Journal of Clinical Neuroscience 13 (2006) 991–994 www.elsevier.com/locate/jocn
Clinical study
Red cell superoxide dismutase activity in sporadic amyotrophic lateral sclerosis Deniz Tuncel a,*, Nursel Aydin b, Pelin Aribal Kocatu¨rk c, ¨ zelc¸i Kavas c, Sevda Sarikaya b Gu¨zin O a
Department of Neurology, Faculty of Medicine, Kahramanmaras University, Kahramanmaras, Turkey b Department of Neurology, Faculty of Medicine, Ankara University, Ankara, Turkey c Department of Pathophysiology, Faculty of Medicine, Ankara University, Ankara, Turkey Received 12 May 2005; accepted 3 October 2005
Abstract Specific biologic markers are not available for definitive diagnosis and monitoring of disease progression in sporadic amyotrophic lateral sclerosis (SALS). Oxidative stress plays a role in ALS pathogenesis. The purpose of this study was to determine the association between Cu/Zn superoxide dismutase (SOD1) activity, diagnosis and prognosis. The present study included 25 SALS patients (SALS group; age 51 ± 12 years) and 10 healthy subjects (age 45 ± 5 years) as a control group. Patients were divided into groups representing four levels of diagnostic certainty of ALS in accordance with the El Escorial Revisited criteria. The disease state was determined using the modified ALS health state scale of Riviere et al. (Arch Neurol 1998:55;526–8). Red-cell SOD1 activity was determined by spectrophotometry. SOD1 activity in red cells was compared statistically with diagnostic criteria and disease state. Red cell SOD1 activity was high in all SALS patients, but there was no significant association between enzyme activity and diagnostic criteria and disease state. In this preliminary study, we did not find any correlation between SOD1 activity level and diagnosis or prognosis. Measured SOD1 activity sometimes supports ALS diagnosis, but it is neither a specific nor a prognostic factor. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Amyotrophic lateral sclerosis; Red cell superoxide dismutase; Diagnosis; Prognosis
1. Introduction Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of the motor neurons, resulting in a loss of spinal, bulbar and cortical motor neurons. ALS causes progressive paralysis that leads to death.1 Approximately 10% of ALS cases are familial (FALS)2 and in about 20% of these, the disease is associated with mutations in the CuZn-superoxide dismutase (SOD1) gene.3 The aetiology of ALS is currently unknown, but there are three major hypotheses to explain the motor neu* Corresponding author. Address: Birlik mahallesi, 5. cadde no: 27/3, C ¸ ankaya 06610, Ankara, Turkey. Tel.: +90 312 495 85 57; fax: +90 344 221 23 71. E-mail address:
[email protected] (D. Tuncel).
0967-5868/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jocn.2005.10.019
ron degeneration that occurs in ALS: (i) the autoimmune hypothesis; (ii) the excitatory amino acid hypothesis; and (iii) the oxidative stress hypothesis, based on mutations in SOD1.4 The SODs are a group of enzymes that catalyze the conversion of the superoxide anion ðO 2 Þ to hydrogen peroxide and oxygen. SOD provides cellular defence 5 against O 2 and its toxic derivatives. The SOD1 mutations in FALS are predominantly missense mutations in which one amino acid is substituted for another; 92 different mutations at 67 different sites have been found to date.6 SOD1 gene mutations account for a few percent of apparent sporadic ALS (SALS) cases.3,4,6–13 The mechanism of motor neuron degeneration in ALS patients harbouring SOD1 mutations is not understood, but the collective evidence suggests a toxic gain of function rather than loss of function.13–15
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At present there is no widely accepted protocol for laboratory testing of patients with suspected ALS. Genetic tests can be used in ALS to confirm the diagnosis, especially in cases with atypical features. Verifying that a patient is a carrier of a particular SOD1 mutation has prognostic value.3,13 Different studies have demonstrated reduced, increased or normal amounts of SOD1 in patients with ALS.7,16–18 In a previous study, we compared red cell SOD1 and catalase activity, and plasma and red cell copper, zinc and magnesium concentrations in SALS patients with corresponding values in a healthy control group. The patients with SALS were found to have increased red cell SOD1 and catalase activity, increased red cell zinc and plasma magnesium concentrations, and decreased red cell copper concentrations.19 The present study is a reanalysis of our previous data using a five-health state classification20 and the El Escorial Revisited21 diagnostic criteria to investigate the diagnostic and prognostic value of SOD1 activity in ALS patients. 2. Materials and methods After receiving approval from the hospital ethics committee, we recruited patients from the Neurology Department, Faculty of Medicine, Ankara University. Written informed consent was obtained from each patient. There were 25 patients (6 female, 19 male) in the SALS group, with a mean age of 51 ± 12 years; and 10 healthy subjects in the control group, with a mean age of 45 ± 5 years. The control group consisted of non-smokers without neurological, chronic or infectious diseases, or iron deficiency. The SALS patients were divided into groups representing four levels of diagnostic certainty of ALS in accordance with the El Escorial Revisited diagnostic criteria:2 clinically definite ALS, clinically probable ALS, clinically probable (laboratory-supported) ALS, and clinically possible ALS. The state of the disease was determined using the modified Table 1 Amyotrophic lateral sclerosis health state scale20 Mild
Moderate
Severe
Terminal
Death
Recent diagnosis, mild deficit in 1/3 regions (speech, arm, leg). Functionally independent in speech, arm activities of daily living, ambulation Mild deficit in all three regions or moderate to severe deficit in one region, with the other two regions being normal or mildly affected Needs assistance in two or three regions, speech dysarthric and/or patient needs assistance to walk and/ or needs assistance with arm activities of daily living Non-functional use of at least two regions and moderate or nonfunctional use of the third region
ALS health state scale of Riviere et al., in which patients are classified as being in mild, moderate, severe, or terminal states, or dead (Table 1).20 The level of red cell SOD1 activity was measured in all patients. For the determination of SOD1 activity, the blood samples of the patients were always drawn at the same time and were put in heparinized tubes. The plasma was kept in polypropylene tubes, and each sample was studied within an hour. Red-cell SOD activity was determined by spectrophotometry.22 SOD1 activity in red cells was statistically compared with diagnostic criteria and health state scale scores. Enzyme activity is expressed as mean ± standard deviation (mean ± SD). The results were analyzed using Pearson’s correlation coefficients. 3. Results The red cell SOD1 activity of patients in the SALS group (4970 ± 646 U/g Hb) was significantly higher than that in controls (3623 ± 276 U/g Hb) (P < 0.001). According to the El Escorial criteria, 17 patients had clinically definite, three had clinically probable, and five had clinically probable (laboratory-supported) ALS. Red cell SOD1 activity was high in all patients, but there was no significant correlation between enzyme activity and diagnostic criteria (Table 2). Patients with ALS were grouped by using the modified Riviere health state scale: nine were classified as mild, eight as moderate, six as severe and two as terminal. Red cell SOD1 activity was high in all patients, but there was no significant correlation between activity and disease state (Table 3).
Table 2 El Escorial2 diagnostic criteria and red cell SOD1 activity El Escorial diagnostic criteria
Number of patients
SOD1 activity (U/g Hb; mean ± SD)
Definite ALS Probable ALS Probable (laboratory-supported)
17 3 5
4966 ± 642 4756 ± 716 5111 ± 732
ALS Total
25
SOD1 = CuZn-superoxide sclerosis.
dismutase,
4970 ± 646 ALS = amyotrophic
lateral
Table 3 Amyotrophic lateral sclerosis health state scale and red cell SOD1 activity ALS health state scale
Number of patients
SOD1 activity (U/g Hb; mean ± SD)
Mild Moderate Severe Terminal Total
9 8 6 2 25
4939 ± 787 4968 ± 641 4954 ± 461 5163 ± 1017 4970 ± 646
SOD1 = CuZn-superoxide sclerosis.
dismutase,
ALS = amyotrophic
lateral
D. Tuncel et al. / Journal of Clinical Neuroscience 13 (2006) 991–994
4. Discussion The aetiology of SALS is unknown, but 20% of FALS pedigrees have a mutation in the Cu/Zn SOD1 gene.2,3 Genetic testing is much more commonly conducted for FALS than SALS. But a mutation in the neurofilamentous heavy chain and SOD1 genes, especially in SALS, raises questions about the distinction between familial and sporadic disease.14 Incomplete disease penetrance appears to be quite common in FALS, and has been described almost as SALS.3,15 The mechanism by which motor neuron degeneration is caused by SOD1 mutations is unknown. The level of measured SOD1 enzyme activity may change according to the type of mutation.7,17 Therefore, two hypotheses have been proposed to explain the disease mechanism of SOD1 mutations: the first is that the mutant SOD1 has an altered substrate affinity that leads to the generation of toxic reaction products, and the second is that the mutant enzyme may be unstably folded, so that it precipitates to form toxic cytoplasmic aggregates.14,15 In a previous study, we found high levels of SOD1 enzyme activity in SALS patients compared with a control group.19 The reason for the high enzyme activity may be related to a different mutation type that occurs in the Turkish population. However, this was a preliminary study with a small number of patients. Further genetic studies should be carried out to confirm the results. Although the essential requirements for a diagnosis of ALS are clearly defined by the El Escorial criteria, some patients can still be misdiagnosed. The differential diagnosis must exclude non-motor neuron diseases and other adultonset motor neuron diseases. Laboratory testing can be used to differentiate other diseases and to confirm the diagnosis. In the present study, we tested for a relationship between El Escorial diagnostic criteria and SOD1 activity level, but did not find a correlation. Although a high level of SOD1 activity may support a diagnosis of ALS, it is not specific for a definitive diagnosis. Furthermore, various levels of SOD1 activity in ALS have been reported in other studies (increased, normal or decreased).7,16–18 The pathophysiology of most motor neuron diseases, including ALS, is poorly understood, and specific biologic markers are not available to monitor disease progression. Although patients undoubtedly want information about their projected survival, it is not easy to provide this information. Biological (including genetic) markers may have prognostic value, and may assist clinicians to identify atypical features. Also, verifying whether a patient carries a particular SOD1 mutation, for example G41S in Italy, H46R in Japan, A4V and L37R in North America, or D90A in Scandinavia, has prognostic value.3,13,23 But there is no correlation between SOD1 enzyme activity and disease severity in ALS patients or SOD1 mutant mice.13,16,18,23–25 Similarly, we did not find a correlation between enzyme activity level and disease state. In conclusion, in this preliminary study, we found no correlation between SOD1 activity level and diagnosis or
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prognosis. The measured SOD1 activity sometimes supports a diagnosis of ALS, but it is neither a specific nor a prognostic factor. Acknowledgement _ The authors of this study are grateful to Demet Iren Bu¨yu¨kkag˘nıcı (biologist) and Birol Gu¨c¸lu¨ (laboratory technician) for their excellent laboratory assistance, and to Kenan Ko¨se for his careful statistical analysis. References 1. Horton WA, Eldridge R, Brody JA. Familial motor neuron disease: evidence for at least 3 different types. Neurology 1976;26:460–5. 2. Haverkamp LJ, Appel V, Appel SH. Natural history of amyotrophic lateral sclerosis in a database population. Brain 1995;118:707–19. 3. Andersen PM. Genetic factors in the early diagnosis of ALS. Amyotroph Lateral Scler Other Motor Neuron Disord 2000;1 (Suppl.1):S31–42. 4. Cha CI, Chung YH, Shin C, et al. Immunocytochemical study on the distribution of nitrotyrosine in the brain of the transgenic mice expressing a human Cu/Zn SOD mutation. Brain Res 2000;853: 156–61. 5. Siddique T, Nijhawan D, Hentati A. Molecular genetic basis of familial ALS. Neurology 1996;47 (Suppl.2):S27–35. 6. Olsen MK, Roberds SL, Ellerbrock BR, et al. Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord. Ann Neurol 2001;50:730–40. 7. Deng H-X, Hentati A, Tainer JA, et al. Amyotrophic lateral sclerosis and structural defects in CuZn superoxide dismutase. Science 1993;261:1047–51. 8. Rosen DR, Siddique T, Patteson D, et al. Mutations in CuZnsuperoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature 1993;364:59–62. 9. Jones CT, Swingler RJ, Brock DJH. Identification of a novel SOD1 mutation in an apparently sporadic amyotrophic lateral sclerosis patient and the detection of Ile 113 Thr in three others. Hum Mol Genet 1994;3:649–50. 10. Rainore L, Pinessi L, Tsuda T, et al. SOD1 missense mutation in an Italian family with ALS. Neurology 1994;44:347–9. 11. Andersen PM, Nilsson P, Keranen M-L, et al. Phenotypic heterogeneity in motor neuron disease patients with CuZn-superoxide dismutase mutation in Scandinavia. Brain 1997;10:1723–37. 12. Shaw CE, Enayat ZE, Chioza BA, et al. Mutations in all five exons of SOD-1 may cause ALS. Ann Neurol 1998;43:390–4. 13. Robberecht L, Van Den Bosch, Vleminckx V. Amyotrophic lateral sclerosis: pathogenesis. Acta Neurol Belg 2000;100:181–7. 14. Shaw CE, Enayat ZE, Powel JF, et al. Familial amyotrophic lateral sclerosis, molecular pathology of a patient with a SOD1 mutation. Neurology 1997;49:1612–6. 15. Andreassen OA, Ferrante RJ, Klivenyi P, et al. Partial deficiency of manganese superoxide dismutase exacerbates a transgenic mouse model of amyotrophic lateral sclerosis. Ann Neurol 2000;47:447–55. 16. Gurney ME, Chin AY, Dal Canto MC, et al. Motor neuron degeneration in mice that express a human CuZn-superoxide dismutase mutation. Science 1994;264:1772–5. 17. Andersen PM, Forsgren L, Binzer M, et al. Autosomal recessive adult-onset amyotrophic lateral sclerosis associated with homozygosity for Asp90Ala CuZn-superoxide dismutase mutation. A clinical and genealogical study of 36 patients. Brain 1996;119: 1153–72. 18. Jacobsson J, Jonsson PA, Andersen PM, et al. Superoxide dismutase in CSF from amyotrophic lateral sclerosis patients with and without CuZn-superoxide dismutase mutations. Brain 2001;124: 1461–6. ¨ , et al. Red cell Cu/Zn-SOD and 19. Kocatu¨rk PA, Tuncel D, Kavas GO catalase activities are increased in ALS. Proceedings of the Second
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