Mechanisms of Ageing and Development, 4 (1975) 415421 © Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

415

L A C K OF E R Y T H R O C Y T E S U P E R O X I D E D I S M U T A S E C H A N G E DURING HUMAN SENESCENCE

C. STEVENS, M. J. GOLDBLATT and J. C. FREEDMAN* Department of Biology, Reed College, Portland, Ore. 97202 (U.S.A.)

(Received July 28, 1975)

SUMMARY Superoxide dismutase (SOD), widely distributed in aerobic organisms, catalyzes dismutation of the superoxide free radical, 02% to oxygen and hydrogen peroxide and apparently protects against oxygen toxicity. In human erythrocytes, 0 2 arises from autoxidation of oxyhemoglobin and SOD activity is copper-dependent. Since human erythrocyte copper content has been reported to decline in the fifth decade of life, we investigated the age dependence of human erythrocyte SOD activity. The mean SOD activity, assayed by inhibition of epinephrine autoxidation, was 415 4- 66 units/g cells or 50 4- 11 units/mg non-Hb protein. No significant difference was observed between young and old adults, and no correlations were detected with sex, state of health of the donor, or with blood hemoglobin content. The lack of general decline of SOD activity with age narrows the possible mechanisms for an involvement of 0 2 - in senescence. SOD may yet decline in other longer-lived tissues or, as suggested by Fridovich, a constant low level of damage may be caused by imperfect scavenging of O2- by SOD. I f such a mechanism is operative, it appears not to affect synthesis of SOD in erythrocyte precursor cells into the eighth decade of human life.

INTRODUCTION The discovery by McCord and Fridovich 1 that erythrocuprein and similar metalloproteins in a variety of cells and organisms exhibit superoxide dismutase (SOD) activity raised the question of the physiological significance of the superoxide free radical, 02-. Since Packer 2 has recently linked free radical oxidation of membrane lipids with cellular aging, and since superoxide appears to be capable of damaging cell membranes a, the present study of human erythrocyte SOD activity as a func-

* Reprint address: Department of Physiology, Yale University School of Medicine, New Haven, Conn. 06510.

416 tion of donor age was designed to provide evidence pertaining to Fridovich's 4-6 suggestion that 02- may contribute to cellular and organismal senescence. Superoxide, the highly reactive radical formed by the one electron reduction of oxygen, is a side product of a variety of metabolic reactions 5. Its formation in human erythrocytes during autoxidation of oxyhemoglobin to methemoglobin was reported by Wever et al. v and by Misra and Fridovich 8. Though spontaneous dismutation of 02- is quite rapid 4, McCord et al. 9 proposed that catalyzed dismutation to oxygen and hydrogen peroxide by SOD enhances protection of aerobic organisms against oxidative damage, a hypothesis subsequently supported by results of a number of studies. That SOD is present in aerobic and aerotolerant microorganisms but not in strict anaerobes 9 suggests that it evolved with aerobic life. The induction of SOD in bacteria 1°, yeast 1~ and mammals ~2 by exposure to hyperbaric oxygen results in increased tolerance to oxygen. Conversely, loss or decline of SOD activity results in decreased protection against superoxide13, a4. The report by Herring et al. ~5 that human erythrocyte copper content begins to decline in the fifth decade of life, coupled with the known dependence of SOD activity on copper 16, raised the possibility that SOD activity may likewise decline with human senescence. However, the present result that erythrocyte SOD activity remains constant well into the eighth decade of life indicates that the reported 15 copper decline does not adversely affect SOD activity. METHODS I. E n z y m e isolation

Fresh human blood was obtained in 5 ml vacutainer tubes containing 0.5 ml of 3.8 ~ sodium citrate with 0.1 mg potassium sorbate. Erythrocytes were separated from the plasma and buffy coat by centrifugation at 3,000 × g for 5 minutes and washed twice in isotonic NaCI buffered with 10 mM sodium phosphate, pH 7.4, by centrifugation at 10,000 × g for 5 minutes. Packed cells were weighed, lysed by addition of an equal volume of deionized water, and hemoglobin was precipitated with chloroform and ethanol by the Tsuchihashi method as described by McCord and Fridovich 1. Superoxide dismutase activity was measured in the diluted supernatants which resulted from this fractionation. II. B i o c h e m i c a l determinations SOD activity determinations were based upon its ability to inhibit autoxidation of epinephrine at pH 10.2 as described by Misra and Fridovich 17. Each of four supernatants was diluted with deionized water to obtain a series of five or six inhibitions between 75 and 18 percent. Percent inhibition was then plotted on double reciprocal coordinates as a function of dilution. Taking 50 ~ inhibition of the uninhibited rate of autoxidation as one unit of activity, the data from the four samples were consolidated into a single curve by replotting the reciprocal of percent inhibition against the reciprocal of activity units (Fig. 1). Inhibitions obtained with the remaining supernatants were converted to activity units from this curve.

417 0.10

0.08

8

.--

0.04

0.02

O 0

I/units

Fig. 1. Standard curve for assay of superoxide dismutase activity based on inhibition of epinephrine autoxidation. O. O, ×, • represent four diluted supernatants. Each point was determined in triplicate. The mean reproducibility between triplicate assays was 3 ~ . Dilution of extraction solution alone had no effect on the uninhibited rate of epinephrine autoxidation. Diluted supernatants gave the same percent inhibition for at least four hours after isolation. Enzyme activity is expressed as units per gram of erythrocytes and as specific activity, in units per mg of non-hemoglobin protein. The protein content of each supernatant was determined in triplicate by the method of Lowry et al. ~8. Hemoglobin content of whole blood and supernatants cootaining SOD activity was determined by the Drabkin cyanmethemoglobin method 19. Extracellular space determination utilized inulin-C 14, extracted from packed cells with 5 ~ trichloroacetic acid, as previously described 20. Water content of packed cells was determined gravimetrically after drying at 70°C for 48 hours. RESULTS

I. Rate of epinephrine autoxidation The uninhibited rate of absorbance increase of 3 × 10 -4 M epinephrine in 0.05 M sodium carbonate buffer, p H 10.2, was 0.038 ± 0.006 OD/min. (SD, n = 9) when recorded at 480 nm at 30°C. This value agrees with the data presented in Figs. ! and 4 of Misra and Fridovich 17. (The assay protocol given at the end of their paper contains an inexplicably lower control rate, 0.025 at 30 °C.) Although this control rate of epinephrine autoxidation varied by as much as 10 ~ from day to day it never varied by more than 5 ~ on a given day. In order to control one source of experimental variability, the temperature sensitivity of epinephrine autoxidation was determined with the results shown in Fig. 2. II. Erythrocyte volume and extracellular space Neither erythrocyte water content nor extracellu[ar space varies significantly

418 -0,9

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-I.2

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-I.6

3.o

gt

3',2

liT

3'3

x I0 3

~

3'.4

3'.5

(°K)

Fig. 2. Temperature sensitivity of epinephrine autoxidation to adren0chrome. Points represent two to five determinations on two separate days (O, ~). The activation energy was calculated to be 16.1 kcal./mole in the range of 25°C to 40°C.

with d o n o r age. The mean water content of erythrocytes obtained f r o m individuals under 50 years of age was 0.65 ± 0.01 g/g cells (SE, n = 4) and for individuals over 50 years, 0.65 ± 0.01 g/g cells (SE, n = 4). The mean erythrocyte extracellular space in cells obtained from individuals under 50 years of age was 3.2 ~ 0.8 ml/100 g cells (SE, n = 4) and for those over 50 years, 2.9 ~ 0.8 (SE, n ~ 4). These results eliminate the possibility that enzyme activity measurements are sigrificantly influenced by agedependent changes in erythrocyte water content or extracellular space. llI. SOD activity The mean SOD activity for individuals of all ages was 415 ! 66 units/g cells (SD, n ~ 29) or 50 +_ 11 units/mg n o n - H b protein (SD, n = 29). The data presented in Table I and Fig. 3 show no significant dependence o f SOD activity on d o n o r age. There is also no significant difference between males and females. The mean specific activity for males was 53 ~ 11 units/mg n o n - H b protein (SD, n = 14) and for females was 47 i 8 units/mg n o n - H b protein (SD, n ~ 13). Superoxide dismutase specific activity was also the same in hospitalized and normal individuals, the mean specific activity for normal individuals being 53 ~- 12 units/rag n o n - H b protein (SD, n = 10) and for hospital patients 48 ± 9 units/mg n o n - H b protein (SD, n = 19). The lack of correlation between S O D activity and sex or state o f health of the d o n o r concurs with previously published immunochemical determinations o f h u m a n erythrocuprein levelsel,z2. The possible influence of the oxygen carrying capacity of the blood on erythrocyte S O D activity was also investigated. The mean S O D activity for the low hemoglobin range, 6.6 to 11.6 g/100 ml blood with mean 10.2 ~_ 1.2 g/100 ml blood (SD, n = 8), was 43 zE 6 units/mg n o n - H b protein and for the high hemoglobin range, 13.7 to

419 TABLE I LACK OF AGE DEPENDENCE OF HUMAN ERYTHROCYTE SUPEROXIDE DISMUTASE (SOD) ACTIVITY Age group Number o f samples

19-30

31-65

9

> 65

10

Mean ± S.D.

SOD activity (units/g cells) 394 ± 30 SOD activity (units/mg nonHb protein) 49 i 12 Whole blood Hb (g/100 ml) 12.6 ± 1.8

10

Range

Mean ± S.D.

Range

Mean ± S.D.

Range

301-534

406 ± 41

331485

448 -4- 94

287-588

39-79

45 ± 5

36-52

55 ~- 10

31-66

9.1 15.0

12.2 ~ 2.1

6.6-14.4

12.8 5_ 2.2

9.1-16.0

16.0 with m e a n of 14.4 ± 0.7 g/100 ml blood (SD, n = 8), the m e a n SOD specific activity was 50 ± 11 units/rag n o n - H b protein. The data presented in Table I also indicate no age dependence o f b l o o d h e m o g l o b i n content. DISCUSSION While e r y t h r o c u p r e i n a m o u n t s , d e t e r m i n e d immunochemically2a,2% a n d S O D activity 23 in n o r m a l h u m a n erythrocytes have been reported, the age dependence of SOD activity has n o t previously been examined. Utilizing the c o n v e n i e n t a n d simple assay based o n i n h i b i t i o n of epinephrine a u t o x i d a t i o n 17, we f o u n d SOD activity to be

120

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0

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6'0

8'0

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Age

Fig. 3. Superoxide dismutase specific activity as a function of donor age. Each point represents one individual and was assayed in triplicate. (1) This point was not included in mean and range calculations in Table I because hemoglobin did not precipitate completely during fractionation. (2) This point represents two individuals. The correlation coefficient is 0.30.

420

not significantly different in young and old adults. In view of current interest in the cellular basis of aging and the accessibility of human erythrocytes for study, we were surprised to find little comparative information in the literature on other erythrocyte enzyme activities as a function of donor age. As with the immunochemical determination21, 22, the biological variation in this sample population's SOD activities exceeds the estimated methodological variation. Our analysis indicates that the biological variation cannot be attributed to the age, sex or state of health of the donor, or the oxygen carrying capacity of the blood. The present finding that SOD activity does not decline with donor age, in spite of the reported age dependent decline of copper 15 in erythrocytes, suggests that the equilibrium between SOD bound copper and other erythrocyte copper is such that a 26 % decline in total erythrocyte copper does not affect SOD activity. Previous investigators 21, remarking upon the lack of correlation between erythrocuprein content and total copper of erythrocytes, have proposed that erythrocuprein is saturated by the more stable of the erythrocyte's two copper pools. Recent studiesZ2, 24 indicate that superoxide dismutase copper accounts for 40 % of total erythrocyte copper, the remaining 60 % being divided betweea a dialysable soluble pool and a non-dialysable stromal pool. The superoxide free radical could contribute to senescence either by a decrease in SOD activity with age or, as Fridovich 4-6 has suggested, by a constant low level of irreparable damage due to the imperfect scavenging of superoxide by SOD. Although erythrocyte SOD activity may not necessarily be representative of longer-lived tissues, our results narrow somewhat the possible mechanisms for an involvement of superoxide in senescence. Apparently the transcriptional and translational processes for superoxide dismutase synthesis in human erythrocyte precursor cells remain intact well into the eighth decade of life. ACKNOWLEDGEMENTS We wish to thank the staff of the Clinical Laboratory at Eastmoreland General Hospital for supplying us with human blood samples and express our appreciation to Will Bloch and James Haaga for helpful discussions and to Irwin Fridovich for commenting on the manuscript. Partial support was provided by the Medical Research Foundation of Oregon.

REFERENCES 1 J. M. McCord and I. Fridovich, Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprein), J. Biol. Chem., 244 (1969) 6049-6055. 2 L. Packer and J. R. Smith, Extension of the lifespan of cultured normal human diploid cells by vitamin E, Proc. Nat. Acad. Sci. U.S., 71 (1974) 4763-4767. 3 J. A. Fee and D. H. Teitelbaum, Evidence that superoxide dismutase plays a role in protecting red blood cells against peroxidative hemolysis, Biochem. Biophys. Res. Commun., 49 (1972) 150-158. 4 I. Fridovich, Superoxide radical and superoxide dismutase, Aeets. Chem. Res., 5 (1972) 321-326. 5 I. Fridovich, Superoxide dismutase, Adv. Enzymol., 41 (1974) 35-97. 6 I. Fridovich, Oxygen: Boon and Bane, Am. Scientist, 63 (1974) 54-59.

421 7 R. Wever, B. Oudega and B. F. Van Gelder, Generation of superoxide radicals during the autoxidation of mammalian oxyhemoglobin, Biochim. Biophys. Acta, 302 (1973) 475-478. 8 H. P. Misra and I. Fridovich, The generation of superoxide radical during the autoxidation of hemoglobin, J. Biol. Chem., 247 (1972) 6960-6962. 9 J. M. McCord, B. B. Keele Jr. and I. Fridovich, An enzyme-based theory of obligate anaerobiosis: the physiological function of superoxide dismutase, Proc. Nat. Acad. Sci. U.S., 68 (1971) 10241027. 10 E. M. Gregory and I. Fridovich, Induction of superoxide dismutase by molecular oxygen, J. Bacteriol., 114 (1973) 1193-1197. 11 S. A. Goscin, Ph.D. Thesis, Duke University, 1973. 12 J. Crapo and D. Tierney, Superoxide dismutase and oxygen toxicity, Clin. Res., 21 (1973) 222. 13 J. M. McCord, C. O. Beauchamp, S. Goscin, H. P. Misra and I. Fridovich, Superoxide and superoxide dismutase, in T. E. King, H. S. Mason and M. Morrison, (eds.), Oxidases and RelatedRedox Systems (Proc. 2nd Intern. Symp.), Vol. 1, Univ. Park. Press, Baltimore, 1973, pp. 5l 61. 14 F. J. Yost and I. Fridovich, Superoxide dismutaseand phagocytosis, Arch. Biochem. Biophys., 161 (1974) 395-401. 15 W. G. Herring, B. S. Leavell, L. M. Paixao and J. H. Yoe, Trace metals in human plasma and red cells. I. Observations of normal subjects, Am. J. Clin. Nutr., 8 (1960) 846-854. See also N. Massari, C. Guardamagna and A. M. Bertolini, Variations of erythrocytic stromatic copper in the course of aging, G. Gerontol., 8 (1960) 21-27. 16 H. J. Forman and I. Fridovich, On the stability of bovine superoxide dismutase. The effect of metals, J. BioL Chem., 248 (1973) 2645-2649. 17 H. P. Misra and I. Fridovich, The role of superoxide anion in autoxidation of epinephrine and a simple assay for superoxide dismutase, J. Biol. Chem., 247 (1972) 3170-3175. 18 O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurement with the folin phenol reagent, J. BioL Chem., 193 (1951) 265-275. 19 International Committee for Standardization in Hematology, Recommendations and requirements for haemoglobinometry in human blood, Scand. J. Clin. Lab. Invest., 17 (1965) 617-720. 20 J. C. Freedman, Ph.D. Thesis, Univ. of Penna, 1973. 21 G. S. Shields, H. Markowitz, W. H. Klassen, G. E. Cartwright and M. M. Wintrobe, Studies on copper metabolism. XXXI. Erythrocyte copper, J. Clin. Invest., 40 (1961) 2007-2015. 22 M.J. Stansell and H. F. Deutsch, The levels of catalase and erythrocuprein in human erythrocytes, Clin. Chim. Acta, 14 (1966) 598-607. See also J. W. Hartz, S. Funakoshi and H. F. Deutsch, The levels of superoxide dismutase and catalase in human tissues as determined immunochemically, Clin. Chim. Acta, 46 (1973) 125-132. 23 F. Lavelle, K. Puget and A. M. Michelson, Superoxide dismutase. Function and concentration of erythrocuprein in the normal human, Compt. Rend., Ser. D, 278 (1974) 2695-2698. 24 H. A. Lichtman and R. I. Weed, Divalent cation content of normal and ATP-depleted erythrocytes and erythrocyte membranes, in M. Bessis, R. Weed and P. Leblond (eds.), Red Cell Shape, Springer Verlag, New York, 1973, pp. 79-95.