Mechanisms of Ageing and Development, 8 (1978) 265-267 ©Elsevier Sequoia S.A., Lausanne - Printed in the Netherlands
265
SPECIFIC ACTIVITY OF HUMAN ERYTHROCYTE SUPEROXIDE DISMUTASE AS A FUNCTION OF DONOR AGE. A BRIEF NOTE
HANS JOENJE, RUNE R. FRANTS, FRI~ ARWERT and ALDUR W. ERIKSSON Institute of Human Genetics, Free University, Amsterdam (The Netherlands) (Received April 13, 1978)
SUMMARY
Superoxide dismutase was assayed both immunochemically and by enzymatic activity in erythrocytes of human donors, 1 to 98 years of age. No change was observed in enzyme activity per unit enzyme antigen as a function of donor age.
150
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DONOR AGE (YEARS)
Fig. 1. Human erythrocyte SOD levels as a function of donor age. Washed erythrocytes were lysed in H 2 0 and diluted to about 10 g hemoglobin/100 ml, Enzyme antigen was determined by the radial immunodiffusion technique of Mancini et al. [8] using agar plates containing sheep antiserum prepared against cytoplasmic SOD, purified from human liver. The mean value is 100 arbitrary units. o, females; o , males.
266 TABLE I ERYTHROCYTE SOD ACTIVITY PER ANTIGENIC UNIT IN YOUNG AND OLD INDIVIDUALS Age (years)
(A ) activity a
(B) antigenic units b
A/B
2 4 5 11
86 96 107 118
105 94 121 108
0.82 1.02 0.88 1.09
0.95 ± 0.13 e
57 63 68 69
102 107 86 96
92 89 102 91
1.11 1.20 0.84 1.05
1.05 ± 0.16 e
aSOD activity was measured in triplicate essentially by the xanthine-xanthine oxidase-NBT method of Beauchamp and Fridovich [9], after extraction of hemolysates with ethanol/chloroform as described by Winterbourn et al. [10] ; values were corrected for Hb content of the hemolysates. The standard deviation of the method for activity measurements was about 5%. The mean activity of all 8 individuals is 100 arbitrary units. bSOD was quantitated immunochemically in hemolysates by the radial immunodiffusion technique of Mancini et al. [8] and corrected for Hb content of the hemolysates. The standard deviation of the method was ~11%. Levels of antigenic activity are expressed as percentage of the mean value. eThe difference between the two means was not significant (P > 0.3).
Superoxide radicals ( 0 7 ) , and their reaction products OH. (hydroxyl radicals) and 102 (singlet excited molecular oxygen), are generated in aerobically living cells and are potentially deleterious to cellular components [1, 2]. Protection against O7 mediated damage is provided by superoxide dismutases which catalyze the dismutation of O7 to H202 and oxygen [1]. Free radical reactions may contribute to the senescence o f an organism by causing partly irreversible damage [3, 4 ] . Therefore, the cellular superoxide dismutase (SOD) level may constitute an important factor influencing the rate of senescence. A number of laboratories have reported on SOD levels as a function of organism age. Reiss and Gershon [5] found that cytoplasmic SOD activity in rat liver drops dramatically (about 2.5 ×) as a function of age, a phenomenon that could be ascribed to a decreased activity per unit enzyme antigen. However, no reduction of SOD activity in liver of old rats was found by Kellogg III and Fridovich [6]. Stevens etal. [7] using the autoxidation o f epinephrine to assay SOD activity in human erythrocyte extracts, were unable to show any change in SOD activity per milligram non-hemoglobin protein as a function of donor age. However, these results do not exclude the possibility that human erythrocytes accumulate partly active SOD molecules, as found by Reiss and Gershon [5] for rat liver. To establish this, SOD must be assayed immunochemically as well. As part of a population study in Finland we have quantitated erythrocyte SOD by the radial immunodiffusion technique o f Mancini et al. [8]. Figure 1 shows that no significant change is observable in SOD antigenic levels as a function o f donor age. The mean SOD level was 100 -+ 16 (S.D., n = 50) for the total population studied and 109 -+ 21 (S.D., n = 18) for the age group 1-20 years, 94 -+ 12 (S.D., n = 18) for the age group
267 21--48, and 98 -+ 12 (S.D., n = 19) for the age group 51-98 years. Differences between the age groups and between males and females were not significant [cf. 7]. In four young (mean age 5 years) and four old (mean age 64 years) individuals, erythrocyte SOD levels were measured both by activity and immunochemically. The
specific activities of SOD were not significantly different in these two age groups (see Table i). These observations, along with those of Stevens et al. [7], show that the activity of erythrocyte SOD per antigenic unit does not change detectably during human ageing.
REFERENCES 1 I. Fridovich, Superoxide dismutases, A, Rev. Biochem., 44 (1975) 147-159. 2 B. Halliwell, Superoxide dismutase, catalase and glutathione peroxidase: solutions to the problems of living with oxygen, New Phytol., 73 (1974) 1075-1086. 3 D. Harman, Aging: A theory based on free radical and radiation chemistry, J. Gerontol., 11 (1956) 298-300. 4 D. Harman, Free radical theory of aging: effect of the amount and degree of unsaturation of dietary fat on mortality rate, J. Gerontol., 26 (1971) 451--457. 5 U. Reiss and D. Gershon, Rat-liver superoxide dismutase. Purification and age-related modifications, Eur. J. Biochern., 63 (1976) 617-623. 6 E. W. Kellogg III and I. Fridovich, Superoxide dismutase in the rat and mouse as a function of age and longevity, J. Gerontol., 31 (1976)405408. 7 C. Stevens, M. J. Goldblatt and J. C. Freedman, Lack of erythrocyte superoxide dismutase change during human senescence, Mech. AgeingDev., 4 (1975) 415421. 8 G. Mancini, J. P. Vaerman, A. O. Carbonara and J. F. Heremans, in H. Peeters (ed.), Protides o f the Biological Fluids, Xlth Colloq., Bruges, Elsevier, Amsterdam, 1964, pp. 370-373. 9 C. Beauchamp and I. Fridovich, Superoxide dismutase: improved assays and an assay applicable to acrylamide gels,AnaL Biochem., 44 (1971) 276-287. 10 C. C. Winterbourn, R. E. Hawkins, M. Brian and R. W. Carrell, The estimation of red cell superoxide dismutase activity, J. Lab. Clin. Med., 85 (1975) 337-341.
265
SPECIFIC ACTIVITY OF HUMAN ERYTHROCYTE SUPEROXIDE DISMUTASE AS A FUNCTION OF DONOR AGE. A BRIEF NOTE
HANS JOENJE, RUNE R. FRANTS, FRI~ ARWERT and ALDUR W. ERIKSSON Institute of Human Genetics, Free University, Amsterdam (The Netherlands) (Received April 13, 1978)
SUMMARY
Superoxide dismutase was assayed both immunochemically and by enzymatic activity in erythrocytes of human donors, 1 to 98 years of age. No change was observed in enzyme activity per unit enzyme antigen as a function of donor age.
150
140f
[]
N N
[] SS
•
D
N
110 •
•°
100
@
•
•
•
[] •
•
E •
[] •
•
•
• []
[]
•
[]
40
30~10
o,
,~,
~'o
-~o
,~
,~
~o
~
8'o
~o
'
IO0
DONOR AGE (YEARS)
Fig. 1. Human erythrocyte SOD levels as a function of donor age. Washed erythrocytes were lysed in H 2 0 and diluted to about 10 g hemoglobin/100 ml, Enzyme antigen was determined by the radial immunodiffusion technique of Mancini et al. [8] using agar plates containing sheep antiserum prepared against cytoplasmic SOD, purified from human liver. The mean value is 100 arbitrary units. o, females; o , males.
266 TABLE I ERYTHROCYTE SOD ACTIVITY PER ANTIGENIC UNIT IN YOUNG AND OLD INDIVIDUALS Age (years)
(A ) activity a
(B) antigenic units b
A/B
2 4 5 11
86 96 107 118
105 94 121 108
0.82 1.02 0.88 1.09
0.95 ± 0.13 e
57 63 68 69
102 107 86 96
92 89 102 91
1.11 1.20 0.84 1.05
1.05 ± 0.16 e
aSOD activity was measured in triplicate essentially by the xanthine-xanthine oxidase-NBT method of Beauchamp and Fridovich [9], after extraction of hemolysates with ethanol/chloroform as described by Winterbourn et al. [10] ; values were corrected for Hb content of the hemolysates. The standard deviation of the method for activity measurements was about 5%. The mean activity of all 8 individuals is 100 arbitrary units. bSOD was quantitated immunochemically in hemolysates by the radial immunodiffusion technique of Mancini et al. [8] and corrected for Hb content of the hemolysates. The standard deviation of the method was ~11%. Levels of antigenic activity are expressed as percentage of the mean value. eThe difference between the two means was not significant (P > 0.3).
Superoxide radicals ( 0 7 ) , and their reaction products OH. (hydroxyl radicals) and 102 (singlet excited molecular oxygen), are generated in aerobically living cells and are potentially deleterious to cellular components [1, 2]. Protection against O7 mediated damage is provided by superoxide dismutases which catalyze the dismutation of O7 to H202 and oxygen [1]. Free radical reactions may contribute to the senescence o f an organism by causing partly irreversible damage [3, 4 ] . Therefore, the cellular superoxide dismutase (SOD) level may constitute an important factor influencing the rate of senescence. A number of laboratories have reported on SOD levels as a function of organism age. Reiss and Gershon [5] found that cytoplasmic SOD activity in rat liver drops dramatically (about 2.5 ×) as a function of age, a phenomenon that could be ascribed to a decreased activity per unit enzyme antigen. However, no reduction of SOD activity in liver of old rats was found by Kellogg III and Fridovich [6]. Stevens etal. [7] using the autoxidation o f epinephrine to assay SOD activity in human erythrocyte extracts, were unable to show any change in SOD activity per milligram non-hemoglobin protein as a function of donor age. However, these results do not exclude the possibility that human erythrocytes accumulate partly active SOD molecules, as found by Reiss and Gershon [5] for rat liver. To establish this, SOD must be assayed immunochemically as well. As part of a population study in Finland we have quantitated erythrocyte SOD by the radial immunodiffusion technique o f Mancini et al. [8]. Figure 1 shows that no significant change is observable in SOD antigenic levels as a function o f donor age. The mean SOD level was 100 -+ 16 (S.D., n = 50) for the total population studied and 109 -+ 21 (S.D., n = 18) for the age group 1-20 years, 94 -+ 12 (S.D., n = 18) for the age group
267 21--48, and 98 -+ 12 (S.D., n = 19) for the age group 51-98 years. Differences between the age groups and between males and females were not significant [cf. 7]. In four young (mean age 5 years) and four old (mean age 64 years) individuals, erythrocyte SOD levels were measured both by activity and immunochemically. The
specific activities of SOD were not significantly different in these two age groups (see Table i). These observations, along with those of Stevens et al. [7], show that the activity of erythrocyte SOD per antigenic unit does not change detectably during human ageing.
REFERENCES 1 I. Fridovich, Superoxide dismutases, A, Rev. Biochem., 44 (1975) 147-159. 2 B. Halliwell, Superoxide dismutase, catalase and glutathione peroxidase: solutions to the problems of living with oxygen, New Phytol., 73 (1974) 1075-1086. 3 D. Harman, Aging: A theory based on free radical and radiation chemistry, J. Gerontol., 11 (1956) 298-300. 4 D. Harman, Free radical theory of aging: effect of the amount and degree of unsaturation of dietary fat on mortality rate, J. Gerontol., 26 (1971) 451--457. 5 U. Reiss and D. Gershon, Rat-liver superoxide dismutase. Purification and age-related modifications, Eur. J. Biochern., 63 (1976) 617-623. 6 E. W. Kellogg III and I. Fridovich, Superoxide dismutase in the rat and mouse as a function of age and longevity, J. Gerontol., 31 (1976)405408. 7 C. Stevens, M. J. Goldblatt and J. C. Freedman, Lack of erythrocyte superoxide dismutase change during human senescence, Mech. AgeingDev., 4 (1975) 415421. 8 G. Mancini, J. P. Vaerman, A. O. Carbonara and J. F. Heremans, in H. Peeters (ed.), Protides o f the Biological Fluids, Xlth Colloq., Bruges, Elsevier, Amsterdam, 1964, pp. 370-373. 9 C. Beauchamp and I. Fridovich, Superoxide dismutase: improved assays and an assay applicable to acrylamide gels,AnaL Biochem., 44 (1971) 276-287. 10 C. C. Winterbourn, R. E. Hawkins, M. Brian and R. W. Carrell, The estimation of red cell superoxide dismutase activity, J. Lab. Clin. Med., 85 (1975) 337-341.
