Neurochemistry International 56 (2010) 687–693

Time course of oxidant markers and antioxidant defenses in subgroups ofamyotrophic lateral sclerosis patients

Emanuela Cova a,*, Paolo Bongioanni b, Cristina Cereda a, Maria Rita Metelli c, Laura Salvaneschi d,Stefano Bernuzzi d, Stefania Guareschi a,e, Bruno Rossi b, Mauro Ceroni a,e

a Laboratory of Experimental Neurobiology, IRCCS, Foundation Neurological Institute ‘‘C. Mondino’’, Via Mondino, 2, 27100 Pavia, Italyb Neurorehabilitation Unit, Azienda Ospedaliero-Universitaria Pisana, Via Paradisa, 2, 56100 Pisa, Italyc Department of Experimental Pathology, University of Pisa, Via Roma, 67, 56100 Pisa, Italyd Immunohematological and Transfusional Service and Centre of Transplantation Immunology, Foundation ‘‘San Matteo’’, IRCCS, viale Golgi, 9 Pavia, Italye Department of Neurosciences, University of Pavia, via Mondino, 2, 27100 Pavia, Italy

A R T I C L E I N F O

Article history:

Received 18 November 2009

Received in revised form 8 January 2010

Accepted 3 February 2010

Available online 10 February 2010

Keywords:

Amyotrophic lateral sclerosis

Antioxidant defenses

Enzyme activity

Reactive oxygen species

A B S T R A C T

Oxidative stress markers have been found in nervous and peripheral tissues of familial and sporadic

amyotrophic lateral sclerosis patients. Here, we evaluated the activity of some antioxidant enzymes

glutathione peroxidase, glutathione reductase and Cu–Zn superoxide dismutase in erythrocyte, the

marker of non-enzymatic antioxidant response (total antioxidant status), as well as plasma reactive

oxygen species, at the enrolment and during disease progression in 88 patients affected by the sporadic

form of amyotrophic lateral sclerosis. Our study has been performed along 72 months by grouping the

patients according to the ALS functional rating score or rate of disease progression. Our results showed a

significant impairment of erythrocytes glutathione peroxidase activity in all groups of patients that

remained low during disease time course. SOD1 activity significantly decreased along disease course in

subjects with a more impaired functional status. A decreasing activity of all assayed enzymes was found

in patients who have a faster disease progression rate. By this work we have the evidence that different

ALS phenotypes present with different profile of enzymatic and non-enzymatic antioxidant response.

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Oxidative stress has been linked to neuronal cell death thatoccurs in various neurodegenerative diseases. Post-mortem braintissues from patients with neurodegenerative disorders, such asParkinson’s disease, Alzheimer’s disease and Amyotrophic LateralSclerosis (ALS) showed increased oxidative stress in specific brainregions (Andersen, 2004). ALS is a progressive neurodegenerativedisease characterized by selective loss of both cortical and spinalmotor neurons. Approximately 10% of ALS cases are inherited(familial ALS, FALS) and 20% of these are linked to mutations in thegene encoding for Cu–Zn superoxide dismutase (SOD1) (E.C. No.1.15.1.1), a protein converting superoxide anion into hydrogenperoxide (Rosen et al., 1993). Although in the remaining 90% ofpatients the aetiology is yet unknown, the overlapping clinicalpicture and disease course of familial and sporadic forms suggestthe involvement of common pathogenesis. Since increased levelsof oxidative stress have been found not only in nervous but also inperipheral tissues of familial and sporadic ALS patients (SALS)(Halliwell, 2001), many studies have been addressed to relate thepresence of increased oxidative stress to reduced antioxidant

* Corresponding author. Tel.: +39 0382380248; fax: +39 0382380356.

E-mail address: emanuela.cova@mondino.it (E. Cova).

0197-0186/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuint.2010.02.004

defenses also in peripheral tissues of these patients (Simpson et al.,2004; Garofalo et al., 1995; Przedborski et al., 1996b; Cohen et al.,1996; Moumen et al., 1997; Apostolski et al., 1998; Ilzecka, 2003;Oteiza et al., 1997; Tuncel et al., 2006; Nikolic-Kokic et al., 2006;Babu et al., 2008; Bowling et al., 1993). However, the majority ofthese data showed conflicting results, due to patients’ heteroge-neity in terms of disease time onset and course. Recently, Babuet al. (2008) found that the activity of some antioxidant enzymes,linked to the metabolism of glutathione, was lower in ALS patientswith longer disease duration. In this work we assayed the activityof some antioxidant enzymes implicated in the defense againstincreased oxidative stress. In fact although it is not yet establishedif oxidative stress is a cause or a consequence of the neurodegen-erative process (Andersen, 2004), the different capacity of eachsubject to respond to increased oxidative stress may be account tothe heterogeneity of the SALS patients in terms of clinical course,disease duration and response to pharmacological treatment. Tothis purpose, SOD1, glutathione peroxidase (GPX) (E.C. No.1.11.1.9), glutathione reductase (GR) (E.C. No. 1.8.1.10) activityhas been studied in erythrocytes of 88 SALS patients during diseasetime course and dividing the patients in subgroups accordingto impairment status and disease progression rate. SOD1,which catalyzes the dismutation of the highly reactive superoxide

Table 1Characteristics of ALS patients at enrolment.

Total Males Females

Numbers 88 49 39

Age of onset (years) Range: 27–79 Range: 31–79 Range: 27–79

Mean� SE: 60.7�1.3 Mean� SE: 58.2�1.8 Mean� SE: 64�1.8

Illness duration (months) Range: 4–58 Range: 4–57 Range: 4–58

Mean� SE: 23.2�2.1 Mean� SE: 24.6�2.2 Mean� SE: 21.8�2

ALSFRS-R score Mild = 44 Mild = 30 Mild = 14

Mild (30–40) Medium = 43 Medium = 19 Medium = 24

Medium (11–29) Severe = 1 Severe = 0 Severe = 1

Severe (0–10)

SE, standard error.

E. Cova et al. / Neurochemistry International 56 (2010) 687–693688

anion O2� to less reactive species H2O2 (hydrogen peroxide) has

been studied for its implication with a portion of FALS cases (Rosenet al., 1993; Ezzi et al., 2007). GPX detoxifies H2O2 utilizingglutathione (GSH) and it is the major source of protection againstoxidative stress in human brain (Mates et al., 1999). GR convertsback oxidized glutathione to GSH restoring the ratio of reduced tooxidized GSH. Moreover, non-enzymatic antioxidant defenses(total antioxidant status, TAS) have been studied in the plasma ofthe same patients. As indicator of altered oxidative stress, westudied the plasmatic level of reactive oxygen species (ROS).Hence, the aim of this work is to evaluate if the enzymes activity,TAS and ROS levels are correlated with the impairment of thefunctional status and disease progression rate. To our knowledge,this is the first study performed overtime and on a such relevantnumber of SALS patients to allow a splitting in subgroups withdifferent disease phenotype.

1. Experimental procedures

Patient and control subjects. Eighty-eight patients without history of familial

disease were recruited at the Neurorehabilitation Unit (Azienda Ospedaliero-

Universitaria Pisana, Pisa, Italy) and enrolled into this study. Patients were

diagnosed with ALS according to the revised El Escorial Criteria (Brooks et al., 2000).

Their characteristics are reported in Table 1.

Fifty healthy subjects, free from any pharmacological treatment, age-matched

with SALS patients (mean: 60.3 � 5.4), recruited at the Neurorehabilitation Unit

(Azienda Ospedaliero-Universitaria Pisana, Pisa, Italy) and at the Immunohematologi-

cal and Transfusional Service and Centre of Transplantation Immunology (IRCCS

Foundation ‘‘San Matteo’’, Pavia, Italy) were used as controls. All patients and controls

were assayed to rule out the presence of inflammatory diseases by white blood cell

counts and red blood cell sedimentation rate, at each blood withdrawal. The study was

approved by Ethics committee and patients and controls signed informed consent prior

to the study.

At the first physical examination, the patient functional status, by following the

revised ALS Functional Rating Scale (ALSFRS-R), score range 0–40 (Cedarbaum and

Stambler, 1997), and disease duration, as reported by patient, were assessed.

Concomitantly, the first blood sample was taken. The enrolled patients were

followed during disease progression. A blood sample was taken every three months

during the period of the study until the patients dropped out or passed away.

Patients were divided into three groups based upon disease severity, as

documented by their ALSFRS-R score (0–40): severe (0–10), medium (11–29) and

mild (30–40). According to the mentioned score we found only 1 patient (female)

with severe disease, 43 (24 females and 19 males) with medium score and 44

patients (14 females and 30 males) showed lower severity grade of the disease

(Table 1).

Table 2Characteristics of ALS patients grouped upon rate of disease progression (ALSFRS score

ALSFRS-R score change/month Total (male/female) Ag

Ra

0–0.1 25 (18/7) 36

0.1–0.2 20 (6/14) 27

>0.2 16 (11/5) 41

SE, standard error.

Patients having similar rate of disease progression, evaluated as ALSFRS-R score

change per month and more than six determinations collected overtime were

grouped. Time course of GPX, GR and SOD1 activity, ROS and TAS level, were

evaluated. Twenty-five patients showed ALSFRS-R score change/month between 0

and 0.1, 20 between 0.1 and 0.2 and 16>0.2. Characteristics of ALS patients grouped

upon rate of disease progression are reported in Table 2. All SALS patients were

treated with riluzole.

Erythrocyte preparation. Whole blood sample was collected and plasma removed

by centrifugation at 3000 � g for 5 min. The erythrocytes were washed three times

by resuspending in 0.9% NaCl solution and centrifuging for 5 min at 3000 � g after

each wash. The cells were lysed in cold redistilled water for 10 min at 4 8C. The

stroma was removed by centrifugation at 300 � g for 5 min and the lysate diluted

with 0.01 mol/l phosphate buffer pH 7.

Determination of erythrocyte GPX, GR and SOD1 activity. To quantify GPX activity

the method described by Paglia and Valentine (1967) was used. GPX catalyzes the

oxidation of glutathione by cumene hydroperoxide. In the presence of GR and

nicotinamide adenine dinucleotide phosphate (NADPH) the oxidized glutathione

(GSSG) is immediately converted to the reduced form with a concomitant oxidation

of NAPDH to NADP+. The decrease in absorbance at 340 nm was measured. The

assay of GR activity is based upon the property of GR to catalyse the reduction of

GSSG in the presence of NADPH, which is oxidized to NADP+. The method for the

determination of SOD1 activity is based upon generation by xanthine and xanthine

oxidase of superoxide radicals which react with 2-(4-iodophenyl)-3-(4-nitrophe-

nol)-5-phenyltetrazolium chloride (INT) to form a red formazan dye. The SOD1

activity is then measured by the degree of inhibition of this reaction. One unit of

SOD1 causes a 50% inhibition of the rate of reduction of INT under the conditions of

the assay. The GPX, GR and SOD1 activities were expressed as units/g of

haemoglobin

Determination of plasmatic ROS and TAS. Plasmatic ROS levels have been assayed

by quantifying hydroperoxides (ROOH) generated by the organic substrates

oxidation (e.g. carbohydrates, lipids, amino acids, proteins, nucleotides, etc.)

produced by ROS. This assay is based on the issue that the amount of

hydroperoxides present in serum is related to the free radicals from which they

are formed. Serum hydroperoxides react with transition metal ions of the proteins

after addition of an acidic buffer and form alkoxyl and peroxyl radicals. A

chromogen (N,N,-diethylparaphenylen-diamine) changes colour after it is oxidized

by peroxyl and alkoxyl radicals. Colour intensity, directly related to the

hydroperoxides levels, was expressed as mg H2O2/dL.

The total antioxidant status (TAS), representing non-enzymatic antioxidants, has

been assayed utilizing 2,20-azinobis(3-ethylbenzothiazoline 6-sulfonate) (ABTS) as

initiator of free radical generation (Re et al., 1999). The incubation of ABTS with a

peroxidase (metmyoglobin) and hydrogen peroxide produced a blue-green colour

which was suppressed proportionally to the plasma antioxidant concentration. The

rate of change of optical density was measured at 600 nm and TAS expressed as

plasma nmol/L.

Statistical analysis. Erythrocyte enzyme activities and plasma ROS, TAS level of

ALS patients were compared to controls, according to severity of disease.

Differences between the mean of erythrocyte GPX, GR and SOD1, activity and

plasma ROS and TAS concentrations in controls and patients were evaluated by the

change/month).

e of onset (years) Illness duration (months)

nge Mean� SE Range Mean� SE

–77 58.4�1.9 4–55 21.6�2.5

–73 61.2�3.3 4–55 20.6�1.8

–79 62.3�2.7 6–52 22.5�3.1

Fig. 1. Determination of erythrocyte GPX (a), GR (b) and SOD1 (c) activity. Each

scatter point represents the value of enzymes activity for each patient at the first

withdrawal. The patients have been divided according the disease severity in mild

(ALSFRS-R: 30–40) and medium (ALSFRS-R: 11–29). The mean of enzymes activity

for each group is represented by the straight line. *P < 0.05 vs. control group.

Fig. 2. Determination of plasmatic ROS (a) and TAS (b) level. Each scatter point

represents the value of ROS or TAS for each patient at the first withdrawal. The

patients have been divided according the disease severity in mild (ALSFRS-R: 30–

40) and medium (ALSFRS-R: 11–29). The mean of ROS and TAS for each group is

represented by the straight line. *P < 0.05 vs. control group.

E. Cova et al. / Neurochemistry International 56 (2010) 687–693 689

analysis of variance (ANOVA) followed by Newman–Keuls’ test. Time course of

these markers was also evaluated in the two groups of patients. For each time point

there were at least 5 determinations. Standard regression analysis was used to

determine the relationship between ROS, TAS concentrations, antioxidant enzymes

activity and disease time course or progression rate. All statistical analyses were

carried out with Graph Prism 5.0 statistical program. A P value <0.05 was

considered statistically significant

2. Results

GPX, GR, SOD1 activity in erythrocytes and plasmatic ROS and TAS

levels. In Fig. 1 each scatter point represents the determinations of

the erythrocyte GPX (a), GR (b) and SOD1 (c) activity assayed at thefirst blood withdrawal. Patients have been grouped according toALSFRS-R score evaluated at the concomitantly physical examina-tion. Means of GPX, GR and SOD1 activities showed that onlyerythrocyte GPX activity was reduced in both groups of patientscompared to control subjects. Surprisingly, plasma ROS meanvalues of patients resulted significantly lower than in controls(Fig. 2a) while TAS level was unchanged compared to controls(Fig. 2b).

GPX, GR, SOD, activity in erythrocytes and plasmatic ROS and TAS

levels during disease progression. The GPX and GR activities did notchange overtime (Fig. 3a and b) and GPX remained significantlylower compared to controls. Even if SOD1 activity was unchangedat the first examination compared to controls, it significantlydecreased during disease progression only in patients with anALSFRS-R score between 11 and 29 (Fig. 3c). ROS concentrationssignificantly increased along disease progression, as evaluated bylinear regression analysis in all groups (Fig. 4a). However, SALSmean values did not exceed control mean value at each consideredtime point. Linear regression analysis did not show any significantovertime variation of TAS level (Fig. 4b).

GPX, GR and SOD1 activities, ROS and TAS levels and disease

progression rate. This analysis highlighted significant differences intime course of different markers according to the rate of diseaseworsening. In particular, GPX decreased in patients with 0.1–0.2

Fig. 3. Linear regression analysis of GPX (a), GR (b) and SOD1 (c) activity vs. time course in patients divided according the disease severity at the diagnosis. Each time point of

the curves represents the mean � SE of all patient determinations (N � 5) collected at the same time lapse after disease onset. SE is always within 10%. A significant correlation

between enzymes activity trend and time is indicated by an asterisk.

E. Cova et al. / Neurochemistry International 56 (2010) 687–693690

and >0.2 disease progression rate (Fig. 5a). Surprisingly, GRactivity that was unvaried in previous analysis, showed aprogressive significant lowering in subjects with faster diseaseworsening rate (Fig. 5b). SOD1 activity augmented overtimealthough not significantly in the group with ALSFRS-R score/month>0.2 (Fig. 5c). ROS level significantly increased overtime (Fig. 6a)and concurrently TAS decreased in patients having diseaseprogression rate 0.1–0.2 and >0.2 (Fig. 6b)

3. Discussion

Previous reports on antioxidant enzyme activities and levels ofoxidative stress in plasma and cerebrospinal fluid of SALS patientshave been demonstrated conflicting and often inconclusive. SinceALS is a heterogeneous disease in terms of clinical, biochemical andgenetic features, it is fair to hypothesize that also the markerslinked to redox homeostasis might differ according to differentclinical state or disease progression rate. Among the several clinicalfeatures, we chose severity at the disease assessment and rate ofdisease progression to find out if they relate to oxidant–

antioxidant imbalance. The large number of recruited patientsand the possibility to follow up almost all of them during diseaseduration allowed us to study these parameters overtime and relatethe progression of the disease to the trend of the marker slopes.This overtime study of GPX, GR and SOD1 activity and ROS, TASlevels represents a novelty in this kind of analysis and allows tohighlight differences in these parameters linked to clinicalphenotype that may be used either as diagnostic or predictivetool for disease progression. In fact there are not previous studieson serial measurements of the considered parameters in the samepatient. It is worthy to note that all patients were under riluzoletreatment, since it would be not ethically correct to stop thepharmacological cure because of the long time of the study.However, since riluzole acts by inhibiting glutamate release frompresynaptic terminals and considering that erythrocytes do notexpress any glutamate receptor, we believe that enzymes’ activityis not directly modified by riluzole intake.

As previously observed by others (Garofalo et al., 1995;Przedborski et al., 1996b) and us (Curti et al., 1996), the meanvalue of SOD1 activity at the diagnosis, is unchanged in all patients

Fig. 4. Linear regression analysis of ROS (a) and TAS (b) determinations vs. time course in patients divided according the disease severity at the diagnosis. Each time point of

the curve represents the mean � SE of all patient determinations (N � 5) collected at the same time lapse after disease onset. SE is always within 10%. A significant correlation

between ROS/TAS level trend and time is indicated by an asterisk.

E. Cova et al. / Neurochemistry International 56 (2010) 687–693 691

subgroups compared to controls. However, in the same tissueother authors found that SOD1 activity was higher (Bonnefont-Rousselot et al., 2000; Tuncel et al., 2006). In addition, theseauthors (Bonnefont-Rousselot et al., 2000) demonstrated thatincreased SOD1 activity was reduced by riluzole treatment, even ifit remained significantly higher compared to control subjects. Inour patients, overtime SOD1 activity showed a significantly timecourse decrease in patients with a ALSFRS-R score between 11 and29. We do not know which is the cause of this reduction but we caninfer that different clinical phenotypes lead to a different overtimeSOD1 activity. This reducing value might indicate that the responseto the oxidative stress is different depending on the degree ofimpairment at the disease assessment. This difference with theother group of patients might also be the reason for the divergingresults reported by different authors since in those works (Garofaloet al., 1995; Przedborski et al., 1996a,b; Bonnefont-Rousselot et al.,2000; Tuncel et al., 2006) the patients were clinically heteroge-neous being not divided in subgroups.

In our patients, GPX activity is significantly reduced andremains significantly lower than in controls overtime indepen-dently from disease severity at diagnosis. GPX activity furtherdecreased in patients with 0.1–0.2 and >0.2 disease progressionrate suggesting that its overtime reduction could contribute tofaster disease progression. Lower GPX activity has been alreadyobserved by other authors in both nervous and peripheral tissuesof SALS patients not treated by riluzole, suggesting that thepharmacological treatment does not considerably modify GPXdecreased activity (Przedborski et al., 1996a,b; Moumen et al.,1997; Apostolski et al., 1998). A reduced GPX activity might becaused by a lessening availability of its substrate, GSH. Althoughwe did not assay GSH in our patients, Babu et al. (2008) found asignificant lower level of GSH in erythrocytes of SALS patients evenif GPX in their study was unchanged.

GR showed significantly lower activity in a recent paper(Siciliano et al., 2007) and higher in two previous works(Apostolski et al., 1998; Nikolic-Kokic et al., 2006). In our SALSpatients, the GR activity is not different at the diagnosis fromcontrols and it does not change during disease progression.However, when overtime enzyme activity was evaluated byconsidering the disease progression rate, the group having fasterworsening rate showed a significant reduced activity. Althoughrecently GR activity was demonstrated reduced according todisease duration (Babu et al., 2008), we did not obtain the sameresults with our patients, by applying the same statistical analysis(Supplementary Fig. 1), even if a role of riluzole treatment on GRactivity in our patients needs to be further explored. Anotherpossible explanation of this discrepancy is that they did notdivided the patients according to progression rate or impairedfunctional state. In fact, when we considered the rate of diseaseprogression we found a decreasing GR activity in the group ofpatients with faster disease worsening (ALSFRS-R score change/month >0.2). Considering all patients together, especially whenthe number is low, could mask possible differences, lead todiverging or not reproducible results.

In this work we also evaluate the redox homeostasis in plasmafrom ALS patients by assaying the ‘‘two faces of the coin’’: ROS andTAS level. Previous data showing a plasmatic ROS increase werecollected only in the familial form of ALS and in differentexperimental models, i.e. transgenic mutated SOD1 mice (Yimet al., 1996) or intracellular lymphoblasts from familial ALSpatients (Said Ahmed et al., 2000). Moreover, our findings havebeen obtained in a large amount of SALS patients and they cannotbe rejected based on bias. A possible explanation may be found inthe recent observations that the wild type SOD1 might beimplicated in ALS onset by acquiring some of the toxic function,as the ability to be secreted, characteristic of the mutated SOD1

Fig. 5. Linear regression analysis of GPX (a), GR (b) and SOD1 (c) activity vs. time

course in patients grouped according similar disease progression rate. Each time

point of the curve represents the mean � SE of all patient determinations (N � 5)

collected at the same time lapse after disease onset. SE for each time point is always

within 10%. Three patient sets have been defined having ALSFRS-R score/month

between 0 and 0.1 (open circle), 0.1 and 0.2 (open square) and >0.2 (open triangle). A

significant correlation between enzymes activity trend and time is indicated by an

asterisk (P < 0.05).

Fig. 6. Linear regression analysis of ROS (a) and TAS (b) determinations vs. time

course in patients grouped according similar disease progression rate. Each time

point of the curve represents the mean � SE of all patient determinations (N � 5)

collected at the same time lapse after disease onset. SE for each time point is always

within 10%. Three patient sets have been defined having ALSFRS-R score/month

between 0 and 0.1 (open circle), 0.1 and 0.2 (open square) and >0.2 (open triangle). A

significant correlation between ROS and TAS determinations and time is indicated by

an asterisk (P < 0.05).

E. Cova et al. / Neurochemistry International 56 (2010) 687–693692

(Ezzi et al., 2007; Urushitani et al., 2006). In addition, we previouslyfound that lymphocytes from SALS patients express lowerintracellular amount of SOD1 protein (Cova et al., 2006; Ceredaet al., 2006) even if the activity is unchanged (Curti et al., 1996).Although we cannot prove this hypothesis at the moment(experiments are in progress), we can not exclude that an higherSOD1 secretion would be responsible of the decreased amount ofROS in plasma and of maintenance of low level during diseaseprogression. Alternatively it is also conceivable that the treatmentwith antioxidants would be responsible of the lower level of ROSobserved in SALS patients. TAS level was similar in each group ofpatients, as previously observed by others (Siciliano et al., 2007).

In conclusion, the use of erythrocyte GPX activity as potentialbiological marker for ALS diagnosis should be further investigatedsince its low level has been proved in all patient subgroups.However, it is also evident that differences have been discovered

by splitting the patients in subgroups, since subjects with higherand, for some aspects, also those with medium disease progressionrate show a progressive impairment of antioxidant defenses. Theseresults suggest a different responsive skill of patients that shouldbe investigated to find out possible protective mechanisms.

Since ALS is a heterogeneous disease, we think by this work andas suggested also by others (Beghi et al., 2007), that in the futurethe search of differences in peripheral markers must be donesplitting the patients into different groups rather than lumpingthem. We suggest a new approach to the study of antioxidantdefenses since the division in subgroups shows different behaviorsaccording to the large variety of the clinical course and impairmentstatus.

Acknowledgments

This work was supported by the Italian Ministry of Health ex.Art. 56 ‘‘Neurodegenerative diseases’’ a.f. 2004 and Ricercacorrente 2007. We thank the Biobank of Neurological Institute‘‘C. Mondino’’ for providing us with blood samples of ALS patients.A special thank to ‘‘our’’ ALS patients that trust our work.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.neuint.2010.02.004.

E. Cova et al. / Neurochemistry International 56 (2010) 687–693 693

References

Andersen, J.K., 2004. Oxidative stress in neurodegeneration: cause or consequence?Nat. Rev. Neurosci. 10 (Suppl.), S18–S25.

Apostolski, S., Marinkovic, Z., Nikolic, A., Blagojevic, D., Spasic, M.B., Michelson,A.M., 1998. Glutathione peroxidase in amyotrophic lateral sclerosis: theeffects of selenium supplementation. J. Environ. Pathol. Toxicol. Oncol. 17,325–329.

Babu, G.N., Kumar, A., Chandra, R., Puri, S.K., Singh, R.L., Kalita, J., et al., 2008.Oxidant–antioxidant imbalance in the erythrocytes of sporadic amyotrophiclateral sclerosis patients correlates with the progression of disease. Neurochem.Int. 52, 1284–1289.

Beghi, E., Mennini, T., Bendotti, C., Bigini, P., Logroscino, G., Chio, A., et al., 2007. Theheterogeneity of amyotrophic lateral sclerosis: a possible explanation of treat-ment failure. Curr. Med. Chem. 14, 3185–3200.

Bonnefont-Rousselot, D., Lacomblez, L., Jaudon, M., Lepage, S., Salachas, F., Bensi-mon, G., et al., 2000. Blood oxidative stress in amyotrophic lateral sclerosis. J.Neurol. Sci. 178, 57–62.

Bowling, A.C., Schulz, J.B., Brown Jr., R.H., Beal, M.F., 1993. Superoxide dismutaseactivity, oxidative damage, and mitochondrial energy metabolism in familialand sporadic amyotrophic lateral sclerosis. J. Neurochem. 61, 2322–2325.

Brooks, B.R., Miller, R.G., Swash, M., Munsat, T.L., World Federation of NeurologyResearch Group on Motor Neuron Diseases, 2000. El Escorial revisited: revisedcriteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph LateralScler Other Motor Neuron Disord 1, 293–299.

Cedarbaum, J.M., Stambler, N., 1997. Performance of the Amyotrophic LateralSclerosis Functional Rating Scale (ALSFRS) in multicenter clinical trials. J.Neurol. Sci. 152 (Suppl. 1), S1–S9.

Cereda, C., Cova, E., Di Poto, C., Galli, A., Mazzini, G., Corato, M., et al., 2006. Effect ofnitric oxide on lymphocytes from sporadic amyotrophic lateral sclerosispatients: toxic or protective role? Neurol. Sci. 27, 312–316.

Cohen, O., Kohen, R., Lavon, E., Abramsky, O., Steiner, I., 1996. Serum Cu/Znsuperoxide dismutase activity is reduced in sporadic amyotrophic lateralsclerosis patients. J. Neurol. Sci. 143, 118–120.

Cova, E., Cereda, C., Galli, A., Curti, D., Finotti, C., Di Poto, C., et al., 2006. Modifiedexpression of Bcl-2 and SOD1 proteins in lymphocytes from sporadic ALSpatients. Neurosci. Lett. 399, 186–190.

Curti, D., Malaspina, A., Facchetti, G., Camana, C., Mazzini, L., Tosca, P., et al., 1996.Amyotrophic lateral sclerosis: oxidative energy metabolism and calcium ho-meostasis in peripheral blood lymphocytes. Neurology 47, 1060–1064.

Ezzi, S.A., Urushitani, M., Julien, J.P., 2007. Wild-type superoxide dismutase acquiresbinding and toxic properties of ALS-linked mutant forms through oxidation. J.Neurochem. 102, 170–178.

Garofalo, O., Figlewicz, D.A., Thomas, S.M., Butler, R., Lebuis, L., Rouleau, G., et al.,1995. Superoxide dismutase activity in lymphoblastoid cells from motor neu-rone disease/amyotrophic lateral sclerosis (MND/ALS) patients. J. Neurol. Sci.129 (Suppl.), 90–92.

Halliwell, B., 2001. Role of free radicals in the neurodegenerative diseases: thera-peutic implications for antioxidant treatment. Drugs Aging 18, 685–716.

Ilzecka, J., 2003. Total antioxidant status is increased in the serum of amyotrophiclateral sclerosis patients. Scand. J. Clin. Lab. Invest. 63, 297–302.

Mates, J.M., Perez-Gomez, C., Nunez de Castro, I., 1999. Antioxidant enzymes andhuman diseases. Clin. Biochem. 32, 595–603.

Moumen, R., Nouvelot, A., Duval, D., Lechevalier, B., Viader, F., 1997. Plasmasuperoxide dismutase and glutathione peroxidase activity in sporadic amyo-trophic lateral sclerosis. J. Neurol. Sci. 151, 35–39.

Nikolic-Kokic, A., Stevic, Z., Blagojevic, D., Davidovic, B., Jones, D.R., Spasic, M.B.,2006. Alterations in anti-oxidative defence enzymes in erythrocytes fromsporadic amyotrophic lateral sclerosis (SALS) and familial ALS patients. Clin.Chem. Lab. Med.: CCLM/FESCC 44, 589–593.

Oteiza, P.I., Uchitel, O.D., Carrasquedo, F., Dubrovski, A.L., Roma, J.C., Fraga, C.G., 1997.Evaluation of antioxidants, protein, and lipid oxidation products in blood fromsporadic amyotrophic lateral sclerosis patients. Neurochem. Res. 22, 535–539.

Paglia, D.E., Valentine, W.N., 1967. Studies on the quantitative and qualitativecharacterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70,158–169.

Przedborski, S., Donaldson, D., Jakowec, M., Kish, S.J., Guttman, M., Rosoklija, G.,et al., 1996a. Brain superoxide dismutase, catalase, and glutathione peroxidaseactivities in amyotrophic lateral sclerosis. Ann. Neurol. 39, 158–165.

Przedborski, S., Donaldson, D.M., Murphy, P.L., Hirsch, O., Lange, D., Naini, A.B., et al.,1996b. Blood superoxide dismutase, catalase and glutathione peroxidase activ-ities in familial and sporadic amyotrophic lateral sclerosis. Neurodegeneration5, 57–64.

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., Rice-Evans, C., 1999.Antioxidant activity applying an improved ABTS radical cation decolorizationassay. Free Radic. Biol. Med. 26, 1231–1237.

Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., et al.,1993. Mutations in Cu/Zn superoxide dismutase gene are associated withfamilial amyotrophic lateral sclerosis. Nature 362, 59–62.

Said Ahmed, M., Hung, W.Y., Zu, J.S., Hockberger, P., Siddique, T., 2000. Increasedreactive oxygen species in familial amyotrophic lateral sclerosis with mutationsin SOD1. J. Neurol. Sci. 176, 88–94.

Siciliano, G., Piazza, S., Carlesi, C., Del Corona, A., Franzini, M., Pompella, A., et al.,2007. Antioxidant capacity and protein oxidation in cerebrospinal fluid ofamyotrophic lateral sclerosis. J. Neurol. 254, 575–580.

Simpson, E.P., Henry, Y.K., Henkel, J.S., Smith, R.G., Appel, S.H., 2004. Increased lipidperoxidation in sera of ALS patients: a potential biomarker of disease burden.Neurology 62, 1758–1765.

Tuncel, D., Aydin, N., Aribal Kocaturk, P., Ozelci Kavas, G., Sarikaya, S., 2006. Red cellsuperoxide dismutase activity in sporadic amyotrophic lateral sclerosis. J. Clin.Neurosci. 13, 991–994.

Urushitani, M., Sik, A., Sakurai, T., Nukina, N., Takahashi, R., Julien, J.P., 2006.Chromogranin-mediated secretion of mutant superoxide dismutase proteinslinked to amyotrophic lateral sclerosis. Nat. Neurosci. 9, 108–118.

Yim, M.B., Kang, J.H., Yim, H.S., Kwak, H.S., Chock, P.B., Stadtman, E.R., 1996. A gain-of-function of an amyotrophic lateral sclerosis-associated Cu, Zn-superoxidedismutase mutant: An enhancement of free radical formation due to a decreasein Km for hydrogen peroxide. Proc. Natl. Acad. Sci. U.S.A. 93, 5709–5714.