《食品化学》课程教学资源（文献资料）Antioxidants in health and disease.pdf

Antioxidants in health and disease I S Young, J V Woodside Abstract Free radical production occurs continuously in all cells as part of normal cellular function. However, excess free radical production originating from endogenous or exogenous sources might play a role in many diseases. Antioxidants prevent free radical induced tissue damage by preventing the formation of radicals, scavenging them, or by promoting their decomposition. This article reviews the basic chemistry of free radical formation in the body, the consequences of free radical induced tissue damage, and the function of antioxidant defence systems, with particular reference to the development of atherosclerosis. (J Clin Pathol 2001;54:176–186) Keywords: free radicals; antioxidants; oxidative stress; coronary heart disease; atherosclerosis An antioxidant can be defined as: “any substance that, when present in low concentrations compared to that of an oxidisable substrate, significantly delays or inhibits the oxidation of that substrate”.1 The physiological role of antioxidants, as this definition suggests, is to prevent damage to cellular components arising as a consequence of chemical reactions involving free radicals. In recent years, a substantial body of evidence has developed supporting a key role for free radicals in many fundamental cellular reactions and suggesting that oxidative stress might be important in the pathophysiology of common diseases including atherosclerosis, chronic renal failure, and diabetes mellitus. The aim of this review is to consider mechanisms of free radical formation in the body, the consequences of free radical induced tissue damage, and the function of antioxidant defence systems in health and disease. Free radicals and their chemical reactions A free radical can be defined as any molecular species capable of independent existence that contains an unpaired electron in an atomic orbital.2 The presence of an unpaired electron results in certain common properties that are shared by most radicals. Radicals are weakly attracted to a magnetic field and are said to be paramagnetic. Many radicals are highly reactive and can either donate an electron to or extract an electron from other molecules, therefore behaving as oxidants or reductants. As a result of this high reactivity, most radicals have a very short half life (10−6 seconds or less) in biological systems, although some species may survive for much longer.2 The most important free radicals in many disease states are oxygen derivatives, particularly superoxide and the hydroxyl radical. Radical formation in the body occurs by several mechanisms, involving both endogenous and environmental factors (fig 1). Superoxide (O2 −.) is produced by the addition of a single electron to oxygen, and several mechanisms exist by which superoxide can be produced in vivo.3 Several molecules, including adrenaline, flavine nucleotides, thiol compounds, and glucose, can oxidise in the presence of oxygen to produce superoxide, and these reactions are greatly accelerated by the presence of transition metals such as iron or copper. The electron transport chain in the inner mitochondrial membrane performs the reduction of oxygen to water. During this process free radical intermediates are generated, which are generally tightly bound to the components of the transport chain. However, there is a constant leak of a few electrons into the mitochondrial matrix and this results in the formation of superoxide.4 The activity of several other enzymes, such as cytochrome p450 oxidase in the liver and enzymes involved in the synthesis of adrenal hormones, also results in the leakage of a few electrons into the surrounding cytoplasm and hence superoxide formation. There might also be continuous production of superoxide by vascular endothelium to neutralise nitric oxide,5 6 production of superoxide by other cells to regulate cell growth and diVerentiation,7 and the production of superoxide by phagocytic cells during the respiratory burst.8 Any biological system generating superoxide will also produce hydrogen peroxide as a result of a spontaneous dismutation reaction. In addition, several enzymatic reactions, including those catalysed by glycolate oxidase and Figure 1 Major sources of free radicals in the body and the consequences of free radical damage. Free radical production Transition metals Fe2+, Cu+ Modified DNA bases Tissue damage Lipid peroxidation Protein damage O2 –., H2O2 OH. Endogenous sources •mitochondrial leak •respiratory burst •enzyme reactions •autooxidation reactions Environmental sources •cigarette smoke •pollutants •UV light •ionising radiation •xenobiotics 176 J Clin Pathol 2001;54:176–186 Department of Clinical Biochemistry, Institute of Clinical Science, Grosvenor Road, Belfast, Northern Ireland, BT12 6BJ, UK I S Young Department of Surgery, Royal Free and University College London Medical School, 67–73 Riding House Street, London, W1P 7LD, UK J V Woodside Correspondence to: Professor Young, Department of Clinical Biochemistry, Institute of Clinical Science, Royal Group of Hospitals, Grosvenor Road, Belfast BT12 6BJ, UK I.Young@qub.ac.uk Accepted for publication 5 June 2000 www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

D-amino acid oxidase, might produce hydrogen peroxide directly.9 Hydrogen peroxide is not a free radical itself, but is usually included under the general heading of reactive oxygen species (ROS). It is a weak oxidising agent that might directly damage proteins and enzymes containing reactive thiol groups. However, its most vital property is the ability to cross cell membranes freely, which superoxide generally cannot do.10 Therefore, hydrogen peroxide formed in one location might diVuse a considerable distance before decomposing to yield the highly reactive hydroxyl radical, which is likely to mediate most of the toxic eVects ascribed to hydrogen peroxide. Therefore, hydrogen peroxide acts as a conduit to transmit free radical induced damage across cell compartments and between cells. In the presence of hydrogen peroxide, myeloperoxidase will generate hypochlorous acid and singlet oxygen, a reaction that plays an important role in the killing of bacteria by phagocytes.11 The hydroxyl radical (OH. ), or a closely related species, is probably the final mediator of most free radical induced tissue damage.12 All of the reactive oxygen species described above exert most of their pathological eVects by giving rise to hydroxyl radical formation. The reason for this is that the hydroxyl radical reacts, with extremely high rate constants, with almost every type of molecule found in living cells including sugars, amino acids, lipids, and nucleotides. Although hydroxyl radical formation can occur in several ways, by far the most important mechanism in vivo is likely to be the transition metal catalysed decomposition of superoxide and hydrogen peroxide.13 All elements in the first row of the d-block of the periodic table are classified as transition metals. In general, they contain one or more unpaired electrons and are therefore themselves radicals when in the elemental state. However, their key property from the point of view of free radical biology is their variable valency, which allows them to undergo reactions involving the transfer of a single electron. The most important transition metals in human disease are iron and copper. These elements play a key role in the production of hydroxyl radicals in vivo.13 Hydrogen peroxide can react with iron II (or copper I) to generate the hydroxyl radical, a reaction first described by Fenton in 1894: Fe2+ + H2O2 → Fe3+ + OH. + OH− This reaction can occur in vivo, but the situation is complicated by the fact that superoxide (the major source of hydrogen peroxide in vivo) will normally also be present. Superoxide and hydrogen peroxide can react together directly to produce the hydroxyl radical, but the rate constant for this reaction in aqueous solution is virtually zero. However, if transition metal ions are present a reaction sequence is established that can proceed at a rapid rate: Fe3+ + O2 − → Fe2+ + O2 Fe2+ + H2O2 → Fe3+ + OH. + OH− net result: O2 − + H2O2 → OH− + OH. + O2 The net result of the reaction sequence illustrated above is known as the Haber-Weiss reaction. Although most iron and copper in the body are sequestered in forms that are not available to catalyse this reaction sequence, it is still of importance as a mechanism for the formation of the hydroxyl radical in vivo. The actual reactions, however, may be somewhat more complex than those described above and it is possible that other reactive intermediates such as the ferryl and perferryl radicals might also be formed.12 Approximately 4.5 g of iron can be found in the average adult man, most of which is contained in the haemoglobin molecule and other haem containing proteins. Dietary iron is absorbed preferentially from the proximal part of the small intestine in the divalent form and is transferred to the circulation in which it is carried by transferrin.14 Under most circumstances iron remains tightly bound to one of several proteins, including transferrin, lactoferrin, haem proteins, ferritin, or haemosiderin. In addition, however, it seems likely that a small iron pool will be maintained as complexes with a variety of small molecules, such as nucleotides and citrate within the cytoplasm and subcellular organelles.14 This pool is probably capable of catalysing an iron driven Fenton reaction in vivo. Certainly, these complexes can promote hydroxyl radical formation in vitro.15 Redox reactive iron can be measured using the bleomycin iron assay,16 although it remains unclear to what extent iron detected by this assay correlates with any discrete anatomical or physiological pool. In normal circumstances, no bleomycin reactive iron is detectable in plasma from healthy subjects, implying that transferrin or ferritin bound iron is not available to drive hydroxyl radical production.17 However, transferrin will release its iron at an acidic pH, particularly in the presence of small molecular weight chelating agents such as ADP, ATP, and citrate.15 Such conditions are found in areas of active inflammation and during ischaemia reperfusion injury,18 and it is therefore likely that hydroxyl radicals contribute to tissue damage in these settings. Iron is released from ferritin by reducing agents including ascorbate and superoxide itself,19 20 and hydrogen peroxide can release iron from a range of haem proteins.21 Therefore, although the iron binding proteins eVectively chelate iron and prevent any appreciable redox eVects under normal physiological conditions, this protection can break down in disease states. The role of copper is analogous to that described above for iron.22 23 Although free radical production occurs as a consequence of the endogenous reactions described above and plays an important role in normal cellular function, it is important to remember that exogenous environmental factors can also promote radical formation. Ultraviolet light will lead to the formation of singlet oxygen and other reactive oxygen species in the skin.24 Atmospheric pollutants such as ozone and nitrogen dioxide lead to radical formation and antioxidant depletion in the bronchoalveolar lining fluid, and this may Antioxidants in health and disease 177 www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

exacerbate respiratory disease.25–27 Cigarette smoke contains millimolar amounts of free radicals, along with other toxins.28 Various xenobiotics also cause tissue damage as a consequence of free radical generation, including paraquat,29 paracetamol,30 bleomycin,31 and anthracyclines.32 Antioxidant defence systems Because radicals have the capacity to react in an indiscriminate manner leading to damage to almost any cellular component, an extensive range of antioxidant defences, both endogenous and exogenous, are present to protect cellular components from free radical induced damage. These can be divided into three main groups: antioxidant enzymes, chain breaking antioxidants, and transition metal binding proteins2 (fig 2). THE ANTIOXIDANT ENZYMES Catalase Catalase was the first antioxidant enzyme to be characterised and catalyses the two stage conversion of hydrogen peroxide to water and oxygen: catalase–Fe(III) + H2O2 → compound I compoundI+H2O2 → catalase–Fe(III) + 2H2O+O2 Catalase consists of four protein subunits, each containing a haem group and a molecule of NADPH.33 The rate constant for the reactions described above is extremely high (∼107 M/sec), implying that it is virtually impossible to saturate the enzyme in vivo. Catalase is largely located within cells in peroxisomes, which also contain most of the enzymes capable of generating hydrogen peroxide. The amount of catalase in cytoplasm and other subcellular compartments remains unclear, because peroxisomes are easily ruptured during the manipulation of cells. The greatest activity is present in liver and erythrocytes but some catalase is found in all tissues. Glutathione peroxidases and glutathione reductase Glutathione peroxidases catalyse the oxidation of glutathione at the expense of a hydroperoxide, which might be hydrogen peroxide or another species such as a lipid hydroperoxide34: ROOH + 2GSH → GSSG + H2O + ROH Other peroxides, including lipid hydroperoxides, can also act as substrates for these enzymes, which might therefore play a role in repairing damage resulting from lipid peroxidation. Glutathione peroxidases require selenium at the active site, and deficiency might occur in the presence of severe selenium deficiency.35 Several glutathione peroxidase enzymes are encoded by discrete genes.36 The plasma form of glutathione peroxidase is believed to be synthesised mainly in the kidney.37 Within cells, the highest concentrations are found in liver although glutathione peroxidase is widely distributed in almost all tissues. The predominant subcellular distribution is in the cytosol and mitochondria, suggesting that glutathione peroxidase is the main scavenger of hydrogen peroxide in these subcellular compartments. The activity of the enzyme is dependent on the constant availability of reduced glutathione.38 The ratio of reduced to oxidised glutathione is usually kept very high as a result of the activity of the enzyme glutathione reductase: GSSG + NADPH + H+ → 2GSH + NADP+ The NADPH required by this enzyme to replenish the supply of reduced glutathione is provided by the pentose phosphate pathway. Any competing pathway that utilises NADPH (such as the aldose reductase pathway) might lead to a deficiency of reduced glutathione and hence impair the action of glutathione peroxidase. Glutathione reductase is a flavine nucleotide dependent enzyme and has a similar tissue distribution to glutathione peroxidase.39 Superoxide dismutase The superoxide dismutases catalyse the dismutation of superoxide to hydrogen peroxide: O2 − + O2 − + 2H+ → H2O2 + O2 The hydrogen peroxide must then be removed by catalase or glutathione peroxidase, as described above. There are three forms of superoxide dismutase in mammalian tissues, each with a specific subcellular location and diVerent tissue distribution. (1) Copper zinc superoxide dismutase (CuZnSOD): CuZnSOD is found in the cytoplasm and organelles of virtually all mammalian cells.40 It has a molecular mass of approximately 32 000 kDa and has two protein subunits, each containing a catalytically active copper and zinc atom. (2) Manganese superoxide dismutase (MnSOD): MnSOD is found in the mitochondria of almost all cells and has a molecular mass of 40 000 kDa.41 It consists of four protein subunits, each probably containing a single manganese atom. The amino acid sequence of MnSOD is entirely dissimilar to that of CuZnSOD Figure 2 Antioxidant defences against free radical attack. Antioxidant enzymes catalyse the breakdown of free radical species, usually in the intracellular environment. Transition metal binding proteins prevent the interaction of transition metals such as iron and copper with hydrogen peroxide and superoxide producing highly reactive hydroxyl radicals. Chain breaking antioxidants are powerful electron donors and react preferentially with free radicals before important target molecules are damaged. In doing so, the antioxidant is oxidised and must be regenerated or replaced. By definition, the antioxidant radical is relatively unreactive and unable to attack further molecules. Free radical production Chain breaking antioxidants •Directly scavenge free radicals •Consumed during scavenging process Lipid phase •Tocopherols •Ubiquinol •Carotenoids •Flavonoids Aqueous phase •Ascorbate •Urate •Glutathione and other thiols Transition metals Fe2+, Cu+ Tissue damage Repair mechanisms O2 –., H2O2 OH. Enzyme antioxidants •Superoxide dismutases •Catalase •Glutathione peroxidase •Caeruloplasmin Metal binding proteins •Transferrin •Ferritin •Lactoferrin 178 Young, Woodside www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

and it is not inhibited by cyanide, allowing MnSOD activity to be distinguished from that of CuZnSOD in mixtures of the two enzymes. (3) Extracellular superoxide dismutase (ECSOD): EC-SOD was described by Marklund in 198242 and is a secretory copper and zinc containing SOD distinct from the CuZnSOD described above. EC-SOD is synthesised by only a few cell types, including fibroblasts and endothelial cells, and is expressed on the cell surface where it is bound to heparan sulphates. EC-SOD is the major SOD detectable in extracellular fluids and is released into the circulation from the surface of vascular endothelium following the injection of heparin.43 EC-SOD might play a role in the regulation of vascular tone, because endothelial derived relaxing factor (nitric oxide or a closely related compound) is neutralised in the plasma by superoxide.44 THE CHAIN BREAKING ANTIOXIDANTS Whenever a free radical interacts with another molecule, secondary radicals may be generated that can then react with other targets to produce yet more radical species. The classic example of such a chain reaction is lipid peroxidation, and the reaction will continue to propagate until two radicals combine to form a stable product or the radicals are neutralised by a chain breaking antioxidant.45 Chain breaking antioxidants are small molecules that can receive an electron from a radical or donate an electron to a radical with the formation of stable byproducts.46 In general, the charge associated with the presence of an unpaired electron becomes dissociated over the scavenger and the resulting product will not readily accept an electron from or donate an electron to another molecule, preventing the further propagation of the chain reaction. Such antioxidants can be conveniently divided into aqueous phase and lipid phase antioxidants. Lipid phase chain breaking antioxidants These antioxidants scavenge radicals in membranes and lipoprotein particles and are crucial in preventing lipid peroxidation. The most important lipid phase antioxidant is probably vitamin E.47 Vitamin E occurs in nature in eight diVerent forms, which diVer greatly in their degree of biological activity. The tocopherols (á, â, ã, and ä) have a chromanol ring and a phytyl tail, and diVer in the number and position of the methyl groups on the ring. The tocotrienols (á, â, ã, and ä) are structurally similar but have unsaturated tails. Both classes of compounds are lipid soluble and have pronounced antioxidant properties.48 They react more rapidly than polyunsaturated fatty acids with peroxyl radicals and hence act to break the chain reaction of lipid peroxidation. In addition to its antioxidant role, vitamin E might also have a structural role in stabilising membranes.49 Frank vitamin E deficiency is rare in humans, although it might cause haemolysis50 and might contribute to the peripheral neuropathy that occurs in abetalipoproteinaemia.51 The absorption, transport, and regulation of plasma concentrations of vitamin E in humans has been reviewed by Kayden and Traber,52 although in general the metabolism of vitamin E is not well described. In cell membranes and lipoproteins the essential antioxidant function of vitamin E is to trap peroxyl radicals and to break the chain reaction of lipid peroxidation.53 Vitamin E will not prevent the initial formation of carbon centred radicals in a lipid rich environment, but does minimise the formation of secondary radicals. á-Tocopherol is the most potent antioxidant of the tocopherols and is also the most abundant in humans. It quickly reacts with a peroxyl radical to form a relatively stable tocopheroxyl radical, with the excess charge associated with the extra electron being dispersed across the chromanol ring. This resonance stabilised radical might subsequently react in one of several ways. á-Tocopherol might be regenerated by reaction at the aqueous interface with ascorbate54 or another aqueous phase chain breaking antioxidant, such as reduced glutathione or urate.55 Alternatively, two á-tocopheroxyl radicals might combine to form a stable dimer, or the radical may be completely oxidised to form tocopherol quinone. The carotenoids are a group of lipid soluble antioxidants based around an isoprenoid carbon skeleton.56 The most important of these is â-carotene, although at least 20 others may be present in membranes and lipoproteins. They are particularly eYcient scavengers of singlet oxygen,57 but can also trap peroxyl radicals at low oxygen pressure with an eYciency at least as great as that of á-tocopherol. Because these conditions prevail in many biological tissues, the carotenoids might play a role in preventing in vivo lipid peroxidation.58 The other important role of certain carotenoids is as precursors of vitamin A (retinol). Vitamin A also has antioxidant properties,59 which do not, however, show any dependency on oxygen concentration. Flavonoids are a large group of polyphenolic antioxidants found in many fruits, vegetables, and beverages such as tea and wine.60–62 Over 4000 flavonoids have been identified and they are divided into several groups according to their chemical structure, including flavonols (quercetin and kaempherol), flavanols (the catechins), flavones (apigenin), and isoflavones (genistein). Epidemiological studies suggest an inverse relation between flavonoid intake and incidence of chronic diseases such as coronary heart disease (CHD).63–65 However, little is currently known about the absorption and metabolism of flavonoids and epidemiological associations might be a consequence of confounding by other factors. Available evidence suggests that the bioavailability of many flavonoids is poor,66–68 and plasma values very low, although there is some evidence that augmenting the intake of flavonoids might improve biochemical indices of oxidative damage.68 69 Apart from flavonoids, other dietary phenolic Antioxidants in health and disease 179 www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com

compounds might also make a small contribution to total antioxidant capacity.70 Ubiquinol-10, the reduced form of coenzyme Q10, is also an eVective lipid soluble chain breaking antioxidant.71 Although present in lower concentrations than á-tocopherol, it can scavenge lipid peroxyl radicals with higher eYciency than either á-tocopherol or the carotenoids, and can also regenerate membrane bound á-tocopherol from the tocopheryl radical.72 Indeed, whenever plasma or isolated low density lipoprotein (LDL) cholesterol is exposed to radicals generated in the lipid phase, ubiquinol-10 is the first antioxidant to be consumed, suggesting that it might be of particular importance in preventing the propagation of lipid peroxidation.73 However, work to clarify further its role has been hampered by the ease with which ubinquinol-10 becomes oxidised during sample handling or analysis. Aqueous phase chain breaking antioxidants These antioxidants will directly scavenge radicals present in the aqueous compartment. Qualitatively the most important antioxidant of this type is vitamin C (ascorbate).74 In humans, ascorbate acts as an essential cofactor for several enzymes catalysing hydroxylation reactions. In most cases, it provides electrons for enzymes that require prosthetic metal ions in a reduced form to achieve full enzymatic activity. Its best known role is as a cofactor for prolyl and lysyl oxidases in the synthesis of collagen. However, in addition to these well defined actions, several other biochemical pathways depend upon the presence of ascorbate.75 In addition to its role as an enzyme cofactor, the other major function of ascorbate is as a key chain breaking antioxidant in the aqueous phase.76 Ascorbate has been shown to scavenge superoxide, hydrogen peroxide, the hydroxyl radical, hypochlorous acid, aqueous peroxyl radicals, and singlet oxygen. During its antioxidant action, ascorbate undergoes a two electron reduction, initially to the semidehydroascorbyl radical and subsequently to dehydroascorbate. The semidehydroascorbyl radical is relatively stable owing to dispersion of the charge associated with the presence of a single electron over the three oxygen atoms, and it can be readily detected by electron spin resonance in body fluids in the presence of increased free radical production.77 Dehydroascorbate is relatively unstable and hydrolyses readily to diketogulonic acid, which is subsequently broken down to oxalic acid. Two mechanisms have been described by which dehydroascorbate can be reduced back to ascorbate; one is mediated by the selenoenzyme thioredoxin reductase78 and the other is a non-enzyme mediated reaction that uses reduced glutathione.79 Dehydroascorbate in plasma is probably rapidly taken up by red blood cells before recycling, so that very little, if any, dehydroascorbate is present in plasma.80 Apart from ascorbate, other antioxidants are present in plasma in high concentrations. Uric acid eYciently scavenges radicals, being converted in the process to allantoin.81 Urate might be particularly important in providing protection against certain oxidising agents, such as ozone.82 Indeed, it has been suggested that the increase in life span that has occurred during human evolution might be partly explained by the protective action provided by uric acid in human plasma.83 Part of the antioxidant eVect of urate might be attributable to the formation of stable non-reactive complexes with iron, but it is also a direct free radical scavenger. Albumin bound bilirubin is also an eYcient radical scavenger,84 and it has been suggested that it might play a particularly crucial role in protecting the neonate from oxidative damage,85 because deficiency of other chain breaking antioxidants is common in the newborn. The other major chain breaking antioxidants in plasma are the protein bound thiol groups. The sulphydryl groups present on plasma proteins can function as chain breaking antioxidants by donating an electron to neutralise a free radical, with the resultant formation of a protein thiyl radical. Albumin is the predominant plasma protein and makes the major contribution to plasma sulphydryl groups, although it also has several other antioxidant properties.86 Albumin contains 17 disulphide bridges and has a single remaining cysteine residue, and it is this residue that is responsible for the capacity of albumin to react with and neutralise peroxyl radicals.87 This property is important in view of the role albumin plays in transporting free fatty acids in the blood. In addition, albumin has the capacity to bind copper ions and will inhibit copper dependent lipid peroxidation and hydroxyl radical formation. It is also a powerful scavenger of the phagocytic product hypochlorous acid, and provides the main plasma defence against this oxidant.88 Because albumin itself is damaged when it acts as an antioxidant, it has been viewed as a sacrificial molecule that prevents damage occurring to more vital species.86 The high plasma concentration of albumin and a relatively short half life mean that any damage suffered is unlikely to be of biological importance. However, in vitro work has shown that protein thiyl radicals can themselves act as a potential source of reactive oxidants. The thiyl radical can abstract an electron from a polyunsaturated fatty acid to initiate the process of lipid peroxidation,89 a reaction that can be inhibited by ascorbate and retinol. The antioxidant eVects of albumin and other proteins have been shown to decrease at high concentrations and it has been suggested that this is because thiyl radicals can oxidatively damage other molecules. The importance of these findings to the antioxidant role of albumin in vivo remains unclear. Reduced glutathione (GSH) is a major source of thiol groups in the intracellular compartment but is of little importance in the extracellular space.90 GSH might function directly as an antioxidant, scavenging a variety of radical species, as well as acting as an essential factor for glutathione peroxidase (discussed above). Thioredoxin might also function as a 180 Young, Woodside www.jclinpath.com Downloaded from jcp.bmj.com on February 16, 2012 - Published by group.bmj.com