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Brain superoxide dismutase catalase and glutathione peroxidase activities in amyotrophic lateral sclerosis.

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ORIGINAL ARTICLES
Brain Superoxide Dismutase, Cadase, and
Glutahone Peroxidase Activities in
Amyotrophc Lateral Sclerosis
Serge Przedborski, MD, PhD,* David Donaldson, BS,* Michael Jakowec, PhD,* Stephen J. Kish, PhD,$
M. Guttman, MD,$ Gorazd Rosoklija, M D , PhD,? and Arthur P. Hays, MDT
Amyotrophic lateral sclerosis is a fatal paralytic disorder of unknown cause. Recent evidence implicated the role of free
radicals in the death of motor neurons in this disease. To investigate this hypothesis further, we measured the activity
of the main free radical scavenging enzymes copperlzinc superoxide dismutase, manganese superoxide dismutase, catalase,
and glutathione peroxidase in postmortem brain samples from 9 patients with sporadic amyotrophic lateral sclerosis
and from 9 control subjects. We examined samples from the precentral gyrus of the cerebral cortex, a region affected
in amyotrophic lateral sclerosis, and from the cerebellar cortex, a region not affected. The two groups did not differ in
age or postmortem delay. In the precentral gyrus from amyotrophic lateral sclerosis samples, glutathione peroxidase
activity as measured by spectrophotometric assay (13.8 & 2.6 nmol/min/mg protein [mean & standard error of mean])
was reduced significantly compared to the activity in the precentral gyrus from control samples (22.7 & 0.5 nmol/min/
mg protein). In contrast, glutathione peroxidase activity was not significantly altered in the cerebellar cortex from
amyotrophic lateral sclerosis patients compared to controls. Copper/zinc superoxide dismutase, manganese superoxide
dismutase (corrected or not corrected for citrate synthase), and catalase were not significantly altered in the precentral
gyrus or cerebellar cortex in the patient samples. This study indicated that glutathione peroxidase activity is reduced
in a brain region affected in amyotrophic lateral sclerosis, thus suggesting that free radicals may be implicated in the
pathogenesis of the disease.
Przedhorski S, Donaldson D, Jakowec M, Kish SJ, Guttman M, Rosoklija G, Hays AP.
Brain superoxide disrnutase, catalase, and glutathione peroxidase activities in
amyotrophic lateral sclerosis. Ann Neurol 1996;39:158- 165
Amyotrophic lateral sclerosis (ALS) is a progressive paralytic neurodegenerative disorder of unknown etiology
characterized mainly by the loss of upper and lower
motor neurons [l]. Mutations of the copper/zinc superoxide dismutase (Cu/Zn-SOD, EC 1.15.1.1) gene
are found in approximately 20% of patients affected
with familial ALS [2, 31. Most studies of these patients
reported a reduction in erythrocyte superoxide dismurase (SOD) activity [4].Since SOD is a key enzyme in
the protective mechanisms against free radical-induced
toxicity [ 5 ] , it is suggested that oxidative stress may
play a physiopathogenic role in familial ALS [4, 61.
Sporadic ALS accounts for more than 90% of all ALS
[ I ] . The clinical syndromes of familial and sporadic
ALS are similar [l], and it is believed that they share
a common mechanism of neuronal death [4, 61. Consistent with this view, clues support that oxidative stress
may be implicated in the physiopathogenesis of both
familial and sporadic ALS [4, 61. Although the oxidative stress hypothesis is supported by identified alterations in SOD in familial ALS, the abnormality responsible for free radical-induced damage in sporadic
ALS is unknown.
Superoxide radicals, hydrogen peroxide (H2O2),and
hydroxyl radicals are oxygen-centered reactive species
[7] that have been implicated in several neurotoxic disorders [S-lo]. They are produced by many normal
biochemical reactions, but their concentrations are kept
in a harmless range by potent protective mechanisms
[I I]. Increased free radical concentrations, resulting
from either increased production or decreased detoxification, can cause oxidative damage to various cellular
components, ultimately leading to cell death (111. In
the present study, we hypothesized that the mechanism
underlying oxidative stress in sporadic ALS is an alteration in one (or more) of the main free radical scaveng-
From the Department of "Neurology and tDivision of Neuropathology, Department of Pathology, Columbia University, New
York, NY, and the $Human Brain Laboratory, Clark Institute of
Psychiatry, Toronto, Ontario, Canada.
Received Jun 19, 1995, and in revised form Aug 11 and Sep 25.
Accepted for publication Oct 13, 1995.
Address correspondence to D r Przedborski, RR-307, Department
of Neurology, College of Physicians and Surgeons, Columbia University, 650 West 168th Street, New York, NY 10032.
158 Copyright 0 1996 by the American Neurological Asmciation
as deprenyl, antispastics, trophic factors, or levodopa for at
least 6 weeks prior to death. For each brain region, control
and ALS samples were randomly matched and assayed simultaneously.
Table I . Clinical Dataa
No. of patients
Age (yr) at the
time of death
Sex
Postmortem delay (hr)
Length of conservationb
Control
ALS
9
56.7 ? 4.5
9
5 women, 4 men
3 women, 6 men
13.9 Z 1.2
11.9 ? 1.8
3.4 2 3.2 yr
61.1 ? 3.0
Chemicals and Equipment
2.5 ? 1.8 yr
“Values represent mean f standard deviation. Differences between
amyotrophic lateral sclerosis (ALS) and control brains were tested
by Student’s t test; none of the tests showed any significant difference
( p > 0.05).
Except for the bathocuprioinedisulfonic acid, which was
from ICN (Costa Mesa, CA), all other compounds were purchased at highest-grade purity from Sigma (St. Louis, MO).
All spectrophotometric assays were performed on Shimatzu
U V 160 and Beckman D U 7400 instruments. Multiwell
plate readings for the enzyme-linked immunosorbent assay
(ELISA) were performed on a Bio-Rad 3550 computerized
microplate reader (Microplate Manager for Windows, version 2.01, Bio-Rad, Richmond, CA).
”At -80°C.
Glutathione Peroxidase Activiq
ing enzymes. To test this hypothesis, we measured in
sporadic ALS and control postmortem brains the activities of Cu/Zn-SOD and manganese SOD (Mn-SOD),
the two intracellular SOD isoenzymes present in eukaryotes [ 5 ] , as well as the activities of catalase (EC
1.1 1.1.6) and glutathione peroxidase (EC 1.11.1.9).
Materials and Methods
Brain Collection and Dissection
Nine brains from patients with ALS and 9 from control patients with nonneurological diseases were obtained from the
Brain Bank, Department of Pathology at Columbia University (n = 7 per group) and from the Human Brain Laboratory, Clarke Institute of Psychiatry (n = 2 per group). In
the ALS group, the cause of death was pneumonia (n = 6),
respiratory failure (n = 2), or starvation (n = 1); in the
control group, it was pulmonary embolism (n = 3), pneumonia (n = 2), acute pancreatitis (n = I), heart failure (n
= I ) , acute myocardial infarction (n = l ) , or septicemia (n
= 1). At autopsy, brains were removed and divided sagittally.
One half of the brain was immediately frozen on dry ice and
stored at -80°C until dissection. O n the day of the dissection, small frozen blocks of cerebellar cortex and of precentral gyrus were cut from each frozen half-brain. The other
half-brain was placed in 10% buffered formalin and was subjected to neuropathological examination. There was no significant difference between the time from death to autopsy
or between the time of autopsy to assay for the ALS and
control groups (Table 1). The clinical diagnosis of ALS was
confirmed pathologically in all 9 patients. In these brains,
neuronal loss was observed in the precentral gyrus, while no
remarkable pathological changes were noted in the cerebellar
cortex. In all control brain, no remarkable pathological
changes were noted either in frontal cerebral cortex or in
cerebellum. For the ALS patients, the mean age (? standard
deviation [SD]) of onset was 57.3 ? 2.9 years, with the
mean duration of disease being 3.4 -+ 0.8 years and ranging
from 2 to 5 years. None of the ALS patients had a family
history for the illness. None of the ALS patients or control
subjects were treated by chronic administration of drugs such
Glutathione peroxidase activity was determined according to
the method described by Sinet and coauthors [12] based on
NADPH oxidation followed at 340 nm at 37°C as described
previously [13], with some modifications [14]. O n the day
of the assay, for each patient and for each region, approximately 250 mg of frozen brain tissue was homogenized by
hand with a glass tissue grinder in 2.5 vol (wt/vol) of 10
mM Tris-hydrochloric acid (HCI) (pH 7.4) buffer containing 0.25 M sucrose. Homogenates were centrifuged at
15,000 g for 10 minutes at 4°C. Supernatants were mixed
with an equal volume of a 4 mM potassium ferricyanide/20
mM potassium cyanide solution to prevent hemoglobin (Hb)
interference with the assay [ 151. Then 100 pl of this solution
was added to the reaction mixture (total volume of 3 ml)
containing 1 m M reduced glutathione, 2 units of glutathione reductase, and 0.2 mM NADPH. After 10 minutes of
preincubation, the absorbance was recorded for 4 minutes
to determine the hydroperoxide-independent oxidation of
NADPH. Thereafter, 1 mM t-butyl hydroperoxide was
added to the mixture, and the rate of NADPH oxidation
was monitored for 4 minutes. For a blank, the sample was
replaced by an equal volume of buffer. Glutathione peroxidase activity was calculated as the overall rate of NADPH
oxidation minus the rate of hydroperoxide-independent
NADPH oxidation minus the blank value.
Plasma-Glutathione Peroxidase
Protein Determination
Quantification of plasma-glutathione peroxidase protein was
performed using a commercial “sandwich” ELISA kit (Bioxytech, Bonneuil sur Marne, France). For each patient and
each region, approximately 100 mg of frozen brain tissue
was homogenized in 2.5 vol (wtivol) of Trisisucrose buffer
and centrifuged as described above. Supernatants were collected and twofold serial dilutions were prepared using 50
mM Tris-HC1 (pH 7.8) buffer containing 1 mglml of bovine casein, 150 m M sodium chloride (NaCI), 0.1% Tween
20, and 0.2% sodium azide. From each dilution, an aliquot
of 100 pl was incubated in a microwell that had been coated
with rabbit anti-human plasma-glutathione peroxidase polyclonal antibody. As per the manufacturer’s protocol, each
microwell was incubated sequentially with the same anti-
Przedborski et al: Free Radical Scavenging Enzymes in ALS
159
human plasma-glutathione peroxidase antibody conjugated
to biotin, streptavidin conjugated to alkaline phosphatase,
and para-nitrophenol-phosphate. Based on information supplied by the manufacturer, these antibodies were obtained
using a synthetic plasma-glutathione peroxidase antigen and
purified by affinity chromatography. The limit of detection
was approximately 2.5 ng/ml. No crossreactivity was found
with cellular-glutathione peroxidase from human erythrocyres. Microplates were read at 405 nm. Optical densities
were converted to nanograms of plasma-glutathione peroxidase using a standard curve generated with purified human
plasma-glutathione peroxidase protein (Kioxytech).
Supevoxide Dismutase Isoenzymes, Catalase, and
Citrate Synthase Determination
For SOD isoenzymes, catalase, and citrate synthase, approximately 250 mg of frozen brain samples was homogenized
in 5 vol (wtlvol) of Tridsucrose buffer as described above.
Homogenates were centrifuged at 2,000 g for 5 minutes at
4°C. Pellets were discarded and supernatants were centrifuged at 15,000 g for 10 minutes at 4°C. Supernatants of
the second centrifugation were stored at - 80°C until assayed
for cytosolic S O D and catalase activity. Pellets were gently
resuspended and used for mitochondria1 purification using
a Ficoll gradient as previously described [16]. At the end
of the purification procedure, mitochondria were subjected
to three cycles of freezelthaw prior to centrifugation at
100,000 g for 1 hour at 4°C. Supernatants were stored at
-80°C until they were assayed for mitochondria1 SOD and
citrate synthase activity.
Total and Mn-SOD activities were determined according
to the method described by Spitz and Oherley [17] based
on nitroblue tetrazolium (NBT) reduction by superoxide
radicals followed at 560 nm at room temperature; xanthinexanthine oxidase was utilized to generate a superoxide radical
flux. The reaction mixture (total volume, 1 id) contained
0.15 mM xanthine, 0.6 tnM NBT, 1 mM diethylenetriaminepentaacetic acid, 1 unit of catalase, 0.13 mg of defatted
bovine serum albumin (BSA), 0.25 m M hathocuprioinedisulfonic acid, and 100 p1 of sample. After 3 minutes of preincubation, the reaction was initiated by the addition of xanthine oxidase (amount necessary to achieve a reference rate
[Le., rate of NBT reduction in the absence of tissue] of 0.02
Aabsorbancelmin). One unit of SOD activity is defined as
the amount of enzyme that inhibits the reaction rate by 50%.
The Mn-SOD was distinguished from the cyanide-sensitive
CulZn-SOD by the inclusion of 5 mM NaCN [18]; the
samples were incubated i n the reaction mixture at room temperature for 30 to 60 minutes before the reaction was started,
to ensure complete inhibition of the CulZn-SOD [19]. C u l
Zn-SOD activity was calculated by subtracting the cyanideresistant SOD activity from the total SOD activity.
Catalase activity was determined according to the method
decomposition foldescribed by Aebi [20] based on HIOz
lowed at 240 nm at room temperature as previously described [13]. Each sample was diluted to 1 : 500 in 50 mM
phosphate buffer (pH 7.0) immediately prior to being assayed. The reaction mixture (total volume, 3 ml) contained
2 ml of the diluted sample and 60 mM H 2 0 2 ,which was
added to initiate the reaction. The blank contained only sam-
160 Annals of Neurology
Vol 39
No 2
February 1996
ple and buffer. Catalase activity was determined by calculating the rate constant of a first-order reaction (k) [20].
Citrate synthase activity was assayed according to the
method of Srere [21] based on formation of mercaptide ions
followed at 412 nm at 30°C as previously described [IG].
The reaction mixture (total volume, 1 ml) contained 0.1
m M 5,5’-dithiohis-(2-nitrobenzoicacid), 0.3 mM acetyl coenzyme A, and 20 pl of sample. After 5 minutes of preincubation, 0.5 mM oxaloacetate was added to initiate the reaction. The increase in absorbance was recorded for 2 minutes.
Protein and Hemoglobin Determination
Protein content was determined by the method of Lowry
and colleagues [22], using BSA as the standdrd. Hb content
was determined by the method of Drabkin and Austin [23],
using cyanmethemoglobin as the standard.
Statistics
All values are expressed as means +- standard error of the
mean (SEM). Differences between the ALS and control
groups were tested by two-tailed Student‘s t test or the
Mann-Whitney rank sum test. For each analysis, data sets
were subjected to normality test and equal variance test to
determine whether Student’s t test or the Mann-Whitney
rank sum test should be used. Pearson’s product correlation
coefficient, and subsequent linear regression, was performed
using a least-squares curve fit model. In all analyses, the null
hypothesis was rejected at the 0.05 level. All statistical analyses were performed using SigmaStat for Windows (version
1.0, Jandel Corporation, San Rafael, CA).
Results
In the present study, the calculated rate of liydroperoxide-dependent oxidation of NADPH was used as a specific measure of glutathione peroxidase activity and
represented more than 85% of the total rate of
NADPH oxidation. In addition, the nonspecific hydroperoxide-independent oxidation of NADPH was low
and similar in the two groups in both the precentral
gyrus (control = 2.73 2 0.66 nmol/min/nig protein, ALS = 2.88 F 0.36 nmollminlmg protein, t
= -0.220, df = 16, p = 0.844) and the cerebellar
cortex (control = 2.79 t 0.45 nmol/min/mg protein, ALS = 3.18 f 0.93 nmol/min/mg protein, t
= -0.377, df = 16, p = 0.711). Glutathione peroxidase activity in the precentral gyrus was significantly
lower in ALS compared to control brains (Fig 1A; t =
-2.49, df = 16, p = 0.0240). In contrast, glutathione
peroxidase activity in the cerebellar cortex was similar
in ALS and control brains (see Fig 1A; t = 0.207, df
= 16, p = 0.839). To account for individual variations, we normalized the activities of glutathione petoxidase in the precentral gyrus (the affected region) by
the activities in the cerebellar cortex (the nonaffected
control region). The mean precentral gyrus-cerebellar
cortex ratio was 5196 smaller in ALS (0.59 ? 0.1 1)
compared to control brains (1.20 f 0.19; t = -2.82,
d f = 16, p = 0.0123). In the ALS group, precentral
37s
30.0
1
-
gyrus glutathione peroxidase activities and precentral
gyrus-cerebellar cortex ratios correlated positively with
duration of the disease ( r 2 > 0.45, n = 9, p < 0.048)
(Fig 2). Glutathione peroxidase activities in the cerebellar cortex did not correlate with the duration of the
disease ( 2 = 0.16, n = 9, p = 0.295). Neither precentral gyrus glutathione peroxidase activities nor precentral gyrus-cerebellar cortex ratios correlated significantly with postmortem delay (v' < 0.10, n = 9, p
> 0.25) or age at onset of the disease (Y' < 0.15, n
I
Controls
ALS
T
1:
=
7.5
0.0
PCG
cc
240
T
T
60
PCG
9, p > 0.20).
To determine whether the reduction in glutathione
peroxidase activity corresponds to a decrease in the glu-
cc
Fig 1. Glutathione peroxiduse (GPx) enzymatic activity (A)
and plasma-glutathione peroxidase (PI-GPx) protein content
(B) measured in sporadic amyotrophic lateral sclerosis (ALS)
and control postmortem brain samples. The activigi of glutathione peroxidase was significantly reduced in the precentral
g r u s (PCG) of sporadic ALS compared to control brains; in
precentral gyrus, plasma-glutathione peroxidase protein content
was similar in ALS and in control groups. In the cerebellar
cortex (CC) none of the measurements was dzfferent in the
ALS compared to the control group. Bars represent means and
the error bars represent standmd error of mean f o r 9 samples
per group and per brain region. The asterisk indicates p =
0.024 (Student? t test).
tathione peroxidase isoenzyme, plasma-glutathione peroxidase, we used an immunoassay to quantify the
amount of plasma-glutathione peroxidase protein in
the precentral gyrus and cerebellar cortex in both ALS
and control brains. We found detectable amounts of
plasma-glutathione peroxidase in all brain samples (see
Fig 1B). In addition, we found that plasma-glutathione
peroxidase protein content was similar in the precentral
gyrus and cerebellar cortex, and no significant difference in plasma-glutathione peroxidase protein content
between ALS and control groups in either brain region
(see Fig 1B).
Inclusion of 5 mM NaCN with the cytosolic fraction from the precentral gyrus and cerebellar cortex
caused reduction in SOD activity greater than 75%,
indicating that most SOD activity in this fraction corresponds to Cu/Zn-SOD. We found no significant difference in Cu/Zn-SOD activity in either brain region
between ALS and control groups (Table 2). The tissue
samples used were frozen; therefore, the integrity of
peroxisomes was not preserved and all of the catalase
activity was found in the cytosolic fraction. Catalase
activity did not differ between ALS and control brains
(see Table 2). Unlike previous experiments [13], we
measured Mn-SOD in purified mitochondria, the organelle that contains most of this SOD isoenzyme [ 5 ] .
As indicated by the activity in citrate synthase, mitochondrial preparations from ALS and control brains
were comparable (see Table 2). As expected, more than
90% of SOD activity measured in the mitochondrial
fraction was resistant to 5 mM NaCN, indicating that
most SOD activity in this fraction corresponds to MnSOD. No significant difference was observed in MnSOD activity in mitochondrial fractions between ALS
and control brains (see Table 2). Likewise, Mn-SOD
activity normalized by the mitochondrial enzyme citrate synthase was not different between ALS and control brains (data not shown).
Since all samples used for these assays contained
some blood, we measured Hb content in tissue homogenates prior to centrifugation. Crude homogenates
contained comparable amounts of Hb in the precentral
gyrus (ALS = 4.05 t 0.75 mg/ml, control = 3.94 &
Przedborski et al: Free Radical Scavenging Enzymes in ALS
161
30.0
-
22.5
-
15.0
-
7.5
-
0.0
-
1.2
-
1.0
-
0.8
-
0.6
-
0.4
-
0.2
-
0.0
-
4
Discussion
It has been demonstrated that the carbonyl content, a
marker of oxidative damage in proteins, is increased by
85% in the frontal cortex of sporadic ALS postmortem
tification of the mechanism by which this oxidative
damage occurs in sporadic ALS will provide important
insight into the cause of the disease. In the present
study, we found that the activity of glutathione peroxidase, a key enzyme in the protective mechanism against
free radicals, is significantly lower in ALS compared to
control precentral gyrus (see Fig 1). A similar change
was not observed in the cerebellum (see Fig l), suggesting that reductions in glutathione peroxidase activity have a specific association with the pathological processes in sporadic ALS.
We also found that glutathione peroxidase activity
in the precentral gyrus, but not in the cerebellar cortex,
positively correlates with the disease duration (see Fig
._.
Fig 2. Scatterplot and linear regression for the disease duration and the activity o f glutathione pesoxidase (GPx) measured in the precentral gyrus (PCG) (A) and the ratio of
glutathione pesoxidase activiq measured in the precentval
gyms to glutathione peroxidase activity measured in the cerebellar cortex (CC) (B). The solid lines show the best leastsquares fit (in A: Y = -7.16 f 6.07X, r' = 0.45,p =
0.048; in B: Y = -0.256 f 0.246X,r 2 = 0.57, p =
0.018), and die dotted lines represent the 9 5 % conjidence
interval.
162 Annals of Neurology Vol 39
and control brains. Thus, approximately 4 mg of Hb
is found per milliliter of homogenate, which in turn
contains 285 mg of wet-weight brain tissue. Since normal blood contains between 120 to 160 mg of H b l
ml, we have 25 to 33 p1 of blood in 285 mg of brain
tissue or 0.09 to 0.12 1.11 of blood per milligram of
brain tissue. Based on this calculation we conclude that
blood contamination of the brain samples accounted
for less than 12% (vol/wt).
No 2
February 1996
following disease of the longest duration. Our review
of the pathological data revealed that the degree of spinal and cortical neurodegeneration was comparable
among these patients; this positive correlation (see Fig
2) suggests that alterations in brain-glutathione peroxidase activity can influence the rate of disease progression.
Our study revealed reductions in glutathione peroxidase activity in the precentral gyrus from 9 ALS patients. In another affected region, the spinal cord, Ince
and coworkers [26]found a marked increase in activity
in 10 ALS patients. The reason for the discrepancy in
the postmortem precentral gyrus and spinal cord in
ALS is unclear. Technical differences are, however, unlikely as the methods used in the two studies were fairly
similar. In our study, we measured free radical scavenging enzyme activities in only a single affected region
Table 2. Superoxide Dismutase (SOD) Isoenzymes, Catalase, and Citrate Synthase Activity in Brain Extracts fFom Amyotrophic
Lateral Sclerosis (ALS) Patients and Control Subjects"
Cyrosolic Fraction
Catalase
(klmg protein)
Mn-SOD
(unitslmg protein)
Citrate Synthase
(nmollminlmg protein)
96.4 2 19.7
98.0 t 22.8
0.185 t 0.057
0.194 t 0.022
92.5 t 20.5
127.1 t 22.5
1.71 i- 0.46
1.88 i- 0.25
110.2 t 22.5
95.8 2 17.1
0.163 t 0.047
0.167 ? 0.038
117.9 2 14.0
137.7 t 28.7
2.12 i- 0.46
2.01 f 0.27
CulZn-SOD
(unitslmg protein)
Precentral gyrus
Controls
ALS
Cerebellar cortex
Controls
ALS
Mitochondria] Fraction
'Values represent mean i standard error of mean of 9 samples per patient group and per brain region. Differences between ALS and control
brains were tested by Student's t test or the Mann-Whitney rank sum test; none of the tests showed any significant difference ( p > 0.05).
k
= rate constant of the first-order reaction (for catalase activity) [20].
of the brain. Thus, it would be important in linking
glutathione peroxidase alterations to the pathogenesis
of ALS to extend our work to other affected and unaffected regions. This future endeavor could also be important in understanding the reasons for the opposite
changes in glutathione peroxidase activity found in
ALS postmortem samples by us and by Ince and collaborators 1261.
Glutathione peroxidase purified from human plasma
is a glycosylated selenoprotein distinct from cellularglutathione peroxidase [27]. Although the two isoenzymes cannot be distinguished by the enzymatic assay,
they can be differentiated by immunoassay [27, 281.
Plasma-glutathione peroxidase has been identified in
several tissues [29] and cell lines [30], but its precise
tissue distribution remains incomplete. Therefore, the
apparent reduction in glutathione peroxidase activity
can be due to changes in either cellular- or plasmaglutathione peroxidase, or both. T o address this issue,
we quantified the amount of plasma-glutathione peroxidase in the ALS and control brain samples using an
immunoassay specific for this isoenzyme. We found
detectable amounts of plasma-glutathione peroxidase
in all samples. This is the first demonstration of measurable amounts of plasma-glutathione peroxidase in
the human brain. In an earlier study [29], no plasmaglutathione peroxidase was detected in the rat brain.
Whether this discrepancy represents interspecies variations or differences in the technique or the antibodies
used remains to be determined. We found that neither
in the precentral gyrus nor in the cerebellar cortex did
the content of plasma-glutathione peroxidase differ between the ALS and control samples (see Fig 1). This
suggests that in the precentral gyrus of ALS patients
the reduction in glutathione peroxidase is related to a
deficit in the cellular-glutathione peroxidase.
We also measured the activities of other major free
radical scavenging enzymes including CuiZn-SOD,
Mn-SOD, and catalase. Consistent with the study by
Bowling and coworkers [24], we found that brain Cu/
Zn-SOD activity in sporadic ALS was comparable to
that in controls. In addition, we found that brain MnSOD activity as well as brain catalase activity were not
significantly changed in sporadic ALS.
Free radical scavenging enzymes are present in high
concentrations in the blood [ 111; hence, contamination
of tissue samples with blood can lead to spurious determination of brain enzyme activities. We measured
blood-glutathione peroxidase (9.59 -+ 0.49 pmol/min/
gm Hb), S O D (28,920 2 1,218 units/gm Hb), and
catalase (309.8 ? 15.3 k/gm Hb) activities in 24 normal volunteers; these values did not differ from those
measured in 17 age-matched sporadic ALS patients
[31]. Based on these values and on normal blood Hb
concentrations, we estimated that glutathione peroxidase activity is 1.34 t 0.07 pmollminigm brain tissue. Since the calculated blood contamination of brain
samples was 12%, brain-glutathione peroxidase activity
originating from blood is 0.161 pmolfminlml blood
(1.34 ? 0.07 pmol/min/ml blood X 0.12). Our brain
value was 22.7 nmollminlmg protein in control brains
and 13.8 nmol/min/mg protein in ALS brains. Since
we had 0.08 to 0.10 gm of protein per gram of brain
tissue, we calculated values of 1.1 to 1.35 pmol/min/
gm brain tissue for ALS and 1.8 to 2.2 Fmoliminigm
brain tissue for controls. Therefore, glutathione peroxidase originating from blood represented 12 to 15% of
measured glutathione peroxidase activity in ALS and 7
to 9% in control brains. Using the same calculation,
we found that the contribution of blood-derived free
radical scavenging enzyme activities may account for
less than 5% in SOD (4,049 2 171 unitdm1 blood)
and less than 28% in catalase (43.4 ? 2.14 k/ml
blood). This suggests that free radical scavenging enzyme activities measured in the present work originated
predominantly from brain tissue. Based on published
values of plasma-glutathione peroxidase concentration
in normal human volunteers (i.e., 3.3 2 0.1 pgirnl
Przedborski et al: Free Radical Scavenging Enzymes in ALS
163
serum) [28], we estimated that the contribution of
blood-derived plasma-glutathione peroxidase may account for less than 10% of its brain measurement.
The main function of glutathione peroxidase is to
detoxify H20,.Although the reactivity of H 2 0 Lis limited and its direct implication in toxic processes is
doubtful, it has been recognized as an important intermediate compound in reactions producing highly reactive damaging species [ 1 I]. Glutathione peroxidase also
catalyzes the reduction of lipid peroxides [7],
preventing lipid peroxidation and thus protecting biological membranes. Several lines of evidence indicate that
a reduction in glutathione peroxidase activity can be
damaging [l 1, 321. For example, a 21 ?h inhibition in
glutathione peroxidase activity in cultured human fibroblasts decreased cell number by 50% [33].This in
vitro model supports the hypothesis that moderate
changes in glutathione peroxidase activity may be noxious. Therefore, the 40% reduction in glutathione peroxidase activity in the ALS precentral gyrus is potentially damaging, and may be implicated in the
pathogenesis of sporadic ALS.
Alternatively, the reduction in glutathione peroxidase activity in ALS might be secondary to the neurodegenerative process, namely the loss of motor neurons.
However, in the human brain, glutathione peroxidase
is mainly if not exclusively localized in glial cells [34],
which are not lost in ALS. In conjunction with this,
there was n o change in the activity of the other free
radical scavenging enzymes, which would be expected
if the reduction of glutathione peroxidase was only
consecutive to motor neuron loss. O n the other hand,
many factors have been recognized to modulate glutathione peroxidase activity in vivo [35]. Thus, if one of
these factors is altered in ALS, a deficit in glutathione
peroxidase activity may result. For instance, a deficiency in selenium can cause a dramatic reduction in
glutathione peroxidase activity 1351. However, no
study has documented a deficit in selenium either in
the brain or in the spinal cord of ALS patients [36].
Glutathione peroxidase and selenium deficiency has
been suggested to be involved in the pathogenesis of
different muscular and neurological disorders [35]. In
addition, some evidence exists for a small (1 !)(yo) reduction in glutathione peroxidase activity in the substantia
nigra of patients with Parkinson’s disease [37]. Moreover, selenium deficiency commorily affects skeletal
muscles in various species including humans [35] and
some studies associated a decrease in glutathione peroxidase activity with muscle cramps and weakness [35].
In conclusion, we speculate that the observed deficit
in glutathione peroxidase activity is implicated in the
pathogenesis of sporadic ALS. Furthermore, we believe
that this deficit could be involved in the oxidative damage observed in sporadic ALS [24].Although our study
needs to be extended to a greater number of postmor-
164
Annals of Neurology
Vol 39
No 2 February 1996
tem samples and a greater number of regions of the
nervous system, it provides strong support to the hypothesis that free radicals are implicated in the neurodegenerative process in sporadic ALS.
This work was supported by The Muscular Dystrophy Association,
the National Institute of Neurological Disorders and Stroke grant 1K08-NSO 1724-0 1, the Lowenstein Foundation, and the Parkinson’s
Disease Foundation. Dr Przedborski is recipient of the Colonel
Berger Junior Investigator Award and the Irving A. Hansen Memorial Foundation Award.
We are grateful to Dr J. Goldman for providing the brain samples
from the Columbia Brain Bank, Dr Yan Liu for her help with brain
sample dissection, Drs N. Latov and S. A. Sadiq for their advice
on ELISA experiments, and Drs I. Tayarani and A. Lemainque from
Bioxytech for their advice on the plasma-glurathione peroxidase
ELISA. We are also grateful to Drs L. 1’. Rowland and R. H.
Brown, Jr., for their constant support and to Drs Timothy Lynch
and Daniel Togasaki for their insightful comments on the manuscript.
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