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Oxidative damage to protein in sporadic motor neuron disease spinal cord.

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logical signs or brain atrophy in patients receiving valproate therapy is of great practical importance. In such
patients valproate discontinuation should be the first
step toward diagnosis, and should be performed before
embarking on expensive and exhaustive investigations.
This principle holds regardless of duration of valproate
administration or measured blood levels. The normality of the EEG background in the face of increasingly
severe symptoms may be useful in differentiating valproate-induced brain atrophy and dementia from other
progressive conditions including biochemical disturbances and epileptic states. Withdrawal of valproate is
usually effective very quickly, with clinical recovery
already evident in 2 or 3 weeks.
1. Herranz JL, Armijo JA, Arteaga R. Clinical side effects of phenobarbital, primidone, phenytoin, carbamazepine, and valproate
during monotherapy in children. Epilepsia 1988;29:794-804
2. Scheffner D, Konig St, Rauterberg-Ruland 1. Fatal liver failure
in 16 children with valproate therapy. Epilepsia 1988;29:530542
3. Coulter DL, Allen RJ. Pancreatitis associated with valproic acid
therapy. Ann Neurol 1980;7:92 (Letter)
4. Brown JK. Valproate Toxicity. Dev Med Child Neurol 1988;30:
5. Triggs WJ, Bohan TP, Lin SN, Wilmore LJ. Valproate-induced
coma with ketosis and carnitine insufficiency. Arch Neurol
1990;47: 113 1-1 133
6. Zaccara G, Paganini M, Campostrini R, et al. Hyperammonemia
and valproate-induced alterations of the state of consciousness.
A report of 8 cases. Eur Neurol 1984;23:104-112
7. McLachlan RS. Pseudoatrophy of the brain with valproic acid
monotherapy. Can J Neurol Sci 1987;14:294-296
8. AicardiJ. Benign rolandic epilepsy. Int Pediatr 1987;2:176-181
9. Loiseau P, Duche B, Cordova S, et al. Prognosis of benign
childhood epilepsy with centrotemporal spikes: a follow-up
study of 168 patients. Epilepsia 1988;29:229-235
10. Zaret BS, Cohen RA. Reversible valproic acid-induced dementiti. a case report. Epilepsia 1986;27:234-260
11. Marescaux C, Warter JM, Micheletti G, et al. Stuporous episodes during treatment with sodium valproate: report of seven
cases. Epilepsia 1982;23:297-305
12. Zaret BS, Beckner RR, Marini AM, et al. Sodium valproate
induced hyperammonemia without clinical hepatic dysfunction.
Neurology 1982;32:206-208
Oxidative Damage to
Protein in Sporadic
Motor Neuron Disease
Spinal Cord
Pamela J. Shaw, MD, FRCP,"
Paul G. Ince, MD, MRCPath,t§
Gavin Falkous, MPhi1,S
and David Mantle, PhDS
The recent discovery that defects in the gene encoding
copper-zinc superoxide dismutase (SOD1)are associated
with some cases of familial motor neuron disease has
heightened interest in the possibility that free radical
mechanisms may contribute to selective motor neuron
injury. Sporadic and familial motor neuron diseases are
clinically and pathologically very similar and may share
common pathophysiological mechanisms. Thus the role
of free radical mechanisms as a contributory factor to
motor neuron injury in the common sporadic form of
motor neuron disease requires urgent exploration, particularly as this may provide an avenue for therapy
aimed at retarding pathological progression. We investigated oxidative damage to proteins in the lumbar spinal
cord by quantifying the protein carbonyl level from 19
patients with sporadic motor neuron disease, 8 neurologically normal control subjects, and 11 neurological disease control subjects, most of whom had slowly progressive neurodegenerative disease. In sporadic motor
neuron disease the mean protein carbonyl level in the
spinal cord was increased by 119% ( p < 0.02) compared
to normal control subjects and by 88% ( p < 0.04) compared to the neurological disease control subjects. These
data contribute to the emerging evidence that oxidative
damage may play a contributory role in the neuronal
death in sporadic motor neuron disease. This mechanism
may be particularly important in a subset of patients
with motor neuron disease.
Shaw PJ, Ince PG, Falkous G, Mantle D. Oxidative
damage to protein in sporadic motor neuron
disease spinal cord. Ann Neurol 1995;38:69 1-695
The cause of selective neuronal death in motor neuron
disease (MND) has not been established. A major recent development has been the discovery that some
From the "Departments of Neurology, TNeuropathoiogy, and
tNeurochemistry and the 5MRC Neurochemicd Pathology Unit,
University of Newcastle upon Tyne, Newcastle upon Tyne, United
Received Mar 14, 1995, and in revised form Jun 21. Accepted for
publication Jun 21, 1995.
Address correspondence to Dr Shaw, University Department of
Neurology, Ward 11, Royal Victoria Infirmary, Newcastle upon
Tyne NE1 4LP, United Kingdom.
Copyright 0 1995 by the American Neurological Association 691
patients with autosomal dominant familial amyotrophic
lateral sclerosis (FALS) have point mutations in the
gene on chromosome 2 1 that encodes copper-zinc
(Cu/Zn) superoxide dismutase (SOD1) [l]. This has
raised considerable interest in the possibility of free
radical-mediated mechanisms of motor neuron injury,
as the normal role of SODl is to catalyze the removal
of superoxide free radicals, which can contribute to
cellular oxidative damage { 2 , 31.
Even in the presence of established SODl mutations, the molecular mechanisms of motor neuron injury and the reasons for the selective vulnerability of
this cell group are not yet understood. Rather than a
simple deficiency of superoxide radical clearance, it
may be that the mutant SODl protein has acquired
some toxic “gain of function” that injures motor neurons by an alternative mechanism {4, 51. FALS patients
represent only about 10% of patients with MND, but
the close clinical and pathological similarity between
familial and sporadic MND suggests that there may be
common underlying pathophysiological mechanisms.
That oxidative stress may be one of these is suggested
by our recent observation of increased iron and increased glutathione peroxidase activity in sporadic
MND spinal cord {b]. Establishing whether oxidative
stress plays a role in motor neuron injury is an important priority in the search for therapies that retard clinical and pathological progression in MND, since there
are multiple targets of oxidative damage at which therapeutic intervention can be aimed.
Introduction of carbonyl groups into amino acid residues of proteins is a hallmark of oxidative modification
and reaction to these groups with carbonyl-specific reagents provides a means of detecting and quantifying
such modification 171. The aim of this study was to
determine whether there was evidence of increased
oxidative damage to proteins in postmortem spinal
cord tissue of sporadic MND patients compared to
normal and neurological disease control groups, by
measuring protein carbonyl derivatives.
Materials and Methods
Central nervous system (CNS) material was obtained at autopsy from 19 patients with sporadic MND, 8 neurologically
normal control subjects, and 11 neurological disease control
subjects. We also included material from 1 subject with familial M N D who had the Glu 100l-+Gly mutation in exon 4
of the S O D l gene. Multiple blocks from the left cerebral
hemisphere and the brainstem, and tranverse sections from
multiple spinal cord segments were sealed in polythene, snapfrozen in melting arcton (ICI) surrounded by liquid nitrogen,
and then stored at - 80°C.
In the sporadic M N D group the diagnosis was established
by clinical criteria [S] and careful investigation including
neurophysiologicd assessement in life and characteristic histopathological changes postmortem. Ten patients were men
and 9 were women, with a mean age of 63.4 5 12.7 years
692 Annals of Neurology Vol 38 No 4 October 1995
(range, 42-89 years). The mean postmortem delay in this
group was 17.8 2 9.0 hours (range, 9-48 hours). The cause
of death in all the M N D patients was respiratory failure,
which was accompanied in 7 patients by bronchopneumonia,
in 1 case by pulmonary embolism, and in 1 by pleural effusion and mesothelioma. The 8 normal control subjects included 7 men and 1 woman, with a mean age of 61.8 ? 16.4
years (range, 32-88 years). The mean postmortem delay was
17.1 ? 9.5 hours (range, 6-36 hours). The causes of death
in this group were as follows: ischemic heart disease (3),
respiratory failure (l), gastrointestinal hemorrhage (l), disseminated carcinoma with bronchopneumonia (2), and rapid
death following a fall with multiple injuries (1). The latter
subject had a skull fracture, brain swelling, and contusions.
However, there was no evidence of preceding CNS pathology and the spinal cord was entirely normal. Neuropathological examination confirmed the absence of any gross or microscopic brain abnormalities in the other 7 control subjects.
The disease control group included patients with the following diagnoses: Alzheimer’s disease (4), senile dementia of
Lewy body type (2), Pick‘s disease (I), olivopontocerebellar
atrophy (l), Friedreich‘s ataxia (l), Huntington’s chorea (l),
and cerebrovascular disease (1). In these patients the mode
of death was frequently similar to that of the M N D patients.
Ten had severe physical incapacity due to their neurological
condition at the time of death, 7 had terminal bronchopneumonia, 1 had hematemesis, 2 were severely cachectic with
pressure sores, and 1patient died during an epileptic seizure.
In the disease control group there were 6 men and 5 women,
with a mean age of 63.9 t 15.8 years (range, 35-88 years).
The mean postmortem delay was 18.5 ? 7.9 hours (range,
10-34 hours).
Sample Preparation and Protein Carbonyl Assay
Samples of spinal cord tissue (0.1-0.5 gm) from the L-5
segment were homogenized (10% wtivol) in 50 mM phosphate buffer, p H 7.5. The protein carbonyl assay used was
based on the method of Levine and colleagues 171. Streptomycin sulfate (10% wt/vol in 50 mM HEPES buffer, p H
7.5) was added to the spinal cord homogenate equivalent to
a final concentration of 1% (wdvol) to precipitate nucleic
acids. The samples were allowed to stand at room temperature for 15 minutes and then centrifuged at 11,000 g for
15 minutes. The pellet was discarded and the supernatant
retained. The protein of the supernatant was determined by
the method of Lowry and associates @I. For each sample the
supernatant was divided into two aliquots, an equal volume
of 20% TCA was added to each aliquot and the samples were
then centrifuged at 3,000 g for 15 minutes, the supernatant
discarded, and the pellet retained. For each sample, the pellet
from one aliquot was reconstituted in 0.5 ml of 10 mM 2,4dinitrophenylhydrazine in 2 M hydrochloric acid (HCI), and
the pellet from the other aliquot in 0.5 ml of 2 M HCI (for
reagent blank assay). The samples were then allowed to stand
at room temperature for 1 hour, 0.5 ml of 20% TCA was
then added and the samples centrifuged at 3,000 g for 5
minutes, and the supernatant discarded. The pellets were
then washed three times with 1 ml of ethano1:ethyl acetate
(1 : 1 vol/vol) to remove unbound reagent, standing the samples for 10 minutes before centrifugation and discarding the
supernatant each time. The protein pellet was redissolved in
1 ml of 6 M guanidine solution in 20 mM potassium phosphate adjusted to p H 2.3 with trifluoroacetic acid and allowed to stand at 37°C for 15 minutes. For each sample the
spectrum was read between 360 and 400 nm in a spectrophotometer and the carbonyl content calculated from the maximum absorption (relative to reagent blank), using a value for
the extinction coefficient E of 22,000 M - ’ cm-’. The carbonyl content was expressed as nmoles of carbonyl per milligram of protein. All the assays were performed without
knowledge of the diagnostic category of the subjects.
Experimental Validztion of Protein Carbonyl Assay
Method as Applied to Spinal Cord Tissue Samples
Spinal cord samples (approximately 0.5 gm) from normal
control subjects were homogenized 10%;wttvol in 50 mM
phosphate buffer, p H 7.5. Samples were centrifuged at
3,000 g for 10 minutes and the insoluble material discarded.
Tissue soluble extracts were gassed to saturation wirh either
N,O for subsequent generation of hydroxyl (OH.) radicals,
or with 0, (following the addition of 20 mM sodium formate
to the extraction buffer as a scavenger of OH. radicals) for
subsequent generation of superoxide (0; .) radicals. Generation of OH. or 0;. radicals in vitro via 6oCogamma radiolysis of aqueous tissue extracts was based on the method of
Davies [lo}. Quantification of free radical dosage rate (equivalent to 99 kradthr) was determined by standard dosimetric
techniques [ 1I]. Samples were irradiated for time periods
between 2 and 16 hours, with subsequent analysis of free
radical-induced protein carbonyl group formation determined as described above (relative to corresponding nonirradiated tissue extracts).
Student’s t test was used to compare the levels of protein
carbonyl between the groups. A p value of <0.05 was considered significant. Linear regression analysis was used to correlate the protein carbonyl levels with age and post mortem
The mean protein carbonyl content of the L-5 spinal
cord samples from the MND group was 1.60 (standard
error of mean ISEM), 0.28) nmol/mg of protein, which
was significantly higher than the values for the normal
control group (mean, 0.73 [SEM, 0.22) nmol/mg of
protein p < 0.02) and the disease control group (mean,
0.85 [SEM, 0.20) nmol/mg of protein, p < 0.04). Interestingly, there was no detectable protein carbonyl
in the single patient with a known SOD1 mutation
(exon 4 Glu 1 0 h G l y ) . There was no significant difference in the protein carbonyl level between the normal and disease control groups (p < 0.67). No significant correlation was found between protein carbonyl
levels and age or postmortem delay in any of the three
groups. The Figure shows the protein carbonyl values
for the individual subjects within each of the three
groups. Seven (36.8%) of the MND patients had protein carbonyl levels of 2 standard deviations (SDs) or
more above the mean of the normal control group.
0 ’
The protein carbonyl levels (nmoltmg protein) in L-5 spinal cord
samples for individuals in the motor neuron disease (MND),
nomral control, and disease control groups. Means and standard
errors of the mean are indicated alongside the data points for
each group.
The Table shows the results of protein carbonyl determinations in normal spinal cord samples exposed to
increasing dosages of hydroxyl and superoxide radicals.
The level of protein carbonyl measured in the nonirradiated spinal cord tissue extracts did not increase with
increasing duration of the experiment up to 16 hours,
indicating that no further radical-induced protein oxidation takes place in vitro once tissue samples have
been homogenized, The level of protein carbonyl determined in irradiated samples increased in a nearlinear manner with increasing exposure to free radicals
over 2 to 16 hours. This provides validation of the
assay principle used for the main experimental series.
The protein oxidation induced by OH- was greater
than that induced by 0;. at all time points in the
course of the experiment.
As far as we are aware, this is the first study to measure
an index of oxidative damage to proteins in spinal cord
tissue in MND. We found that the mean protein carbonyl content in the L-5 spinal cord segment was increased by 119% in the MND patients compared to
neurologically normal control subjects and by 88%
compared to the neurological disease control subjects.
Seven of the MND patients had protein carbonyl levels
2 SD or more above the mean for the normal control
group, whereas 12 had levels overlapping with the normal range, suggesting that oxidative damage to proteins
may be particularly important in a subgroup of these
patients. The inclusion of a disease control group was
important to ensure that any increase in protein car-
BriefCommunication: Shaw et d : Spinal Protein Carbonyl in MND
Protein Carbonyl Content (nmol carbonyltmg protein) of Normal Human Spinal Cord Extracts after “Co Gamma Irradiation
Irradiation dose (krad)
Sample treatment
1. OH. exposed
2. Sample gassed with N,O, not irradiated
3. 0;. exposed
4. Sample gassed with O,, not irradiated
196 (2 hours)
392 (4 hours)
784 (8 hours)
1,568 (16 hours)
bony1 content in the MND patients was not simply a
reflection of the mode of death and agonal status. The
mode of death in the disease control group, which
largely consisted of patients with progressive neurodegenerative disease, was similar to that of the MND
patients. It thus appears that a factor specific to the
MND disease process produces the elevation in spinal
cord protein carbonyl. The results of this study are
consistent with the findings of Bowling and colleagues
1121, who found that the protein carbonyl content in
the frontal cortex of 11 patients with sporadic MND
was increased by 85% compared to normal control
values. In the latter study no increase in frontal cortex
protein carbonyl was found in 3 patients with FALS
and SODl mutations and similarly, we found no increase in the spinal cord in the single patient with an
exon 4 SODl mutation in this study.
The increase in protein carbonyl content in spinal
cord and frontal cortex sugests that oxidative damage
may play a contributory role in the neuronal death
in sporadic MND. Other circumstantial evidence also
supports this hypothesis. For example, the activity of
the selenoprotein, free radical scavenging enzyme glutathione peroxidase, and the level of selenium itself are
increased in MND spinal cord 161. SODl messenger
RNA is increased in motor neurons of MND patients
compared to control subjects 1131. All of these findings
may represent a compensatory response to oxidative
stress. In addition the iron level is increased in MND
spinal cord 161. Iron accumulation may be a secondary
change associated with various neurodegenerative diseases and the reasons for it are at present uncertain
1141. However, iron is also a well-recognized catalyst
for oxidative pathology and may contribute to cellular
Free radical-induced cell death is likely to occur
through impairment of function of macromolecules
and organelles that are especially sensitive to oxidative
damage. The exact molecular mechanisms by which
reactive oxygen species (ROS) might selectively injure
motor neurons are at present uncertain. Neurotoxicity
of ROS has, however, been clearly demonstrated in
spinal cord motor neurons in vivo and in vitro 115,
161 and there is some evidence to suggest that motor
694 Annals of Neurology Vol 38 N o 4 October 1995
neurons may be more vulnerable than other groups of
neurons 1161. Some specific metabolic feature of motor neurons may render them more vulnerable, such as
the presence of a macromolecule sensitive to oxidative
damage which is also essential for motor neuron survival, or unique aspects of glutamatergic neurotransmission in the motor system. Some cellular components appear particularly sensitive to protein thiol
oxidation including membrane pumps involved in
maintenance of Ca2+ homeostasis {17); glutamine synthetase, an enzyme essential for the removal and recycling of synaptic glutamate [lS]; and the glutamate
transporter system essential for the reuptake of glutamate from the synaptic cleft {191. Thus free radical
mechanisms are closely interlinked with other potential
pathways of neuronal injury involving Ca2+ homeostasis and glutamate receptor activation {20}.
Further work, using human postmortem material as
well as motor neuron culture, spinal cord explant, and
transgenic experimental model systems, is necessary to
attempt to unravel whether oxidative damage in MND
is primary or secondary to some other pathophysiological process. The evidence that has emerged to date,
including the results of this study, suggest that trial of
antioxidant therapy, in an attempt to retard pathological progression, would be worthwhile in MND.
Dr Shaw is supported by the Wellcome Trust as a Senior Research
Fellow in Clinical Science.
We thank Prof Barry Halliwell for helpful discussion of this work.
1. Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn
superoxide disrnutase are associated with familial amyotrophic
lateral sclerosis. Nature 1993;362:59-62
2. McCord JM, Fridovich I. Superoxide disrnutase. J Biol Chem
3. Halliwell B. Reactive oxygen species and the central nervous
system. J Neurochern 1992;59:1609-1623
4. Borchelt DR, Lee MK, Slunt HS, et al. Superoxide dismutase
1 with mutations linked to familial amyotrophic lateral sclerosis
possesses significant activity. Proc Natl Acad Sci USA 1994;91:
5. Gurney ME, Pu H, Chiu AY, et al. Motor neuron degeneration
in mice that express a human Cu/Zn superoxide dismutase mutation. Science 1994;265: 1772- 177 5
6. Ince PG, Shaw PJ, Candy JM, et al. Iron, selenium and glutathione peroxidase activity are elevared in sporadic moror neuron
disease. Neurosci Lett 1994;182:87-90
7. Levine RL, Garland D, Oliver CN, et al. Determination of carbony1 content in oxidatively modified proteins. Methods Enzymol 1990;186:464-478
8. Brooks BR. El Escorial World Federation of Neurology criteria
for the diagnosis of amyotrophic lateral sclerosis. J Neurol Sci
9. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ. Protein measurement with folin phenol reagent. J Biol Chem 1951;193:
10. Davies KJA. Protein damage and degradation by oxygen radicals. I. General aspects. J Biol Chem 1987;262:9895-9901
11. Fricke H, Hart EJ. Radiation dosimetry. New York: Academic,
12. Bowling AC, Schultz JB, Brown RH, Bed MF. Superoxide
dismutase activity, oxidative damage and mitochondrial energy
metabolism in familial and sporadic amyotrophic lateral sclerosis.
J Neurochem 1993;61:2322-2325
13. Bergeron C, Muntasser S, Somerville MJ, et al. Coppedzinc
superoxide dismutase mRNA levels are increased in sporadic
amyotrophic lateral sclerosis motor neurons. Brain Res 1994;
14. Jenner P. Oxidative damage in neurodegenerative disease. Lancet 1994;344:796-798
15. Liu D, Yang R, Yan X, McAdoo DJ. Hydroxyl radicals generated in vivo kill neurons of the rat spinal cord: electrophysiological, histological and neurochemical results. J Neurochem 1994;
16. Michikawa M, Lim K, McLarmon J, K m S. Oxygen radicalinduced neurotoxicity in spinal cord neuron cultures. J Neurosci
Res 1994;37:62-70
17. Orrenius S. Oxidative stress studied in intact mammalian cells.
Philos Trans R SOCLon [Biol) 1985;311:223-227
18. Schor NF. Inactivation of mammalian brain glutamine synthetase by oxygen radicals. Brain Res 1988;456:17-21
19. Volterra A, Trotti D, Tromba C, et al. Glutamate uptake inhibition by oxygen free radicals in rat cortical astrocytes.J Neurosci
20. Coylefl, Puttfarcken P. &dative stress, glutamate and neurodegenerative disorders. Science 1993;262:689-695
BriefCommunication: Shaw et al: Spinal Protein Carbonyl in MND 695
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