Ann. din. Biochem. 13 (1976) 393-398
Superoxide Dismutase (Erythrocuprein) and Free Radicals in Clinical Chemistry
J. M. C.
GUTTERIDGE
Department of Clinical Chemistry, Whittington Hospital, London N19
Erythrocuprein (superoxide dismutase) has recently been shown to have an enzymic function towards superoxide anions. The discovery of superoxide dismutase, its mode of action, and estimation are reviewedalong with a brief introduction to oxygen activation and free-radical chemistry. The formation, activity, and destruction of oxygen free radicals in white blood cells, red blood cells, and subcellular particles are discussed. (a) The production of superoxide anions by white cells during phagocytosis is thought to be advantageous for the overall bactericidal event. (b) Normal red blood cells generate low levels of superoxide anions. Increased levelsof free-radical production could playa significantrole in acceleratingcell ageing (haemolysis). (c) Subcellular particles produce superoxide anions. These as well as organic peroxides have been implicated in drug hydroxylation reactions involving cytochrome P-450. Superoxide dismutase is a recent enzymic function ascribed to the proteins previously known as cuproproteins. Erythrocuprein (haemocuprein) was first isolated in 1938 as a blue-green protein from ox blood. It had a molecular weight of 35 000 and contained 0.38 % copper (Mann and Keiling, 1938). Because it had no discernible activity its name was derived from its source and copper content. Similar cuproproteins were isolated from diverse sources and carefully characterised. The nomenclature of these proteins merely reflected the tissue of origin and the content of copper-hence cerebrocuprein, erythrocuprein, and hepatocuprein (Porter and Ainsworth, 1959; Kimmel et al., 1959; Porter et al., 1964). While much work was spent on characterising these macromolecules as interesting proteins, it was discovered that as well as 2 atoms of copper they contained 2 atoms per molecule of zinc (Carrico and Deutsch, 1910). A more serendipitous line of inquiry finally led to the discovery by McCord and Fridovitch (1969) of an enzymic function for erythrocuprein. They were able to show that this protein had specific catalytic activity toward free radical ions-superoxide anions (02'-) converting two molecules (dismutation) to hydrogen peroxide and oxygen (Fig. la). a
O' 2"
+
O' 2"
+
2H + -+ 02
+
H202
superoxide dismutase
FREE RADICALS
When two atoms or molecules (represented as X and Y) are covalently bonded to form the molecule XY, stability of the molecule is achieved by electron pairing, each donating and sharing an electron. X:Y-+X'
+ v-
Breaking of this bond is known as homolytic fission
(homolysis), giving rise to two free radicals X' and Y', Free radicals are therefore odd-electron atoms or molecules. A bold point (') is used to indicate the presence of the unpaired electron. The energy required for the dissociation of a covalent bond may be provided by (a) thermal sources, (b) electromagnetic radiation, or (e) redox coupling with a source of free radicals. The majority of free radicals are electrically neutral, though free radical ions such as the superoxide anion do exist. They are usually extremely reactive and paramagnetic-a property utilised for detecting their presence. A reaction which proceeds by a free-radical mechanism can be inhibited by compounds which combine with free radicals; these are known as free-radical scavengers or antioxidants. What is a superoxide anion and where is it found in biological systems? Oxygen is not a reactive molecule; in order to take part in reactions it has to undergo a stepwise reduction; this is usually achieved by the univalent addition of electrons. Complete reduction of oxygen to water is achieved by the addition of 4 electrons. The first step in the activation of oxygen requires an input of energy and results in the formation of the hydroperoxyl radical (HO' 2), the ion of which is the superoxide anion (02'-). Further additions of electrons give sequentially peroxide (02=) and the hydroxyl radical (OH') with energy liberation. Once produced, the superoxide anions can form hydrogen peroxide (by either enzymic or spontaneous dismutation (Fig. la). The hydrogen peroxide can then itself react with a superoxide anion to give the hydroxyl radical (OH") (Fig. l c) (Harber and Weiss, 1934). The enzyme
c superoxide dismutase is clearly the first line of 393
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394 J. M. C. Gutteridge
"antioxidant" defence in aerobically metabolising cells against "activated" oxygen. To test this hypothesis Fridovitch carried out a detailed study in bacteria, examining aerobes, aerotolerant anaerobes, and obligate anaerobes for the presence of superoxide dismutase (Gregory and Fridovitch, 1973). As a general rule superoxide dismutases were present in aerobic bacteria, absent in obligate anaerobes, but present in low levels and inducible by molecular oxygen in aerotolerant anaerobes. Studies in bacteria led to the discovery of quite different trace-metal-dependent superoxide dismutases. They were shown to contain both an iron--containing ferrienzyme (Yost and Fridovitch, 1973) and a managanese-d.ependent enzyme (Keele et al., 1970). The manganese enzyme was reddish rather than blue in colour and had a higher molecular weight and different primary structure, though its catalytic properties remained unchanged. Several isoenzyrnes have been demonstrated in extracts from bacteria and chicken liver using polyacrylamide gels. Chicken liver contained both the copper enzyme and the manganese enzyme, the former in the cell cytosol, the latter entirely in the mitochondria (Weisiger and Fridovitch, 1973). The metals associated with the different superoxide dismutases are all transitional trace metalsthat is, they occur in period 4 and have variable valencies. Electrons are readily transferred from the third to the fourth electron shell in oxidising conditions and from the fourth to the third during reducing conditions. The catalytic mechanisms of this enzyme depend on the sequential reduction and re-oxidation of the metal at the active centre (Fridovitch, 1974). MEASUREMENT OF SUPEROXIDE DISMUTASE ACTIVITY
It is not possible to measure enzymic activity by either disappearance of substrate or formation of an end product under normal laboratory conditions. This is because the substrate has to be continuously produced in the reaction mixture and itself undergoes spontaneous dismutation to hydrogen peroxide. An indirect assay system is therefore used in which a scavenger is added to intercept the generated superoxide anion. If superoxide dismutase is then introduced into the system it will compete with both the spontaneous dismutation of 02'- and also interception of the radical by the scavenger molecule. Superoxide anions can be generated in the reaction by either photoreduction of riboflavin and oxygen (Baeuchamp and Fridovitch, 1971) or enzymically with xanthine oxidase (McCord and
Fridovitch, 1969). The latter technique utilises the capability of xanthine oxidase to bring about the univalent reduction of oxygen (Fig. Id). Scavengers d
Xanthine
+
02
---+
0'2
+
Uric Acid
xanthine oxidase
are selected which are reduced by 02"- and also act as indicators by undergoing a colour change in the process. Those most frequently used are cytochrome C and nitro blue tetrazolium (McCord and Fridovitch, 1969; Beauchamp and Fridovitch, 1971). One unit of superoxide dismutase activity has been defined as the amount of enzyme which will inhibit 50 % of the rate of reduction of the indicating scavenger under specified conditions. FORMATION OF ACTIVATED OXYGENS IN VIVO
The toxicity of oxygen has been recognised for some time now (Raiha, 1955; Mengel and Kann, 1966); but the mechanism by which the deleterious effects were brought about are only now being fully understood. In this respect the discovery of an enzyme capable of dismutasing oxygen free radicals was a major breakthrough. It is almost certain that wherever oxygen is aerobically metabolised by univalent electron reductions active oxygens are formed. The generation of free radicals and active oxygen have now been shown to accompany various normal physiological and abnormal pathological processes in the body. (a) White blood cells
The recognition by Babior, Kipnes and Curnutte (1973) that phagocytosing leucocytes generated superoxide anions which were released into the surrounding medium led to the postulation that either the superoxide anion or its dismutation to hydrogen peroxide were themselves important components of the bactericidal event (Curnutte and Babior, 1974; Johnston et al., 1975). A second possibility is that superoxide anions or hydrogen peroxide are involved in a free radical oxidation of intra- or extracellular unsaturated lipids. Lipid peroxidation would then result in the formation of numerous cytotoxic compounds (Stossel et al., 1974; Shohet et al., 1974; Gutteridge, 1974). Many peroxidic and aldehydic compounds are formed during lipid peroxidation; these have extremely potent antibacterial properties (Gutteridge et al., 1974, 1976). Much of the data supporting the involvement of lipid peroxidation in leucocytes as an essential part of bacterial killing is derived from experiments carried out on white blood cells from patients with
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Superoxide dismutase (erythrocuprein) and free radicals in clinical chemistry the rare hereditary disorder, chronic granulomatous disease. White blood cells from patients with this disease are defective in killing ingested bacteria. Along with many biochemical abnormalities detected in these cells they have been shown to be defective in generating superoxide anions and hydrogen peroxide and deficient in the enzyme glutathione peroxidase (Curnutte et al., 1974; Holmes et al., 1967; Holmes et al., 1970). Some evidence against the advantagous involvement of lipid peroxidation in bacterial killing during phagocytosis could be interpreted from the results of Serfass and Ganther (1975). A deficiency of glutathione peroxidase was produced in rat leucocytes by feeding a seleniumdeficient diet. The trace metal selenium is an integral part of glutathione peroxidase (an enzyme which destroys lipid peroxides and hydrogen peroxide). This resulted in an impaired leucocyte ability to kill ingested yeast cells. The above authors suggest this was due to the damaging effect of lipid peroxides on cell function. Liberation by white blood cells of both hydrogen peroxide and superoxide anions into extracellular fluids (which contain insignificant amounts of either superoxide dismutase or catalase) could result in the formation of the highly reactive hydroxyl radical OH' (McCord, 1974; Salin and McCord, 1975). McCord suggests that this species could be the agent responsible for inflammatory conditions such as those found in arthritic joints. He was able to show that enzymic generation of OH' in vitro could degrade purified hyaluronic acid and bovine synovial fluid. (b) Red blood cells
The red blood cell is of particular interest since its function is one of oxygen transport and its premature ageing (haemolysis) in various clinical conditions well documented. It has been shown that red blood cells in certain clinical conditions are more susceptible to oxidative stress than normal cells (Dodge et al., 1967; Stocks et al., 1972). The standard stress test consists in incubating washed red blood cells in vitro with dilute hydrogen peroxide under carefully controlled conditions (Stocks and Dormandy, 1971). The generation of free radicals within the system attacks the cell membrane lipids, resulting in their peroxidation, The degree of oxidation is related to the formation of a lowmolecular-weight water-soluble carbonyl malonyldialdehyde, which is measured colorimetrically. In normal red blood cells a continuous production of superoxide anions is brought about by the autoxidation of oxyhaemoglobin to methaemoglobin (Misra and Fridovitch,1972). Under normal con-
395
ditions it has been estimated that about 3 % of the total body haemoglobin is converted to methaemoglobin each day (Carrell et al., 1975). Rapid reduction back to haemoglobin is brought about by the enzyme methaemoglobin reductase. The above authors observed that increased formation of methaemoglobin associated with such factors as increased heat, certain drugs, and the abnormal unstable haemoglobins is invariably accompanied by increased superoxide radical production and release. A deleterious role for activated oxygen in the condition erythropietic protophorphyria has been proposed by Lamola et al. (1973). They suggest that photo-irradiation in the presence of free porphyrins leads to the production of singlet oxygen (02·), which oxidises cholesterol leading to premature haemolysis of the red blood cell. Incubation of red blood cells from vitamin-Edeficient animals with hyperbaric oxygen results in haemolysis and oxidation of lipids (Mengel et al., 1964; Mengel and Kann, 1966). In our own laboratory lipid peroxidation with hyperbaric oxygen has been achieved using normal (vitamin-E-deficient) human umbilical-cord red blood cells (Gutteridge, 1974).
(c) Subcellular particles The reduction of oxygen to water via the respiratory chain is a fundamental biochemical process for energy conservation. During oxidoreduction reactions in mitochondria and microsomes univalent reduction of oxygen does occur with the production of the superoxide radical (Zimmerman et al., 1973; Aust et 01., 1972). The ability of microsomal cytochrome P--450 to carry out hydroxylation reactions may also depend on the production of superoxide anions (Strobel and Coon, 1971). This interpretation is based on the finding that superoxide dismutase when added to the cytochrome P--450 catalysed reaction inhibited drug hydroxylation. As well as oxygen activation a process of substrate activation may also occur. Recent reports suggest that the ability of liver microsomal suspensions to utilise organic peroxides for the hydroxylation of various substrates is catalysed by cytochrome P--4S0 (Rahimtula and O'Brien, 1975; Hrycay and O'Brien, 1972; Kadlubar et al., 1973). This peroxidase activity of cytochrome P--450 with organic peroxides has also been shown to occur with hydrogen peroxide (Coon et al., 1975). Studies in our own laboratory have shown that an incubating ox-brain homogenate (Stocks et al., 1974) undergoes rapid lipid peroxidation if electron transport is maintained.
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396 J. M. C. Gutteridge DESTRUCTION OF ACTlVATED OXYGEN IN VIVO
The highly reactive chemical nature of free radicals can lead to molecular damage in biological material. Free radicals are known to damage proteins, particularly if they contain sulphydryl groups, and unsaturated lipids (Little and O'Brien, 1967; Jacob and Lux, 1968; Slater, 1972). The first line of defence in oxygen-metabolising cells against active oxygen is the enzyme superoxide dismutase, which brings about the formation of oxygen and hydrogen peroxide (Fig. I a). The hydrogen peroxide can then be destroyed by the catalytic activity of catalase (Fig. I b) or glutathione peroxidase (Fig. Ie) (Nitowsky 2H202
b
--+
02
catalase
e
2GSH
+
H202
--+
+
2H20
GSSG
+
2H20
glutathione peroxidase
and Tildon, 1956; Gross et al., 1967). Should a superoxide anion react with hydrogen peroxide it could result in the formation of the highly reactive hydroxyl radical (OH') (Fig. lc), Its removal would seem to depend on a second line of defence such as the chemical antioxidants vitamin E, ascorbic acid, purine bases, and related compounds.
functions of this copper protein are not yet clear and may be related to free radical scavenging or dismutation of superoxide anions (Stocks et al., 1976). Increased serum levels of the acute reacting protein caeruloplasmin in rheumatoid arthritis (Lorber et al., 1968) may give some support to McCord's postulation that free radicals are involved in inflammatory states. Free radicals are transient and the most elusive of chemical species. In spite of this the discovery of superoxide dismutase a few years ago has initiated great interest and a detailed search for functions and abnormalities related to activated oxygens, free radicals, and superoxide-disrnutasing enzymes. As a speculative footnote, it seems extremely surprising that the body should rely on chemical scavenging for the inactivation of one of the most active oxidants known-the hydroxyl radical (OH"). Perhaps there are specific proteins involved in the inactivation or dismutation of hydroxyl radicals yet to be characterised? The implication of activated oxygens in disease offers an entirely new field of study for clinical scientists, some of whom find themselves involved in free radical chemistry.
SUMMARY AND SPECULATIVE CONCLUSIONS
REFERENCES
The discovery of a ubiquitous free radical dismutasing enzyme in oxygen-metabolising cells suggests by implication that activated oxygens and free radicals are produced in living systems. Should the cell's defences be impaired or the production of radicals significantly increased, damage could result in premature ageing and cell degeneration. Red blood cells are well protected with antioxidants such as vitamin E and the enzymes superoxide dismutase, catalase, and glutathione peroxidase. In spite of this, activated oxygene are implicated in certain haemolytic conditions, and red blood cells from different clinical groups are more susceptible to peroxidative stress than normal controls (Carrell et al., 1975; Stocks et al., 1972). As well as cellular abnormalities, changes in extracellular fluids may be important, as these do not contain appreciable amounts of superoxide dismutase and catalase (McCord, 1974). In extracellular fluids therefore other proteins may have an important antioxidant function. The copper--containing protein (enzyme) present in serum caeruloplasmin (copper oxidase) acts as an antioxidant by inhibiting lipid peroxidation in an incubating ox-brain homogenate (Stocks et al., 1974). This can be partly explained by its ferroxidase activity (Curzon and O'Reilly, 1960). Other antioxidant
Aust, S. D., Roerig, D. L.. Pederson. T. C. Evidence or superoxide generation by NADPH-eytochrome C reductase of rat liver microsomes, Biochem. biophys. Res. Commun., 47 (1972) 1133. Babior, B. M., Kipnes, R. S., Curnutte, J. T. The production by Ieucocytes of superoxide, A potential bactericidal agent, J. din. Invest., 52 (1973) 741. Beauchamp, c., Fridovitch, 1. Superoxide dismutase; improved assays and an assay applicable to acrylamide gels. Analyt, Biochem., 44 (1971) 276.
Carrell, R. W., Winterbourn, C. c, Racbmilewitz, E. A. Activated oxygen and haemolysis (Annotation). Brit. J. Haemat., 30 (1975) 259.
Carrico, R. J., Deutsch, H. F. The presence of zinc in human cytocuprein and some properties of the apoprotein, J. bioi. Chem., 245 (1970) 723.
Coon, M. J., Nordblom, G. D., White, R. E., Haugen, D. A. Purified liver microsomal cytochrome P-4S0: Catalytic mechanism and characterization of multiple forms. Biochem. Soc. Trans. 3 (1975) 813. Curnutte, J. T., Babior, B. M. The effect of bacteria and serum on superoxide production by granulocytes. J. clin. Invest. 53 (1974) 1662.
Curnutte, J. T., Whitten, D. M., Babior, B. M. Defective superoxide production by granulocytes from patients with chronic granulomatous disease. New Engl. J. Med. 290 (1974) 593.
Curzon, G., O'Reilly, S. A coupled iron caeruloplasmin oxidation system. Blochem. Biophys. Res. Commun. Z (1960) 284.
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Superoxide dismutase (erythrocuprein) and free radicals in clinical chemistry 397 Dodge, J. T., Cohen, G., Kayden, H. J., Phillips, G. B. Peroxidative haemolysis of red blood cells from patients with Abeta Iipoproteinemia (Acanthocytosis), J. din. Invest. 4(j (1967) 357. Fridovitch, I. In Advances in Enzymology, Vol. 41, Ed. Meister, London, Interscience, 1974. Gregory, E. M., Fridovitch, I. Oxygen toxicity and the superoxide dismutase. J. Bact. 114 (1973) 1193. Gross, R. T., Bracci, R., Rudolph, N., Schroeder, E., Kochen, J. A. Hydrogen peroxide toxicity and detoxification in the erythrocytes of the new born infants. Blood 29 (1967) 381. Gutteridge, J. M. C. Consequences of lipid autoxidation in biological material. Ph.D. Thesis, London University, 1974. Gutteridge, J. M. c., Lamport, P., Dormandy, T. L. Autoxidation as a cause of antibacterial activity in unsaturated fatty acids. J. med. Microbiol. 7 (1974) 387. Gutteridge, J. M. c., Lamport, P., Dormandy, T. L. The antibacterial effect of water-soluble compounds from autoxidising linolenic acid. J. med. Microbiol. 9 (1976) 105. Harber, F., Weiss, J. The catalytic decomposition of hydrogen peroxide by iron salts, Proc. roy. Soc. A 146 (1934) 332. Holmes, B., Page, A. R., Good, R. A. Studies of the metabolic activity of leucocytes from patients with a genetic abnormality of phagocytic function. J. elin. Invest. 4(j (1967) 1422. Holmes, B., Park, B. H., Malawista, S. E., Quie, P. G., Nelson, D. L., Good, R. A. Chronic granulomatous disease in females: A deficiency of leucocyte glutathione peroxidase. New Engl.J. Med. 283 (1970) 217. Hrycay, E. G., O'Brien, P. J. Cytochrome P--450 as a microsomal peroxidase in steroid hydroperoxide reduction. Arch. Biochem. Biophys. 153 (1972) 480. Jacob, H. S., Lux, S. E. Degradation of membrane phospholipids and thiols in peroxide hemolysis: Studies in Vitamin E deficiency. Blood 32 (1968) 549. Johnston, R. B., Jr., Keele, B. B., Jr., Misra, H. P., Lehmeyer, J. E., Webb, L. S., Baehner, R. L., Rajogopalan, K. V. The role of superoxide anion generation in phagocytic bactericidal activity. J. clln, Invest. SS (1975) 1357. Kadlubar, F. F., Morton, K. C., Ziegler, D. M. Microsomal--catalysed hydroperoxide-dependent c-oxidation of amines. Biochem. Biophys. Res. Commun. 54 (1973) 1255. Keele, B. B., Jr., McCord, J. M., Fridovitch, I. Superoxide dismutase from Escherichia coli B, a new manganese containing enzyme. J. bioi Chem. 24S (1970) 6176. Kimmel, J. R., Markowitz, H., Brown, D. M. The isolation of the copper-containing protein cerebrocuprein from normal human brain. J. bioi. Chem, 234 (1959) 46. Lamola, A. A., Yamane, T., Trozzolo, A. M. Cholesterol hydroperoxide formation in red cell membranes and photohernolysis in erythropoietic protoporphyria, Science 179 (1973) 1131. Little, G., O'Brien, P. J. Effectiveness of a lipid peroxide
in oxidising proteins and nonprotein thioI. Biochem. J. 106 (1967) 419. Lorber, A., Cutler, L. S., Chang, C. C. Serum copper levels in rheumatoid arthritis. Relationship of elevated copper to protein alterations. Arthr. and Rheum. 11 (1968) 65. Mann, T., Keilin, D. Haemocruprein and hepatocuprein. Copper-protein compounds of blood and liver of mammals, Proc. roy. Soc. B 126 (1938) 303. Mengel, C. E., Karin, H. E., Smith, W., Horton, B. Effects of in vivo hyperoxia on erythrocytes. Hemolysis in mice exposed tohyerbaric oxygenation. Proc. Soc. expo Bioi. (N. Y.) 116 (1964) 259. Mengel, C. E., Kann, H. E., Jr. Effects of in vivo hyperoxia on erythrocytes. III. In vivo peroxidation of erythrocyte lipid. J. din. Invest. 4S (1966) 1150. McCord, J. M., Fridovitch, I. Superoxide dismutase, an enzymic function for erythrocuprein (haemocuprein), J. bioi. Chern. 244 (1969) 6049. McCord, J. M. Free radicals and inflammation: Protection of synovial fluid by superoxide dismutase. Science ISS (1974) 529. Misra, H. P., Fridovitch, I. The generation of superoxide radical during the autoxidation of hemoglobin. J. bioi. Chem. 247 (1972) 6960. Nitowsky, H. M., Tildon, J. T. Some studies of tocopherol deficiency in infants and children. III. Relation of blood catalase activity and other factors to hemolysis of erythrocytes and hydrogen peroxide. Amer. J. din. Nutr. 4 (1956). Porter, H., Ainsworth, S. The isolation of the coppercontaining protein cerebrocuprein from normal human brain. J. Neurochem, S (1959) 91. Porter, H., Sweeney, M., Porter, E. M. Human hepatocuprein. Isolation of a copper-protein from the subcellular soluble fraction of adult human liver. Arch. Biochem. lOS (1964) 319. Rahimtula, A. D., O'Brien, P. J. Hydroperoxide dependent o-dealkylation reactions catalysed by liver microsomal cytochrome P--450. Biochem. Biophys, Res. Commun. 62 (1975) 268. Riiihii, N. Hemolysis of human blood caused by oxygen and its prevention with Vitamin E. Acta paediat, (Uppsala) 44 (1955) 128. Salin, M. L., McCord, J. M. Free radicals and inflammation: Protection of phagocytosing leukocytes by superoxide dismutase. J. clin. Invest. 56 (1975) 1319. Serfass, R. E., Ganther, H. E. Defective microbiocidal activity in glutathione peroxidase-deficient neutrophils of selenium-deficient rats. Nature (Lond.) 255 (1975) 640. Shohet, S. B., Pitt, J., Bachner, R. L., Poplack, D. G. Lipid peroxidation in the killing of phagocytized pneumococci. Infect, Immunity 10 (1974) 1321. Slater, T. F. Free Radical Mechanisms in Tissue Injury. London, Pion, 1972. Stocks, J., Dorrnandy, T. L. The autoxidation of human red ceUs Lipids induced by hydrogen peroxide. Brit. J. Haemat, 20 (1971) 95. Stocks, J., Offerman, E. L., Modell, C. H., Dormandy, T. L. The susceptibility to autoxidation of human red cell lipids in health and disease. Brit. J. Haemat, 2.3
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398 J. M. C. Gutteridge (1972) 713. Stocks, J., Gutteridge, J. M. C., Sharp, R., Dorrnandy, T. L. Assay using brain homogenate for measuring the antioxidant activity of biological fluids. Clin. Sci. mol. Med. 47 (1974) 215. Stocks, J., Gutteridge, J. M. C., Sharp, R., Dormandy, T. L. The inhibition of lipid autoxidation by human serum and its relation to serum proteins and tocopherol. Clin. Sci. mol. Med. 43 (1974) 223. Stocks, J., Scudder, P., Gutteridge, J. M. C., Dormandy, T. L. (1976). To be published. Stossel, T. P., Mason, R. J., Smith, A. L. Lipid peroxidation by human blood phagocytes. J. din. Invest. 54 (1974) 638.
Strobel, H. W., Coon, M. J. Effect of superoxide generation and dismutation on hydroxylation reactions catalysed by liver microsomal cytochrome P--450. J. bioi. Chem. 246 (1971) 7826. Weisiger, R. A., Fridovitch, 1. Superoxide dismutase. Organelle specificity. J. biol. Chern. 248 (1973) 3582. Yost, F. J., Jr., Fridovitch, I. An iron-containing superoxide dismutase from Escherichia coli. J. bioi. Chem, 248 (1973) 4905. Zimmerman, R., Flohe, L., Weser, N., Hartman, H. J. Inhibition of lipid peroxidation in isolated inner membranes of rat liver mitochrondia by superoxide dismutase. FEBS lett. 29 (1973) 117. Accepted for publication 15 March 1976.
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Superoxide Dismutase (Erythrocuprein) and Free Radicals in Clinical Chemistry
J. M. C.
GUTTERIDGE
Department of Clinical Chemistry, Whittington Hospital, London N19
Erythrocuprein (superoxide dismutase) has recently been shown to have an enzymic function towards superoxide anions. The discovery of superoxide dismutase, its mode of action, and estimation are reviewedalong with a brief introduction to oxygen activation and free-radical chemistry. The formation, activity, and destruction of oxygen free radicals in white blood cells, red blood cells, and subcellular particles are discussed. (a) The production of superoxide anions by white cells during phagocytosis is thought to be advantageous for the overall bactericidal event. (b) Normal red blood cells generate low levels of superoxide anions. Increased levelsof free-radical production could playa significantrole in acceleratingcell ageing (haemolysis). (c) Subcellular particles produce superoxide anions. These as well as organic peroxides have been implicated in drug hydroxylation reactions involving cytochrome P-450. Superoxide dismutase is a recent enzymic function ascribed to the proteins previously known as cuproproteins. Erythrocuprein (haemocuprein) was first isolated in 1938 as a blue-green protein from ox blood. It had a molecular weight of 35 000 and contained 0.38 % copper (Mann and Keiling, 1938). Because it had no discernible activity its name was derived from its source and copper content. Similar cuproproteins were isolated from diverse sources and carefully characterised. The nomenclature of these proteins merely reflected the tissue of origin and the content of copper-hence cerebrocuprein, erythrocuprein, and hepatocuprein (Porter and Ainsworth, 1959; Kimmel et al., 1959; Porter et al., 1964). While much work was spent on characterising these macromolecules as interesting proteins, it was discovered that as well as 2 atoms of copper they contained 2 atoms per molecule of zinc (Carrico and Deutsch, 1910). A more serendipitous line of inquiry finally led to the discovery by McCord and Fridovitch (1969) of an enzymic function for erythrocuprein. They were able to show that this protein had specific catalytic activity toward free radical ions-superoxide anions (02'-) converting two molecules (dismutation) to hydrogen peroxide and oxygen (Fig. la). a
O' 2"
+
O' 2"
+
2H + -+ 02
+
H202
superoxide dismutase
FREE RADICALS
When two atoms or molecules (represented as X and Y) are covalently bonded to form the molecule XY, stability of the molecule is achieved by electron pairing, each donating and sharing an electron. X:Y-+X'
+ v-
Breaking of this bond is known as homolytic fission
(homolysis), giving rise to two free radicals X' and Y', Free radicals are therefore odd-electron atoms or molecules. A bold point (') is used to indicate the presence of the unpaired electron. The energy required for the dissociation of a covalent bond may be provided by (a) thermal sources, (b) electromagnetic radiation, or (e) redox coupling with a source of free radicals. The majority of free radicals are electrically neutral, though free radical ions such as the superoxide anion do exist. They are usually extremely reactive and paramagnetic-a property utilised for detecting their presence. A reaction which proceeds by a free-radical mechanism can be inhibited by compounds which combine with free radicals; these are known as free-radical scavengers or antioxidants. What is a superoxide anion and where is it found in biological systems? Oxygen is not a reactive molecule; in order to take part in reactions it has to undergo a stepwise reduction; this is usually achieved by the univalent addition of electrons. Complete reduction of oxygen to water is achieved by the addition of 4 electrons. The first step in the activation of oxygen requires an input of energy and results in the formation of the hydroperoxyl radical (HO' 2), the ion of which is the superoxide anion (02'-). Further additions of electrons give sequentially peroxide (02=) and the hydroxyl radical (OH') with energy liberation. Once produced, the superoxide anions can form hydrogen peroxide (by either enzymic or spontaneous dismutation (Fig. la). The hydrogen peroxide can then itself react with a superoxide anion to give the hydroxyl radical (OH") (Fig. l c) (Harber and Weiss, 1934). The enzyme
c superoxide dismutase is clearly the first line of 393
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394 J. M. C. Gutteridge
"antioxidant" defence in aerobically metabolising cells against "activated" oxygen. To test this hypothesis Fridovitch carried out a detailed study in bacteria, examining aerobes, aerotolerant anaerobes, and obligate anaerobes for the presence of superoxide dismutase (Gregory and Fridovitch, 1973). As a general rule superoxide dismutases were present in aerobic bacteria, absent in obligate anaerobes, but present in low levels and inducible by molecular oxygen in aerotolerant anaerobes. Studies in bacteria led to the discovery of quite different trace-metal-dependent superoxide dismutases. They were shown to contain both an iron--containing ferrienzyme (Yost and Fridovitch, 1973) and a managanese-d.ependent enzyme (Keele et al., 1970). The manganese enzyme was reddish rather than blue in colour and had a higher molecular weight and different primary structure, though its catalytic properties remained unchanged. Several isoenzyrnes have been demonstrated in extracts from bacteria and chicken liver using polyacrylamide gels. Chicken liver contained both the copper enzyme and the manganese enzyme, the former in the cell cytosol, the latter entirely in the mitochondria (Weisiger and Fridovitch, 1973). The metals associated with the different superoxide dismutases are all transitional trace metalsthat is, they occur in period 4 and have variable valencies. Electrons are readily transferred from the third to the fourth electron shell in oxidising conditions and from the fourth to the third during reducing conditions. The catalytic mechanisms of this enzyme depend on the sequential reduction and re-oxidation of the metal at the active centre (Fridovitch, 1974). MEASUREMENT OF SUPEROXIDE DISMUTASE ACTIVITY
It is not possible to measure enzymic activity by either disappearance of substrate or formation of an end product under normal laboratory conditions. This is because the substrate has to be continuously produced in the reaction mixture and itself undergoes spontaneous dismutation to hydrogen peroxide. An indirect assay system is therefore used in which a scavenger is added to intercept the generated superoxide anion. If superoxide dismutase is then introduced into the system it will compete with both the spontaneous dismutation of 02'- and also interception of the radical by the scavenger molecule. Superoxide anions can be generated in the reaction by either photoreduction of riboflavin and oxygen (Baeuchamp and Fridovitch, 1971) or enzymically with xanthine oxidase (McCord and
Fridovitch, 1969). The latter technique utilises the capability of xanthine oxidase to bring about the univalent reduction of oxygen (Fig. Id). Scavengers d
Xanthine
+
02
---+
0'2
+
Uric Acid
xanthine oxidase
are selected which are reduced by 02"- and also act as indicators by undergoing a colour change in the process. Those most frequently used are cytochrome C and nitro blue tetrazolium (McCord and Fridovitch, 1969; Beauchamp and Fridovitch, 1971). One unit of superoxide dismutase activity has been defined as the amount of enzyme which will inhibit 50 % of the rate of reduction of the indicating scavenger under specified conditions. FORMATION OF ACTIVATED OXYGENS IN VIVO
The toxicity of oxygen has been recognised for some time now (Raiha, 1955; Mengel and Kann, 1966); but the mechanism by which the deleterious effects were brought about are only now being fully understood. In this respect the discovery of an enzyme capable of dismutasing oxygen free radicals was a major breakthrough. It is almost certain that wherever oxygen is aerobically metabolised by univalent electron reductions active oxygens are formed. The generation of free radicals and active oxygen have now been shown to accompany various normal physiological and abnormal pathological processes in the body. (a) White blood cells
The recognition by Babior, Kipnes and Curnutte (1973) that phagocytosing leucocytes generated superoxide anions which were released into the surrounding medium led to the postulation that either the superoxide anion or its dismutation to hydrogen peroxide were themselves important components of the bactericidal event (Curnutte and Babior, 1974; Johnston et al., 1975). A second possibility is that superoxide anions or hydrogen peroxide are involved in a free radical oxidation of intra- or extracellular unsaturated lipids. Lipid peroxidation would then result in the formation of numerous cytotoxic compounds (Stossel et al., 1974; Shohet et al., 1974; Gutteridge, 1974). Many peroxidic and aldehydic compounds are formed during lipid peroxidation; these have extremely potent antibacterial properties (Gutteridge et al., 1974, 1976). Much of the data supporting the involvement of lipid peroxidation in leucocytes as an essential part of bacterial killing is derived from experiments carried out on white blood cells from patients with
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Superoxide dismutase (erythrocuprein) and free radicals in clinical chemistry the rare hereditary disorder, chronic granulomatous disease. White blood cells from patients with this disease are defective in killing ingested bacteria. Along with many biochemical abnormalities detected in these cells they have been shown to be defective in generating superoxide anions and hydrogen peroxide and deficient in the enzyme glutathione peroxidase (Curnutte et al., 1974; Holmes et al., 1967; Holmes et al., 1970). Some evidence against the advantagous involvement of lipid peroxidation in bacterial killing during phagocytosis could be interpreted from the results of Serfass and Ganther (1975). A deficiency of glutathione peroxidase was produced in rat leucocytes by feeding a seleniumdeficient diet. The trace metal selenium is an integral part of glutathione peroxidase (an enzyme which destroys lipid peroxides and hydrogen peroxide). This resulted in an impaired leucocyte ability to kill ingested yeast cells. The above authors suggest this was due to the damaging effect of lipid peroxides on cell function. Liberation by white blood cells of both hydrogen peroxide and superoxide anions into extracellular fluids (which contain insignificant amounts of either superoxide dismutase or catalase) could result in the formation of the highly reactive hydroxyl radical OH' (McCord, 1974; Salin and McCord, 1975). McCord suggests that this species could be the agent responsible for inflammatory conditions such as those found in arthritic joints. He was able to show that enzymic generation of OH' in vitro could degrade purified hyaluronic acid and bovine synovial fluid. (b) Red blood cells
The red blood cell is of particular interest since its function is one of oxygen transport and its premature ageing (haemolysis) in various clinical conditions well documented. It has been shown that red blood cells in certain clinical conditions are more susceptible to oxidative stress than normal cells (Dodge et al., 1967; Stocks et al., 1972). The standard stress test consists in incubating washed red blood cells in vitro with dilute hydrogen peroxide under carefully controlled conditions (Stocks and Dormandy, 1971). The generation of free radicals within the system attacks the cell membrane lipids, resulting in their peroxidation, The degree of oxidation is related to the formation of a lowmolecular-weight water-soluble carbonyl malonyldialdehyde, which is measured colorimetrically. In normal red blood cells a continuous production of superoxide anions is brought about by the autoxidation of oxyhaemoglobin to methaemoglobin (Misra and Fridovitch,1972). Under normal con-
395
ditions it has been estimated that about 3 % of the total body haemoglobin is converted to methaemoglobin each day (Carrell et al., 1975). Rapid reduction back to haemoglobin is brought about by the enzyme methaemoglobin reductase. The above authors observed that increased formation of methaemoglobin associated with such factors as increased heat, certain drugs, and the abnormal unstable haemoglobins is invariably accompanied by increased superoxide radical production and release. A deleterious role for activated oxygen in the condition erythropietic protophorphyria has been proposed by Lamola et al. (1973). They suggest that photo-irradiation in the presence of free porphyrins leads to the production of singlet oxygen (02·), which oxidises cholesterol leading to premature haemolysis of the red blood cell. Incubation of red blood cells from vitamin-Edeficient animals with hyperbaric oxygen results in haemolysis and oxidation of lipids (Mengel et al., 1964; Mengel and Kann, 1966). In our own laboratory lipid peroxidation with hyperbaric oxygen has been achieved using normal (vitamin-E-deficient) human umbilical-cord red blood cells (Gutteridge, 1974).
(c) Subcellular particles The reduction of oxygen to water via the respiratory chain is a fundamental biochemical process for energy conservation. During oxidoreduction reactions in mitochondria and microsomes univalent reduction of oxygen does occur with the production of the superoxide radical (Zimmerman et al., 1973; Aust et 01., 1972). The ability of microsomal cytochrome P--450 to carry out hydroxylation reactions may also depend on the production of superoxide anions (Strobel and Coon, 1971). This interpretation is based on the finding that superoxide dismutase when added to the cytochrome P--450 catalysed reaction inhibited drug hydroxylation. As well as oxygen activation a process of substrate activation may also occur. Recent reports suggest that the ability of liver microsomal suspensions to utilise organic peroxides for the hydroxylation of various substrates is catalysed by cytochrome P--4S0 (Rahimtula and O'Brien, 1975; Hrycay and O'Brien, 1972; Kadlubar et al., 1973). This peroxidase activity of cytochrome P--450 with organic peroxides has also been shown to occur with hydrogen peroxide (Coon et al., 1975). Studies in our own laboratory have shown that an incubating ox-brain homogenate (Stocks et al., 1974) undergoes rapid lipid peroxidation if electron transport is maintained.
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396 J. M. C. Gutteridge DESTRUCTION OF ACTlVATED OXYGEN IN VIVO
The highly reactive chemical nature of free radicals can lead to molecular damage in biological material. Free radicals are known to damage proteins, particularly if they contain sulphydryl groups, and unsaturated lipids (Little and O'Brien, 1967; Jacob and Lux, 1968; Slater, 1972). The first line of defence in oxygen-metabolising cells against active oxygen is the enzyme superoxide dismutase, which brings about the formation of oxygen and hydrogen peroxide (Fig. I a). The hydrogen peroxide can then be destroyed by the catalytic activity of catalase (Fig. I b) or glutathione peroxidase (Fig. Ie) (Nitowsky 2H202
b
--+
02
catalase
e
2GSH
+
H202
--+
+
2H20
GSSG
+
2H20
glutathione peroxidase
and Tildon, 1956; Gross et al., 1967). Should a superoxide anion react with hydrogen peroxide it could result in the formation of the highly reactive hydroxyl radical (OH') (Fig. lc), Its removal would seem to depend on a second line of defence such as the chemical antioxidants vitamin E, ascorbic acid, purine bases, and related compounds.
functions of this copper protein are not yet clear and may be related to free radical scavenging or dismutation of superoxide anions (Stocks et al., 1976). Increased serum levels of the acute reacting protein caeruloplasmin in rheumatoid arthritis (Lorber et al., 1968) may give some support to McCord's postulation that free radicals are involved in inflammatory states. Free radicals are transient and the most elusive of chemical species. In spite of this the discovery of superoxide dismutase a few years ago has initiated great interest and a detailed search for functions and abnormalities related to activated oxygens, free radicals, and superoxide-disrnutasing enzymes. As a speculative footnote, it seems extremely surprising that the body should rely on chemical scavenging for the inactivation of one of the most active oxidants known-the hydroxyl radical (OH"). Perhaps there are specific proteins involved in the inactivation or dismutation of hydroxyl radicals yet to be characterised? The implication of activated oxygens in disease offers an entirely new field of study for clinical scientists, some of whom find themselves involved in free radical chemistry.
SUMMARY AND SPECULATIVE CONCLUSIONS
REFERENCES
The discovery of a ubiquitous free radical dismutasing enzyme in oxygen-metabolising cells suggests by implication that activated oxygens and free radicals are produced in living systems. Should the cell's defences be impaired or the production of radicals significantly increased, damage could result in premature ageing and cell degeneration. Red blood cells are well protected with antioxidants such as vitamin E and the enzymes superoxide dismutase, catalase, and glutathione peroxidase. In spite of this, activated oxygene are implicated in certain haemolytic conditions, and red blood cells from different clinical groups are more susceptible to peroxidative stress than normal controls (Carrell et al., 1975; Stocks et al., 1972). As well as cellular abnormalities, changes in extracellular fluids may be important, as these do not contain appreciable amounts of superoxide dismutase and catalase (McCord, 1974). In extracellular fluids therefore other proteins may have an important antioxidant function. The copper--containing protein (enzyme) present in serum caeruloplasmin (copper oxidase) acts as an antioxidant by inhibiting lipid peroxidation in an incubating ox-brain homogenate (Stocks et al., 1974). This can be partly explained by its ferroxidase activity (Curzon and O'Reilly, 1960). Other antioxidant
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in oxidising proteins and nonprotein thioI. Biochem. J. 106 (1967) 419. Lorber, A., Cutler, L. S., Chang, C. C. Serum copper levels in rheumatoid arthritis. Relationship of elevated copper to protein alterations. Arthr. and Rheum. 11 (1968) 65. Mann, T., Keilin, D. Haemocruprein and hepatocuprein. Copper-protein compounds of blood and liver of mammals, Proc. roy. Soc. B 126 (1938) 303. Mengel, C. E., Karin, H. E., Smith, W., Horton, B. Effects of in vivo hyperoxia on erythrocytes. Hemolysis in mice exposed tohyerbaric oxygenation. Proc. Soc. expo Bioi. (N. Y.) 116 (1964) 259. Mengel, C. E., Kann, H. E., Jr. Effects of in vivo hyperoxia on erythrocytes. III. In vivo peroxidation of erythrocyte lipid. J. din. Invest. 4S (1966) 1150. McCord, J. M., Fridovitch, I. Superoxide dismutase, an enzymic function for erythrocuprein (haemocuprein), J. bioi. Chern. 244 (1969) 6049. McCord, J. M. Free radicals and inflammation: Protection of synovial fluid by superoxide dismutase. Science ISS (1974) 529. Misra, H. P., Fridovitch, I. The generation of superoxide radical during the autoxidation of hemoglobin. J. bioi. Chem. 247 (1972) 6960. Nitowsky, H. M., Tildon, J. T. Some studies of tocopherol deficiency in infants and children. III. Relation of blood catalase activity and other factors to hemolysis of erythrocytes and hydrogen peroxide. Amer. J. din. Nutr. 4 (1956). Porter, H., Ainsworth, S. The isolation of the coppercontaining protein cerebrocuprein from normal human brain. J. Neurochem, S (1959) 91. Porter, H., Sweeney, M., Porter, E. M. Human hepatocuprein. Isolation of a copper-protein from the subcellular soluble fraction of adult human liver. Arch. Biochem. lOS (1964) 319. Rahimtula, A. D., O'Brien, P. J. Hydroperoxide dependent o-dealkylation reactions catalysed by liver microsomal cytochrome P--450. Biochem. Biophys, Res. Commun. 62 (1975) 268. Riiihii, N. Hemolysis of human blood caused by oxygen and its prevention with Vitamin E. Acta paediat, (Uppsala) 44 (1955) 128. Salin, M. L., McCord, J. M. Free radicals and inflammation: Protection of phagocytosing leukocytes by superoxide dismutase. J. clin. Invest. 56 (1975) 1319. Serfass, R. E., Ganther, H. E. Defective microbiocidal activity in glutathione peroxidase-deficient neutrophils of selenium-deficient rats. Nature (Lond.) 255 (1975) 640. Shohet, S. B., Pitt, J., Bachner, R. L., Poplack, D. G. Lipid peroxidation in the killing of phagocytized pneumococci. Infect, Immunity 10 (1974) 1321. Slater, T. F. Free Radical Mechanisms in Tissue Injury. London, Pion, 1972. Stocks, J., Dorrnandy, T. L. The autoxidation of human red ceUs Lipids induced by hydrogen peroxide. Brit. J. Haemat, 20 (1971) 95. Stocks, J., Offerman, E. L., Modell, C. H., Dormandy, T. L. The susceptibility to autoxidation of human red cell lipids in health and disease. Brit. J. Haemat, 2.3
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