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High sensitivity to autoxidation in neonatal calf erythrocytes: possible mechanism of accelerated cell aging
Mechanisms of Ageing and Development 122 (2001) 69 – 76 www.elsevier.com/locate/mechagedev
High sensitivity to autoxidation in neonatal calf erythrocytes: possible mechanism of accelerated cell aging S. Imre a,*, M. Csornai b, M. Balazs c a
Department of Pathophysiology, Medical Uni6ersity of Debrecen, Nagyerdei krt. 98, PO Box 23, H-4012 Debrecen, Hungary b Department of Neurology and Psychiatry, Medical Uni6ersity of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, Hungary c Department of Biophysics, Medical Uni6ersity of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, Hungary
Received 14 May 2000; received in revised form 8 September 2000; accepted 15 September 2000
Abstract The suspension viscosity, formation of methaemoglobin and production of malondialdehyde (MDA) associated with the non-enzymatic oxidation of polyunsaturated fatty acids during auto-oxidation conditions in vitro have been compared in erythrocytes from young calves (2, 4 and 6 weeks of age) and mature cattle. The autoxidation conditions were designed to simulate the oxidative stress to which neonatal erythrocytes are exposed in vivo. Characterisation of lipid peroxidation was also undertaken by a combination of lipid fluorescent measurements and quantification of the superoxide dismutase (SOD) activities of the erythrocytes. The results demonstrated that high SOD activities in the erythrocytes of the neonatal calf was insufficient to afford protection against the increased autoxidation of haemoglobin and subsequent accumulation of lipid peroxidation products. High levels of methaemoglobin formation and lipid peroxidation were able to provide an explanation for an observed reduction in rheological adaptability (increased suspension viscosity) and an accelerated aging of the neonatal cells under in vivo conditions. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Superoxide dismutase; Lipid peroxidation; Autoxidation; Rheology; Calf erythrocytes
* Corresponding author. Tel.: +36-52-417159. E-mail address: [email protected] (S. Imre). 0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 1 6 - 5
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1. Introduction It is well established that the erythrocyte of the new-born human infant differs from that of its adult in a selection of both biochemical and biophysical parameters (Matovcik and Mentzer, 1985). These differences, by themselves or in concert, result in a foreshortened survival of the neonatal cell. By comparison the bovine erythrocyte displays very pronounced and distinctive properties during the newborn period that set it apart from the human (Schatzmann and Scheidegger, 1975). It is generally accepted that there is a negative correlation between the lipid peroxidation capacity and the maximum life-span potential (Cutler, 1985). This is particularly true of the erythrocyte which is at increased risk as a result of its exposure to high concentrations of oxygen and an inability to replace damaged components by resynthesis (Carrell et al., 1975). Within the erythrocyte the constant conversion of oxyhaemoglobin to methaemoglobin (Carrell et al. 1975) results in an associated and consistent production of superoxide anions. Since erythrocytes are regularly exposed to both extracellular and intracellular sources of free radicals, the former arising from granulocytes and macrophages as well as other metabolically active cells, it is not surprising that they are equipped with mechanisms against toxic elemental species. The first defence against oxygen reduction products is the enzyme superoxide dismutase (SOD). This step, involving the dismutation of superoxide, plays a crucially important role in the metabolism of the red cell as it removes a free radical capable of initiating chain reactions (Chiu et al., 1989). Inability of the red cell to defend itself adequately against free radicals may allow the accumulation of the products of haemoglobin denaturation (hemichrome and Heinz-body formation) and membrane lipid peroxidation (Stern, 1989). Decreased cell deformability and acceleration of the processes that give rise to erythrocyte senescence then ensue. Based on in vitro methodology and in vivo observations, the present report is concerned with an investigation that compares the sensitivities of erythrocytes from neonatal and adult cattle to autoxidative effects.
2. Materials and methods Blood samples from 22 adult cows were obtained from the local slaughter house and blood samples from 31 calves at 2, 4 and 6 weeks of age were obtained from the Institute of Animal Health, Debrecen. Clotting of the blood was prevented by the addition of acid citrate dextrose (ACD) at a standard rate of dilution. The blood was then stored at 4°C in sealed plastic vials. Following centrifugation for 10 min at 1000 ×g at 4°C, the plasma and buffy coat were removed. The red blood cells were then mixed at 4°C with isotonic saline and re-centrifuged. Following careful removal of the supernatant, the washing/centrifugation procedure was repeated a further two times. Autoxidation of the isolated erythrocytes was promoted by incubation of a 10% (v/v) suspension of erythrocytes in an isotonic saline solution containing 10 mM
S. Imre et al. / Mechanisms of Ageing and De6elopment 122 (2001) 69–76
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Veronal – Na – HCl buffer, pH 7.4, in an atmosphere of air for 24 h in a thermoshaker at 37°C. Lipid peroxidation was characterised in two ways: 1. by quantification of the lipid peroxide decomposition product malondialdehyde (MDA) within the red cells before and after incubation at 37°C based on reaction to thiobarbituric acid (TBA) (Stocks and Dormandy, 1971); modifications were made to the standard measurement of TBA reactants involving the TBA – MDA chromophore being detected as a function of incubation time without the addition of hydrogen peroxide; 2. by measurement of lipid fluorescence at an excitation maximum of 360 nm and an emission maximum of 440 nm through the production of a fluorescent amino-iminopropene derivative following the reaction of the MDA carbonyl groups with protein or phospholipid amino groups (Bidlack and Tapple, 1973). This method has a particular application to the measurement of lipid peroxidation in vivo in red blood cells (Goldstein et al., 1979). Fluorescent measurements were made on a Hitachi-Perkin Elmer spectrofluorimeter. Erythrocyte methaemoglobin formation following incubation of the erythrocytes for 24 h at 4°C was quantified by the method of Brewer et al. (1960). Erythrocyte deformability was monitored by measurement of erythrocyte suspension viscosity at 37°C (Stoltz et al., 1999). Viscosity measurements were performed using a cone-plate viscometer (Wells-Brookfield, MA) at a shear rate of 42 106 and 212 s − 1. Isotonic saline containing 10 mM Veronal–Na–HCl buffer, pH 7.4, was used as the suspending medium. The erythrocyte concentration of the samples was adjusted to 45% (v/v) on the basis of the uncorrected hematocrit as determined by standard capillary centrifugation. As the shear rate, the composition of suspending medium and the erythrocyte concentration were standardized, measured values of the suspension viscosity reflected the changes in erythrocyte deformability. SOD activity was assayed by a standard procedure through its ability to inhibit the autoxidation of L-epinephrine at alkaline pH (Misra and Fridovich, 1972). Haemolysis, extraction of haemoglobin by chloroform–ethanol precipitation and haemoglobin quantification were carried out as described by Bartosz et al. (1978). Aliquots of the final supernatants of the haemolysates were used for such analyses. The activity of SOD was expressed relative to the haemoglobin content of the haemolysate. Student’s t-test was used for all statistical analyses.
3. Results Table 1 shows the levels of MDA (nmol MDA per g haemoglobin) obtained for the erythrocytes before and after incubation for the calves at the various stages of development and for the adult cattle. As can be seen the background level of MDA, i.e. its level before erythrocyte incubation, was very much lower in the erythrocytes from the 2-week old calf than for all the other groups. Clearly there was a
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Table 1 The effect of autoxidative test on the levels of MDA (nmol MDA/g haemoglobin) in 2, 4 and 6 week old calf and adult cattle erythrocytes (mean 9S.E.M. of 36 observations) Incubation (h)
0a 24b
Calf
Adult cattle
2 week
4 week
6 week
1.0 9 0.3 15.0 9 1.2
5.0 90.7 8.0 90.7
6.3 90.5 8.5 90.4
6.8 90.6 8.6 90.6
a Significance of difference of ‘background’ MDA levels, i.e. before incubation: 2 vs. 4 weeks, PB0.001; 4 vs. 6 weeks, PB0.05; 6 weeks vs. adult, NS. b Significance of difference of MDA levels between 0 and 24 h incubation: 2 weeks, PB0.001; 4 weeks, PB0.01; 6 weeks, PB0.05; adult, PB0.05.
continuum of increasing MDA levels maximising within the adult. Incubation of the red cells induced significant increases in MDA levels of all age groups. However, by far the largest increase in MDA as a result of incubation occurred within the erythrocytes from the calves at 2 weeks of age, the accumulation being such that the MDA level far exceeded that for all the other groups of animals in which the levels of MDA attained were very similar. The levels of fluorescence measurable within the lipid extracts from the erythrocytes, methaemoglobin formation following incubation of the erythrocytes for 24 h at 4°C, i.e. a measurement of spontaneous intracellular haemoglobin autoxidation and the activity levels of SOD in 2, 4 and 6 week old calf and adult cattle erythrocytes are shown in Table 2. As can be seen fluorescence was maximum within extracts from erythrocytes from 4-week old calves with levels differing significantly from the measurements obtainable for all the other groups of animals. Table 2 Fluorescence activity, methaemoglobin and SOD levels in 2, 4 and 6 week old calf and adult cattle erythrocytes Calf
Fluorescence (Units)a Methaemoglobin (%)b SOD (Units/g Hb)c
Adult cattle
2 week
4 week
6 week
27.09 1.2 41.099.0 58009 270
33.0 91.2 14.3 92.4 6280 9300
30.0 9 1.3 7.0 95.0 5700 9 310
26.0 9 2.0 10.0 93.6 4500 9700
a Fluorescence activity levels (arbitrary spectrophotometer values) for lipid extracts from calf and adult erythrocytes (means 9 S.E.M. of 12 observations). Significance of difference: 2 vs. 4 weeks, PB0.01; 4 vs. 6 weeks, PB0.05; 6 weeks vs. adult, PB0.05. b Methaemoglobin levels (% total haemoglobin) in calf and adult erythrocytes following 24 h incubation at 4°C (means9S.E.M. of 11 observations). Significance of difference: 2 vs. 4 weeks, PB0.001; 4 vs. 6 weeks, NS; 6 weeks vs. adult, NS. c Levels of SOD (U per g haemoglobin) of calf and adult erythrocytes (means 9 S.E.M. of 20 observations). Significance of difference between calf and adult, PB0.01.
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Table 3 The effect of autoxidative test on the suspension viscosity (cP) of 2 week old calf and adult cattle erythrocytes Shear rate (s−1) Calf
Adult cattle
Before incubation After incubation 42 106 212
4.29 0.2 4.19 0.2 3.9 90.1
5.5 9 0.2 5.4 90.2 5.2a 90.1
Before incubation
After incubation
3.4 9 0.4 3.3 9 0.3 3.1b 90.2
4.7 9 0.6 4.6 9 0.5 4.4a,c 90.5
a
Significantly different (PB0.01) following incubation. Significantly different (PB0.05) before incubation. c Significantly different (PB0.05) after incubation. b
By far the highest level of methaemoglobin accumulation was observed in the erythrocytes from calves at 2 weeks of age, the level being significantly higher from those observed for the other age groups between which there were no significant differences. The erythrocytes displayed substantially high levels of SOD, this feature being displayed particularly by the calf. With increasing age there was a decline in SOD level such that in the adult levels were significantly lower compared to that of the calf. Measurements of the viscosities of the erythrocyte suspensions are given in Table 3. Although there was a consistent reduction in viscosity as a function of increased shear rate, these differences were not significant. However, as can be seen, exposure of the erythrocytes to autoxidation during the 24-h incubation period resulted in a significant enhancement of their suspension viscosities because of their decreased deformability. This effect has been much greater in neonatal calves than in adult animals.
4. Discussion The mechanisms that result in a shorter survival time for erythrocytes from neonatal animals compared with the adult are not clear. However, from studies involving cross-transfusions it has been able to be shown that the diminished life-span demonstrated by the neonatal erythrocyte is the result of features intrinsic to the cell as opposed to any feature of the foetal circulation (Bratteby et al., 1968). Lipid peroxidation and its major end product, namely MDA, have been shown to bring about a wide range of responses by the erythrocyte. Thus in studies with erythrocytes from adult animals it was demonstrated that lipid peroxidation or treatment with MDA caused responses that most importantly included reduction in fluidity, decreased deformability and reduced cell survival time (Rice-Evans and Hochstein, 1981; Pfafferott et al., 1982; Bruch and Thayer, 1983; Jain et al., 1983). Other studies (Hebbel and Miller, 1984, 1988) have shown that MDA treatment of
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erythrocytes resulted in increased phagocytosis by the human marrow macrophages. With respect to cattle, it has been shown that autoxidation in calf erythrocytes is associated with a more pronounced loss of ATP content and filterability in comparison to the adult (Imre and Sa´ri, 1979). From the results obtained presently via direct measurement it was indicated that the erythrocytes of the young neonatal calf contain less MDA than erythrocytes of the adult. However, indirect measurement based on the fluorescence emissions of lipid derived products would suggest otherwise. A similar discrepancy based on similar direct and indirect indices of peroxidation has been obtained for erythrocytes of the new-born human (Jain, 1986). As a result of the 24 h incubation at 37°C the present study clearly indicated an increased MDA-forming capacity within the erythrocytes of the neonatal calf, especially at 2 weeks of age, when compared to the adult. The observations would suggest that in the neonatal calf a very active metabolic rate is accompanied by a limited antioxidant capacity in the erythrocyte. Increased vulnerability to peroxidation damage therefore would be expected. The role of vitamin E in preventing autoxidation of membrane lipids has been widely documented (Machlin and Bendich, 1987). Inadequacy of vitamin E in the erythrocytes from young animals may be of considerable importance with respect to oxidative resistance. Additionally the new-born may display an excessive generation of superoxide radicals, reduced activities of the antioxidative enzymes glutathione peroxidase and catalase and a decreased ability to renew fatty acids (Lubin and Oski, 1972; Agostoni et al., 1980). With respect to the latter feature, it has recently been shown that, under conditions of oxidative stress, calf erythrocytes demonstrate a particularly marked diminution in their levels of polyunsaturated fatty acids (Imre and Farkas, 1996). The high rate of methaemoglobin formation presently observed in erythrocytes from 2-week old calves during incubation at 4°C for 24 h can best be interpreted as the consequence of the persistence of foetal haemoglobin within a small population of the neonatal erythrocytes. It is interesting to note that Watkins et al. (1986) have shown that purified foetal haemoglobin generates greater amounts of superoxide, peroxide and hydroxyl radicals when compared with adult haemoglobin. Such a property of the foetal haemoglobin can destabilise haemoglobin, and form subsequent oxidative products (Rachmilewitz et al., 1971; Advani et al., 1992) thereby contributing further to the increased oxidative susceptibility and response from the neonatal erythrocytes. Compared with the adult, the erythrocyte of the new-born animal displays a significantly reduced ratio of surface area to volume with consequential effects on a range of physical parameters (Linderkamp et al. 1983). In volume the neonatal erythrocyte was estimated to be some 21% greater, its surface area 13% larger and its width 11% larger. Such differences could also account for the observations (Imre et al., 1978 Linderkamp et al., 1986) that the neonatal erythrocyte retained much more of its haemoglobin bound to its membrane when compared to the adult. This increased association with the membrane would undoubtedly enhance the oxidative threat posed by haemoglobin.
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Undoubtedly the neonatal calf erythrocyte displays all the pro- and antioxidant features pertinent to its function and demonstrated by its adult counterpart. However, within the neonatal erythrocyte it would appear that the balance of pro-oxidant features outweighs that of the antioxidant features rendering it particularly susceptible to oxidative damage and unable to reverse or stop a genetically programmed accelerated aging process.
Acknowledgements This study was supported by Grant No. 1490 from OTKA, Hungary. The technical assistance of M.T. Egri is gratefully acknowledged. We are indebted to Professor Dr J. Tanyi (Institute of Animal Health, Debrecen) for the supply of calf blood, and to Professor R.C. Noble (Department of Biochemical Science, Scottish Agricultural College, Ayr, Scotland, UK) for helpful discussions.
References Advani, R., Mentzer, W., Andrews, D., Schrier, S.L., 1992. Oxidation of Hemoglobin-F is associated with the ageing process of neonatal red blood cells. Paediatr. Res. 32, 165 – 168. Agostoni, A., Gerli, G.C., Beretta, L., 1980. Superoxide dismutase, catalase and glutathione peroxidase activities in maternal and cord blood erythrocytes. J. Clin. Chem. Clin. Biochem. 18, 771 – 773. Bartosz, G., Tannert, Ch., Fried, R., Leyko, W., 1978. Superoxide dismutase activity decreases during erythrocyte aging. Experientia 34, 1464. Bidlack, W.R., Tapple, A.R., 1973. Fluorescence products of phospholipids during lipid peroxidation. Lipids 8, 203–207. Bratteby, L.E., Garby, L., Wadman, B., 1968. Studies on erythrokinetics in infancy. XII. Survival in adult recipients of cord blood red cells labelled in vitro with di-isopryl fluorophosphate (DF32P). Acta Paediatr. Scand. 57, 305–310. Brewer, G.J., Tarlov, A.R., Alving, A.S., 1960. Methaemoglobin reduction test. Bull. World Health Org. 22, 633–640. Bruch, R.C., Thayer, W.S., 1983. Differential effect of lipid peroxidation on membrane fluidity as determined by electron spin resonance probes. Biochim. Biophys. Acta 733, 216 – 222. Carrell, R.W., Winterbourn, C.C., Rachmilewitz, E.A., 1975. Activated oxygen and haemolysis. Br. J. Haematol. 30, 259–264. Chiu, D., Kuypers, F., Lubin, B., 1989. Lipid peroxidation in human red cells. Semin. Hematol. 26, 257–276. Cutler, R.G., 1985. Peroxide-producing potential of tissues: Inverse correlation with longevity of mammalian species. Proc. Natl. Acad. Sci. USA 82, 4798 – 4802. Goldstein, B.D., Rozen, M.G., Amoruso, M.A., 1979. Relation of fluorescence in lipid containing red cell membrane extracts to in vivo lipid peroxidation. J. Lab. Clin. Med. 93, 687 – 694. Hebbel, R.P., Miller, W.J., 1984. Phagocytosis of sickle erythrocytes: Immunological and oxidative determinants of hemolytic anaemia. Blood 64, 733 – 741. Hebbel, R.P., Miller, W.J., 1988. Unique promotion of erythrophagocytosis by malondialdehyde. Am. J. Hematol. 29, 222–225. Imre, S., Plotkin, J., Thiele, O.W., 1978. The effect of magnesium on the hemolysis of calf and adult cattle erythrocytes. Blut 37, 201–209. Imre, S., Sa´ri, B., 1979. Comparative study on the metabolism and filterability of red blood cells of the calf and adult cattle. Acta Phys. Acad. Sci. Hung. 53, 23 – 30.
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Imre, S., Farkas, T., 1996. High sensitivity to autoxidation as a marker of reduced life potential. In: Viidik, A., Hofecker, G. (Eds.), Vitality, Mortality and Aging, Vienna Aging, Series V. Proceedings of the 10th Wiener Symposium on Experimental Gerontology, 29 – 30 April 1994, Wien, Austria. Facultas Universitatsverlag, pp. 181– 185. Jain, S.K., Mohandas, N., Clark, M.R., Shohet, S.B., 1983. Effect of malonyldialdehyde, a product of lipid peroxidation on deformability, cellular dehydration and 51Cr-survival of erythrocytes. Br. J. Haematol. 53, 247–255. Jain, S.K., 1986. Presence of phosphatidylserine in the outer membrane bilayer of new-born human erythrocytes. Biochem. Biophys. Res. Commun. 136, 914 – 920. Linderkamp, O., Wu, P.Y.K., Meiselman, H.J., 1983. Geometry of neonatal and adult red blood cells. Pediatr. Res. 17, 250–253. Linderkamp, O., Nash, G.B., Wu, P.Y.K., Meiselman, H.J., 1986. Deformability and intrinsic material properties of neonatal red blood cells. Blood 67, 1244 – 1250. Lubin, B., Oski, F.A., 1972. Red cell metabolism in the new-born infant. IV. Irreversible oxidant-induced injury. J. Pediatr. 81, 698–704. Machlin, L.J., Bendich, A., 1987. Free radical tissue damage: Protective role of antioxidant nutrients. FASEB J. 1, 441–445. Matovcik, L.M., Mentzer, W.C., 1985. The membrane of the human neonatal red cell. In: Schrier, S.L. (Ed.), Clinics in Haematology, vol. 14, Saunders, London, pp. 203 – 221. Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the auto oxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170 – 3174. Pfafferott, C., Meiselman, H.J., Hochstein, P., 1982. The effect of malonyldialdehyde on erythrocyte deformability. Blood 59, 12–15. Rachmilewitz, E.A., Peisach, J., Blumberg, W.E., 1971. Studies on the stability of oxyhemoglobin A and its constituent chains and their derivatives. Biol. Chem. 246, 3356 – 3366. Rice-Evans, C., Hochstein, P., 1981. Alterations in erythrocyte membrane fluidity by phenylhydrazineinduced peroxidation of lipids. Biophys. Res. Commun. 100, 1537 – 1542. Schatzmann, H.J., Scheidegger, H.R., 1975. Postnatal decline of (Ca2 + +Mg2 + ) activated membrane ATP-ase in cattle red cells. Experientia 31, 1260 – 1261. Stern, A., 1989. Drug-induced oxidative denaturation in red blood cells. Semin. Hematol. 26, 301 – 306. Stocks, J., Dormandy, T.L., 1971. The autoxidation of human red cell lipids induced by hydrogen peroxide. Br. J. Haematol. 20, 95–111. Stoltz, J.F., Singh, M., Riha, P., 1999. Hemorheology in Practice, IOS Press, Ohmsha, Amsterdam. Watkins, J.A., Claster, S., Caughey, W.S., 1986. Exchanged production of oxy radicals and peroxide by hemoglobins S and F. Fed. Proc. 45, 1640A.
.
High sensitivity to autoxidation in neonatal calf erythrocytes: possible mechanism of accelerated cell aging S. Imre a,*, M. Csornai b, M. Balazs c a
Department of Pathophysiology, Medical Uni6ersity of Debrecen, Nagyerdei krt. 98, PO Box 23, H-4012 Debrecen, Hungary b Department of Neurology and Psychiatry, Medical Uni6ersity of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, Hungary c Department of Biophysics, Medical Uni6ersity of Debrecen, Nagyerdei krt. 98, H-4012 Debrecen, Hungary
Received 14 May 2000; received in revised form 8 September 2000; accepted 15 September 2000
Abstract The suspension viscosity, formation of methaemoglobin and production of malondialdehyde (MDA) associated with the non-enzymatic oxidation of polyunsaturated fatty acids during auto-oxidation conditions in vitro have been compared in erythrocytes from young calves (2, 4 and 6 weeks of age) and mature cattle. The autoxidation conditions were designed to simulate the oxidative stress to which neonatal erythrocytes are exposed in vivo. Characterisation of lipid peroxidation was also undertaken by a combination of lipid fluorescent measurements and quantification of the superoxide dismutase (SOD) activities of the erythrocytes. The results demonstrated that high SOD activities in the erythrocytes of the neonatal calf was insufficient to afford protection against the increased autoxidation of haemoglobin and subsequent accumulation of lipid peroxidation products. High levels of methaemoglobin formation and lipid peroxidation were able to provide an explanation for an observed reduction in rheological adaptability (increased suspension viscosity) and an accelerated aging of the neonatal cells under in vivo conditions. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Superoxide dismutase; Lipid peroxidation; Autoxidation; Rheology; Calf erythrocytes
* Corresponding author. Tel.: +36-52-417159. E-mail address: [email protected] (S. Imre). 0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 1 6 - 5
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S. Imre et al. / Mechanisms of Ageing and De6elopment 122 (2001) 69–76
1. Introduction It is well established that the erythrocyte of the new-born human infant differs from that of its adult in a selection of both biochemical and biophysical parameters (Matovcik and Mentzer, 1985). These differences, by themselves or in concert, result in a foreshortened survival of the neonatal cell. By comparison the bovine erythrocyte displays very pronounced and distinctive properties during the newborn period that set it apart from the human (Schatzmann and Scheidegger, 1975). It is generally accepted that there is a negative correlation between the lipid peroxidation capacity and the maximum life-span potential (Cutler, 1985). This is particularly true of the erythrocyte which is at increased risk as a result of its exposure to high concentrations of oxygen and an inability to replace damaged components by resynthesis (Carrell et al., 1975). Within the erythrocyte the constant conversion of oxyhaemoglobin to methaemoglobin (Carrell et al. 1975) results in an associated and consistent production of superoxide anions. Since erythrocytes are regularly exposed to both extracellular and intracellular sources of free radicals, the former arising from granulocytes and macrophages as well as other metabolically active cells, it is not surprising that they are equipped with mechanisms against toxic elemental species. The first defence against oxygen reduction products is the enzyme superoxide dismutase (SOD). This step, involving the dismutation of superoxide, plays a crucially important role in the metabolism of the red cell as it removes a free radical capable of initiating chain reactions (Chiu et al., 1989). Inability of the red cell to defend itself adequately against free radicals may allow the accumulation of the products of haemoglobin denaturation (hemichrome and Heinz-body formation) and membrane lipid peroxidation (Stern, 1989). Decreased cell deformability and acceleration of the processes that give rise to erythrocyte senescence then ensue. Based on in vitro methodology and in vivo observations, the present report is concerned with an investigation that compares the sensitivities of erythrocytes from neonatal and adult cattle to autoxidative effects.
2. Materials and methods Blood samples from 22 adult cows were obtained from the local slaughter house and blood samples from 31 calves at 2, 4 and 6 weeks of age were obtained from the Institute of Animal Health, Debrecen. Clotting of the blood was prevented by the addition of acid citrate dextrose (ACD) at a standard rate of dilution. The blood was then stored at 4°C in sealed plastic vials. Following centrifugation for 10 min at 1000 ×g at 4°C, the plasma and buffy coat were removed. The red blood cells were then mixed at 4°C with isotonic saline and re-centrifuged. Following careful removal of the supernatant, the washing/centrifugation procedure was repeated a further two times. Autoxidation of the isolated erythrocytes was promoted by incubation of a 10% (v/v) suspension of erythrocytes in an isotonic saline solution containing 10 mM
S. Imre et al. / Mechanisms of Ageing and De6elopment 122 (2001) 69–76
71
Veronal – Na – HCl buffer, pH 7.4, in an atmosphere of air for 24 h in a thermoshaker at 37°C. Lipid peroxidation was characterised in two ways: 1. by quantification of the lipid peroxide decomposition product malondialdehyde (MDA) within the red cells before and after incubation at 37°C based on reaction to thiobarbituric acid (TBA) (Stocks and Dormandy, 1971); modifications were made to the standard measurement of TBA reactants involving the TBA – MDA chromophore being detected as a function of incubation time without the addition of hydrogen peroxide; 2. by measurement of lipid fluorescence at an excitation maximum of 360 nm and an emission maximum of 440 nm through the production of a fluorescent amino-iminopropene derivative following the reaction of the MDA carbonyl groups with protein or phospholipid amino groups (Bidlack and Tapple, 1973). This method has a particular application to the measurement of lipid peroxidation in vivo in red blood cells (Goldstein et al., 1979). Fluorescent measurements were made on a Hitachi-Perkin Elmer spectrofluorimeter. Erythrocyte methaemoglobin formation following incubation of the erythrocytes for 24 h at 4°C was quantified by the method of Brewer et al. (1960). Erythrocyte deformability was monitored by measurement of erythrocyte suspension viscosity at 37°C (Stoltz et al., 1999). Viscosity measurements were performed using a cone-plate viscometer (Wells-Brookfield, MA) at a shear rate of 42 106 and 212 s − 1. Isotonic saline containing 10 mM Veronal–Na–HCl buffer, pH 7.4, was used as the suspending medium. The erythrocyte concentration of the samples was adjusted to 45% (v/v) on the basis of the uncorrected hematocrit as determined by standard capillary centrifugation. As the shear rate, the composition of suspending medium and the erythrocyte concentration were standardized, measured values of the suspension viscosity reflected the changes in erythrocyte deformability. SOD activity was assayed by a standard procedure through its ability to inhibit the autoxidation of L-epinephrine at alkaline pH (Misra and Fridovich, 1972). Haemolysis, extraction of haemoglobin by chloroform–ethanol precipitation and haemoglobin quantification were carried out as described by Bartosz et al. (1978). Aliquots of the final supernatants of the haemolysates were used for such analyses. The activity of SOD was expressed relative to the haemoglobin content of the haemolysate. Student’s t-test was used for all statistical analyses.
3. Results Table 1 shows the levels of MDA (nmol MDA per g haemoglobin) obtained for the erythrocytes before and after incubation for the calves at the various stages of development and for the adult cattle. As can be seen the background level of MDA, i.e. its level before erythrocyte incubation, was very much lower in the erythrocytes from the 2-week old calf than for all the other groups. Clearly there was a
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Table 1 The effect of autoxidative test on the levels of MDA (nmol MDA/g haemoglobin) in 2, 4 and 6 week old calf and adult cattle erythrocytes (mean 9S.E.M. of 36 observations) Incubation (h)
0a 24b
Calf
Adult cattle
2 week
4 week
6 week
1.0 9 0.3 15.0 9 1.2
5.0 90.7 8.0 90.7
6.3 90.5 8.5 90.4
6.8 90.6 8.6 90.6
a Significance of difference of ‘background’ MDA levels, i.e. before incubation: 2 vs. 4 weeks, PB0.001; 4 vs. 6 weeks, PB0.05; 6 weeks vs. adult, NS. b Significance of difference of MDA levels between 0 and 24 h incubation: 2 weeks, PB0.001; 4 weeks, PB0.01; 6 weeks, PB0.05; adult, PB0.05.
continuum of increasing MDA levels maximising within the adult. Incubation of the red cells induced significant increases in MDA levels of all age groups. However, by far the largest increase in MDA as a result of incubation occurred within the erythrocytes from the calves at 2 weeks of age, the accumulation being such that the MDA level far exceeded that for all the other groups of animals in which the levels of MDA attained were very similar. The levels of fluorescence measurable within the lipid extracts from the erythrocytes, methaemoglobin formation following incubation of the erythrocytes for 24 h at 4°C, i.e. a measurement of spontaneous intracellular haemoglobin autoxidation and the activity levels of SOD in 2, 4 and 6 week old calf and adult cattle erythrocytes are shown in Table 2. As can be seen fluorescence was maximum within extracts from erythrocytes from 4-week old calves with levels differing significantly from the measurements obtainable for all the other groups of animals. Table 2 Fluorescence activity, methaemoglobin and SOD levels in 2, 4 and 6 week old calf and adult cattle erythrocytes Calf
Fluorescence (Units)a Methaemoglobin (%)b SOD (Units/g Hb)c
Adult cattle
2 week
4 week
6 week
27.09 1.2 41.099.0 58009 270
33.0 91.2 14.3 92.4 6280 9300
30.0 9 1.3 7.0 95.0 5700 9 310
26.0 9 2.0 10.0 93.6 4500 9700
a Fluorescence activity levels (arbitrary spectrophotometer values) for lipid extracts from calf and adult erythrocytes (means 9 S.E.M. of 12 observations). Significance of difference: 2 vs. 4 weeks, PB0.01; 4 vs. 6 weeks, PB0.05; 6 weeks vs. adult, PB0.05. b Methaemoglobin levels (% total haemoglobin) in calf and adult erythrocytes following 24 h incubation at 4°C (means9S.E.M. of 11 observations). Significance of difference: 2 vs. 4 weeks, PB0.001; 4 vs. 6 weeks, NS; 6 weeks vs. adult, NS. c Levels of SOD (U per g haemoglobin) of calf and adult erythrocytes (means 9 S.E.M. of 20 observations). Significance of difference between calf and adult, PB0.01.
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Table 3 The effect of autoxidative test on the suspension viscosity (cP) of 2 week old calf and adult cattle erythrocytes Shear rate (s−1) Calf
Adult cattle
Before incubation After incubation 42 106 212
4.29 0.2 4.19 0.2 3.9 90.1
5.5 9 0.2 5.4 90.2 5.2a 90.1
Before incubation
After incubation
3.4 9 0.4 3.3 9 0.3 3.1b 90.2
4.7 9 0.6 4.6 9 0.5 4.4a,c 90.5
a
Significantly different (PB0.01) following incubation. Significantly different (PB0.05) before incubation. c Significantly different (PB0.05) after incubation. b
By far the highest level of methaemoglobin accumulation was observed in the erythrocytes from calves at 2 weeks of age, the level being significantly higher from those observed for the other age groups between which there were no significant differences. The erythrocytes displayed substantially high levels of SOD, this feature being displayed particularly by the calf. With increasing age there was a decline in SOD level such that in the adult levels were significantly lower compared to that of the calf. Measurements of the viscosities of the erythrocyte suspensions are given in Table 3. Although there was a consistent reduction in viscosity as a function of increased shear rate, these differences were not significant. However, as can be seen, exposure of the erythrocytes to autoxidation during the 24-h incubation period resulted in a significant enhancement of their suspension viscosities because of their decreased deformability. This effect has been much greater in neonatal calves than in adult animals.
4. Discussion The mechanisms that result in a shorter survival time for erythrocytes from neonatal animals compared with the adult are not clear. However, from studies involving cross-transfusions it has been able to be shown that the diminished life-span demonstrated by the neonatal erythrocyte is the result of features intrinsic to the cell as opposed to any feature of the foetal circulation (Bratteby et al., 1968). Lipid peroxidation and its major end product, namely MDA, have been shown to bring about a wide range of responses by the erythrocyte. Thus in studies with erythrocytes from adult animals it was demonstrated that lipid peroxidation or treatment with MDA caused responses that most importantly included reduction in fluidity, decreased deformability and reduced cell survival time (Rice-Evans and Hochstein, 1981; Pfafferott et al., 1982; Bruch and Thayer, 1983; Jain et al., 1983). Other studies (Hebbel and Miller, 1984, 1988) have shown that MDA treatment of
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erythrocytes resulted in increased phagocytosis by the human marrow macrophages. With respect to cattle, it has been shown that autoxidation in calf erythrocytes is associated with a more pronounced loss of ATP content and filterability in comparison to the adult (Imre and Sa´ri, 1979). From the results obtained presently via direct measurement it was indicated that the erythrocytes of the young neonatal calf contain less MDA than erythrocytes of the adult. However, indirect measurement based on the fluorescence emissions of lipid derived products would suggest otherwise. A similar discrepancy based on similar direct and indirect indices of peroxidation has been obtained for erythrocytes of the new-born human (Jain, 1986). As a result of the 24 h incubation at 37°C the present study clearly indicated an increased MDA-forming capacity within the erythrocytes of the neonatal calf, especially at 2 weeks of age, when compared to the adult. The observations would suggest that in the neonatal calf a very active metabolic rate is accompanied by a limited antioxidant capacity in the erythrocyte. Increased vulnerability to peroxidation damage therefore would be expected. The role of vitamin E in preventing autoxidation of membrane lipids has been widely documented (Machlin and Bendich, 1987). Inadequacy of vitamin E in the erythrocytes from young animals may be of considerable importance with respect to oxidative resistance. Additionally the new-born may display an excessive generation of superoxide radicals, reduced activities of the antioxidative enzymes glutathione peroxidase and catalase and a decreased ability to renew fatty acids (Lubin and Oski, 1972; Agostoni et al., 1980). With respect to the latter feature, it has recently been shown that, under conditions of oxidative stress, calf erythrocytes demonstrate a particularly marked diminution in their levels of polyunsaturated fatty acids (Imre and Farkas, 1996). The high rate of methaemoglobin formation presently observed in erythrocytes from 2-week old calves during incubation at 4°C for 24 h can best be interpreted as the consequence of the persistence of foetal haemoglobin within a small population of the neonatal erythrocytes. It is interesting to note that Watkins et al. (1986) have shown that purified foetal haemoglobin generates greater amounts of superoxide, peroxide and hydroxyl radicals when compared with adult haemoglobin. Such a property of the foetal haemoglobin can destabilise haemoglobin, and form subsequent oxidative products (Rachmilewitz et al., 1971; Advani et al., 1992) thereby contributing further to the increased oxidative susceptibility and response from the neonatal erythrocytes. Compared with the adult, the erythrocyte of the new-born animal displays a significantly reduced ratio of surface area to volume with consequential effects on a range of physical parameters (Linderkamp et al. 1983). In volume the neonatal erythrocyte was estimated to be some 21% greater, its surface area 13% larger and its width 11% larger. Such differences could also account for the observations (Imre et al., 1978 Linderkamp et al., 1986) that the neonatal erythrocyte retained much more of its haemoglobin bound to its membrane when compared to the adult. This increased association with the membrane would undoubtedly enhance the oxidative threat posed by haemoglobin.
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Undoubtedly the neonatal calf erythrocyte displays all the pro- and antioxidant features pertinent to its function and demonstrated by its adult counterpart. However, within the neonatal erythrocyte it would appear that the balance of pro-oxidant features outweighs that of the antioxidant features rendering it particularly susceptible to oxidative damage and unable to reverse or stop a genetically programmed accelerated aging process.
Acknowledgements This study was supported by Grant No. 1490 from OTKA, Hungary. The technical assistance of M.T. Egri is gratefully acknowledged. We are indebted to Professor Dr J. Tanyi (Institute of Animal Health, Debrecen) for the supply of calf blood, and to Professor R.C. Noble (Department of Biochemical Science, Scottish Agricultural College, Ayr, Scotland, UK) for helpful discussions.
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