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Clinica Chimica Acta 308 Ž2001. 191–193 www.elsevier.comrlocaterclinchim
Letter to the Editor
Keywords: Extracellular-superoxide dismutase; Infant; Cerebrospinal fluid
Extracellular-superoxide dismutase in cerebror children spinal fluid from infantsr Extracellular-superoxide dismutase ŽEC-SOD. is a secretory glycoprotein with affinity for heparin-like substances, and is the major SOD isozyme in extracellular fluids w1x. EC-SOD in plasma is heterogeneous with regard to affinity for heparin and can be divided into five fractions ŽI–V. w2x. The small component of plasma EC-SOD, form V, might be the primary form synthesized in the body, and forms I–IV with reduced heparin affinity have been shown to be the result of endo- and exoproteolytic truncation at the C-terminal end w3x. Therefore, the plasma EC-SOD level and its heparin affinity might reflect the physiological and pathological conditions of the vascular system as well as the expression of this enzyme w4,5x. Previously, we showed that the ECSOD level in childrenryouths was significantly higher than that in their parents w6x. Especially in the infantsrchildren group, plasma EC-SOD levels reached a peak at about 1 year old, and then gradually decreased w7x. We speculated that the age-dependent decrease of EC-SOD above 1 year old might not be due to a decrease in EC-SOD expression but to an increase in the ability of the vascular wall to bind EC-SOD. Maturation of the amounts and characteristics of glycosaminoglycans might contribute to the increase of the ability to bind to EC-SOD. The EC-SOD levels in extracellular fluids other than plasma have scarcely been investigated, although this enzyme was initially found not only in plasma but also in cerebrospinal fluid ŽCSF., lymph and ascites w8x. The aim of this study was to examine the age-related changes of EC-SOD level in CSF and its heparin affinity.
CSF samples were collected from infantsrchildren with suspected cerebrospinal meningitis. Written informed consent for participation in the study was obtained from the parents of each child. Forty CSF samples showing no abnormalities Ž23 days old to 17 years old; male, n s 21; female, n s 19. were used in this experiment. The EC-SOD concentration was determined by enzyme-linked immunosorbent assay ŽELISA. as described previously w9x. There were no gender-related differences in the standard deviation Ž F s 0.992, p s NS. and mean level of CSF ECSOD Žmale: 29.9 " 9.8 ngrml, female: 30.1 " 9.7 ngrml, t s 0.047, p s NS.. Fig. 1a shows the significant age-dependent increase in EC-SOD level in CSF Ž F s 8.00, p - 0.01; correlation coefficient r s 0.417, p - 0.0001 vs. log phase age.. We reasoned that if EC-SOD level in CSF directly reflects the expression level of this enzyme, CSF EC-SOD might be the primary form synthesized and not a proteolytically truncated form. We next investigated the heparin affinity of EC-SOD in two CSF samples with heparin-HPLC. CSF EC-SOD was eluted at 0.70 molrl NaCl similarly to recombinant EC-SOD Žkindly provided by Symbicom, Umea, ˚ Sweden. as shown in Fig. 1b. Recently, we reported that human EC-SOD in brain tissue has high affinity for heparin w10x. It is possible that the proteolytic activity in brain homogenate would be less than that in homogenates from other tissues. These results suggested that EC-SOD in CSF did not undergo proteolytic truncation and retained its high affinity for heparin, while that of plasma EC-SOD was reduced by proteolytic removal of the heparin-binding domain w3,11x. Levels of other SOD isozymes, Cu,Zn–SOD and Mn–SOD, in CSF have been examined. Okabe et al .
0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 4 7 7 - 6
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Letter to the Editor
Fig. 1. Age-related changes in EC-SOD level in CSF Ža. and comparison of heparin-HPLC of EC-SOD in CSF Žb.. The open and closed circles in ‘a’ represent samples from female and male subjects, respectively. CSF samples from two children ŽCSF-1 and CSF-2. and r-EC-SOD ŽStd. were analyzed by heparin-HPLC. The closed circles and broken lines in ‘b’ show the EC-SOD concentrations in HPLC fractions determined by ELISA and the NaCl concentration in the mobile phase of HPLC, respectively.
w12x reported that SOD activity in CSF from adult subjects gradually increased with age up to the sixth decade. On the other hand, Yoshida et al. w13x reported that there were no age-related changes in levels of either SOD isozyme in CSF. They also showed that levels of both SOD isozymes were elevated in cerebrovascular diseases, and speculated that the elevations of SOD levels in the CSF might be due to leakage from damaged nervous tissues andror induction of these isozymes in these lesions. It has been reported that secretion of rabbit pulmonary EC-SOD into the extracellular compartment increased after birth w14x. EC-SOD is a secretory protein, and the level of this isozyme in CSF might
reflect the expression andror level of secretion in nervous tissues, whereas Cu,Zn–SOD and Mn–SOD are intracellular enzymes. The results of the present study suggested that EC-SOD concentration in CSF might reflect the level of EC-SOD expression andror secretion in the brain and the levels of EC-SOD expression andror secretion in infantsrchildren increase in an age-dependent manner. Acknowledgements This project was supported in part by a Grant-inAid for Scientific Research from the Ministry of
Letter to the Editor
Education, Science, Sports and Culture of Japan, and a grant from the Gifu Life Science Research Promotion Council ŽTA..
w12x
w13x
References w1x Marklund SL. Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci U S A 1982;79:7634–8. w2x Adachi T, Yamada H, Futenma A, Kato K, Hirano K. Heparin-induced release of extracellular-superoxide dismutase form ŽV. to plasma. J Biochem 1995;117:586–90. w3x Adachi T, Morihara N, Yamazaki N, Yamada H, Futenma A, Kato K, et al. An arginine-213 to glycine mutation in human extracellular-superoxide dismutase reduces susceptibility to trypsin-like proteinases. J Biochem 1996;120:184–8. w4x Landmesser U, Merten R, Spiekermann S, Buttner K, Drexler ¨ H, Hornig B. Vascular extracellular superoxide dismutase activity in patients with coronary artery disease. Relation to endothelium-dependent vasodilation. Circulation 2000;101: 2264–70. w5x Adachi T, Yamazaki N, Tasaki H, Toyokawa T, Yamashita K, Hirano K. Changes in the heparin affinity of extracellular-superoxide dismutase in patients with coronary artery atherosclerosis. Biol Pharm Bull 1998;21:1090–3. w6x Adachi T, Wang J, Wang XL. Age-related change of plasma extracellular-superoxide dismutase. Clin Chim Acta 2000; 290:169–78. w7x Adachi T, Masuda K, Hara H, Yamamoto M, Mitsui N, Oh-ishi T, et al. Plasma extracellular-superoxide dismutase in healthy newborns and infantsrchildren. Clin Chim Acta 2000;295:203–5. w8x Marklund SL, Holme E, Hellner L. Superoxide dismutase in extracellular fluids. Clin Chim Acta 1982;126:41–51. w9x Adachi T, Ohta H, Yamada H, Futenma A, Kato K, Hirano K. Quantitative analysis of extracellular-superoxide dismutase in serum and urine by ELISA with monoclonal antibody. Clin Chim Acta 1992;212:89–102. w10x Adachi T, Yamamoto M, Hara H. Heparin-affinity of human extracellular-superoxide dismutase in the brain. Biol Pharm Bull 2001;24:191–3. w11x Karlsson K, Edlund A, Sandstrom ¨ J, Marklund SL. Prote-
w14x
193
olytic modification of the heparin-binding affinity of extracellular superoxide dismutase. Biochem J 1993;290:623–6. Okabe T, Hamaguchi K, Inafuku T, Hara M. Aging and superoxide dismutase activity in cerebrospinal fluid. J Neurol Sci 1996;141:100–4. Yoshida E, Mokuno K, Aoki S, et al. Cerebrospinal fluid levels of superoxide dismutases in neurological diseases detected by sensitive enzyme immunoassay. J Neurol Sci 1994;124:25–31. Nozik-Grayck E, Dieterle CS, Piantadosi CA, Enghild JJ, Oury TD. Secretion of extracellular superoxide dismutase in neonatal lungs. Am J Physiol: Lung Cell Mol Physiol 2000;279:L977–84.
Tetsuo Adachi ) , Masayuki Yamamoto Hirokazu Hara Laboratory of Clinical Pharmaceutics Gifu Pharmaceutical UniÕersity 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan E-mail address: [email protected] Kazuhiro Masuda Department of Radiology Saitama Children’s Medical Center Saitama, Japan Norimasa Mitsui Department of Clinical Laboratory Saitama Children’s Medical Center Saitama, Japan Tsutomu Oh-ishi, Minoru Okazaki DiÕision of Infectious Diseases Immunology and Allergy Saitama Children’s Medical Center Saitama, Japan
)
Corresponding author. Tel.: q81-58-237-3931; fax: q81-58237-5979.
Letter to the Editor
Keywords: Extracellular-superoxide dismutase; Infant; Cerebrospinal fluid
Extracellular-superoxide dismutase in cerebror children spinal fluid from infantsr Extracellular-superoxide dismutase ŽEC-SOD. is a secretory glycoprotein with affinity for heparin-like substances, and is the major SOD isozyme in extracellular fluids w1x. EC-SOD in plasma is heterogeneous with regard to affinity for heparin and can be divided into five fractions ŽI–V. w2x. The small component of plasma EC-SOD, form V, might be the primary form synthesized in the body, and forms I–IV with reduced heparin affinity have been shown to be the result of endo- and exoproteolytic truncation at the C-terminal end w3x. Therefore, the plasma EC-SOD level and its heparin affinity might reflect the physiological and pathological conditions of the vascular system as well as the expression of this enzyme w4,5x. Previously, we showed that the ECSOD level in childrenryouths was significantly higher than that in their parents w6x. Especially in the infantsrchildren group, plasma EC-SOD levels reached a peak at about 1 year old, and then gradually decreased w7x. We speculated that the age-dependent decrease of EC-SOD above 1 year old might not be due to a decrease in EC-SOD expression but to an increase in the ability of the vascular wall to bind EC-SOD. Maturation of the amounts and characteristics of glycosaminoglycans might contribute to the increase of the ability to bind to EC-SOD. The EC-SOD levels in extracellular fluids other than plasma have scarcely been investigated, although this enzyme was initially found not only in plasma but also in cerebrospinal fluid ŽCSF., lymph and ascites w8x. The aim of this study was to examine the age-related changes of EC-SOD level in CSF and its heparin affinity.
CSF samples were collected from infantsrchildren with suspected cerebrospinal meningitis. Written informed consent for participation in the study was obtained from the parents of each child. Forty CSF samples showing no abnormalities Ž23 days old to 17 years old; male, n s 21; female, n s 19. were used in this experiment. The EC-SOD concentration was determined by enzyme-linked immunosorbent assay ŽELISA. as described previously w9x. There were no gender-related differences in the standard deviation Ž F s 0.992, p s NS. and mean level of CSF ECSOD Žmale: 29.9 " 9.8 ngrml, female: 30.1 " 9.7 ngrml, t s 0.047, p s NS.. Fig. 1a shows the significant age-dependent increase in EC-SOD level in CSF Ž F s 8.00, p - 0.01; correlation coefficient r s 0.417, p - 0.0001 vs. log phase age.. We reasoned that if EC-SOD level in CSF directly reflects the expression level of this enzyme, CSF EC-SOD might be the primary form synthesized and not a proteolytically truncated form. We next investigated the heparin affinity of EC-SOD in two CSF samples with heparin-HPLC. CSF EC-SOD was eluted at 0.70 molrl NaCl similarly to recombinant EC-SOD Žkindly provided by Symbicom, Umea, ˚ Sweden. as shown in Fig. 1b. Recently, we reported that human EC-SOD in brain tissue has high affinity for heparin w10x. It is possible that the proteolytic activity in brain homogenate would be less than that in homogenates from other tissues. These results suggested that EC-SOD in CSF did not undergo proteolytic truncation and retained its high affinity for heparin, while that of plasma EC-SOD was reduced by proteolytic removal of the heparin-binding domain w3,11x. Levels of other SOD isozymes, Cu,Zn–SOD and Mn–SOD, in CSF have been examined. Okabe et al .
0009-8981r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 8 9 8 1 Ž 0 1 . 0 0 4 7 7 - 6
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Letter to the Editor
Fig. 1. Age-related changes in EC-SOD level in CSF Ža. and comparison of heparin-HPLC of EC-SOD in CSF Žb.. The open and closed circles in ‘a’ represent samples from female and male subjects, respectively. CSF samples from two children ŽCSF-1 and CSF-2. and r-EC-SOD ŽStd. were analyzed by heparin-HPLC. The closed circles and broken lines in ‘b’ show the EC-SOD concentrations in HPLC fractions determined by ELISA and the NaCl concentration in the mobile phase of HPLC, respectively.
w12x reported that SOD activity in CSF from adult subjects gradually increased with age up to the sixth decade. On the other hand, Yoshida et al. w13x reported that there were no age-related changes in levels of either SOD isozyme in CSF. They also showed that levels of both SOD isozymes were elevated in cerebrovascular diseases, and speculated that the elevations of SOD levels in the CSF might be due to leakage from damaged nervous tissues andror induction of these isozymes in these lesions. It has been reported that secretion of rabbit pulmonary EC-SOD into the extracellular compartment increased after birth w14x. EC-SOD is a secretory protein, and the level of this isozyme in CSF might
reflect the expression andror level of secretion in nervous tissues, whereas Cu,Zn–SOD and Mn–SOD are intracellular enzymes. The results of the present study suggested that EC-SOD concentration in CSF might reflect the level of EC-SOD expression andror secretion in the brain and the levels of EC-SOD expression andror secretion in infantsrchildren increase in an age-dependent manner. Acknowledgements This project was supported in part by a Grant-inAid for Scientific Research from the Ministry of
Letter to the Editor
Education, Science, Sports and Culture of Japan, and a grant from the Gifu Life Science Research Promotion Council ŽTA..
w12x
w13x
References w1x Marklund SL. Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci U S A 1982;79:7634–8. w2x Adachi T, Yamada H, Futenma A, Kato K, Hirano K. Heparin-induced release of extracellular-superoxide dismutase form ŽV. to plasma. J Biochem 1995;117:586–90. w3x Adachi T, Morihara N, Yamazaki N, Yamada H, Futenma A, Kato K, et al. An arginine-213 to glycine mutation in human extracellular-superoxide dismutase reduces susceptibility to trypsin-like proteinases. J Biochem 1996;120:184–8. w4x Landmesser U, Merten R, Spiekermann S, Buttner K, Drexler ¨ H, Hornig B. Vascular extracellular superoxide dismutase activity in patients with coronary artery disease. Relation to endothelium-dependent vasodilation. Circulation 2000;101: 2264–70. w5x Adachi T, Yamazaki N, Tasaki H, Toyokawa T, Yamashita K, Hirano K. Changes in the heparin affinity of extracellular-superoxide dismutase in patients with coronary artery atherosclerosis. Biol Pharm Bull 1998;21:1090–3. w6x Adachi T, Wang J, Wang XL. Age-related change of plasma extracellular-superoxide dismutase. Clin Chim Acta 2000; 290:169–78. w7x Adachi T, Masuda K, Hara H, Yamamoto M, Mitsui N, Oh-ishi T, et al. Plasma extracellular-superoxide dismutase in healthy newborns and infantsrchildren. Clin Chim Acta 2000;295:203–5. w8x Marklund SL, Holme E, Hellner L. Superoxide dismutase in extracellular fluids. Clin Chim Acta 1982;126:41–51. w9x Adachi T, Ohta H, Yamada H, Futenma A, Kato K, Hirano K. Quantitative analysis of extracellular-superoxide dismutase in serum and urine by ELISA with monoclonal antibody. Clin Chim Acta 1992;212:89–102. w10x Adachi T, Yamamoto M, Hara H. Heparin-affinity of human extracellular-superoxide dismutase in the brain. Biol Pharm Bull 2001;24:191–3. w11x Karlsson K, Edlund A, Sandstrom ¨ J, Marklund SL. Prote-
w14x
193
olytic modification of the heparin-binding affinity of extracellular superoxide dismutase. Biochem J 1993;290:623–6. Okabe T, Hamaguchi K, Inafuku T, Hara M. Aging and superoxide dismutase activity in cerebrospinal fluid. J Neurol Sci 1996;141:100–4. Yoshida E, Mokuno K, Aoki S, et al. Cerebrospinal fluid levels of superoxide dismutases in neurological diseases detected by sensitive enzyme immunoassay. J Neurol Sci 1994;124:25–31. Nozik-Grayck E, Dieterle CS, Piantadosi CA, Enghild JJ, Oury TD. Secretion of extracellular superoxide dismutase in neonatal lungs. Am J Physiol: Lung Cell Mol Physiol 2000;279:L977–84.
Tetsuo Adachi ) , Masayuki Yamamoto Hirokazu Hara Laboratory of Clinical Pharmaceutics Gifu Pharmaceutical UniÕersity 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan E-mail address: [email protected] Kazuhiro Masuda Department of Radiology Saitama Children’s Medical Center Saitama, Japan Norimasa Mitsui Department of Clinical Laboratory Saitama Children’s Medical Center Saitama, Japan Tsutomu Oh-ishi, Minoru Okazaki DiÕision of Infectious Diseases Immunology and Allergy Saitama Children’s Medical Center Saitama, Japan
)
Corresponding author. Tel.: q81-58-237-3931; fax: q81-58237-5979.