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Lipid peroxides in blood plasma and enzymatic antioxidative defence of erythrocytes in Down's syndrome
191
Clinica Chimicu Acta, 154 (1986) 191-194 Elsevier
CCA 03387
Lipid peroxides in blood plasma and enzymatic antioxidative defence of erythrocytes in Down’s syndrome J&ef Kedziora a, Grzegorz Bartosz ‘, Jolanta Gromadzihska b, Maria SMIodowska b, Wojciech Wesowicz b and Jozef Scianowski b Departmenis of a Physiology and b Department of Biochemistry Instiiuie of Fundamental Sciences,‘ WAM Medical Academy, Plac 9 Maja No. I, 90-647 Lbdi and’ Department of Biophysics, Institute of Biochemistry and Biophysics, University of I.&di. 90 - 237 L&ii (Poland) (Received
May 9th, 1985; revision September
9th, 1985)
Key words: Down’s syndrome; Erythrocyte; Superoxide dismutase; Glutaihione peroxidase; Catalase; Selenium; Lipid peroxides
Summary
The level of lipid peroxides was significantly increased in the blood of patients with Down’s syndrome. In erythrocytes increased activities of superoxide dismutase and glutathione peroxidase were confirmed while catalase activity was similar to that of healthy controls. The concentration of selenium in erythrocytes of Down’s syndrome patients was reduced, in spite of increased glutathione peroxidase activity. These results confirm the hypothesis of an altered oxidative metabolism in Down’s syndrome.
Introduction
The molecular mechanisms leading from the chromosomal aneuploidy to mental deficiency and other pathological consequences in Down’s syndrome (DS) await elucidation. It has been suggested that an increased (Cu, Zn)-superoxide dismutase (SOD) activity in cells of DS patients may be a key phenomenon [l]. It might result in an increased rate of hydrogen peroxide production detectable by elevated peroxide levels in cells and body fluids. These speculations have stimulated interest in the activities of protective enzymes, chiefly glutathione peroxidase. An increase in the activity of this enzyme has been found in red blood cells of DS patients [2-41 though some authors have found normal activity [5,6]. On the other hand, normal activities have been reported from other tissues [7]. The aim of the present study was to reexamine the activities of erythrocyte antioxidative enzymes in DS, and to
0009-8981/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
192
determine the level of lipid peroxides in the blood plasma of DS patients as an index of the oxidative status of body fluids. Materials and methods
The subjects were institutionalized DS patients, 5 females and 9 males (karyotypes 47,Xx,21 + or 47,XY,21 + , respectively). Results obtained for the patients were compared with those obtained for an appropriate control group of age-matched healthy donors of both sexes (23 females, 58 males). Blood was taken by clean venepuncture and anticoagulated with heparin. Superoxide dismutase activity was estimated by the adrenaline method [8], catalase activity by spectrophotometric monitoring of the rate of substrate decomposition [9] and glutathione peroxidase by the method of Paglia and Valentine [lo] as modified by Hopkins and Tudhope [ll]. Selenium was determined according to Watkinson [12] and lipid peroxides according to Yagi [13]. Results and discussion
In agreement with the data of virtually all authors (e.g. [4,6,14]), including our own previous data obtained by another method [15], SOD activity was increased by approximately 50% in erythrocytes of DS patients (Table I). The catalase level was normal, in agreement with data of other authors based on activity determination [16,17] but at variance with recent results of immunochemical estimation of the catalase protein content in DS erythrocytes [14]. This difference may be’due to ‘increased inactivation of catalase in DS in mature erythrocytes or perhaps to increased oxidative stress at an earlier stage of cell maturation. The present results confirm our previous observation made on another group of
TABLE
1
Activities of antioxidant enzymes and selenium plasma of 14 patients with Down’s syndrome
content
of erythrocytes,
SOD
Catalase
GPx
(U/g Hb x10-q
(U/g Hb x lo-‘)
(U/g
and lipid peroxide
Hb) cells)
levels in blood
ROOH (nmol MDA/ml)
DS Mean SD
2.22 *0.17
50.6 f 9.2
24.4 It3.8
1.60 f 0.28
5.07 f 0.72
Normal controls Mean SD
1.43 * 0.21
49.5 *7.8
18.5 *3.5
2.15 f 0.46
4.21 f 0.72
Pa
i 0.001
n.s.
< 0.001
< 0.001
< 0.001
a Estimated
using the two-tailed
Student’s
t test.
193
patients [2] that the glutathione peroxidase activity is increased in erythrocytes in DS. However, the total selenium content of erythrocytes was decreased (Table I). These results are in partial agreement with the data of Neve et al [4] who found normal selenium levels in DS erythrocytes and a significant correlation between erythrocyte selenium and glutathione peroxidase activity. Our own results in a relatively small number suggest no correlation between the selenium content and glutathione peroxidase activity. This is not perhaps surprising since glutathione peroxidase accounts for only 10% of total erythrocyte selenium [18]. It suggests that selenium is not a factor limiting glutathione peroxidase activity in erythrocytes of DS. The difference between our results and those of N&e et al [4] may reflect the different nutritional status of the two groups of patients. The increased level of lipid peroxides in the blood plasma of DS patients may be related to the increased lipid peroxidation in the cerebral cortex of DS fetuses [7]. It supports the hypothesis of an increased oxidative stress in this disease [19,20]. Such stress might result from an increased generation of ‘active oxygen species’ and/or decreased antioxidative defence. The present results confirm that erythrocyte antioxidant enzymes are increased or normal in DS. However, in the blood plasma the proteins transferrin, lactoferrin and ceruloplasmin are known to be the main factors protecting lipids from undergoing peroxidation [21]. We have demonstrated a decreased transferrin level in the blood plasma of DS patients [22]. This could conceivably be a factor in the increased peroxidation. Increased oxidative stress might underlie the premature aging [23,24] and other pathological phenomena in DS. The results of the present study point to the need for further investigations of the antioxidative mechanisms in DS and raise the distant hope of an effective antioxidant therapy. References 1 Epstein CJ, Epstein LB, Weil J, Cox DR. Trisomy 21: mechanisms and models. Ann NY Acad Sci. 1982; 396: 107-118. 2 Kedziora J, tukaszewicz R, Koter M, Bartosz G, Pawlowska B, Aitkin D. Red blood cell glutathione peroxidase in sample trisomy 21 and translocation 21/22. Experientia 1982; 38: 543-544. 3 Sinet PM, Michelson AM, Bazin A, Lejeune J, Jerome H. Increase in glutathione peroxidase activity in erythrocytes from trisomy 21 patients. Biochem Biophys Res Commun 1975; 67: 910-915. 4 Ntve J, Sinet PM, Molle L, Nicole A. Selenium, zinc and copper in Down’s syndrome (trisomy 21): blood levels and relations with glutathione peroxidase and superoxide dismutase. Clin Chim Acta 1983; 133: 209-214. 5 Brooksbank BWL, Balazs R. Superoxide dismutase and lipoperoxidation in Down’s syndrome foetal brain. Lancet 1983; i: 881-882. 6 Vertongen F, Ntve J, Cauchie P, Molle L. Zinc, copper, selenium and glutathione peroxidase in plasma and erythrocytes of Down’s syndrome (trisomy 21) patients. Interpretation of some variations. In: Bratter P, Schramel P, eds. Trace element - analytical chemistry in medicine and biology, Vol. 3. Berlin and New York: Walter de Gruyter & Co., 1984: 175-181. 7 Brooksbank BWL, Balazs R. Superoxide dismutase, glutathione peroxidase and lipoperoxidation in Down’s syndrome fetal brain. Dev Brain Res 1984; 16: 37-44. 8 Concetti A, Massei P, Rotilio G, Brunori M, Rachmilewitz EA. Superoxide dismutase in red blood cells-Method of assay and enzyme content in normal subjects and in patients with /3-thalassemia (major and intermedia). J Lab Clin Med 1976; 87: 1057-1064.
194 9 Marklund S, Nordensson I, Back 0. Normal CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in Werner’s syndrome. J Gerontol 1981; 36: 40-409. 10 Paglia DE, Valentine WN. Studies on quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158-169. 11 Hopkins J, Tudhope GR. Glutathione peroxidase in human red cells in health and disease. Br J Haematol 1973; 25: 563-575. 12 Watkinson JH. Fluorometric determination of selenium in biological material with 2,3-diaminonaphthalene. Anal Chem 1966; 38: 92-97. 13 Yagi K. A simple fluorometric assay for lipoperoxide in blood plasma. Biochem Med 1976; 15: 212-216. 14 Ohno H, Iizuka S, Kondon T, Yamamura K, Sekiya C, Taniguchi N. The levels of superoxide dismutase, catalase, and carbonic anhydrase in erythrocytes of patients with Down’s syndrome. Klin Wochenschr 1984; 62: 287-288. 15 Kedziora J, Jeske J, Witas H, Bartosz G, Leyko W. The level of superoxide dismutase in erythrocytes of children with Down syndrome (trisomy G and unbalanced translocation G21/2~). Acta Biol Med Germ 1977; 36: 779-782. 16 Michelson AM, Puget K, Durosay P. Clinical aspects of the dosage of erythrocuprein. In: Superoxide and superoxide dismutases. Michelson AM, ed. New York/San Francisco/Land: 1977: Academic Press, 467-499. 17 Pantelakis SN, Karaklis AG, Alexiou D, Vardas E, Valaes T. Red cell enzymes in trisomy 21. Am J Human Genet 1970; 22: 184-193. 18 Schmidt K, Heller W. Selenkonzentration und Aktivitat der Glutathion Peroxidase im Lysat menschlither Erythrozyten. Blut 1976; 33: 247-251. 19 Sinet PM. Metabolism of oxygen derivatives in Down’s syndrome Ann N Y Acad Sci 1982; 396: 83-94. 20 Bartosz G, Kpdziora J. Erythrocyte anomalies in Down’s syndrome. Med Hypothesis 1983; 11: 471-477. 21 Stocks J, Gutteridge JMC, Sharp RJ, Dormandy TL. The inhibition of lipid autoxidation by human serum and its relationship to serum proteins and alpha-tocopheral. Clin Sci Mol Med 1974; 47: 223-233. 22 Kedziora J, Witas H, Bartosz G, Leyko W, Jeske J, Roiynkowa, D. Down’s syndrome - transferrin parallels plasma iron changes. Experientia 1978; 34: 712. 23 Lott IT. Down’s syndrome, aging, and Alzheimer’s disease: a clinical review. Ann N Y Acad Sci 1982; 396: 15-27. 24 Walford RL. Immunological studies of Down’s syndrome and Alzheimer’s disease. Ann N Y Acad Sci 1982; 396: 95-106.
Clinica Chimicu Acta, 154 (1986) 191-194 Elsevier
CCA 03387
Lipid peroxides in blood plasma and enzymatic antioxidative defence of erythrocytes in Down’s syndrome J&ef Kedziora a, Grzegorz Bartosz ‘, Jolanta Gromadzihska b, Maria SMIodowska b, Wojciech Wesowicz b and Jozef Scianowski b Departmenis of a Physiology and b Department of Biochemistry Instiiuie of Fundamental Sciences,‘ WAM Medical Academy, Plac 9 Maja No. I, 90-647 Lbdi and’ Department of Biophysics, Institute of Biochemistry and Biophysics, University of I.&di. 90 - 237 L&ii (Poland) (Received
May 9th, 1985; revision September
9th, 1985)
Key words: Down’s syndrome; Erythrocyte; Superoxide dismutase; Glutaihione peroxidase; Catalase; Selenium; Lipid peroxides
Summary
The level of lipid peroxides was significantly increased in the blood of patients with Down’s syndrome. In erythrocytes increased activities of superoxide dismutase and glutathione peroxidase were confirmed while catalase activity was similar to that of healthy controls. The concentration of selenium in erythrocytes of Down’s syndrome patients was reduced, in spite of increased glutathione peroxidase activity. These results confirm the hypothesis of an altered oxidative metabolism in Down’s syndrome.
Introduction
The molecular mechanisms leading from the chromosomal aneuploidy to mental deficiency and other pathological consequences in Down’s syndrome (DS) await elucidation. It has been suggested that an increased (Cu, Zn)-superoxide dismutase (SOD) activity in cells of DS patients may be a key phenomenon [l]. It might result in an increased rate of hydrogen peroxide production detectable by elevated peroxide levels in cells and body fluids. These speculations have stimulated interest in the activities of protective enzymes, chiefly glutathione peroxidase. An increase in the activity of this enzyme has been found in red blood cells of DS patients [2-41 though some authors have found normal activity [5,6]. On the other hand, normal activities have been reported from other tissues [7]. The aim of the present study was to reexamine the activities of erythrocyte antioxidative enzymes in DS, and to
0009-8981/86/$03.50
0 1986 Elsevier Science Publishers
B.V. (Biomedical
Division)
192
determine the level of lipid peroxides in the blood plasma of DS patients as an index of the oxidative status of body fluids. Materials and methods
The subjects were institutionalized DS patients, 5 females and 9 males (karyotypes 47,Xx,21 + or 47,XY,21 + , respectively). Results obtained for the patients were compared with those obtained for an appropriate control group of age-matched healthy donors of both sexes (23 females, 58 males). Blood was taken by clean venepuncture and anticoagulated with heparin. Superoxide dismutase activity was estimated by the adrenaline method [8], catalase activity by spectrophotometric monitoring of the rate of substrate decomposition [9] and glutathione peroxidase by the method of Paglia and Valentine [lo] as modified by Hopkins and Tudhope [ll]. Selenium was determined according to Watkinson [12] and lipid peroxides according to Yagi [13]. Results and discussion
In agreement with the data of virtually all authors (e.g. [4,6,14]), including our own previous data obtained by another method [15], SOD activity was increased by approximately 50% in erythrocytes of DS patients (Table I). The catalase level was normal, in agreement with data of other authors based on activity determination [16,17] but at variance with recent results of immunochemical estimation of the catalase protein content in DS erythrocytes [14]. This difference may be’due to ‘increased inactivation of catalase in DS in mature erythrocytes or perhaps to increased oxidative stress at an earlier stage of cell maturation. The present results confirm our previous observation made on another group of
TABLE
1
Activities of antioxidant enzymes and selenium plasma of 14 patients with Down’s syndrome
content
of erythrocytes,
SOD
Catalase
GPx
(U/g Hb x10-q
(U/g Hb x lo-‘)
(U/g
and lipid peroxide
Hb) cells)
levels in blood
ROOH (nmol MDA/ml)
DS Mean SD
2.22 *0.17
50.6 f 9.2
24.4 It3.8
1.60 f 0.28
5.07 f 0.72
Normal controls Mean SD
1.43 * 0.21
49.5 *7.8
18.5 *3.5
2.15 f 0.46
4.21 f 0.72
Pa
i 0.001
n.s.
< 0.001
< 0.001
< 0.001
a Estimated
using the two-tailed
Student’s
t test.
193
patients [2] that the glutathione peroxidase activity is increased in erythrocytes in DS. However, the total selenium content of erythrocytes was decreased (Table I). These results are in partial agreement with the data of Neve et al [4] who found normal selenium levels in DS erythrocytes and a significant correlation between erythrocyte selenium and glutathione peroxidase activity. Our own results in a relatively small number suggest no correlation between the selenium content and glutathione peroxidase activity. This is not perhaps surprising since glutathione peroxidase accounts for only 10% of total erythrocyte selenium [18]. It suggests that selenium is not a factor limiting glutathione peroxidase activity in erythrocytes of DS. The difference between our results and those of N&e et al [4] may reflect the different nutritional status of the two groups of patients. The increased level of lipid peroxides in the blood plasma of DS patients may be related to the increased lipid peroxidation in the cerebral cortex of DS fetuses [7]. It supports the hypothesis of an increased oxidative stress in this disease [19,20]. Such stress might result from an increased generation of ‘active oxygen species’ and/or decreased antioxidative defence. The present results confirm that erythrocyte antioxidant enzymes are increased or normal in DS. However, in the blood plasma the proteins transferrin, lactoferrin and ceruloplasmin are known to be the main factors protecting lipids from undergoing peroxidation [21]. We have demonstrated a decreased transferrin level in the blood plasma of DS patients [22]. This could conceivably be a factor in the increased peroxidation. Increased oxidative stress might underlie the premature aging [23,24] and other pathological phenomena in DS. The results of the present study point to the need for further investigations of the antioxidative mechanisms in DS and raise the distant hope of an effective antioxidant therapy. References 1 Epstein CJ, Epstein LB, Weil J, Cox DR. Trisomy 21: mechanisms and models. Ann NY Acad Sci. 1982; 396: 107-118. 2 Kedziora J, tukaszewicz R, Koter M, Bartosz G, Pawlowska B, Aitkin D. Red blood cell glutathione peroxidase in sample trisomy 21 and translocation 21/22. Experientia 1982; 38: 543-544. 3 Sinet PM, Michelson AM, Bazin A, Lejeune J, Jerome H. Increase in glutathione peroxidase activity in erythrocytes from trisomy 21 patients. Biochem Biophys Res Commun 1975; 67: 910-915. 4 Ntve J, Sinet PM, Molle L, Nicole A. Selenium, zinc and copper in Down’s syndrome (trisomy 21): blood levels and relations with glutathione peroxidase and superoxide dismutase. Clin Chim Acta 1983; 133: 209-214. 5 Brooksbank BWL, Balazs R. Superoxide dismutase and lipoperoxidation in Down’s syndrome foetal brain. Lancet 1983; i: 881-882. 6 Vertongen F, Ntve J, Cauchie P, Molle L. Zinc, copper, selenium and glutathione peroxidase in plasma and erythrocytes of Down’s syndrome (trisomy 21) patients. Interpretation of some variations. In: Bratter P, Schramel P, eds. Trace element - analytical chemistry in medicine and biology, Vol. 3. Berlin and New York: Walter de Gruyter & Co., 1984: 175-181. 7 Brooksbank BWL, Balazs R. Superoxide dismutase, glutathione peroxidase and lipoperoxidation in Down’s syndrome fetal brain. Dev Brain Res 1984; 16: 37-44. 8 Concetti A, Massei P, Rotilio G, Brunori M, Rachmilewitz EA. Superoxide dismutase in red blood cells-Method of assay and enzyme content in normal subjects and in patients with /3-thalassemia (major and intermedia). J Lab Clin Med 1976; 87: 1057-1064.
194 9 Marklund S, Nordensson I, Back 0. Normal CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase in Werner’s syndrome. J Gerontol 1981; 36: 40-409. 10 Paglia DE, Valentine WN. Studies on quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967; 70: 158-169. 11 Hopkins J, Tudhope GR. Glutathione peroxidase in human red cells in health and disease. Br J Haematol 1973; 25: 563-575. 12 Watkinson JH. Fluorometric determination of selenium in biological material with 2,3-diaminonaphthalene. Anal Chem 1966; 38: 92-97. 13 Yagi K. A simple fluorometric assay for lipoperoxide in blood plasma. Biochem Med 1976; 15: 212-216. 14 Ohno H, Iizuka S, Kondon T, Yamamura K, Sekiya C, Taniguchi N. The levels of superoxide dismutase, catalase, and carbonic anhydrase in erythrocytes of patients with Down’s syndrome. Klin Wochenschr 1984; 62: 287-288. 15 Kedziora J, Jeske J, Witas H, Bartosz G, Leyko W. The level of superoxide dismutase in erythrocytes of children with Down syndrome (trisomy G and unbalanced translocation G21/2~). Acta Biol Med Germ 1977; 36: 779-782. 16 Michelson AM, Puget K, Durosay P. Clinical aspects of the dosage of erythrocuprein. In: Superoxide and superoxide dismutases. Michelson AM, ed. New York/San Francisco/Land: 1977: Academic Press, 467-499. 17 Pantelakis SN, Karaklis AG, Alexiou D, Vardas E, Valaes T. Red cell enzymes in trisomy 21. Am J Human Genet 1970; 22: 184-193. 18 Schmidt K, Heller W. Selenkonzentration und Aktivitat der Glutathion Peroxidase im Lysat menschlither Erythrozyten. Blut 1976; 33: 247-251. 19 Sinet PM. Metabolism of oxygen derivatives in Down’s syndrome Ann N Y Acad Sci 1982; 396: 83-94. 20 Bartosz G, Kpdziora J. Erythrocyte anomalies in Down’s syndrome. Med Hypothesis 1983; 11: 471-477. 21 Stocks J, Gutteridge JMC, Sharp RJ, Dormandy TL. The inhibition of lipid autoxidation by human serum and its relationship to serum proteins and alpha-tocopheral. Clin Sci Mol Med 1974; 47: 223-233. 22 Kedziora J, Witas H, Bartosz G, Leyko W, Jeske J, Roiynkowa, D. Down’s syndrome - transferrin parallels plasma iron changes. Experientia 1978; 34: 712. 23 Lott IT. Down’s syndrome, aging, and Alzheimer’s disease: a clinical review. Ann N Y Acad Sci 1982; 396: 15-27. 24 Walford RL. Immunological studies of Down’s syndrome and Alzheimer’s disease. Ann N Y Acad Sci 1982; 396: 95-106.