Plasma superoxide dismutase and glutathione peroxidase activity in sporadic amyotrophic lateral sclerosis

Journal of Neurological Sciences 151 (1997) 35–39

Plasma superoxide dismutase and glutathione peroxidase activity in sporadic amyotrophic lateral sclerosis a b b a,c Radouane Moumen , Andre´ Nouvelot , Dominique Duval , Bernard Lechevalier , a,c , Fausto Viader * a

Equipe d’ Universite´ ‘‘ Hydrolases et cytotoxicite´ ’’, Universite´ de Caen, Caen, France b CNRS UMR 6551, Universite´ de Caen, Caen, France c Services de Neurologie, CHU de Caen, Caen, France Received 1 November 1996; revised 13 March 1997; accepted 15 March 1997

Abstract To determine the possible role of oxydative stress in the pathology of amyotrophic lateral sclerosis (SALS), we measured the plasma activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX), together with GPX and malone dialdehyde (MDA, a marker of lipoperoxydation) plasma concentrations in a sample of 21 SALS patients and 7 normal control (NC) subjects. MDA concentration and SOD activity were significantly higher, whereas GPX activity was significantly lower in SALS patients than in NC. Increased MDA concentration provides indirect confirmation of excess lipoperoxydation. Increased plasma SOD activity might reflect the involvement of extra-cellular SOD (SOD3), a hitherto unreported finding in SALS. Impaired GPX activity, which has already been found in red blood cells and brain tissue of SALS patients, might play a part in the pathogenesis of this disease.  1997 Elsevier Science B.V. Keywords: Amyotrophic lateral sclerosis; Glutathione peroxidase; Malone dialdehyde; Superoxide dismutase

1. Introduction Amyotrophic lateral sclerosis (ALS) is a degenerative disease of unknown ætiology. It is sporadic in around 90% of cases and inherited as an autosomal dominant trait in the remaining 10%. There is substantial evidence to suggest, as proposed by Mitchell et al. as early as 1987 (Mitchell et al., 1987), that oxidative stress is implicated in the pathogenesis of ALS. About 20% of cases of familial ALS (FALS) are associated with missense point mutations of the gene coding for copper–zinc superoxide dismutase (Cu–Zn SOD or SOD1) (Rosen et al., 1993). In most, but not all (Borchelt et al., 1994) of these patients, red blood cell (RBC) SOD activity has been found to be decreased. This decrease is not generally thought to directly underlie *Corresponding author. Address: Service de Neurologie, Vastel, CHU ˆ de Nacre, F-14033-Caen, France; Tel: 133 2 31064625; Fax: 133 2 Cote 31064627. 0022-510X / 97 / $17.00  1997 Elsevier Science B.V. All rights reserved PII S0022-510X( 97 )00109-3

the pathological changes of the disease, since both the remaining FALS and sporadic ALS (SALS) cases have the same clinical and pathological features with normal RBC SOD activity (Puymirat et al., 1994; Robberecht et al., 1994). Additional evidence for free radical involvement in SALS has been found: 1) SOD1 mRNA increases in alpha motoneurons of the anterior horn of the spinal cord (Bergeron et al., 1994); 2) increased density of glutathione binding sites in the spinal cord (Lanius et al., 1993); 3) decreased SOD activity in cerebrospinal fluid (Bracco et al., 1991; Iwasaki et al., 1993); 4) increased serum freeradical levels (Ihara et al., 1995); 5) decreased glutathione peroxidase (GPX) activity in whole blood (Mitchell et al., 1993), in RBC (Przedborski et al., 1996a) and in the precentral gyrus at post-mortem study (Przedborski et al., 1996b). We studied plasma SOD and GPX activities, together with GPX and malondialdehyde (a marker of lipoperoxidation) plasma concentrations, in a sample of SALS patients

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and normal control subjects. Our aims were 1) to learn whether plasma studies would disclose more abnormalities than RBC studies undertaken so far in SALS; 2) to try to correlate our results with the clinical type of disease, i.e. either of bulbar (BSALS) or spinal (SpSALS) onset, based on the motor territory first involved.

2. Materials and methods

2.1. Patients and normal control subjects Twenty-one patients (15 men, 6 women) with probable or definite SALS according to the ‘‘El Escorial’’ WFN criteria (El Escorial, 1994) were studied. SpSALS patients (n512) were aged 35 to 72 years (mean 57.5612), while BSALS patients were older (age 57 to 76 years, mean 6667). All gave informed consent. Normal control subjects (NC) (5 men, 2 women) were recruited from the Transfusion Center of Caen and were aged 40 to 60 years (mean 50.568).

2.2. Blood sampling and measurements Blood was sampled in EDTA and immediately centrifuged at 2500 rpm for 15 min. Butylhydroxytoluene (0.001%) was then added to plasma to prevent oxidation and stored at 2808C until use. SOD activity was measured using the commercially available SOD-525 Bioxytech kit (Bioxytech, France). This method measures the SOD catalyzed auto-oxidation of a chromophore, as described in detail by Robberecht et al. (1994). GPX activity was measured by 2 different methods: 1) the method of Mill (1958) which is based on the determination of the concentration of non-consumed reduced glutathione; and 2) the method of Paglia and Valentine (1967) which is based on the measurement of the quantity of NADPH used in the reduction of glutathione, obtained

from oxidised glutathione through a reaction catalyzed by glutathione reductase. GPX concentration was measured through an immunoenzymatic method using the commercially available kit ‘‘pl.GPXIEA’’ (Bioxytech, France). MDA concentration was measured by the method described by Uchiyama and Mihara (1978) (MDA reacts with TBA to give pick complex [MDA TBA2]): 3.0 ml of phosphoric acid solution (1%) were pipetted into the required number of screw-capped glass test tubes. 500 ml of either distilled water (blank), standards, and plasma were then pipetted into the respective tubes, and vortexmixed. 1.0 ml of TBA solution (0.6%) was added to all tubes. All tubes were tightly capped and their contents were vortex-mixed for 20 s. The tubes were placed in a boiling water bath for 60 min and then cooled in an ice–water bath to stop the reaction. 3 ml of n-butanol were added to each tube and vortex mixed in order to extract the color complex. The tubes were then centrifuged at 2500 rpm during 10 min. MDA concentration was estimated from the difference between absorption of supernatant at 532 nm and at 520 nm. Total protein content was measured by the method of Lowry et al. (1951). All values are expressed as means6SD and have been compared by both the Student’s t-test and the Mann– Whitney rank sum test.

3. Results All results are shown in Table 1 and Figs. 1–3. The principal findings can be summarized as follows: 1) There is no difference between SALS patients and controls with regard to protein content or plasma GPX concentration. 2) MDA concentrations (Fig. 1) and SOD activity (Fig. 2) are significantly higher in SALS patients than in NC. SOD activity is higher in BSALS than in SpSALS. In both cases, the lowest BSALS values are greater than the

Table 1 Results of different tests in plasma

Protein mg / ml GPX ng / g prot. Activity SOD U SOD 525 / g protein Activity GPX 10 22 U / g prot. MDA m M

BSALS

SpSALS

NC

Student’s t-test

(n512) M SD

(n59) M SD

(n57) M SD

Significance BSALS / NC

SpSALS / NC

BSALS / SpSALS

ns ns

ns ns

ns ns

7667 10426174

7468 9576329

7664 11356220

159649

110634

83617

P,0.001

P,0.005

P,0.05

3896108 4.9661.46

350691 4.5961.31

7036123 2.2360.38

P,0.001 P,0.001

P,0.001 P,0.001

ns ns

B / Sp SALS, bulbar / spinal sporadic amyotrophic lateral sclerosis; GPX, glutathione peroxydase; MDA, malondialdehyde; M, mean; NC, normal control; ns, non significant; SOD, superoxide dismutase; SD, standard deviation.

R. Moumen et al. / Journal of Neurological Sciences 151 (1997) 35 – 39

Fig. 1. Plasma malone dialdehyde (MDA) concentration in bulbar onset sporadic amyotrophic lateral sclerosis (BSALS), spinal onset sporadic amyotrophic lateral sclerosis (SpSALS) and normal control (NC).

highest value of NC. 3) GPX activity (Fig. 3) is significantly lower in SALS patients than in NC, with no difference between the 2 groups of patients. The highest value of SpSALS patients is below the lowest value of NC.

4. Discussion SOD activity measurements in SALS patients have so far given somewhat contradictory results. In RBC, both Puymirat et al. (1994) and Robberecht et al. (1994) have found normal levels. On the other hand, Ihara et al. (1995) have found increased RBC SOD activity in some SALS cases. Bracco et al. (1991) and Iwasaki et al. (1993) both found decreased CSF SOD activity in SALS patients. In

Fig. 2. Plasma superoxide dismutase (SOD) activity in bulbar onset sporadic amyotrophic lateral sclerosis (BSALS), spinal onset sporadic amyotrophic lateral sclerosis (SpSALS) and normal control (NC).

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Fig. 3. Plasma glutathione peroxidase (GPX) activity in bulbar onset sporadic amyotrophic lateral sclerosis (BSALS), spinal onset sporadic amyotrophic lateral sclerosis (SpSALS) and normal control (NC).

post mortem studies of nervous tissue, Bowling et al. (1993) found normal SOD activity in the brain, whereas Uchino et al. (1994) found decreased SOD1 and SOD2 activities in several cell groups (pyramidal cells in the cerebral cortex, and anterior and posterior horn cells of the spinal cord). According to O’Reilly et al. (1995), SOD1 activity is unchanged in spinal motoneurons, but decreased in the neighbouring astrocytes. Finally, Bergeron et al. (1994) found high SOD1 mRNA expression in spinal motoneurons. Our finding of elevated SOD activity in the plasma of SALS patients raises several questions. First, as our patients were, on average, older than control subjects, one might first ask whether these results could reflect a physiological increase in plasma SOD activity with age. We are unaware of relevant references to variations of plasma SOD activity with age, but Bracco et al. (1991) found that SOD activity actually decreases with age in the CSF, and Puymirat et al. (1994) found no change in RBC activity with age in their control subjects. Finally, SOD activity was not correlated with age within any of our groups of subjects (NC, BSALS, SpSALS [data not shown]). It seems thus unlikely that age can explain our findings. Furthermore, there was no difference in the average storage time of the samples taken from both patients and control subjects. Could the differences between our results and previous reports be explained by technical problems? Among those who found normal RBC SOD activity, Robberecht et al. (1994) used the same technique as us, while Puymirat et al. used Spitz and Oberley’s method. On the other hand, Ihara et al. (1995) found increased SOD activity in the some of their patients by using a spin-trapping method, that differs from ours. Whether SOD activity is found to be normal or not does not seem to depend on the method used. It seems thus unlikely that discrepancies between our

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results and some of the previously reported findings are accounted for by technical differences. In fact, unlike previous authors, we measured SOD activity in plasma rather than in RBC and thus the type of SOD we measured may not be the same as in previous studies. There are 3 types of SOD: Cu–Zn SOD (SOD1), which is mainly intra-cellular, Mn SOD (SOD2), mitochondrial, and extracellular SOD (SOD3), which is a glycosylated form of SOD1 (Marklund et al., 1982). While our method does not distinguish among these different types, plasma SOD is likely to be SOD3. Discrepancies between RBC and plasma SOD changes might reflect the fact that the different types of SODs can be differentially implicated in SALS, as suggested by Shaw et al. (1995). BSALS patients had relatively higher SOD activity than SpSALS. Since BSALS patients usually have prominent swallowing difficulties, differences could be related to superimposed dietary factors. However, as all our patients were seen within the first months of their disease, their nutritional state was not significantly impaired at the time of blood sampling, as is suggested by the normality of their serum protein concentration (Table 1). GPX activity measurements in SALS have also given conflicting results. In the anterior horn of the spinal cord, Fitzmaurice et al. (1995) found normal GPX activity, while Ince et al. (1994) found it increased and Mitchell et al. (1991) decreased. Lanius et al. (1993) reported increased density of binding sites for glutathione in the spinal cord. More recently, Shaw et al. (1995) were unable to find detectable GPX activity in the spinal cord of both SALS patients and control subjects. However, GPX activity has been found to be decreased in SALS, in whole blood (Mitchell et al., 1993), in RBC (Przedborski et al., 1996a) and in the motor cortex (Przedborski et al., 1996b). Our findings are in accordance with these latter results. The increase in MDA concentration reflects enhanced lipoperoxydation and indirectly confirms a dysfunction of free radical scavenging systems. Whatever the mechanism of increased plasma SOD activity, this might result in excess H 2 O 2 production, which deficient GPX is unable to metabolize, finally leading to uncontrolled hydroxyl radical production and lipoperoxidation. Such an interpretation stresses the need to elucidate the cause of GPX dysfunction, and possibly to direct therapeutic efforts to enhance the activity of this enzyme.

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