Longitudinal SPECT study in Alzheimer’s
disease: relation to apolipoprotein E
M Lehtovirta, J Kuikka, S Helisalmi, P Hartikainen, A Mannermaa, M Ryynänen,
P Sr Riekkinen, H Soininen
Objectives—In mild Alzheimer’s disease,
blood flow has highlighted deficits in the
posterior association cortex, and later in
the disease process, the deficit spreads to
involve the frontal cortex. The ?4 allele of
apolipoprotein E is a risk factor for Alzhe-
imer’s disease. The eVect of apolipopro-
perfusion was studied. The hypothesis was
that those patients with Alzheimer’s dis-
ease who carry the ?4 allele would have
more severe cerebral hypoperfusion.
Methods—Thirty one patients with Alzhe-
imer’s disease and eight age and sex
matched control subjects were examined
in a three year longitudinal study. Patients
with Alzheimer’s disease were divided into
subgroups according to their number of ?4
alleles. Regional cerebral blood flow ratios
referred to the cerebellum were examined
E genotypes were determined by digestion
of polymerase chain reaction products
with the restriction enzyme Hha1.
Results—All patients with Alzheimer’s
disease had bilateral temporoparietal hy-
poperfusion compared with control sub-
jects. The two ?4 allele subgroups had the
lowest ratios at the baseline assessment in
the parietal and occipital cortices, and at
the follow up in the temporal, parietal,
and occipital cortices. They had the high-
est reduction in percentage terms in the
temporal and occipital cortices compared
with the other subgroups. However, the
global clinical severity did not diVer at the
baseline or follow up examinations be-
tween the subgroups.
Conclusion—Apolipoprotein E polymor-
phism is involved in the pathogenesis and
heterogeneity of Alzheimer’s disease as
the most severe cerebral hypoperfusion
was found in the ?4 allele subgroups. This
might have implications for therapeutic
approaches in Alzheimer’s disease.
(J Neurol Neurosurg Psychiatry 1998;64:742–746)
99mTc-HMPAO SPECT. Apolipoprotein
disease; apolipoprotein E;
Clinical, neurochemical, pathological, and ge-
netic studies suggest that Alzheimer’s disease is
a heterogeneous entity.1–4The ?4 allele of
apolipoprotein E is a risk factor for Alzheimer’s
dementia.5–8The apolipoprotein E gene, lo-
cated on chromosome 19, has three major alle-
les: ?2, ?3, and ?4. Apolipoprotein E is present
in senile plaques, neurofibrillary tangles, and
ropathological changes seen in Alzheimer’s
disease. DiVerent binding properties of the
apolipoprotein isoforms to ?-amyloid and tau
protein also suggests that it is involved in the
pathogenesis of Alzheimer’s disease.9In addi-
tion, the extent of the deficit of acetylcholine
containing neurons in brains of patients with
Alzheimer‘s disease is related to the number of
?4 alleles present.10 11
Changes in the cerebral metabolism and per-
fusion can be detected by single photon
emission computed tomography (SPECT).The
decreased metabolism and reduced cerebral
perfusion in Alzheimer’s disease are known to
occur predominantly in temporal and parietal
regions which show the greatest
neurochemical postmortem abnormalities.15It
has been suggested that mildly aVected patients
whereas more severely demented patients also
show decreased blood flow in other regions—for
example,the occipital areas.14 16In a recent PET
study, subjects with age associated memory
impairment, one ?4 allele, and a family history
of Alzheimer’s disease had abnormally low and
asymmetric rates of glucose metabolism in a
preselected parietal region even before the onset
of dementia.17Another PET study on cogni-
tively normal subjects, with a family history of
Alzheimer’s disease reported that the ?4 ho-
mozygotes had significantly reduced rates of
glucose metabolism in the same posterior cingu-
late,parietal,temporal,and prefrontal regions as
seen in patients with probable Alzheimer’s
This study attempted to examine the role of
apolipoprotein E polymorphism in Alzheimer’s
disease with perfusion SPECT included in a
three year longitudinal follow up. Our hypoth-
esis was that those patients with Alzheimer’s
disease carrying the ?4 allele of apolipoprotein
E would have more severe deficits in their cer-
ebral perfusion compared with patients with-
out any ?4 alleles.
andaccelerates the onset of
Patients and methods
We studied 31 patients fulfilling the NINCDS-
ADRDA criteria of probable Alzheimer’s
disease19and eight age and sex matched cogni-
tively intact control subjects in a longitudinal
J Neurol Neurosurg Psychiatry 1998;64:742–746742
P Sr Riekkinen
Department of Clinical
Unit of Clinical
Genetics of the
and University of
Dr Maarit Lehtovirta,
Department of Neurology,
Kuopio University Hospital,
FIN 70211 Kuopio, Finland.
Fax 00358 17 173019.
Received 2 June 1997 and in
revised form 27 October
Accepted 29 October 1997
study, in which the follow up examination was
three years after the baseline assessment. Table
1 presents their clinical characteristics. The
patients with Alzheimer’s disease were at the
baseline either undergoing diagnostic examin-
ation or had been recently diagnosed. The eth-
ics committee of the Kuopio University Hospi-
tal approved the study and all the subjects and
caregivers of demented patients gave informed
consent for participation in the study.
The patients with Alzheimer’s disease under-
physical and clinical neurological examination,
assessment of clinical severity with mini mental
state examination (MMSE)20and brief cogni-
tive rating scale (BCRS),21
extrapyramidal signs with the Webster scale,22
assessment of depressive symptoms by the
laboratory tests to exclude secondary causes of
dementia, brain MRI and SPECT, conven-
tional and quantitative EEG, and event related
potentials.All patients with Alzheimer’s disease
scored less than 4 on the modified ischaemic
SINGLE PHOTON EMISSION COMPUTED
A dose of 370 to 555 MBq 99m Tc-HMPAO
(Amersham International, London, UK) was
intravenously injected into a subject’s vein in a
dark and quiet room. High resolution SPECT
was carried out with a three head Siemens
MultiSPECT 3 gamma camera equipped with
high resolution collimators.25Ten minutes after
injection of the tracer, the radioactivity distri-
bution of the brain was acquired in a 128×128
matrix mode. Data sets were acquired at 3°
intervals for 35 seconds each, with a total of 40
sets (120° per camera head). Three and a half
millimeter thick transaxial (oriented in orbito-
meatal line), sagittal, and coronal slices were
reconstructed using a Butterworth filter (a cut
oV frequency of 0.5 cm−1) and a uniform
attenuation correction of 0.12 cm−1. The imag-
ing resolution was 8–9 mm.25Two consecutive
slices were summed to obtain a slice thickness
of 7 mm and visually surveyed on a TV screen.
A semiautomatic brain quantification program
of Siemens Gammsonics Inc (Hofman, IL,
USA) was used to analyse the regions of inter-
est. Firstly, the slices were rotated and
realigned so that transaxial (x direction), sagit-
tal (y direction), and coronal (z direction) ones
were in 90° angles to each other. Secondly, the
regions of interest were drawn on to aligned
transaxial slices on the right and then mirrored
on the left. The method has been described
earlier in detail.26Regional count densities
(regional cerebral blood flow) were calculated
for frontal, temporal, parietal, and occipital
cortices. The regional counts were related to
the cerebellar counts and regional cerebral
blood flow is expressed as this ratio for each
region. Cerebellum was used to normalise the
other cortical areas, because it is generally
spared by major pathological involvement.27
The rater was not aware of the clinical data of
DETERMINATION OF APOLIPOPROTEIN E
DNA was prepared from blood leucocytes of
patients with Alzheimer’s disease by standard
procedures. Apolipoprotein E genotypes were
analysed using the polymerase chain reaction
(PCR)described earlier28 29
modifications.30Apolipoprotein E genotypes
were identified through HhaI digestion. Di-
gested DNA fragments were analysed via poly-
acrylamide gel electrophoresis and separated
fragments of DNA were visualised by staining
with ethidium bromide.
STATISTICAL ANALYSIS OF DATA
The data were analysed using SPSS for
Windows version 6.1.3. software. We used
analysis of variance (ANOVA) to detect diVer-
ences in means over the study groups at the
baseline and the follow up examination. The
Duncan method was applied in post hoc analy-
sis when appropriate. Multivariate analysis for
repeated measures (MANOVA) with Bonferro-
ni’s correction was used to detect the change in
perfusion ratios over time between the study
groups. Patients with Alzheimer’s disease were
divided into three subgroups according to the
number of apolipoprotein E alleles; two ?4
alleles, one ?4 allele, and no ?4 alleles. A ?2test
was used for evaluation of the categorical data.
The level of significance was set at p<0.05.
Patients with Alzheimer’s disease and control
subjects did not diVer in age, education, or sex.
Alzheimer’s disease subgroups had a similar
duration of dementia and equal clinical severity
as assessed with MMSE at the baseline and
follow up, the decline in MMSE was about six
points in three years. However, the ho-
mozygous ?4 allele subgroup had an earlier
onset of the disease and was younger than the
other subgroups (table 1).
Cerebral perfusion ratios measured with
SPECT for all patients with Alzheimer’s
disease were significantly reduced in tempo-
roparietal regions compared with control
subjects. Moreover, some diVerences were evi-
dent in the perfusion ratios for subgroups of
Alzheimer’s disease. The homozygous ?4 allele
subgroup had the lowest ratios at the baseline
in the parietal and occipital cortices, and at the
follow up in the temporal, parietal, and occipi-
tal cortices. The diVerence was significant at
the follow up in the right and left occipital
(AD) according to ?4 allele
Clinical characteristics of control subjects and patients with Alzheimer’s disease
Patients with AD
Age at onset (y)
MMSE base line
MMSE follow up
*p<0.05 v 2 ?4, ANOVA; ***p<0.001 v all other groups, ANOVA.
Values are expressed as mean (SD).
Longitudinal SPECT study in Alzheimer’s disease
cortices compared with the no ?4 allele
subgroup and control subjects. Also, at the fol-
low up, the one ?4 allele subgroup had a
significantly reduced perfusion ratio in the left
occipital cortex compared with the no ?4 allele
subgroup (table 2).
Multivariate analysis of variance for repeated
tests showed a significant eVect of the three
year follow up time on all regions studied. A
significant interaction eVect of time and diVer-
ent apolipoprotein E groups was found in the
right occipital cortex and the left parietal
cortex. In the right occipital cortex, the
perfusion ratio was significantly reduced in the
homozygous ?4 allele subgroup compared with
the no ?4 allele subgroup and in the left parietal
cortex in the one ?4 allele subgroup compared
with the control subjects (figure). The figure 1
shows, in percentage terms, the change be-
tween the baseline and follow up examination
in the diVerent study groups. The homozygous
?4 allele subgroup had the highest reduction in
the perfusion ratios in the temporal and the
occipital cortices compared with the other sub-
Patients with Alzheimer’s disease show cerebral
hypoperfusion, mainly in the temporal and
parietal cortices,12–14but 15%-20% of patients
cortices.31 32Occipital areas have been used as
reference regions for normalising perfusion
values, although PET and a few SPECT stud-
ies have also shown occipital changes in
patients with Alzheimer’s disease.14 33 34In our
three year longitudinal study, patients with
Alzheimer’s disease had decreased perfusion
ratios in the parietal and temporal regions
compared with the control subjects. When
patients with dementia were divided into
subgroups according to the ?4 allele, the
homozygous ?4 allele subgroup had the lowest
perfusion ratios in the parietal and occipital
cortices at the baseline, and at the follow up
this was evident also in the temporal cortices.
The ?4 allele of apolipoprotein E is a well
established risk factor for Alzheimer’s disease.
In both patients with Alzheimer’s disease and
non-demented elderly subjects, memory func-
tions and medial temporal lobe structures,
such as the hippocampus, seem to be particu-
larly vulnerable to the deleterious eVects of the
?4 allele of apolipoprotein E.29 35–37Two PET
studies in non-demented relatives at risk for
familial dementia showed reduced rates of glu-
cose metabolism in the same regions where this
occurs in patients with Alzheimer’s disease.17 18
These findings suggest that identification of ?4
homozygous subjects with memory impair-
ment might help to identify those at highest
according to the number of the ?4 alleles
Regional cerebral blood flow related to cerebellum:control subjects and subgroups with Alzheimer’s disease (AD)
Patients with AD
Control subjects (n=8)2 ?4 (n=8)1 ?4 (n=13)0 ?4 (n=10) all AD (n=31)
Right frontal cortex:
Left frontal cortex:
Right temporal cortex:
Left temporal cortex:
Right parietal cortex:
Left parietal cortex:
Right occipital cortex:
Left occipital cortex:
*p<0.05 v controls, †v 0 ?4 subgroup, ANOVA.
Values are expressed as mean (SD).
Percentage changes between the baseline and the follow up examinations in control subjects
and the subgroups with Alzheimer’s disease according to the number of the ?4 alleles.
RFC=right frontal cortex;LFC=left frontal cortex;RTC=right temporal cortex;LTC=left
temporal cortex;RPC=right parietal cortex;LPC=left parietal cortex;ROC=right occipital
cortex;LOC=left occipital cortex.MANOVA p<0.05.* DiVers from control subjects;o
diVers from no ?4 subgroup.
RFC LFC RTC LTC RPC LPC
0 ε4 alleles 1 ε4 allele 2 ε4 alleles
risk for dementia, and locate a possible group
for treatment strategies to prevent the disease.
The homozygous ?4 subgroup had the
greatest reduction in percentage terms in the
follow up in the temporal and occipital cortices
compared with other subgroups (figure). An
earlier two year longitudinal SPECT study
reported that the decreases in regional cerebral
function, compared with control values varied
from 1% to 4.2% in the diVerent regions.38In
our three year follow up study, the perfusion
values for all patients with Alzheimer’s disease
compared with controls, decreased on average,
between 2.4% and 9%. In our study, at the
baseline and follow up examinations,the extent
of global disease was equally severe in the
Alzheimer subgroups. Moreover, the
mozygous ?4 allele subgroup did not diVer
from other subgroups in visuospatial functions
as examined with a copy a cube and clock set-
ting test39and the block design subtest of the
Wechsler adult intelligence scale.40However,
the homozygous ?4 allele subgroup had the
lowest scores on immediate and delayed tests
assessing verbal and visual memory. Verbal
memory was examined with a list learning test
using shopping items41and the Wechsler logical
memory test using one story.42Visual memory
was examined with the Heaton visual repro-
duction test.43The delayed recall of the story
and the figures and a yes/no recognition of the
words in the list were asked after a 30 minute
delay filled with other psychometric tests.
Occipital hypoperfusion is only occasionally
found in most studies of Alzheimer’s disease.
Recently, a PET study of three patients with
pathologically verified pure diVuse Lewy body
disease and three patients with combined Lewy
body and Alzheimer’s disease pathology dis-
closed pronounced declines in association and
primary somatomotor cortices in both groups.
However, the patients with pure Lewy body
disease also exhibited hypometabolism in the
cortex.44Patients with Lewy body disease are
thought to have a more severe cholinergic defi-
cit than patients with Alzheimer’s disease.45
Also, recent data suggest that the magnitude of
the cholinergic depletion is greater in ?4
disease.10 11The cerebral blood flow is regu-
lated by cholinergic, noradrenergic, and sero-
tonergic pathways.46As the density of choliner-
gic innervation is lower in the primary sensory
areas—such as the occipital cortex—than in
structures,47it is possible that the more severe
cholinergic depletion in the ?4 homozygotes
may be reflected in early phases as hypoper-
fusion in those regions with reduced choliner-
In conclusion, we found that the ho-
mozygous ?4 patients with Alzheimer’s disease
had the lowest cerebral perfusion in the tempo-
ral, parietal, and occipital cortices compared
with those patients with Alzheimer’s with one
or no ?4 alleles. Apolipoprotein E polymor-
phism seems to be a major contributor to the
heterogeneity of Alzheimer’s disease, and this
might have implications for therapeutic ap-
proaches in this disorder.
This study was supported by grant from the Health Research
Council of the Academy of Finland and the North Savo
Regional Fund of the Finnish Cultural Foundation.
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