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Impaired blood superoxide dismutase in the traumatic paraplegic patients Liao W, Jin XY, Wang BH, Cui XS, Wang JL, Zhang J, Zhou TJ. Impaired blood superoxide dismutase in the traumatic paraplegic patients. Acta Neurol Scand 1992: 86: 329-331.
W. Liao’, X. Y. Jin R. H. Wang I , X. S. Cui I , J. L. Wan! J. Zhang’, T. J. Zhou2
’
Blood superoxide dismutase (SOD) activity and blood copper, zinc-superoxide dismutase (Cu.Zn-SOD) content were measured by luminol chemiluminescence assay and by single radial immunodflusion assay, respectively, in 50 patients with paraplegia due to traumatic injury to the spinal cord by the Tangshan Earthquake on July 28, 1976, compared with 20 age-matched healthy subjects. We found that blood SOD activity and blood Cu.Zn-SOD content in the paraplegic patients were significantly lower than those in healthy subjects (~(0.01). In healthy subjects, blood Cu.Zn-SOD fully expressed the enzymatic activity, whereas only 77% of blood Cu.Zn-SOD in the paraplegic patients expressed the enzymatic activity, indicating that in the patients, part of blood Cu.Zn-SOD protein is in a state without function. Also the serum lipid peroxide level in the paraplegic patients was higher than that in healthy subjects (p < 0.05). These findings suggest decreased endogenous blood protection against oxygen derived free radicals in these paraplegic patients.
Increased free radical generation and lipid peroxidation have been implicated in various pathological conditions, such as in ionizing radiation, carcinogenesis, inflammation, and in aging. Living organisms can protect themselves from the injurious effects of free radicals in many ways. Superoxide dismutase represents one of the major antioxidant enzymes. It is a cytoplasmic enzyme that catalyses the dismutation of superoxide anion (0g ) to hydrogen peroxide (H202), thus protecting the organism against the toxic reactions of O F . Whereas free radicals e.g. superoxide anion are very toxic and the ranges are very short (a few micrometers), the transformation to hydrogen peroxide gives rise to long range actions. In this study, blood superoxide dismutase (SOD) activity, blood copper, zincsuperoxide dismutase (Cu.Zn-SOD) content and serum lipid peroxide (LPO) were measured in 50 patients with paraplegia due to traumatic injury to the spinal cord by the Tangshan Earthquake on July 28, 1976. We found decrease in blood SOD activity and in blood Cu.Zn-SOD content, and increase in serum lipid peroxide level in these patients.
Clinical Research Laboratory, Department of Surgery, Beijing Charity Hospital, China RehabilitationResearch Centre, People’s Republic of China
Key words: superoxide dismutase (SOD): lipid peroxidation; free radicals; paraplegia Dr. Wei Liao, Present address: Department of Internal Medicine, Malmo General Hospital, S-214 01, Malmo, Sweden Accepted for publication February 13, 1992
Patients and methods
Subjects. Fifty of the traumatic paraplegics, 37 men and 13 women, between the age of 26 and 66 years were studied. Blood sampling was made for the present study 13 years after the accident. All patients are survivors of the Tangshan Earthquake on July 28,1976. They have been paralyzed due to traumatic injury to the spinal cord of thoracic or lumbar by the earthquake since then. The muscle strength of lower extremities in all patients was below 2 scale, based on the criteria of the MRC (Medical Research Council of Great Britain). All patients were resided in the Fengnan Sanatorium for Paraplegics, Tangshan, during the study period. Twenty age-matched healthy subjects served as controls. Blood SOD activity assay. 10 pl of blood taken from earlobe capillary were immediately lysed with 240 pl of distilled water. The SOD activity were then measured with luminol chemiluminescenceassay according to (l), and the measurements were accomplished within 24 h. Briefly, to 1-3 p1 of the blood lysate, 10 p1 of 0.1 mg/ml xanthine oxidase was added, followed by 980 pl of the mixture of hypoxanthine/
329
Liao et al. luminol (0.1 mmol/L). The luminescence was measured on LKB-Wallac 1250 Luminometer 60 s after initiation of the reaction. This referred to as enzymatic activity. One unit of enzymatic activity was defined as the amount of enzyme that produces a 50 % inhibition of luminescence. Blood hemoglobin (Hb) level were determined at same time. In order to compare with the Cu.Zn-SOD content measured by single radial immunodiffusion assay all results were calculated in terms of pg equivalents of human blood Cu.Zn-SOD, and expressed as ug SOD/g.Hb.
Table 2. Comparisonof blood SOD activity with blood Cu.Zn-SOD content in the healthy subjects and in the traumatic paraplegic patients
Blood Cu.Zn-SOD content assay. 20 p1 of blood taken from earlobe capillary were immediately lysed with 40 pl of distilled water. 1Oul of lysate were then applied to the agar plate containing rabbit antihuman Cu.Zn-SOD antibody (kindly provided by the Molecular Biological Research Lab, Navy General Hospital, Beijing) for measuring Cu.Zn-SOD content by single radial immunodiffusion assay using human blood Cu.Zn-SOD as standard. After 24 h diffusion, the diameter of the precipative ring was measured, and the Cu.Zn-SOD amount of blood sample are calculated according to standard curve. The results are expressed as ug SOD/g.Hb.
activity and blood Cu.Zn-SOD content in normal subjects (p < 0.01) (Table 1). In healthy subjects, blood SOD activity measured by luminol chemiluminescence assay was consistent with blood Cu.Zn-SOD content measured by using immunodiffusion assay (Table 2), indicating blood Cu.Zn-SOD protein fully expressed the enzymatic activity. However, in the paraplegic patients, blood SOD activity was significantly lower than blood Cu.Zn-SOD immunoreactive protein levels (p < 0.01) (Table 2), only 77% of Cu.Zn-SOD protein expressing the enzymatic activity (based on that Cu.Zn-SOD in healthy subjects expressed 100% of the enzymatic activity). This suggests that in the patients, part of blood Cu.Zn-SOD protein is in a state without function. The serum lipid peroxide level in the patients was found higher than that in healthy subjects (p < 0.05) (Table 1). However, no correlation between the SOD activity and the serum lipid peroxide level was found in the patients (r = 0.059, p > 0.05).
Lipid peroxide assay. Serum lipid peroxidation was determined by the thiobarbituric acid reaction according to the method of Yagi (2) using 1,1,3,3,tetramethoxypropane as standard. The assay measures oxidation by determining the content of malondialdehyde (MDA). The results were expressed as equivalent nmol/L of MDA. Statistical analyses. Experimental results were analysed for their statistical significance by Wilcoxon rank sum test for unpaired data (Table 1) or for matched pairs of data (Table 2). A p value less than 0.05 was considered as significant. Results
The results showed that both blood SOD activity and blood Cu.Zn-SOD content in the paraplegic patients were significantly lower than blood SOD Table 1. Blood SOD activity, blood Cu.Zn-SOD content and serum LPO levels in the traumatic paraplegic patients Control Numbers of cases SOD activity (ug/g.Hb) Cu.Zn-SOD content (ug/g. Hb) LPO (nmolll)
*
P value
Impaired blood superoxide dismutase in the traumatic paraplegic patients Liao W, Jin XY, Wang BH, Cui XS, Wang JL, Zhang J, Zhou TJ. Impaired blood superoxide dismutase in the traumatic paraplegic patients. Acta Neurol Scand 1992: 86: 329-331.
W. Liao’, X. Y. Jin R. H. Wang I , X. S. Cui I , J. L. Wan! J. Zhang’, T. J. Zhou2
’
Blood superoxide dismutase (SOD) activity and blood copper, zinc-superoxide dismutase (Cu.Zn-SOD) content were measured by luminol chemiluminescence assay and by single radial immunodflusion assay, respectively, in 50 patients with paraplegia due to traumatic injury to the spinal cord by the Tangshan Earthquake on July 28, 1976, compared with 20 age-matched healthy subjects. We found that blood SOD activity and blood Cu.Zn-SOD content in the paraplegic patients were significantly lower than those in healthy subjects (~(0.01). In healthy subjects, blood Cu.Zn-SOD fully expressed the enzymatic activity, whereas only 77% of blood Cu.Zn-SOD in the paraplegic patients expressed the enzymatic activity, indicating that in the patients, part of blood Cu.Zn-SOD protein is in a state without function. Also the serum lipid peroxide level in the paraplegic patients was higher than that in healthy subjects (p < 0.05). These findings suggest decreased endogenous blood protection against oxygen derived free radicals in these paraplegic patients.
Increased free radical generation and lipid peroxidation have been implicated in various pathological conditions, such as in ionizing radiation, carcinogenesis, inflammation, and in aging. Living organisms can protect themselves from the injurious effects of free radicals in many ways. Superoxide dismutase represents one of the major antioxidant enzymes. It is a cytoplasmic enzyme that catalyses the dismutation of superoxide anion (0g ) to hydrogen peroxide (H202), thus protecting the organism against the toxic reactions of O F . Whereas free radicals e.g. superoxide anion are very toxic and the ranges are very short (a few micrometers), the transformation to hydrogen peroxide gives rise to long range actions. In this study, blood superoxide dismutase (SOD) activity, blood copper, zincsuperoxide dismutase (Cu.Zn-SOD) content and serum lipid peroxide (LPO) were measured in 50 patients with paraplegia due to traumatic injury to the spinal cord by the Tangshan Earthquake on July 28, 1976. We found decrease in blood SOD activity and in blood Cu.Zn-SOD content, and increase in serum lipid peroxide level in these patients.
Clinical Research Laboratory, Department of Surgery, Beijing Charity Hospital, China RehabilitationResearch Centre, People’s Republic of China
Key words: superoxide dismutase (SOD): lipid peroxidation; free radicals; paraplegia Dr. Wei Liao, Present address: Department of Internal Medicine, Malmo General Hospital, S-214 01, Malmo, Sweden Accepted for publication February 13, 1992
Patients and methods
Subjects. Fifty of the traumatic paraplegics, 37 men and 13 women, between the age of 26 and 66 years were studied. Blood sampling was made for the present study 13 years after the accident. All patients are survivors of the Tangshan Earthquake on July 28,1976. They have been paralyzed due to traumatic injury to the spinal cord of thoracic or lumbar by the earthquake since then. The muscle strength of lower extremities in all patients was below 2 scale, based on the criteria of the MRC (Medical Research Council of Great Britain). All patients were resided in the Fengnan Sanatorium for Paraplegics, Tangshan, during the study period. Twenty age-matched healthy subjects served as controls. Blood SOD activity assay. 10 pl of blood taken from earlobe capillary were immediately lysed with 240 pl of distilled water. The SOD activity were then measured with luminol chemiluminescenceassay according to (l), and the measurements were accomplished within 24 h. Briefly, to 1-3 p1 of the blood lysate, 10 p1 of 0.1 mg/ml xanthine oxidase was added, followed by 980 pl of the mixture of hypoxanthine/
329
Liao et al. luminol (0.1 mmol/L). The luminescence was measured on LKB-Wallac 1250 Luminometer 60 s after initiation of the reaction. This referred to as enzymatic activity. One unit of enzymatic activity was defined as the amount of enzyme that produces a 50 % inhibition of luminescence. Blood hemoglobin (Hb) level were determined at same time. In order to compare with the Cu.Zn-SOD content measured by single radial immunodiffusion assay all results were calculated in terms of pg equivalents of human blood Cu.Zn-SOD, and expressed as ug SOD/g.Hb.
Table 2. Comparisonof blood SOD activity with blood Cu.Zn-SOD content in the healthy subjects and in the traumatic paraplegic patients
Blood Cu.Zn-SOD content assay. 20 p1 of blood taken from earlobe capillary were immediately lysed with 40 pl of distilled water. 1Oul of lysate were then applied to the agar plate containing rabbit antihuman Cu.Zn-SOD antibody (kindly provided by the Molecular Biological Research Lab, Navy General Hospital, Beijing) for measuring Cu.Zn-SOD content by single radial immunodiffusion assay using human blood Cu.Zn-SOD as standard. After 24 h diffusion, the diameter of the precipative ring was measured, and the Cu.Zn-SOD amount of blood sample are calculated according to standard curve. The results are expressed as ug SOD/g.Hb.
activity and blood Cu.Zn-SOD content in normal subjects (p < 0.01) (Table 1). In healthy subjects, blood SOD activity measured by luminol chemiluminescence assay was consistent with blood Cu.Zn-SOD content measured by using immunodiffusion assay (Table 2), indicating blood Cu.Zn-SOD protein fully expressed the enzymatic activity. However, in the paraplegic patients, blood SOD activity was significantly lower than blood Cu.Zn-SOD immunoreactive protein levels (p < 0.01) (Table 2), only 77% of Cu.Zn-SOD protein expressing the enzymatic activity (based on that Cu.Zn-SOD in healthy subjects expressed 100% of the enzymatic activity). This suggests that in the patients, part of blood Cu.Zn-SOD protein is in a state without function. The serum lipid peroxide level in the patients was found higher than that in healthy subjects (p < 0.05) (Table 1). However, no correlation between the SOD activity and the serum lipid peroxide level was found in the patients (r = 0.059, p > 0.05).
Lipid peroxide assay. Serum lipid peroxidation was determined by the thiobarbituric acid reaction according to the method of Yagi (2) using 1,1,3,3,tetramethoxypropane as standard. The assay measures oxidation by determining the content of malondialdehyde (MDA). The results were expressed as equivalent nmol/L of MDA. Statistical analyses. Experimental results were analysed for their statistical significance by Wilcoxon rank sum test for unpaired data (Table 1) or for matched pairs of data (Table 2). A p value less than 0.05 was considered as significant. Results
The results showed that both blood SOD activity and blood Cu.Zn-SOD content in the paraplegic patients were significantly lower than blood SOD Table 1. Blood SOD activity, blood Cu.Zn-SOD content and serum LPO levels in the traumatic paraplegic patients Control Numbers of cases SOD activity (ug/g.Hb) Cu.Zn-SOD content (ug/g. Hb) LPO (nmolll)
*
P value
