Biomedical importance
Free radicals are formed in the body under normal conditions. They cause damage to nucleic acids, proteins, and lipids in cell membranes and plasma lipoproteins. This can cause cancer, atherosclerosis and coronary artery disease, and autoimmune diseases. Epidemiological and laboratory studies have identified a number of protective antioxidant nutrients: selenium, vitamins C and E, β-carotene, and other carotenoids, and a variety of polyphenolic compounds derived from plant foods. Many people take supplements of one or more antioxidant nutrients. However, intervention trials show little benefit of antioxidant supplements except among people who were initially deficient, and many trials of β-carotene and vitamin E have shown increased mortality among those taking the supplements.
Free Radical Reactions Are Self Perpetuating Chain Reactions
Free radicals are highly reactive molecular species with an unpaired electron; they persist for only a very short time (of the order of 10−9 to 10−12 seconds) before they collide with another molecule and either abstract or donate an electron in order to achieve stability. In so doing, they generate a new radical from the molecule with which they collided. The main way in which a free radical can be quenched, so terminating this chain reaction, is if two radicals react together, when the unpaired electrons can become paired in one or other of the parent molecules. This is a rare occurrence, because of the very short half-life of an individual radical and the very low concentrations of radicals in tissues.
The most damaging radicals in biological systems are oxygen radicals (sometimes called reactive oxygen species)— especially superoxide, •O2−, hydroxyl, •OH, and perhydroxyl, •O2H. Tissue damage caused by oxygen radicals is often called oxidative damage, and factors that protect against oxygen radical damage are known as antioxidants.
Radicals Can Damage DNA, Lipids, & Proteins
Interaction of radicals with bases in DNA can lead to chemical changes that, if not repaired, may be inherited in daughter cells. Radical damage to unsaturated fatty acids in cell membranes and plasma lipoproteins leads to the formation of lipid peroxides, precursors of highly reactive dialdehydes that can chemically modify proteins and nucleic acid bases. Proteins are also subject to direct chemical modification by interaction with radicals. Oxidative damage to tyrosine residues in proteins can lead to the formation of dihydroxy phenylalanine that can undergo nonenzymic reactions leading to further formation of oxygen radicals (Figure 1).
Fig1. Tissue damage by radicals.
The total body radical burden can be estimated by measuring the products of lipid peroxidation. Lipid peroxides can be measured by the ferrous oxidation in xylenol orange (FOX) assay. Under acidic conditions, they oxidize Fe2+ to Fe3+, which forms a chromophore with xylenol orange. The dialdehydes formed from lipid peroxides can be measured by reaction with thiobarbituric acid, with which they form a red fluorescent adduct—the results of this assay are generally reported as total thiobarbituric acid reactive substances, TBARS. Peroxidation of n-6 polyunsaturated fatty acids leads to the formation of pentane, and of n-3 polyunsaturated fatty acids to ethane, both of which can be measured in exhaled air.
Radical Damage May Cause Mutations, Cancer, Autoimmune Disease, & Atherosclerosis
Radical damage to the DNA of germline cells in ovaries and testes can lead to heritable mutations; in somatic cells, the result may be initiation of cancer. The dialdehydes formed as a result of radical-induced lipid peroxidation in cell membranes can also modify bases in DNA.
Chemical modification of amino acids in proteins, either by direct radical action or as a result of reaction with the products of radical-induced lipid peroxidation, leads to proteins that are recognized as nonself by the immune system. The resultant antibodies may also cross-react with normal tissue proteins, so initiating autoimmune disease.
Chemical modification of the proteins or lipids in plasma low-density lipoprotein (LDL) leads to abnormal LDL that is not recognized by the liver LDL receptors, and so is not cleared by the liver. Instead, the modified LDL is taken up by macro phage scavenger receptors. Lipid-engorged macrophages infiltrate under blood vessel endothelium (especially when there is already some damage to the endothelium), where they die as a consequence of the toxic levels of unesterified cholesterol they have accumulated. This leads to the development of atherosclerotic plaques, which, in extreme cases, can more or less completely occlude a blood vessel.
There Are Multiple Sources of Oxygen Radicals in the Body
Ionizing radiation (x-rays and UV) can lyse water, leading to the formation of hydroxyl radicals. Transition metal ions, including Cu+, Co2+, Ni2+, and Fe2+ can react nonenzymically with oxygen or hydrogen peroxide, again leading to the formation of hydroxyl radicals. Nitric oxide (an important compound in cell signaling, originally described as the endothelium-derived relaxation factor) is itself a radical, and, more importantly, can react with superoxide to yield peroxynitrite, which decays to form hydroxyl radicals (Figure 2).
Fig2. Sources of radicals.
The respiratory burst of activated macrophages (see Chapter 53) is characterized by the increased utilization of glucose via the pentose phosphate pathway to reduce NADP+ to NADPH, and increased utilization of oxygen to oxidize NADPH to produce cytotoxic oxygen (and halogen) radicals that kill phagocytosed microorganisms. The respiratory burst oxidase (NADPH oxidase) is a flavoprotein that reduces oxygen to superoxide:
NADPH + 2O2 →NADP++ 2•O2 − + 2H+
Plasma markers of radical damage to lipids increase consider ably in response to even a mild infection.
The oxidation of reduced flavin coenzymes in the mitochondrial and microsomal electron transport chains proceeds through a series of steps in which the flavin semiquinone radical is stabilized by the protein to which it is bound, and forms oxygen radicals as transient intermediates. Because of the unpredictable nature of these radical intermediates, there is considerable “leakage” of radicals, and some 3 to 5% of the daily consumption of 30 mol of oxygen by an adult human being is converted to singlet oxygen, hydrogen peroxide, and superoxide, perhydroxyl, and hydroxyl radicals, rather than undergoing complete reduction to water. This results in daily production of about 1.5 mol of reactive oxygen species.
There Are Various Mechanisms of Protection Against Radical Damage
The metal ions that undergo nonenzymic reactions to form oxygen radicals are not normally free in solution, but are bound to either the proteins for which they provide a pros thetic group, or to specific transport and storage proteins, so that they are unreactive. Iron is bound to transferrin, ferritin, and hemosiderin, copper to ceruloplasmin, and other metal ions are bound to metallothionein. This binding to transport proteins that are too large to be filtered in the kidneys also prevents loss of metal ions in the urine.
Superoxide is produced both accidentally and also as the reactive oxygen species required for a number of enzyme catalyzed reactions. A family of superoxide dismutases catalyze the reaction between superoxide and protons to yield oxygen and hydrogen peroxide:
•O2 − + 2H+ → H2O2
The hydrogen peroxide is then removed by catalase and various peroxidases: 2H2O2 → 2H2 O + O2. Most enzymes that produce and require superoxide are contained in the peroxisomes, together with superoxide dismutase, catalase, and peroxidases.
The peroxides that are formed by radical damage to lipids in membranes and plasma lipoproteins are reduced to hydroxy fatty acids by glutathione peroxidase, a selenium-dependent enzyme (hence the importance of adequate selenium intake to maximize antioxidant activity), and oxidized glutathione, which is reduced by NADPH-dependent glutathione reductase. Lipid peroxides are also reduced to fatty acids by reaction with vitamin E, forming the tocopheroxyl radical, which is relatively stable, since the unpaired electron can be located in any one of three positions in the molecule (Figure 3). The tocopheroxyl radical persists long enough to undergo reduction back to tocopherol by reaction with vitamin C at the surface of the cell or lipoprotein. The resultant monodehydroascorbate radical then undergoes enzymic reduction back to ascorbate or a nonenzymic reaction of 2 mol of monodehydroascorbate to yield 1 mol each of ascorbate and dehydroascorbate.
Fig3. The roles of vitamins E and C in reducing lipid peroxides, and stabilization of the tocopheroxyl radical by delocalization of the unpaired electron.
Ascorbate, uric acid, and a variety of polyphenols derived from plant foods act as water-soluble radical trapping antioxidants, forming relatively stable radicals that persist long enough to undergo reaction to nonradical products. Ubiquinone and carotenes similarly act as lipid soluble radical-trapping antioxidants in membranes and plasma lipoproteins.
The Antioxidant Paradox— Antioxidants Can Also Be Pro-Oxidants
Although ascorbate is an antioxidant, reacting with superoxide and hydroxyl to yield monodehydroascorbate and hydrogen peroxide or water, it can also be a source of superoxide radicals by reaction with oxygen, and hydroxyl radicals by reaction with Cu2+ ions (Table 1). However, these pro-oxidant actions require relatively high concentrations of ascorbate, which are unlikely to be reached in tissues, since once the plasma concentration of ascorbate reaches about 30 mmol/L, the renal threshold is reached, and at intakes above about 100 to 120 mg/d the vitamin is excreted in the urine quantitatively with intake.
Table1. Antioxidant and Pro-Oxidant Roles of Vitamin C
A considerable body of epidemiological evidence suggests that carotene is protective against lung and other cancers. However, two major intervention trials in the 1990s showed an increase in death from lung (and other) cancer among people who were given supplements of β-carotene. The problem is that although β-carotene is indeed a radical-trapping antioxidant under conditions of low partial pressure of oxygen, as in most tissues, at high partial pressures of oxygen (as in the lungs) and especially in high concentrations, β-carotene is an autocatalytic pro-oxidant, and hence can initiate radical damage to lipids and proteins.
Epidemiological evidence also suggests that vitamin E is protective against atherosclerosis and cardiovascular disease. However, meta-analysis of intervention trials with vitamin E shows increased mortality among those taking (high dose) supplements. These trials have all used α-tocopherol, and it is possible that the other vitamers of vitamin E that are present in foods, but not the supplements, may be important. In vitro, plasma lipoproteins form lower levels of cholesterol ester hydroperoxide when incubated with sources of low concentrations of perhydroxyl radicals when vitamin E has been removed than when it is present. The problem seems to be that vitamin E acts as an antioxidant by forming a stable radical that persists long enough to undergo metabolism to nonradical products. This means that the radical also persists long enough to penetrate deeper in to the lipoprotein, causing further radical damage, rather than interacting with a water soluble antioxidant at the surface of the lipoprotein.
Nitric oxide and other radicals are important in cell signaling, and especially in signaling for programmed cell death (apoptosis) of cells that have suffered DNA and other dam age. It is likely that high concentrations of antioxidants, rather than protecting against tissue damage, may quench the signaling radicals, and so permit the continued survival of damaged cells, so increasing, rather than decreasing, the risk of cancer development.
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