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A simple, inexpensive and specific method for the determination of hemoglobin in tissue homogenates is described. It is based on the specific binding of hemoglobin by haptoglobin and the stabilizing effect of this binding on the peroxidase activity of hemoglobin. The haptoglobin is added as serum and no purification is needed. The method gives reliable estimates of hemoglobin contents above 10 mg/l.

Eighteen persons, dentists and nurses, with urinary mercury levels higher than the group median value of all dental personnel in the country of Västerbotten were compared with a group consisting of 15 persons with low urinary mercury levels working in the same clinics. A statistically significant difference between the high urinary mercury group and the low urinary mercury group could be seen in the plasma mercury level. In each group a statistically significant relation could be seen between the plasma mercury level and the total number of amalgam surfaces. The two groups did not differ with regard to the levels of plasma selenium and erythrocyte glutathione peroxidase, and no correlation between these two variables and the plasma mercury levels could be found. To evaluate organ functions, a large number of supplementary analyses were performed. These analyses did not indicate any influence on organ functions. Although the persons in the present study were occupationally exposed to mercury, none of the biologic variables analyzed seemed to be affected. Even among dental personnel who handle amalgam professionally the number of amalgam surfaces is a major contributory factor to the P-mercury level.

A further characterization of human extracellular superoxide dismutase is reported. The study was especially aimed at the interaction with substances known to interfere with CuZn superoxide dismutase and other superoxide dismutases. Extracellular superoxide dismutase is efficiently inhibited by cyanide and is about 3 times more sensitive than is human CuZn superoxide dismutase. The sensitivity to azide is much lower, but still about 3 times higher than that of human CuZn superoxide dismutase. Extracellular superoxide dismutase is about as rapidly inactivated by hydrogen peroxide as is CuZn superoxide dismutase. The sensitivity to diethyldithiocarbamate is very high and more than an order of magnitude larger than that of CuZn superoxide dismutase. Sodium dodecyl sulphate, under conditions suggested as being suitable for distinguishing between the insensitive CuZn superoxide dismutase and the sensitive Mn superoxide dismutase, efficiently inactivated extracellular superoxide dismutase. No antigenic similarities between extracellular superoxide dismutase and CuZn superoxide dismutase could be demonstrated. Anti-(extracellular superoxide dismutase) did not bind CuZn superoxide dismutase, and anti-(CuZn superoxide dismutase) did not bind extracellular superoxide dismutase.

The autoxidation of pyrogallol was investigated in the presence of EDTA in the pH range 7.9–10.6. The rate of autoxidation increases with increasing pH. At pH 7.9 the reaction is inhibited to 99% by superoxide dismutase, indicating an almost total dependence on the participation of the superoxide anion radical, O2·−, in the reaction. Up to pH 9.1 the reaction is still inhibited to over 90% by superoxide dismutase, but at higher alkalinity, O2·− -independent mechanisms rapidly become dominant. Catalase has no effect on the autoxidation but decreases the oxygen consumption by half, showing that H2O2 is the stable product of oxygen and that H2O2 is not involved in the autoxidation mechanism. A simple and rapid method for the assay of superoxide dismutase is described, based on the ability of the enzyme to inhibit the autoxidation of pyrogallol. A plausible explanation is given for the non-competitive part of the inhibition of catechol O-methyltransferase brought about by pyrogallol.

Extracellular-superoxide dismutase (EC-SOD) is heterogenous in the vasculature with regard to heparin affinity and can be separated into three fractions: A, without affinity; B, with weak affinity; and C, with relatively strong heparin affinity. The plasma clearance of intravenously injected 125I-labeled and unlabeled human EC-SOD C was studied in rabbits. About 90% of injected 125I-EC-SOD C was eliminated from the blood within 5-10 min. Injection of heparin after 10 or 20 min led to an immediate release of all sequestered 125I-EC-SOD C back to the blood plasma. Later injections of heparin led to diminished release, although release could still be demonstrated after 72 h. A half-time of approximately 10 h could be calculated for heparin-releasable 125I-EC-SOD C. Unlabeled EC-SOD C, determined as enzymic activity and with ELISA, was likewise sequestered and released to the same degree as 125I-labeled EC-SOD C by heparin as tested at 20 min and 5 h. The immediacy of the heparin-induced release indicates that the sequestered enzyme had been bound to endothelial cell surfaces. The length of the half-time suggests that the putative cell surface binding has a physiological function and is not primarily a step in enzyme degradation. The distribution of sequestered 125I-labeled EC-SOD C to different organs was determined at times between 10 min and 24 h. Of the organs, the liver contained the most 125I-EC-SOD C, followed by kidney, spleen, heart, and lung. At all investigated times, the content in the analyzed organs was nearly as large as the amount that could be promptly released to plasma by intravenous heparin. This indicates that almost all 125I-EC-SOD C in the organs was present on endothelial cell surfaces and was not bound by other tissue cell surfaces, or was present within the cells.

A procedure is described for the determination of plasma or serum haemoglobin employing the peroxidase activity of the haemoprotein using 2,2'-azino-di-(3-ethyl-benzthiazolinsulphonate-6) as chromogen. The method gives equal results for free haemoglobin, methaemoglobin and haemoglobin complexed to haptoglobin. It is designed to measure haemoglobin in the range 0--12 mumol/l. The peroxidase activity of myoglobin is similar to that of haemoglobin, whereas haemin in free solution, bound to haemopexin or to albumin (methaemalbumin) shows much lower activity. The precision within run is satisfactory, +/- 5%.

Intravenous heparin has previously been shown to release the high-heparin-affinity fraction C of extracellular-superoxide dismutase (EC-SOD, EC 1.15.1.1) to plasma in man and other mammals. This paper reports on further studies of the phenomenon in the pig. A dose-response curve of the effect of heparin revealed that 1000 IU/kg body weight is needed for maximal release of EC-SOD C. This dose is an order of magnitude larger than that needed for the maximal release to plasma of factors such as lipoprotein lipase, hepatic lipase, and diamine oxidase, which are distributed between plasma and endothelium similarly to EC-SOD C. Thus EC-SOD C appears to have an unusually high affinity for endothelial cell-surface sulfated glycosaminoglycans relative to the affinity for heparin. There was no significant difference in releasing potency between unfractionated heparin and heparin subfractions with high or low affinity for antithrombin III. The heparin structure conferring high-affinity binding to antithrombin III is thus not specifically involved in binding to EC-SOD C. The non-biosynthetic compound dextran sulfate 5000 was an order of magnitude more efficient than heparin. Protamine displayed dual effects. Given alone in high dose it released EC-SOD to plasma, probably due to binding to endothelial cell-surface sulfated glycosaminoglycans displacing fraction C of the enzyme. Whe given after heparin, in a dose just below that expected to neutralize the heparin, protamine reversed the heparin-induced EC-SOD release.

Serum from healthy volunteers contained very little CuZn Superoxide dismutase. Larger amounts were found in serum from patients with impaired renal function, and there was a good correlation between serum creatinine and serum CuZn superoxide dismutase content. Almost all superoxide dismutase activity of human serum was cyanide sensitive. and was found to be given by a factor(s) with a molecular mass of approximately 130 000. The factor was found in all human extracellular fluids investigated: plasma, serum, lymph, ascites and cerebrospinal fluid. The factor was not recognized by radioimmunoassay for human CuZn superoxide dismutase, and it was not inhibited by rabbit antibodies against human CuZn superoxide dismutase. A similar high-molecular mass factor was found in plasma from all mammals investigated: horse, cow, pig, dog, cat, rabbit, rat and mouse. The activities differed much among species and were mostly higher than those found in human serum/plasma.

Alloxan diabetes may be mediated by an iron-catalyzed formation of hydroxyl radicals. The iron chelators desferrioxamine and diethylenetriaminepentacetic acid (DETAPAC) which inhibit hydroxyl radical formation in vitro were tested against alloxan diabetes in mice. DETAPAC inhibited while desferrioxamine stimulated the hyperglycemic response to alloxan. The diverging treatment results are discussed.

In various types of autoimmune disease, an increased frequency of spontaneous chromosome breaks has been reported. Plasma from such patients induces chromosome breaks in normal cells. Exposure of plasma to superoxide radicals increases the breakage activity, and addition of superoxide dismutase protects against it. The New Zealand black mouse is an animal model of autoimmune disease which displays the breakage phenomenon. To test for the possibility that the breakage is related to deficient protection against superoxide radicals, the activities of superoxide dismutase isoenzymes were determined in tissues and blood from New Zealand black mice and compared with the activities of normal BALB/c mice. No differences between the strains were revealed in tissue EC-superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase activity. The erythrocyte superoxide dismutase activities were also equal. The plasma EC-superoxide dismutase activity was 35% lower in the New Zealand black mice than in the BALB/c mice. Between euthymic BALB/c mice and nude mice, previously reported to be deficient in tissue superoxide dismutase activity, no difference could be demonstrated.

The effect of genetically determined glutathione deficiency on the fibroblast content of CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase was investigated. No significant differences between glutathione-deficient and -proficient human fibroblasts were revealed. There was a large variation in the content of the investigated enzymes in fibroblasts grown and analysed on different occasions. Whereas the contents of CuZn superoxide dismutase, catalase and glutathione peroxidase did not deviate much from what has been found in other human cell-lines and tissues, the fibroblasts were found to contain exceptional amounts of Mn superoxide dismutase.

We investigated if superoxide dismutase and catalase could reduce myocardial infarct size in an open chest occlusion-reperfusion model. Thirty pigs were used for the experiment. The left anterior descending artery was ligated for 60 min followed by a 5 h reperfusion period. After randomisation and blinding the two enzymes or placebo were injected into the left atrium as a bolus immediately before and at the end of the occlusion and as a continuous infusion over the first hour of the reperfusion period. The total dose for each enzyme was 8 mg/kg bw. Tetrazolium staining was used to determine infarct size. The study code was not broken until all calculations and exclusions had been made. Nine animals died from intractable ventricular fibrillation, most commonly during the occlusion. Another three were excluded for technical reasons. We found that superoxide dismutase and catalase reduced infarct size in relation to myocardium at risk from a mean of 89% to 63% (P less than 0.01). Initial plasma half life for the two enzymes after the bolus infusions were calculated to be 30 min.

Enzyme secretion from incubated guinea pig submandibular gland slices was induced by adrenaline, noradrenaline, dopamine, 5-hydroxytryptamine or dibutyryl cyclic AMP combined with theophylline. Effects of alpha- and beta-blocking agents on stimulated peroxidase and amylase discharge were studied. Propranolol (beta-blocker), but not phenoxybenzamine (alpha-blocker), markedly inhibited the secretion of both enzymes induced by adrenaline or noradrenaline. Dopamine and 5-hydroxytryptamine were found to be potent enzyme secretagogues. Both propranolol and phenoxybenzamine antagonized the effects of these two amines. The secretagogic effect of dibutyryl cyclic AMP in combination with theophylline was not affected by either propranolol or phenoxybenzamine. L-DOPA and 5-hydroxytryptophan were without effect on enzyme secretion. It is concluded that peroxidase and amylase are secreted in parallel from guinea pig submandibular gland and that enzyme release is mainly mediated via the beta-adrenergic receptor.

The high heparin affinity subtype C of the secretory enzyme extracellular superoxide dismutase (EC-SOD) exists in the body mainly complexed with extracellular sulfated glycosaminoglycans (SGAGs). Addition of sulfated polysaccharides to EC-SOD C resulted in a prompt partial inhibition of the enzymic activity, in most cases amounting to 10-17%, but with the large dextran sulfate 500,000 amounting to 35%. Complex formation between heparin and EC-SOD C could also be observed as increases in apparent molecular weight of the enzyme. The findings suggest that the binding sites for SGAGs on EC-SOD C are localized far from the active site and that EC-SOD in vivo associated with SGAGs should retain the major part of its enzymic activity. Studies with amino acid-specific reagents suggested that both lysine and arginine residues are involved in the binding of SGAGs. In particular, modification of only a few lysine residues/subunit resulted in loss of high SGAG affinity, whereas arginine modification resulted in loss of not only SGAG affinity but also enzymic activity. We propose that this is due to modification of Arg-186, which is homologous to the highly conserved arginine in the entrance to the active site of the copperzinc-SODs.

1. 1. The catalytic properties of two horseradish peroxidases, isoenzymes A2 and C2, with p I 3.9 and 8.8, have been compared. 2. 2. The rate of formation of Compound I has been determined for the hydrogen, methyl, ethyl, n -propyl, and hydroxymethyl hydroperoxides and for p -nitroperoxybenzoic acid. The elongation of the alkyl group hampered the reaction with the acidic peroxidase and stimulated the reaction with the basic peroxidase. The acidic peroxidase generally reacted more slowly. Its reaction with H 2 O 2 showed a higher energy of activation than that of the slightly basic peroxidase. 3. 3. The rate of reaction with hydrogen peroxide was slightly but definitely influenced by pH for both peroxidases. 4. 4. The rates of reaction of twelve hydrogen donors (phenols, aromatic amines, acidic substances) with the secondary peroxidase-peroxide complex of the two peroxidases could be arranged in three groups according to the chemical nature of the donors. The acidic peroxidase was consistently more active at pH 4.5 than at pH 7.0 whereas the slightly basic peroxidase showed the reverse behavior in the presence of phenols. 5. 5. It is concluded that the two peroxidases are kinetically different with distinct substrate profiles and that they may fulfill different physiological functions.

The neuronal ceroid-lipofuscinoses are characterized by a widespread deposit in the body of pigments believed to be end products of lipid peroxidation damaged organelles. CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase, enzymes which conceivably might protect against lipid peroxidation, were investigated in blood cells from patients afflicted with infantile, late infantile and juvenile neuronal ceroid-lipofuscinosis. In some cases the enzymic activities were slightly lower than the activities of the controls, but no deficiencies which might be of etiological importance were revealed.

To test the possible role of superoxide radicals in the diabetogenic action of streptozotocin, blood glucose levels were measured in mice after a single high-dose (150 mg/kg body weight) or multiple low-dose (40 mg/kg for 5 days) injections of streptozotocin. Pre-treatment 6 h before streptozotocin with 250–300 mg/kg superoxide dismutase coupled to polyethylene glycol reduced the hyperglycaemic response in mice injected with a single dose of streptozotocin. The blood glucose levels after multiple low doses of streptozotocin were not affected by superoxide dismutase-polvethvlene glycol. Enzymatically inactive superoxide dismutase did not affect the development of hyperglycaemia. The results suggest that superoxide radicals may play a role in the diabetogenic action of streptozotocin injected as a high-dose single bolus.

Werner's syndrome is often regarded as a segmental progeroid syndrome. It has been suggested that free radical damage involving oxygen contributes to aging. Furthermore, lymphocytes from Werner's syndrome patients show in vitro increased chromosome breakage and are protected by exogenous superoxide dismutase and catalase. We prepared erythrocytes and lymphocytes from three patients with Werner's syndrome and determined four important enzymes protecting against oxygen toxicity; CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase. All enzymic activities were found to be normal.

CuZn superoxide dismutase and Mn superoxide dismutase were analysed in hypothalamus, nucleus caudatus, hippocampus and cortex gyrus cinguli from 14 patients with dementia of Alzheimer type and from 16 controls. Ample amounts of both enzymes were demonstrated in the brains and there was little difference between the various parts of the brain. There were only small differences in the enzymic activities between the two groups indicating that deficiencies of these enzymes are not a very likely cause of brain degeneration in dementias of Alzheimer type.

Ceruloplasmin and extracellular-superoxide dismutase are similar in physical properties. Both are found in extracellular fluids and both are scavengers of the superoxide radical. The relationship between the two proteins was further explored in the present investigation. Ceruloplasmin preparations were found to be commonly contaminated with extracellular-superoxide dismutase. In one preparation, 80% of the superoxide dismutase activity was due to extracellular-superoxide dismutase. Ceruloplasmin, freed from contaminating superoxide dismutase, was found to catalytically dismute the superoxide anion radical with a rate constant of about 1.0 × 10 4 M − s −1 per copper atom. Under physiological conditions with a low rate of superoxide production, ceruloplasmin preferentially reacts stoichiometrically with the superoxide radical with a rate constant of about 2 × 10 5 M −1 s −1 per copper atom. Under such conditions, the reaction does not result in hydrogen peroxide formation. From the kinetic data obtained it was calculated that in normal human plasma, extracellular-superoxide dismutase will scavenge about twice as much superoxide as ceruloplasmin. Using immobilized antibodies toward extracellular superoxide dismutase and ceruloplasmin, no antigenic cross-reactivity between the two proteins could be detected.

Hydrogen peroxide efficiently protects horseradish peroxidase against inactivation by hydroxymethylhydroperoxide, whereas the hydrogen donor substrate guaiacol has little protective effect. These results, and direct studies of the effects of hydroxymethylhydroperoxide on peroxidase compound II, indicate that compound II and possibly also compound I are quite resistant to hydroxymethylhydroperoxide. The inactivation of peroxidase caused by hydroxymethylhydroperoxide seems to be due to a reaction of the peroxide with free Fe 3+ peroxidase. Albumin, which protects thiol enzymes against hydroxymethylhydroperoxide, does not protect peroxidase. Neither does 2-mercaptoethanol, which also protects thiol enzymes, appear to hamper the inactivation reaction. This seems to indicate that the inactivation is not caused by a free radical released from hydroxymethylhydroperoxide. Hydroxymethylhydroperoxide inactivates peroxidase by attacking the hematin group, the ring of which is opened by way of at least three intermediates. The most stable intermediate is green (“compound IV”). H 2 C 2 attacks the methemoglobin-peroxide compound faster than hydroxymethylhydroperoxide does. These reactions are much slower than the attack of hydroxymethylhydroperoxide on peroxidase. It is suggested that the inactivation of peroxidase takes place as a side reaction in the process of forming compound I from hydroxymethylhydroperoxide and peroxidase.

1. 1.|Many reports attribute biochemical actions to bis(hydroxymethyl)peroxide. Mixtures of hydrogen peroxide and formaldehyde are known to exert a greater effect on biochemical systems than either substance alone. In both cases the effects are probably caused by hydroxymethylhydroperoxide, formed from bis(hydroxymethyl)-peroxide by hydrolysis or from hydrogen peroxide and formaldehyde by a condensation. 2. 2.|Hydroxymethylhydroperoxide is a fairly potent inhibitor of fumarate hydratase ( l -malate hydro-lyase, EC 4.2.1.2), lactate dehydrogenase ( l -lactate:NAD+ oxidoreductase, EC 1.1.1.27), and aspartate aminotransferase ( l -aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1), whereas glucose oxidase (β- d -glucose:O2 oxidoreductase, EC 1.1.3.4) and especially acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) are less affected. 3. 3.|2-Mercaptoethanol and albumin protect against the inhibitory action of hydroxymethylhydroperoxide without reducing the amount of peroxide. 4. 4.|Fumarate hydratase and lactate dehydrogenase, exposed to hydroxymethyl-hydroperoxide, are not reactivated by 2-mercaptoethanol, whereas aspartate amino-transferase slowly partially regains its activity.

Endogenous peroxidase activity was studied at the electronhistochemical level at various stages of the secretory cycle in the guinea pig submandibular gland. The histochemical findings were correlated to quantitative assays of peroxidase and amylase activity in the glands.

Tryptic digestion of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) leads to a gradually increased peroxidatic activity towards phenolic compounds. The reactivity with ethylhydroperoxide increases 2–3 times after brief digestion but decreases thereafter. The catalatic activity and the reactivity with methylhydroperoxide disappear rapidly. The results indicate a loosening of the structure around the hematins, increasing the reactivity with peroxide and hydrogen donor substrates formerly sterically hindered. This change may occur to some extent without loss of the catalase-specific ability to react rapidly with hydroperoxides. The ability to peroxidize ethanol is apparently not loss before the loss of high reactivity with hydroperoxides. After brief exposure of catalase to pH > 12 leading to its dissociation into subunits, the enzyme recombines when the pH is lowered (Samejima, T., McCabe, W. J. and Tsi Yang, J. (1968) Arch. Biochem. Biophys. 127, 354–360). After this procedure most (∼ 70%) of the catalatic activity is recovered. The reactivity with ethylhydroperoxide is doubled and the peroxiding actifvity towards phenolic compounds is strongly increased. The results indicate an increased accessibility of the hematins of recombined catalase. However, they do not become reducible by dithionite. After longer exposure to high pH, all enzymic activities are decreased and the hematins become reducible.

Alloxan injection into animals causes pancreatic P-cell necrosis, insulin deficiency and diabetes mellitus [I]. From protection experiments in vivo alloxan diabetogenicity was inferred [2] to be mediated by the sequenced generation of dialuric acid, superoxide radicals (O;-), hydrogen peroxide (H,O,) and hydroxyl radicals (OH’). In support of this hypothesis in vitro experiments with isolated pancreatic islets and islet cells showed that the cytotoxicity of alloxan is counteracted by superoxide dismutase, catalase, and nonenzymic scavangers of the hydroxyl radical [3]. The strong and indiscriminate reactivity of OH’ with cell constituents can explain why alloxan rapidly affects as diverse phenomena as insulin synthesis [4], insulin release [S], glucose oxidation and oxygen consumption [6], univalent cation pumping [7] and trypan blue exclusion [8,9] in mammalian islet cells, and the mannitol permeability [lo] in fish islets. The generation of OH’ from O;and H202 (Haber-Weiss reaction) in biological systems appears to require catalysis by traces of transitional metals [l l-141 and may be seen as the summation of the reduction of ferric ion by superoxide (Fe3’ t O;+ Fe’+ t 0,) and the Fenton reaction (H,Oz t Fe’+ + OH. + OHt Fe3”); the analogous reactions with Cu+/Cu*’ as catalyst have also been proposed [ 151. Therefore, to test further the free radical hypothesis of alloxan diabetogenicity we have investigated how metal ions and chelators influence the toxic action of alloxan on Rb’ accumulation by isolated pancreatic islets. The effect of alloxan was found to be inhibited by diethylenetriaminepentaacetic acid (Detapac), a chelator with marked ability to suppress the metalcatalyzed Haber-Weiss reaction in biochemical systems

The most common cause of amyotrophic lateral sclerosis (ALS) is mutations in superoxide dismutase-1 (SOD1). Since there is evidence for the involvement of non-neuronal cells in ALS, we searched for signs of SOD1 abnormalities focusing on glia. Spinal cords from nine ALS patients carrying SOD1 mutations, 51 patients with sporadic or familial ALS who lacked such mutations, and 46 controls were examined by immunohistochemistry. A set of anti-peptide antibodies with specificity for misfolded SOD1 species was used. Misfolded SOD1 in the form of granular aggregates was regularly detected in the nuclei of ventral horn astrocytes, microglia, and oligodendrocytes in ALS patients carrying or lacking SOD1 mutations. There was negligible staining in neurodegenerative and non-neurological controls. Misfolded SOD1 appeared occasionally also in nuclei of motoneurons of ALS patients. The results suggest that misfolded SOD1 present in glial and motoneuron nuclei may generally be involved in ALS pathogenesis. Electronic supplementary material The online version of this article (doi:10.1007/s00401-011-0805-3) contains supplementary material, which is available to authorized users.

Isolierte Submandibularisdrusen des Meerschweinchens wurden in physiologischer Pufferlosung mit sekretionsstimulierenden Substanzen inkubiert. Dopamin, Noradrenalin, Adrenalin und 5-HT induzieren die Sekretion von Peroxydase und Amylase, wahrend der Serotoninantagonist BOL 148 (2-Bromo-d-Lysergsaure Dietylamid) eine beinahe vollstandige Sekretionshemmung hervorruft. BOL 148 hat sich also nicht als spezifisch 5-HT-hemmend an der Meerschweinchenspeicheldruse erwiesen.

Male rats were exposed to 100% O 2 for 24 h in a temperature-controlled cage and compared to a control group, similarly exposed to air. No effect of O 2 was observed on plasma testosterone concentrations or on testis testosterone concentrations. The rat testis was found to contain a comparatively high level of superoxide dismutase activity, an enzyme believed to protect against the toxicity of oxygen. Oxygen did not induce the enzyme in testis under these conditions.

The contents of extracellular superoxide dismutase, CuZn superoxide dismutase and Mn superoxide dismutase were determined in tissues from nine mammalian species. The pattern of CuZn superoxide dismutase distribution was similar in all species, with high activity in metabolically active organs such as liver and kidney and low activity in, for example, skeletal muscle. Mn superoxide dismutase activity was high in organs with high respiration, such as liver, kidney, and myocardium. Overall the Mn superoxide dismutase activity in organs was almost as high as the CuZn superoxide dismutase activity. The content of extracellular superoxide dismutase was, almost without exception, lower than the content of the other isoenzymes. The pattern of tissue distribution was distinctly different from those of CuZn superoxide dismutase and Mn superoxide dismutase. The tissue distribution of extracellular superoxide dismutase differed among species, but in general there was much in lungs and kidneys and little in skeletal muscle. In man, pig, sheep, cow, rabbit and mouse the overall tissue extracellular superoxide dismutase activities were similar to each other, whereas dog, cat and rat tissues contained distinctly less. There was no general correlation between the tissue extracellular superoxide dismutase activity of any of the various species and the variable plasma activity. The ratio between the plasma and the overall tissue activities was high, for some species over unity, providing further evidence for the notion that one role of extracellular superoxide dismutase is as a plasma protein.

When discussing aging and the aged it is of importance to separate normal or physiological aging or orthoinvolution from pathological aging or pathoinvolution. The borderline between normal and pathological aging, however, is not sharp. Normal aging will usually not induce severe mental impairment if no other complications arise, even if the individual reaches a very old age.

Exogenous superoxide dismutase, catalase and scavengers of the hydroxyl radical protect pancreatic-islet cells against the toxic actions of alloxan in vitro [Grankvist et al. (1979) Biochem. J. 182, 17--25]. To test whether the extraordinary sensitivity of islet cells to alloxan is due to a deficiency of endogenous enzymes protecting against oxygen-reduction products, we assayed CuZn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in mouse islets and other tissues. To correct for any blood contamination, haemoglobin was also measured in the tissue samples. Pancreatic islets were found to belong to tissues with relatively little activity of the protective enzymes. However, the deviation from other tissues in this respect is probably not large enough to explain the especially great susceptibility of islet cells to alloxan.

(1983). Diethyldithiocarbamate Inhibition of CuZn Superoxide Dismutase in Human Erythrocytes: No Increase in Radiation Haemolysis. International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine: Vol. 43, No. 1, pp. 103-109.

Extracellular-superoxide dismutase (EC-SOD; 1.15.1.1,) is the major SOD isoenzyme in extra- cellular fluids such as plasma, lymph1 and synovial fluid2 but also occurs in tissues.3,4 EC-SOD is heterogenous with regard to binding to Heparin-Sepharose and can be separated into three fractions: A, without affinity; B with weak affinity; and C, with relatively high affinity.5 Most EC-SOD in the vascular system apparently is bound to endothelial cell surfaces.6 Membrane-bound heparan sulfate is the likely receptor for the enzyme, and EC-SOD fraction C is the form that binds.6 EC-SOD is a tetrameric glycoprotein with an apparent subunit molecular weight of around 3 0 kDa.5 Like the CuZn SOD’s, EC-SOD contains one Cu and one Zn atom per subunit.5 Still, despite the similarities, the amino acid compositions of human CuZn SOD and EC-SOD are clearly different,5 and no cross-reaction between polyclonal rabbit antibodies directed towards the enzymes have been observed.7

The aim of this study was to devise conditions for manipulation of the activity of selenium-dependent glutathione peroxidase in cell lines by means of variation in culture medium contents of selenite and fetal calf serum. Nine different cell lines were studied. A low glutathione peroxidase activity was, in most cases, obtained by the use of a medium with a low (2%) serum content. Selenite induced in most of the cell lines an increase in glutathione peroxidase activity, with a plateau ranging from 10 nM to 300–1000 nM. Growth-retarding effects of selenite became apparent at 300–2000 nM, showing a large cell line variation. Supplementation with 50–100 nM selenite for 1 week should generally be suitable for maximal glutathione peroxidase induction. The selenium contents of serum batches were highly variable, pointing to the importance of using only one well-defined, preferably low-selenium, batch. The glutathione peroxidase activities varied considerably between cell lines and the selenite-induced increases ranged from negligible to more than 10-fold. The availability of cell lines with such variable responses should be valuable for experiments aimed at evaluating the importance of glutathione peroxidase and selenium compounds independently of glutathione peroxidase for the protection against oxidative insult.

Extracellular-superoxide dismutase (EC 1.15.1.1., abbreviation EC-SOD) is a tetrameric glycoprotein with an apparent subunit molecular weight of around 30 kDa.1 Like the CuZn SODs, EC-SOD contains one Cu and one Zn atom per subunit1 and is inhibited by cyanide, azide, and diethyldithiocar-bamate,2 and H2O2 is the product of the catalysed reaction.3 Still, despite the similarities, the amino acid compositions of human CuZn SOD and EC-SOD are clearly different,’ and no cross-reaction between polyclonal rabbit antibodies directed towards the enzymes have been observed.2 EC-SOD is the major SOD isoenzyme in extracellular fluids like plasma, lymph,4 and synovial fluid,5 but also occurs in tissues.6,7 The total plasma EC-SOD concentration varies greatly between species, whereas the tissue concentrations are more equal.7

Human milk and saliva from newborn infants were analyzed for their content of lactoperoxidase and thiocyanate. The activity of lactoperoxidase in infant saliva was variable but generally higher than that found in calf saliva. In contrast, the activity in human colostrum was low (∼5%) compared with that found in cow's milk. The enzyme was resistant to gastric juice. Thiocyanate was demonstrated in infant saliva in concentrations about one-third of that in adult saliva. The amounts of lactoperoxidase and thiocyanate in infant saliva are quite sufficient to inhibit bacterial growth in in vitro systems. The importance of this system in vivo has not yet been demonstrated. The availability of this system to both newborn calves and humans (in calves provided largely by colostrum and in human babies by saliva) might be indirect evidence of its importance.

Experiments with isolated pancreatic islets or dispersed islet cells from non-inbred ob/ob mice were performed to test the hypothesis that free radicals, notably OH., mediate the diabetogenic toxicity of alloxan. Accumulation of 86Rb+ by whole islets and exclusion of Trypan Blue by dispersed cells were used as previously validated criteria of islet-cell viability. Alloxan alone drastically inhibited the Rb+ accumulation and significantly decreased the frequency of cells excluding Trypan Blue. Enzymic scavengers of O2.- and H2O2 or non-enzymic scavengers of OH. or singlet oxygen were added to the incubation medium and tested for their ability to protect against these effects of alloxan. Superoxide dismutase, catalase, dimethyl sulphoxide, benzoate, and mannitol counteracted the effects of alloxan in both cytotoxicity assays. Significant protection of the Rb+-accumulating capacity was also afforded by butanol, caffeine, theophylline, NADH, NADPH and, to a small extent, NAD+. Urea has a poor affinity for OH. and did not protect against alloxan. No effect was obtained with the singlet-oxygen scavenger, histidine. Except for the protection by NADH and NADPH, which may be due to a direct reaction with alloxan in the medium, the results strongly support the hypothesis. beta-Cells may be particularly vulnerable to alloxan because their metabolic specialization facilitates reduction of the drug and perhaps of other substrates for O2.–yielding redox cycles.

(1985). Oxygen Toxicity and Protective Systems. Journal of Toxicology: Clinical Toxicology: Vol. 23, No. 4-6, pp. 289-298.

Short-term testicular ischemia was induced unilaterally by ligating the testicular artery for 60 or 100 minutes in adult rats. After 7 days, the rats were fixed by vascular perfusion. The effect of ischemia and reperfusion was quantified using morphometric techniques. Occlusion of the testicular artery for 60 and 100 minutes resulted in a mild and moderate damage, respectively, to the seminiferous tubules. The contralateral control testis was not affected. To study the role of oxygen radicals in ischemia-reperfusion-induced testicular damage, rats were treated with superoxide dismutase and catalase. These radical scavengers did not influence the extent of testicular damage.

The secretory protein extracellular-superoxide dismutase is the major superoxide dismutase (SOD) isoenzyme in the extracellular space. There is evidence to suggest that most extracellular-superoxide dismutase in the vascular system is bound to endothelial cell surfaces. Venous stasis and physical exercise is known to induce release of several endothelium-associated factors into the plasma. However, venous stasis and physical exercise were not found to induce any release of extracellular-superoxide dismutase into plasma. The plasma activity of the intracellular isoenzyme CuZn SOD was doubled by venous stasis.

A superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1), distinct from previously known superoxide dismutases, has been isolated from human lung tissue. It is probably of the same nature as a previously demonstrated high molecular weight superoxide dismutating factor in human extracellular fluids. The enzyme has a molecular weight around 135,000 and is composed of four equal noncovalently bound subunits. Each molecule appears to have four copper atoms. No iron or manganese was found in the enzyme. Cyanide inhibits the enzyme efficiently. The enzyme brings about a first-order dismutation of the superoxide radical, the rate constant for the catalyzed reaction being about 1 X 10(9) M-1 s-1 per copper atom. The enzyme has hydrophobic properties. Affinity for various lectins indicates the presence of carbohydrate. Upon chromatography on heparin-Sepharose it is divided into three fractions, one with no, one with weak, and one with strong affinity for heparin.

The effect of ethanol consumption on serum concentration of alpha-tocopherol, erythrocyte activities of superoxide dismutase, glutathione peroxidase, and catalase were studied in 34 male alcoholics and 35 age-matched controls. Serum concentration of alpha-tocopherol was 30% lower in the alcoholics as compared to the controls (p less than 0.001). No significant difference was found in erythrocyte activities of Cu-Zn-containing superoxide dismutase, glutathione peroxidase, or catalase between the groups. Of the 12 alcoholics with subnormal serum alpha-tocopherol, 50% had concomitant neurological clinical scores and cerebellar atrophy, and their neurological scores were significantly higher (82%) than for alcoholics with normal alpha-tocopherol levels (p less than 0.03). However, no significant correlation was observed between levels of alpha-tocopherol and neurological clinical scores or cerebellar atrophy. When entering the study, alcoholics and controls were each randomized into two separate groups, receiving vitamin E supplementation (100 mg/day) or placebo capsules for 10 days, respectively. In the four subgroups, alpha-tocopherol levels increased only in alcoholics receiving vitamin E supplementation (23%) (p less than 0.001). The reduced serum levels of alpha-tocopherol in alcoholics may be normalized by vitamin E supplementation.

A complementary DNA (cDNA) clone from a human placenta cDNA library encoding extracellular superoxide dismutase (EC-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been isolated and the nucleotide sequence determined. The cDNA has a very high G+C content. EC-SOD is synthesized with a putative 18-amino acid signal peptide, preceding the 222 amino acids in the mature enzyme, indicating that the enzyme is a secretory protein. The first 95 amino acids of the mature enzyme show no sequence homology with other sequenced proteins and there is one possible N-glycosylation site (Asn-89). The amino acid sequence from residues 96-193 shows strong homology (approximately 50%) with the final two-thirds of the sequences of all known eukaryotic CuZn SODs, whereas the homology with the P. leiognathi CuZn SOD is clearly lower. The ligands to Cu and Zn, the cysteines forming the intrasubunit disulfide bridge in the CuZn SODs, and the arginine found in all CuZn SODs in the entrance to the active site can all be identified in EC-SOD. A comparison with bovine CuZn SOD, the three-dimensional structure of which is known, reveals that the homologies occur in the active site and the divergences are in the part constituting the subunit contact area in CuZn SOD. Amino acid sequence 194-222 in the carboxyl-terminal end of EC-SOD is strongly hydrophilic and contains nine amino acids with a positive charge. This sequence probably confers the affinity of EC-SOD for heparin and heparan sulfate. An analysis of the amino acid sequence homologies with CuZn SODs from various species indicates that the EC-SODs may have evolved from the CuZn SODs before the evolution of fungi and plants.