidants

Transferrin binds iron in the Fe III state, and ceruloplasmin (copper storage and transport protein) oxidizes iron from the Fe II state to the Fe III state.

Measurement of reaction products

Lipids are the common substrate for attack by reactive oxygen species (ROS). The breakdown products include malondialdehyde (MDA), conjugated dienes, short-chain alkanes, and lipid hydroperoxides. The most commonly used test to assess MDA concentrations is the thiobarbituric acid reactive substances (TBARS) test. Up to 98% of MDA that reacts with TBARS is formed after collection, causing a severe limitation of this test.

When ROS attack arachidonic acids on cell membranes, isoprostanes (prostaglandin like compounds) are created. The most frequently studied is the aF2-isoprostane. The free form can be measured in urine and plasma, esterified complexes can be measured in tissue, or metabolites can be measured in urine. Blood contains appreciable quantities of arachidonic acid, and this is not a problem when urine is used to run the test.

The ferrous oxidation of xylenol (FOX assay) is based on the oxidation of ferrous iron to ferric iron. The ferric ions can be detected with xylenol, a ferricsensitive dye.

The PUFA test (oxidation of polyunsaturated fatty acids) results from exhalation of ethane gas. There are several limitations with this test, including contamination by air pollution, contamination by isoprene gas (present in human breath), and production of the gases by bacteria.

References

Marino PL. Oxidant Injury. In: The Veterinary ICU Book. Wingfield WE, Raffe MR, editors. Tenton NewMedia, Jackson, WY. 202, pp 24-39.

McMichael M, Moore RM. Ischemia-reperfusion injury pathophysiology, partI. JVECC 14(4) 2004, 231-241.

McMichael M. Ischemia-reperfusion injury: assessment and treatment, partII. JVECC 14(4) 2004, 242-252.

Cassutto BH, Gfeller RW. Use of intravenous lidocaine to prevent reperfusion injury and subsequent multiple organ dysfunction syndrome. JVECC 13(3) 2003, pp 137-148.