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Ratio of 8-Hydroxyguanine in Intact DNA to Free 8-Hydroxyguanine Is Increased in Alzheimer Disease Ventricular Cerebrospinal Fluid
Mark A. Lovell, PhD;
William R. Markesbery, MD
Arch Neurol. 2001;58:392-396.
ABSTRACT
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Background Markers of oxidative stress are increased in cerebrospinal fluid (CSF)
of patients with Alzheimer disease (AD), although none of those reported are
appropriate diagnostic markers because of the overlap between patients with
AD and control subjects.
Objective To determine the ratio of 8-hydroxyguanine (8-OHG) levels in intact
DNA to free 8-OHG in the ventricular CSF of patients with AD and age-matched
control subjects. The most prominent marker of DNA oxidation is 8-OHG.
Methods Free 8-hydroxy-2'-deoxyguanosine (8-OHdG) was isolated from ventricular
CSF taken at autopsy from 18 subjects with AD and 7 control subjects using
solid-phase extraction columns. Levels were measured as the hydrolysis product,
8-OHG, using gas chromatography/mass spectrometry with selective ion monitoring.
Intact DNA was isolated from the same CSF and the levels of 8-OHG were determined
in the intact structures. Stable-labeled 8-OHG was used for quantification.
Results A statistically significant (P<.05) 108-fold increase
in the ratio of 8-OHG in intact DNA to free 8-OHG was observed in patients
with AD. Analysis of the data distribution indicated that the lowest AD ratio
was 3.5 times higher than the highest control ratio; there was no overlap
of the 2 populations.
Conclusion Although the data for each individual measurement demonstrates overlap
between patients with AD and control subjects, the ratio of 8-OHG intact in
DNA to free 8-OHG demonstrates a delineation between patients with AD and
control 8-OHG subjects and may be useful as a marker of disease progression
or the efficacy of therapeutic antioxidant intervention.
INTRODUCTION
INCREASING evidence supports the role of oxidative stress in the pathogenesis
of neuronal degeneration in several neurological disorders, including stroke,
amyotrophic lateral sclerosis, Parkinson disease, head trauma, and Alzheimer
disease (AD). Several studies show that the AD brain has the potential for
increased oxidative stress due to elevations of brain iron levels, particularly
redox active iron.1, 2 Studies
of oxidative damage show increased levels of protein oxidation, lipid peroxidation,
and markers of lipid peroxidation, including 4-hydroxynonenal2
and acrolein,3, 4 F2-isoprostanes,5 and F4-neuroprotanes6
in AD. Increased levels of 4-hydroxynonenal, F2-isoprostanes, and
F4-neuroprostanes are present in ventricular cerebrospinal fluid
(CSF) in AD. Markers of oxidative stress are present in neurofibrillary tangles
and senile plaques in the brain in AD. Two markers of oxidative stress in
nuclear and mitochondrial DNA are increased in normal aging7
and in AD.8, 9, 10
Attack of DNA by reactive oxygen species, specifically the hydroxyl radical,
leads to the hydroxylation of DNA bases,11
the most prominent of which is 8-hydrodeoxyguanine (8-OHdG). Levels of 8-OHdG
are elevated in mitochondrial DNA in the cerebral cortex of patients with
AD compared with controls.10 Our laboratory
demonstrated statistically significant (P<0.5)
elevations of 8-hydroxyguanine (8-OHG) as the hydrolysis product of 8-OHdG,
8-hydroxyadenine, and 5-hydroxyuracil in the parietal, temporal, and frontal
lobes in patients with AD compared with age-matched control subjects.8 Of the 6 oxidatively modified base adducts analyzed,
8-OHG demonstrated the highest absolute levels, indicating its prominence
as a marker of oxidative stress in AD. Consistent with observations of elevated
8-OHG in the AD brain, we recently demonstrated a statistically significant
(P<0.5) decrease in levels of base excision repair
enzymes responsible for the repair of oxidized guanine.12
In another study, we showed statistically significant (P<0.5) elevations of 8-OHG in DNA extracted from ventricular CSF
in patients with AD compared with age-matched control subjects.13
Analysis of levels of free 8-OHG, resulting from excision from damaged DNA
by base-specific glycosylases, demonstrated a statistically significant depletion
of the free repair product in AD CSF. Although mean levels of free and intact
8-OHG were significantly (P<0.5) different in
8-OHG AD subjects compared with control subjects, overlap of the data showed
that the individual measures alone were not appropriate as diagnostic markers
of AD.
This study demonstrates that the ratio of levels of 8-OHdG in intact
DNA to free 8-OHG in ventricular CSF removed at autopsy (determined using
stable-labeled 8-OHG and gas chromatography/mass spectrometry with selective
ion monitoring) is statistically significantly elevated in AD subjects. The
data distribution suggests that the ratio delineates AD and control subjects.
The ratio also suggests that AD subjects are subject to a dual insult of increased
oxidative stress and decreased repair capacities. The combination of these
factors (rather than either individually) may contribute to the neurodegeneration
observed in the brain in AD.
PATIENTS, MATERIALS, AND METHODS
CSF SAMPLING
Approximately 20 to 40 mL of CSF was removed from the lateral ventricles
at autopsy using an 18-gauge spinal needle and virgin polyethylene syringes
from 18 AD subjects (7 men, 11 women) and 7 control subjects (4 men, 3 women).
Data for 7 AD subjects and 5 control subjects are from a previous article13 and are combined with the results of analysis of
intact and free 8-OHG from 11 additional AD subjects and 2 additional control
subjects. Taken separately, data from the new subjects confirm our prior findings.
Demographic data for all subjects are shown in Table 1. The samples were immediately centrifuged to remove particulate
matter, and the supernatant was placed in fresh polyethylene tubes and stored
at -80°C until used for analysis. The mean ± SEM age was
79.9 ± 2.5 years for AD subjects and 80.0 ± 2.6 years for control
subjects. The mean ± SEM postmortem interval was 2.7 ± 0.2 hours
for AD subjects and 3.0 ± 0.3 hours for control subjects. Neither age
nor postmortem interval was significantly different (P<.05)
between AD and control subjects. All AD subjects demonstrated progressive
intellectual decline and met the criteria of the National Institute of Neurological
and Communicative Disorders and Stroke–Alzheimer's Disease and
Related Disorders Associations Work Group for the clinical diagnosis of probable
AD.14 Histopathologic diagnosis was based on
the analysis of multiple sections of neocortex, hippocampus, amygdala, entorhinal
cortex, basal ganglia, brainstem, and cerebellum stained with hematoxylin-eosin,
modified Bielschowsky silver stain, 10D-5 (for ß-amyloid, Athena Neurosciences,
South San Francisco, Calif),15 ubiquitin, and  -synuclein
immunochemistry. Braak staging, an index of neuropathologic severity of AD,16, 17 was carried out using the Gallyas
stain. All AD subjects met accepted criteria for histopathologic diagnosis
of AD.18, 19 Control subjects were
individuals without a history of dementia or other neurological disorders
who underwent annual mental status testing as a part of our normal volunteer
control group study. All control subjects had test scores within the normal
range. Neuropathologic evaluation of control brains revealed only age-associated
gross and histopathologic alterations. Subjects were excluded from this study
who had been on a respirator or had prolonged terminal hypoxia, recent or
old infarcts, intracranial hemorrhages, drug intoxication, alcoholism, or
central nervous system neoplasms. Hypoxic changes were not found in any brain
region on microscopic examination in any of the subjects used in this study.
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Table 1. Demographic Data*
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ISOLATION OF FREE 8-OHG FROM VENTRICULAR CSF
Free 8-OHdG was isolated from ventricular CSF samples as previously
described,13 using a modification of the procedure
of Shigenaga et al.20 Briefly, the CSF samples
(8-33.4 mL) were thawed at room temperature and thoroughly vortexed for 2
minutes, then 20 µL aliquots were removed for protein content determinations
using the Pierce bicinchoninic acid method. To allow normalization of levels
of free 8-OHG to protein content, accurately measured
volumes (8-33.4 mL) of CSF were passed through C18 solid-phase
extraction columns preconditioned with 10 mL of high-pressure liquid chromatography–grade
methanol, 10 mL of 18- distilled and deionized water, and 10 mL of
50-mmol/L (KH2PO4) monobasic potassium phosphate (KH2PO4)(pH 7.4) under slight vacuum. Stable-labeled
8-OHdG (8C13; 7.9N15; 41.2 nmol) was added to the columns
at the same time. The eluent CSF was collected in fresh side-arm test tubes
to prevent cross-contamination of the samples and reserved for isolation of
intact DNA. The columns were then washed with 4 mL of 50-mM KH2PO4 followed by two 2-mL washes with 5% methanol-KH2PO4. The 8-OHdG and standard 8-OHG were eluted from the column with 3
mL of 15% methanol-KH2PO4.The eluent was added to new
C18 columns preconditioned as described above. The columns were
washed with 1 mL of distilled and deionized water and dried for 15 minutes
under slight vacuum. Purified 8-OHdG and standard 8-OHG were eluted with 2
mL of high-pressure liquid chromatography–grade methanol, placed in
5-mL conical glass tubes, and lyophilized. Using standard solutions of DNA
and nonlabeled 8OHdG, the purification allowed passage of approximately 97%
of DNA while retaining nearly all 8-OHdG as determined by UV-visible absorption
spectrometry.
ISOLATION OF DNA FROM VENTRICULAR CSF
Intact DNA was isolated from ventricular CSF using a modified procedure
of Mecocci et al.7 The CSF was mixed with a
1:10 volume of 1mol/L disodium EDTA, 5% sodium dodecyl sulfate,
and 50-mmol/L Tris-hydrochloride (pH 8.0) along with a 1:25 volume of 10-mg/mL
proteinase K. The samples were digested for 2 hours at 37°, and 1:10 volume
of 5-mmol/L sodium chloride was added. The solution was then extracted 3 times
with buffer-saturated phenol containing 5.5-mmol/L 5-hydroxyquinoline to prevent
artifactual oxidation of DNA. The samples were extracted 3 times with 24:1
chloroform-isoamyl alcohol, and an additional 1:10 volume of 5-mol/L sodium
chloride was added. To isolate DNA, the solution was centrifuged through a
5000 molecular weight cutoff filter at a speed of 2000g for 14 hours at 4°C. The resulting DNA was resuspended in 1 mL
of distilled and deionized water and the concentration was determined at 260
nm using a Genesys 5 UV–visible absorbtion spectrophotometer. Ratios
of absorbance at 260 and 280 nm showed a mean ± SEM 260-280 ratio of
1.45 ± 0.05, indicating slight protein contamination. Although the
isolation procedure should precipitate all DNA, the predominant band demonstrated
by polyacrylamide gel electrophoresis was approximately 400 base pairs.
Stable-labeled 8-OHG (41.2 nmol) was added and the samples lyophilized.
After lyophilization, the DNA and free 8-OHdG samples were subjected to formic
acid hydrolysis, which converted 8-OHdG to the free base, 8-OHG, and bis (trimethylsilyl)trifluoroacetamide
derivatization as previously described.13 After
derivatization, the samples were lyophilized and suspended in 20 µL
bis(trimethylsilyl)trifluoroacetamide immediately before analysis.
Analysis of samples was carried out on a model HP6890 gas chromatograph
(Hewlett-Packard; Palo Alto, Calif) equipped with a mass spectrometer operated
in selective ion-monitoring mode as previously described.13
Retention times for 8-OHG were essentially constant. Shot-to-shot variation
was less than 2% for standard 8-OHG samples. Duplicate analysis of 8-OHG from
randomly selected free and intact samples demonstrated variabilities comparable
to those observed for standards. Levels of 8-OHG were quantified using stable-labeled
8-OHdG as an internal standard as described by Dizdaroglu.21
For quantification, peaks of interest were integrated and the area of the
8-OHG peak was compared with the internal standard peak, which has a known
concentration. For 8-OHG in intact DNA, 8-OHG levels (nanomoles) were normalized
to DNA content and converted to nanomoles per milliliter of CSF by multiplying
the DNA content by the average DNA yield per milliliter of CSF. Free 8-OHG
levels (nanomoles) were normalized to protein content (nanomoles per milligram
of protein) and converted to nanomoles per milliliter of CSF by multiplying
by the average protein concentration (milligrams of protein per milliliter
of CSF). Conversion of levels of 8-OHG to terms of volume of CSF allows calculation
of a unitless ratio. Because the gas chromatography/mass spectrometry procedure
uses mass spectrometry as the detector, the signals from the labeled and unlabeled
compounds may be separated. The advantage of using stable-labeled 8-OHG as
an internal standard is that it responds identically to the compound of interest
during hydrolysis and derivatization.
STATISTICAL ANALYSES
The ratio of 8-OHdG in intact DNA to free 8-OHG was calculated, and
statistical analyses were performed using a 2-tailed Student t test and the commercially available ABSTAT software (AndersonBell;
Arvada, Colo). Correlation analyses were carried out using ABSTAT software.
RESULTS
Statistical comparison of mean age, postmortem interval, DNA content
(micrograms per milliliter) and protein content (milligrams per milliliter)
indicated no statistical differences between AD and control subjects (Table 1). There was a statistically significant
decrease in brain weight in subjects with AD (mean ± SEM, 1080 ±
30 g) compared with age-matched controls (1310 ± 50 g) (P<.001). The mean ± SEM ratio of 8-OHG in intact DNA to free
8-OHdG was significantly elevated (108-fold) in ventricular 8-OHG CSF samples
from 18 AD subjects (6.46 ± 1.39) compared with samples from 7 control
subjects (0.06 ± 0.02) (P = .01). Levels of
8-OHG in intact DNA (Table 2)
were significantly (P = .03) increased (18-fold)
in AD subjects' ventricular CSF (912.3 ± 232.7 pmol/mL CSF) compared
with control subjects (50.1 ± 22.3 pmol/mL CSF). Levels of free 8-OHG
were significantly lower in AD subjects (195.9 ± 40.3 pmol/mL CSF)
compared with control subjects (683.7 ± 191.1 pmol/mL CSF) (P = .001) (Table 2). There
were no significant correlations between the ratio of 8-OHG in intact DNA
to free 8-OHG and age, postmortem interval, protein content, DNA content,
or brain weight. There was a positive statistically significant (r = 0.89, P = .05) correlation between intact-to-free
ratio and Braak stage.
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Table 2. Individual Values for Bound and Free 8-Hydroxyguanine (8-OHG)*
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The distribution of individual data points of the ratio of 8-OHG in
intact DNA to free 8-OHG plotted on a log scale ranged from 0.01 to 0.12 for
control subjects and from 0.44 to 23.60 for AD subjects (Figure 1). There is no overlap of the data, with the lowest AD value
(0.44) 3.5 times the highest control value (0.12). Two of the 18 AD subjects
had values close to those observed for control subjects. The other 16 AD subjects
had ratios at least 10 times those of the highest control values. Figure 1 shows the data plotted on a log
scale to allow comparisons of the distributions.
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Individual ratios of 8-hydroxyguanine in intact DNA to free 8-hydroxyguanine
for subjects with Alzheimer disease and control subjects plotted on a logarithmic
scale. The lowest ratio for a subject with Alzheimer disease is 3.5 times
higher than the highest control ratio for a control subject.
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COMMENT
It is possible that oxidative damage to DNA may contribute to the pathophysiologic
alterations found in the brain in AD. The determination of OHdG in urine is
thought to be a good index of in vivo oxidation in DNA damage.22
Because ventricular CSF filters and disposes degraded cellular material from
the brain, it should more accurately reflect levels of brain DNA oxidation
than blood or urine. This study demonstrates a significant, 108-fold increase
in the ratio of 8-OHG in intact DNA to free 8-OHG in ventricular CSF from
AD patients compared with age-matched control subjects, all with short postmortem
intervals. Distribution of the individual data points demonstrates that the
lowest AD value is 3.5 times higher than the highest control value. Inspection
of values of free 8-OHG and 8-OHG in intact DNA for each subject suggests
that for AD subjects, the relatively low level of repair product contributes
to the higher ratios observed. There is a statistically significant positive
correlation between the ratio of intact 8-OHG in DNA to free 8-OHG and Braak
stage (P = .05). Thus, this marker of DNA oxidation
seems to mirror brain degeneration and has the potential to be used as an
index of disease progression.
This study extends our prior study of levels of 8-OHG in intact DNA
and free 8-OHG isolated from ventricular CSF.13
We used stable-labeled 8-OHG and gas chromatography/mass spectrometry with
selective ion monitoring with selective ion monitoring to unequivocally identify
peaks of interest based on chromatographic retention times and mass spectra.
The results of this study for individual measures of free and intact 8-OHG
agree with those of our prior study and demonstrate a statistically significant
elevation of 8-OHG in intact DNA (P = .03) and a
significant depletion of free 8-OHG (P = .001) in
patients with AD compared with age-matched control subjects. The levels of
8-OHG measured in ventricular CSF are comparable to those observed in our
brain nuclear DNA study, which demonstrated elevations of 8-OHdG in AD frontal,
temporal, and parietal lobe structures.8 The
studies of Lyras et al9 and Mecocci et al10 also showed increased DNA 8-OHG in the brain in AD.
Our finding of an elevation of a marker of DNA oxidation concomitant with
a decrease in levels of free repair product correlates well with our previous
studies of CSF oxidation, which showed increased levels of 4-hydroxynonenal,
a neurotoxic marker of lipid peroxidation, in AD ventricular CSF.2
Free 8-OHG measured in this study results from the excision of 8-OHG
through the action of a base-specific glycosylase,23, 24
which functions to cleave the altered base. After removal from the brain,
cleaved bases are subsequently transported via CSF and blood and eventually
excreted in urine,20, 25 and they
may serve as an efficient marker of oxidative DNA damage in vivo.20 A study of free 8-OHG in urine indicated that excretion
decreases with age,26 whereas Mecocci et al10 found that brain 8-OHG in intact DNA increases with
age, suggesting a decline in the repair mechanisms responsible for the excision
of 8-OHG. These observations are consistent with our study showing decreased
activity of the base excision repair enzyme, 8-oxyguanine glycosylase, responsible
for excision of 8-OHG in vulnerable regions of the AD brain.13
Although several markers of oxidative stress have been measured in AD
ventricular CSF, most demonstrate an overlap between AD and control subjects.
Indeed, the distribution of individual measurements of free or 8-OHG in intact
DNA demonstrates considerable overlap. However, when we compared the ratio
of intact to free 8-OHG, there was no overlap between AD and control populations,
with a 3.5-fold difference between the lowest AD and highest control values.
Other studies of CSF samples have failed to demonstrate significant differences
between AD and control subjects for levels of glutathione27
or selenium.28 Levels of CSF F2-isoprostanes
and F4-neuroprostanes show overlaps between AD and control subjects,2 as do levels of CSF tau alone 29, 30
or in combination with Aß1-40 and Aß1-42.31, 32, 33
It has been suggested that the combination of CSF tau and Aß may serve
as a diagnostic marker for AD; however, these studies used living patients
in whom the diagnosis was not absolutely certain. Although the present study
used a method requiring larger volumes of CSF than are practical in living
subjects, we are pursuing an immunoblot method requiring only a small amount
of CSF. Because the study measured markers of oxidative DNA damage, it is
unlikely that the results observed are specific to AD, but they may be present
in other neurodegenerative diseases in which oxidative stress is involved
in the pathogenic mechanism. Although our results represent the analysis of
a relatively small number of subjects, they clearly separate patients with
late-stage AD from nondemented age-matched controls. The potential of the
ratio of 8-OHG in intact DNA to free 8-OHG as a diagnostic marker in AD deserves
further study in living subjects at various stages of AD and in other neurodegenerative
disorders. It also may be helpful in determining oxidative status and the
response of AD patients to therapeutic antioxidant intervention.
AUTHOR INFORMATION
Accepted for publication August 17, 2000.
This study was supported by the National Institutes of Health, Bethesda,
Md, grants 5-P01-AG0 5119, and 5-P50-AG0 5144 and by a grant from the Abercrombie
Foundation, Versailles, Ky.
The authors thank Drs Daron Davis and David Wekstein for CSF procurement,
Jane Meara and Paula Thomason for technical and editorial assistance, and
Cecil Runyons for demographic data.
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