CIinica C&mica Acta, 207 (1992) 261-263 0 1992 Elsevier Science Publishers B.V. All rights reserved. ~-8981~~$05.~
261
CCA 05289
Letter to the Editor
Expression of Con-superoxide dismutase and ~~utat~ione peroxidase in e~t~rocytes from diabetic and non-diabetic subjects (Received 25 March 1992; accepted 26 March 1992)
Dear Editor, Recent studies implicate reactive oxygen species (ROS) in the development of the secondary compli~tions that character&e chronic diabetes. Thus, activity of antioxidant enzymes such as superoxide dismutase is reduced in several organs of the diabetic rat [l] and plasma lipid peroxide levels are higher in diabetic patients with retinopathy than in those without this complication [2]. Erythrocytes may be an important ‘sink’ for ROS such as superoxide anion and hydrogen peroxide which are generated in plasma and which can cross the red cell membrane [3,4]. Erythrocytes express various antioxidant enzymes including CuZn superoxide dismutase (CuZn SOD) which catalyses the disproportionation of superoxide anion and, glutathione peroxidase and catalase which catalyse the detoxication of hydrogen peroxide. These reactions suppress formation of the toxic hydroxyl radical. Studies showing that CuZn SOD is susceptible to glycation and modification by ROS suggests expression of this enzyme may be reduced in erythrocytes from diabetics. Thus, Sakurai et al. [S] reported both glycation of Iysine residues and loss of enzymic activity following incubation of purified CuZn SOD with high concentrations of glucose and Shibata and Ogita [6J found three isoforms of the enzyme in murine erythrocytes, one of which resulted from the actions of hydrogen peroxide. Concentration-dependent inactivation of erythrocyte CuZn SOD by hydrogen peroxide has also been reported [7]. Prolonged exposure of CuZn SOD to relatively high concentrations of hydrogen peroxide caused fragmentation of the enzyme [7]. The combined effect of ROS and hyperglycaemia may have important effects on the level of antioxidant enzyme expression in the erythrocytes of patients with diabetes, particularly in older cells that have been in the vascular space for over 100 days. Data on the levels of antioxidant enzyme activity could be useful as prognostic C~r~e+?&?rceto: Dr. R.C. Strange, Centre of Pathology and Molecular Medicine, North Staffordshire Hospital Centre, Thornburrow Drive, Hartshiif, Stoke on Trent, Staffs., UK.
262
markers. We describe studies on the expression of CuZn SOD and glutathione peroxidase in erythrocytes separated by age in Percoll gradients [S]. Heparinised blood (10 ml) was obtained with ethical approval from 9 non-diabetic controls (glycated Hb ~6.3%; age range 25-50 years) and 15 well-controlled, insulin-dependent diabetic subjects (mean glycated Hb 12.0 + 2.0%; age range 17-69 years). Ten patients were without evidence of diabetic complications but five patients demonstrated either nephropathy or retinopathy. Blood was centrifuged (2,000 x g, 10 min, 4°C) within 1 h, the plasma removed and replaced with an equal vohune of 154 mmoM NaCl. Leucocytes and platelets were removed by elution through cellulose and the haemat~rit adjusted with NaCi to about 0.3. Portions (1.5 ml) of the resultant erythrocyte suspension were gently layered onto continuous Percoll gradients (40 ml) prepared by mixing 56% and 76% Pet-co11and after centrifugation, cells were drawn off in 7 fractions. Cells from diabetic and non-diabetic subjects demonstrated similar decreases in mean cell volume (92-88 fl) and K+/haemoglobin ratio (360-278 PmoYg) and increase in mean cell haemoglobin concentration (31-36 g/100 ml) confirming the gradients were properly separating cells by age [8]. CuZn SOD activity was determined in chloroform-ethanol extracted lysates by monitoring the reduction of nitroblue tetrazolium. Glutathione peroxidase activity was deter-
Superoxide 7,000
dismutase
lunltllo Hbl 1 T
2poo 1
2
3
4
5
6
7
Cell Fractions Fig. 1. CuZn-superoxide dismutase activity in Percoll separated erythrocytes. The insert shows an immunoblot of CuZn-superoxide dismutase in (left to right) standard, fractions 1,2, 5, 6, 7. & controls; *, diabetic subjects.
263
mined using cumene hydroperoxide [9]. To determine whether enzyme mass altered or fragmentation of the enzyme occured, lysates from the Percoll gradients were adjusted to a constant haemoglobin, extracted with chloroform-ethanol and electrophoresed in 12.5% discontinuous polyacrylamide gels, electroblotted onto nitrocellulose membranes and probed with antisera to CuZn SOD [9]. The figure shows the levels of activity of CuZn SOD in erythrocytes from different parts of the Percoll gradient. In samples from the total diabetic group and nondiabetic subjects activity declined progressively with increasing erthrocyte age and was significantly lower in older cells (fraction 7) than in young cells (fraction 1). Activities in erythrocytes from diabetic subjects either with or without complications and non-diabetic subjects of similar age were not significantly different. The immunoblotting studies supported the activity measurements showing that CuZn SOD mass declined with aging in erythrocytes from both diabetic and non-diabetic subjects. Glutathione peroxidase activity in the groups was not significantly different and did not alter during aging. Our data show that erythrocyte expression of CuZn SOD and glutathione peroxidase, two major antioxidant enzymes, is unaltered in diabetics. Unlike studies in streptozotocin-treated rats, levels of CuZn SOD in erythrocytes from human diabetics are not reduced even in older cells exposed to glycation and ROS stress for about 120 days. Richard C. Strangea, Peter Jonesb, John Bicknella and John Scarpelloa ‘Clinical Biochemistry
Research Laboratory,
School of Postgraduate
Medicine. University of Keele, North
Staffor&hire Hospital Center and Metabolic Unit. North StaffordFhire Hospital Centre, Hartshill, Stoke on Trent. Staffordshire and b Department of Mathematics, Keele University, Stafforhhire (UK)
References Loven D, Schedl H, Wilson H, Daabees TT, Stegink LD, Diekus M, Oberley L. Effect of insulin and oral glutathione on glutathione levels and superoxide dismutase activities in organs of rats with streptozocin-induced diabetes. Diabetes 1986;35:503-507. Godin DV, Wohaieb SA, Garnett ME, Goumeniouk AD. Antioxidant enzyme alterations in experimental and clinical diabetes. Mel Cell Biochem 1988;84:223-231. Halliwell B, Gutteridge JMC. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990;280:1-8. Toth KM, Clifford DP, Berger EM, White CW, Repine JE. Intact human erythrocytes prevent hydrogen peroxide-mediated damage to isolated perfused rat lungs and cultured bovine pulmonary artery endothelial cells. J Clin Invest 1984;74:292-295. Sakurai T, Matsuyama M, Tsuchiya S. Glycation of erythrocyte superoxide dismutase reduces its activity. Chem Pharm Bull 1987;35:302-307. Shibata F, Ogita 2. Epigenetic modification of murine Cu,Zn-superoxide dismutase. Electrophoresis 1986;7:426-428. Salo DC, Pacifici RE, Lin SW, Giulivi C, Davies KJA. Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation. J Biol Chem 1990;265:11919-11927. Strange RC, Johnson PH, Lawton A, Moult JA, Tector MJ, Tyminski RJ, Cotton W. Studies on the variability of glutathione S-transferase from human erythrocytes. Clin Chim Acta 1982; 120:25l-260. Strange RC, Cotton W, Fryer AA, Jones P, Bell J, Hume R. Lipid peroxidation and expression of copper-zinc and manganese superoxide dismutase in lungs of premature infants with hyaline membrane disease and bronchopulmonary dysplasia. J Lab Clin Med 1990;116:666-673.
261
CCA 05289
Letter to the Editor
Expression of Con-superoxide dismutase and ~~utat~ione peroxidase in e~t~rocytes from diabetic and non-diabetic subjects (Received 25 March 1992; accepted 26 March 1992)
Dear Editor, Recent studies implicate reactive oxygen species (ROS) in the development of the secondary compli~tions that character&e chronic diabetes. Thus, activity of antioxidant enzymes such as superoxide dismutase is reduced in several organs of the diabetic rat [l] and plasma lipid peroxide levels are higher in diabetic patients with retinopathy than in those without this complication [2]. Erythrocytes may be an important ‘sink’ for ROS such as superoxide anion and hydrogen peroxide which are generated in plasma and which can cross the red cell membrane [3,4]. Erythrocytes express various antioxidant enzymes including CuZn superoxide dismutase (CuZn SOD) which catalyses the disproportionation of superoxide anion and, glutathione peroxidase and catalase which catalyse the detoxication of hydrogen peroxide. These reactions suppress formation of the toxic hydroxyl radical. Studies showing that CuZn SOD is susceptible to glycation and modification by ROS suggests expression of this enzyme may be reduced in erythrocytes from diabetics. Thus, Sakurai et al. [S] reported both glycation of Iysine residues and loss of enzymic activity following incubation of purified CuZn SOD with high concentrations of glucose and Shibata and Ogita [6J found three isoforms of the enzyme in murine erythrocytes, one of which resulted from the actions of hydrogen peroxide. Concentration-dependent inactivation of erythrocyte CuZn SOD by hydrogen peroxide has also been reported [7]. Prolonged exposure of CuZn SOD to relatively high concentrations of hydrogen peroxide caused fragmentation of the enzyme [7]. The combined effect of ROS and hyperglycaemia may have important effects on the level of antioxidant enzyme expression in the erythrocytes of patients with diabetes, particularly in older cells that have been in the vascular space for over 100 days. Data on the levels of antioxidant enzyme activity could be useful as prognostic C~r~e+?&?rceto: Dr. R.C. Strange, Centre of Pathology and Molecular Medicine, North Staffordshire Hospital Centre, Thornburrow Drive, Hartshiif, Stoke on Trent, Staffs., UK.
262
markers. We describe studies on the expression of CuZn SOD and glutathione peroxidase in erythrocytes separated by age in Percoll gradients [S]. Heparinised blood (10 ml) was obtained with ethical approval from 9 non-diabetic controls (glycated Hb ~6.3%; age range 25-50 years) and 15 well-controlled, insulin-dependent diabetic subjects (mean glycated Hb 12.0 + 2.0%; age range 17-69 years). Ten patients were without evidence of diabetic complications but five patients demonstrated either nephropathy or retinopathy. Blood was centrifuged (2,000 x g, 10 min, 4°C) within 1 h, the plasma removed and replaced with an equal vohune of 154 mmoM NaCl. Leucocytes and platelets were removed by elution through cellulose and the haemat~rit adjusted with NaCi to about 0.3. Portions (1.5 ml) of the resultant erythrocyte suspension were gently layered onto continuous Percoll gradients (40 ml) prepared by mixing 56% and 76% Pet-co11and after centrifugation, cells were drawn off in 7 fractions. Cells from diabetic and non-diabetic subjects demonstrated similar decreases in mean cell volume (92-88 fl) and K+/haemoglobin ratio (360-278 PmoYg) and increase in mean cell haemoglobin concentration (31-36 g/100 ml) confirming the gradients were properly separating cells by age [8]. CuZn SOD activity was determined in chloroform-ethanol extracted lysates by monitoring the reduction of nitroblue tetrazolium. Glutathione peroxidase activity was deter-
Superoxide 7,000
dismutase
lunltllo Hbl 1 T
2poo 1
2
3
4
5
6
7
Cell Fractions Fig. 1. CuZn-superoxide dismutase activity in Percoll separated erythrocytes. The insert shows an immunoblot of CuZn-superoxide dismutase in (left to right) standard, fractions 1,2, 5, 6, 7. & controls; *, diabetic subjects.
263
mined using cumene hydroperoxide [9]. To determine whether enzyme mass altered or fragmentation of the enzyme occured, lysates from the Percoll gradients were adjusted to a constant haemoglobin, extracted with chloroform-ethanol and electrophoresed in 12.5% discontinuous polyacrylamide gels, electroblotted onto nitrocellulose membranes and probed with antisera to CuZn SOD [9]. The figure shows the levels of activity of CuZn SOD in erythrocytes from different parts of the Percoll gradient. In samples from the total diabetic group and nondiabetic subjects activity declined progressively with increasing erthrocyte age and was significantly lower in older cells (fraction 7) than in young cells (fraction 1). Activities in erythrocytes from diabetic subjects either with or without complications and non-diabetic subjects of similar age were not significantly different. The immunoblotting studies supported the activity measurements showing that CuZn SOD mass declined with aging in erythrocytes from both diabetic and non-diabetic subjects. Glutathione peroxidase activity in the groups was not significantly different and did not alter during aging. Our data show that erythrocyte expression of CuZn SOD and glutathione peroxidase, two major antioxidant enzymes, is unaltered in diabetics. Unlike studies in streptozotocin-treated rats, levels of CuZn SOD in erythrocytes from human diabetics are not reduced even in older cells exposed to glycation and ROS stress for about 120 days. Richard C. Strangea, Peter Jonesb, John Bicknella and John Scarpelloa ‘Clinical Biochemistry
Research Laboratory,
School of Postgraduate
Medicine. University of Keele, North
Staffor&hire Hospital Center and Metabolic Unit. North StaffordFhire Hospital Centre, Hartshill, Stoke on Trent. Staffordshire and b Department of Mathematics, Keele University, Stafforhhire (UK)
References Loven D, Schedl H, Wilson H, Daabees TT, Stegink LD, Diekus M, Oberley L. Effect of insulin and oral glutathione on glutathione levels and superoxide dismutase activities in organs of rats with streptozocin-induced diabetes. Diabetes 1986;35:503-507. Godin DV, Wohaieb SA, Garnett ME, Goumeniouk AD. Antioxidant enzyme alterations in experimental and clinical diabetes. Mel Cell Biochem 1988;84:223-231. Halliwell B, Gutteridge JMC. The antioxidants of human extracellular fluids. Arch Biochem Biophys 1990;280:1-8. Toth KM, Clifford DP, Berger EM, White CW, Repine JE. Intact human erythrocytes prevent hydrogen peroxide-mediated damage to isolated perfused rat lungs and cultured bovine pulmonary artery endothelial cells. J Clin Invest 1984;74:292-295. Sakurai T, Matsuyama M, Tsuchiya S. Glycation of erythrocyte superoxide dismutase reduces its activity. Chem Pharm Bull 1987;35:302-307. Shibata F, Ogita 2. Epigenetic modification of murine Cu,Zn-superoxide dismutase. Electrophoresis 1986;7:426-428. Salo DC, Pacifici RE, Lin SW, Giulivi C, Davies KJA. Superoxide dismutase undergoes proteolysis and fragmentation following oxidative modification and inactivation. J Biol Chem 1990;265:11919-11927. Strange RC, Johnson PH, Lawton A, Moult JA, Tector MJ, Tyminski RJ, Cotton W. Studies on the variability of glutathione S-transferase from human erythrocytes. Clin Chim Acta 1982; 120:25l-260. Strange RC, Cotton W, Fryer AA, Jones P, Bell J, Hume R. Lipid peroxidation and expression of copper-zinc and manganese superoxide dismutase in lungs of premature infants with hyaline membrane disease and bronchopulmonary dysplasia. J Lab Clin Med 1990;116:666-673.
