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In central nervous system cholesterol cannot be degraded but is secreted into circulation predominantly in the form of its polar metabolite 24(S)-hydroxycholesterol (24S-OH-Chol). Some studies suggested an association between 24S-OH-Chol metabolism and different neurological diseases including dementia. A possible decrease in 24S-OH-Chol plasma levels has been reported late onset Alzheimer's disease (LOAD) and vascular dementia (VD), but results of previous studies are partially contradictory. By high-speed liquid chromatography/tandem mass spectrometry we evaluated the plasma levels of 24S-OH-Chol in a sample of 160 older individuals: 60 patients with LOAD, 35 patients with VD, 25 subjects affected by cognitive impairment no-dementia (CIND), and 40 (144 for genetics study) cognitively normal Controls. We also investigated the possible association between PPARgamma Pro12Ala polymorphism and dementia or 24S-OH-Chol levels. Compared with Controls, plasma 24S-OH-Chol levels were higher in LOAD and lower in VD; a slight not-significant increase in CIND was observed (ANOVA p: 0.001). A positive correlation between 24S-OH-Chol/TC ratio and plasma C reactive protein (CRP) levels was found in the whole sample, independent of possible confounders (multiple regression p: 0.04; r2: 0.10). This correlation was strong in LOAD (r: 0.39), still present in CIND (r: 0.20), but was absent in VD patients (r: 0.08). The PPARgamma Pro12Ala polymorphism was not associated with the diagnosis of LOAD, VD, or CIND; no correlation emerged between the Ala allele and 24S-OH-Chol plasma levels. Our results suggest that plasma 24S-OH-Chol levels might be increased in the first stages of LOAD, and this phenomenon might be related with systemic inflammation. The finding of lower 24S-OH-Chol concentrations in VD might be related with a more advanced stage of VD compared with LOAD in our sample, and/or to different pathogenetic mechanisms and evolution of these two forms of dementia.
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RESEARCH ARTICLE Open Access
Plasma 24S-hydroxycholesterol levels in elderly
subjects with late onset Alzheimers disease or
vascular dementia: a case-control study
Giovanni Zuliani
1,2*
, Michela Perrone Donnorso
3
, Cristina Bosi
1
, Angelina Passaro
1
, Edoardo Dalla Nora
1
,
Amedeo Zurlo
4
, Francesco Bonetti
1
, Alessia F Mozzi
3
and Claudio Cortese
3
Abstract
Background: In central nervous system cholesterol cannot be degraded but is secreted into circulation
predominantly in the form of its polar metabolite 24(S)-hydroxycholesterol (24S-OH-Chol). Some studies suggested
an association between 24S-OH-Chol metabolism and different neurological diseases including demen tia. A
possible decrease in 24S -OH-Chol plasma levels has been reported late onset Alzheimers disease (LOAD) and
vascular dementia (VD), but results of previous studies are partially contradictory.
Methods: By high-speed liquid chromatography/tandem mass spectrometry we evaluated the plasma levels of
24S-OH-Chol in a sample of 160 older individuals: 60 patients with LOAD, 35 patients with VD, 25 subjects affected
by cognitive impairment no-dementia (CIND), and 40 (144 for genetics study) cognitively normal Controls. We also
investigated the possible association between PPARgamma Pro12Ala polymorphism and dementia or 24S -OH-Chol
levels.
Results: Compared with Controls, plasma 24S-OH-Chol levels were higher in LOAD and lower in VD; a slight not-
significant increase in CIND was observed (ANOVA p: 0.001). A positive correlation between 24S-OH-Chol/TC ratio
and plasma C reactive protein (CRP) levels was found in the whole sample, independent of possible confounders
(multiple regression p: 0.04; r
2
: 0.10). This correlation was strong in LOAD (r: 0.39), still present in CIND (r: 0.20), but
was absent in VD patients (r: 0.08). The PPARgamma Pro12Ala polymorphism was not associated with the diagnosis
of LOAD, VD, or CIND; no correlation emerged between the Ala allele and 24S-OH-Chol plasma levels.
Conclusions: Our results suggest that plasma 24S-OH-Chol levels might be increased in the first stages of LOAD,
and this phenomenon might be related with systemic inflammation. The finding of lower 24S-OH-Chol
concentrations in VD might be related with a more advance d stage of VD compared with LOAD in our sample,
and/or to different pathogenetic mecha nisms and evolution of these two forms of dementia.
Background
Late onset Alzheimer s disease (LOAD) is the most
common form of dementia in older individuals living in
Western countries, followed by vascular dementia (VD)
[1,2]. Besides a definite role in the pathogenesis of
atherosclerosis, and of consequence of some types of
VD, disturbances of cholesterol homeostasis mi ght have
a role also in the development of LOAD [3,4]. In central
nervous system (CNS) cholesterol originates a lmost
exclusively from in situ synthesis [5], while circulating
cholesterol is normally prevented from entering the
CNS by the blood-brain-barrier [4]. As cholesterol can-
not be eliminated in CNS, and may be toxic to neurons
when in excess, it is secreted from CNS into the circula-
tion predominantly in the form of its polar metabolite
24(S)-hydroxycholesterol (24S-OH-Chol) [6].
Interestingly , some studie s have suggested a p ossible
association between 24S-OH-C hol metabolism a nd dif-
ferent neurological diseases [7]. As most of th e circula t-
ing 24S-OH-Chol originates in the brain, it has been
* Correspondence: gzuliani@hotmail.com
1
Department of Clinical and Experimental Medicine, Section of Internal
Medicine, Gerontology and Clinical Nutrition; Azienda Ospedaliero-
Universitaria Arcispedale S. Anna, Ferrara, Italy
Full list of author information is available at the end of the article
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© 2011 Zuliani et al; licensee BioMed Central Ltd. This is an Open Access article distribute d under the terms of the Creative Co mmons
Attribution License (http://creativec ommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, pro vided the original work is properly cited.
proposed that the concentrations of 24S-OH-Chol in
cerebrospinal fluid and/or plasma might represent per-
ipheral markers of neuronal degeneration occurring in
primary diseases of CNS [8].
It has been reported that 24S-OH-Chol levels were
higher in cerebrospinal fluid from LOAD patients com-
pared to controls [9,10]. On the contrary, 24S-OH-Chol
concentrations were found to be low in brain samples
obtained from deceased patients with LOAD in
advanced stage [11].
Plasma levels of 24S-OH-Chol should reflect the num-
berofactiveneuronsinthebrainandthus,thevolume
of the grey matter structures. In different neurodegen-
erative disorders plasma 24S-OH-Chol was found to be
reduced, proportionally to the degree of brain at rophy
as measured by MRI [8]. As regards dementia , only a
few studies co nducted on small samples of patients have
investigated this specific topic. At first, slightly higher
24S-OH-Chol l evels have been f ound, bot h in pa tients
with LOAD and VD [12]. Subsequent studies gave dif-
ferent results re porting no rmal [10] or even decreased
24S-OH-Chol levels in patients with LOAD or VD com-
pared with controls [13-15]. Of consequence, findings
about 24S-OH-Chol plasma levels in patients affected by
LOAD and VD are not definite; indeed, it is possible
that brain cholesterol and thus 24S-OH-Chol plasma
levels mi ght change over time along these two neurolo-
gical diseases.
Some studies have also investigated the possible influ-
ence of common DNA polymorphisms on 24S-OH-Chol
plasma levels or the risk of b eing affected by LOAD. In
particular, the genes coding for the 24S-hydroxylase
(CYP46) and for the peroxiso me proliferator-activated
receptor gamma (PPARgamma) have been studied
[16,17].
In the present study we evaluated, b y high-speed
liquid chromatography/tandem mass spect rometry, the
plasma levels of 24S-OH-Chol in a sample of older indi-
viduals affected by LOAD, VD, or cognitive impairment
no dementia (CIND); successively, we investigated t he
possible ass ociation between the PPARgamma Pro12Ala
polymorphism and 24S-OH-Chol levels.
Methods
Subjects
On the whole, 120 consecut ive patients referring to the
Day Hospital service for the study of cognitive decline
(Institute of Internal Medicine, Gerontology and Clinical
Nutrition; Geriatric Unit, S. Anna University-Hospital,
Ferrara, Italy ) wer e enr olled. All data were coll ected
during a three years period (2006-2009). The sample
included:
1) Sixty patients with LOAD (mean age: 78 ± 5.5 SD
years; females: 78%) by the NINCDS-ADRDA criteria
[18]. Only patients with probable LOAD were included;
patients with possible LOAD or with LOAD associated
with significant cerebrovascular disea se on CT scan w ere
excluded in order to increase specificity. The Global
Deterioration Scale ranged from stage 3 to 5.
2) Thirty five patients with VD (mean age: 77 ± 7.6 SD
years; females: 60%) by the NINDS-AIREN criteria [19].
Only patients with probable VD were enrolled. The
Global Deterioration Scale ranged from stage 4 to 6.
3) Twenty five patients with cognitive impairment no
dementia (CIND) (mean age: 74 ± 8.2 SD years; females:
44.5%). CIND was defined as the presence of short/
long-term memory impairment and/or impairment in
other single or multiple cognitive domains in an indivi-
dual who didn t meet the standardized criteria for
dementia. We also required that the patient with CIND
would be still independent in the activities of daily living
(ADLs). Most of these individuals were affected by
amnesic multi-domain cognitive impairment.
No Cerebrospinal fluid (CSF) biomarkers were avail-
able for all the patients (LOAD, VD, and CIND)
enrolled into this study.
Forty cognitively normal older individuals were
enrolled as controls (C). Their mean age was 72 ± 8.7
SD years; females represented 68% of the sample. All
these indivi duals w ere free-living, healthy (no important
comorbidity was found), and were independent on
ADLs (mean Barthel score: 96/100). Their average
MMSE s core was 28/30. In case of memory complains,
a battery of tests was administered, as previously
described [20].
For the evaluation of the PPARgamma Pro12Ala poly-
morphisms, additional 104 normal older individuals
were enrolled (mean age: 77 ± 9.1 SD years; females:
52.6%).
Personal data and medical history were collected b y a
structured interview f rom patients and caregivers. All
patients underwent a general and neurological examina-
tion; clinical-chemistry analyses were performed to
exclude causes of seconda ry cognitive impairment. The
diagnosi s of dementia was made by trained geriatricians;
for ne uropsychological assessment, all patients were
given a battery of tests [20]. Subjects affe cted by severe
congestive heart failure (New York Heart Association
class III-IV), severe liver or kidney disease, severe
chronic obstructive pulmonary disease, and ca ncer we re
excluded. There were no evidences of acute illnesses at
the time o f clinical observation in DH and blood
sampling.
The criteria for the diagnosis of diabetes were: 1) his-
tory of diabetes or current hypoglycaemic therapy; 2)
fasting glycaemia > 126 mg/dl in two or m ore measure-
ments; 3) glycaemia > 200 mg/dl at 120 min after oral
glucoseload.Thecriteriaforthediagnosisof
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hypertension were: 1) history of hypertensi on or anti hy-
pertensive the rapy at visit time; 2) blood pressure >
140/90 mmHg in three or more measurements. The
diagnosis of coronary heart disease (CHD) was made in
the presence of a documented history of previous myo-
cardial infarction or angina. No patients were taking a
statin at the time of enrolment into the study. All sub-
jects (and/or their caregiver if demented) were informed
about t he research project during the first visit, and
gave their written consent in order to participate to the
study. The study was approved by the local ethic com-
mittee (S. Anna University Hospital, Ferrara, I taly) and
was conducte d in accordance with the Helsinki Declara-
tion as revised in 1989.
Brain CT scan
All p atients (LOAD, VD, and CIND) underwent a brain
CT. The instrument used was a third generation SIE-
MENS SOMATON HQ. The slice thickness was 10
mm. Radiograms were evaluated by trained radiologists
not informed about the clinical characteristics of the
patient. CT scan information was used to support the
clinical diagnosis, and to exclude possible brain patholo-
gies associated with cognitive impairment. CT scan was
also used for a qualitative evaluation of brain morphol-
ogy. Brain atrophy was asse ssed through direct/indirect
signs of neuronal loss. Lacunar infarcts were defined as
demarcated lesions with a diameter < 15 mm involving
deep regions such as internal capsulae, basal ganglia,
corona radiata or thalamus. Cortical infarctions were
defined as the presen ce of larg e cortical- subcortical
infarctions resulting from the occlusion of large cerebral
arteries.
Plasma 24S-OH-Chol concentrations measurement
Blood was drown in the morning after a 12-hours over-
night fasting, and after the patient has been sedentary in
sitting or supine position for at least 10 minutes. After
having been aliquoted, plasma was frozen and stored at
-80°C until the tests were performed.
Stock solutions of 24S-OH-Chol and racemic
24OHChol-d
6
internal standard (IS), obtained from
Avanti Polar Lipids, were prepared in toluene at a con-
cent ration of 1 mg/mL. Samples were purified following
a procedure previously described by DeBarber et al.
[21], after adding 5 μLof5μg/mL isopropanol IS work-
ing solution, to 500 μL of serum. The dried organic
extract residue was reconstituted in 125 μL of acetoni-
trile/methanol (30/70 v/v). 24S-OH-Chol concentration
was determined using a linear 7-point calibration curve
ranging from 7.85 to 500 ng/mL, prepared evaporating
an aliquot of the 24S-OH-Chol working isopropanol
solutions und er flow of pure N
2
and redissolving t he
dried sample with an appropriate volume of acetonitrile/
methanol (30/70 v/v). 24S-OH-Chol assay was per-
formed using an ultra performance liquid cro matograph
(Dionex Ultimate 3000 RS) coupled with a mass spec-
trometer (AB SCIEX Triple Quadrupole API 3000),
equipped wit h an atmospheric pressure chemical ioniza-
tion (APCI) source and operating in multiple reaction
monitoring in positive mode. Tra nsition from m/z 385.4
to m/z 3 67.5 ion was monitored for 24S-OH-Chol an d
transition from m/z 391.4 to m/z 373.5 ion for
24OHChol-d
6.
Chromatographic separation was per-
formed using a Pinnacle DB C18 column (1, 9 um parti-
cle size; 100 × 2.1 mm i.d.; Restek) at a temperature of
50°C, with a mobile phase consisted of MeOH/CH
3
CN/
H
2
O 45/45/10+ 0.1% acetic a cid (phase A) and CH
2
Cl/
MeOH (80/20 v/v) (phase B). The eluition gradient was
programmed a s follows (flow rate, 0.8 mL/min): 100%
mobile phase A for 5 minute, increased to 100% B in 2
minutes, 5 minutes at 100% B, decreased to 100% A in
1 minute and then reequilibrated with 100% A for 10
minute until the next sample was injected. The linear
regression constant (r) was more than 0.999 in the lin-
ear ra nge 7.85-500 mg/mL. Calibrators accuracy and
interday precision (CV%) ranged respectively from
95.81%-108.5% and 0.6%-11.96%. Recovery was between
102,68% and 95,92%. Intra- and interday precision were
respectively less than 5% and 8% for calculated 24S-OH-
Chol. The detection limit was 1 ng/mL and the quantifi-
cation limit 5 ng/mL. 24S-OH-Chol was reported as
plasma levels (ng/ml).
Plasma cholesterol measurement
Plasma concentrat ions of total cholesterol (TC) were
measured by standard enzymatic procedures (CHOD-
PAP Method, Boehringer, Mannheim, Germany).
PPARgamma Pro12Ala genotyping
Genomic DNA was extracted from blood leukocytes by
the salting out method. The Pro12Ala PPARgamma var-
iant was detected by polymerase chain reaction-re stric-
tion fragment length polymorphism (PCR-RFLP)
analysis. This analysis a mutagenic PCR primer is used
tointroduceaBstUIrestrictionsiteonlywhenaC®G
substi tution at nucleotide 34 of the PPARgamma 2 gene
is present [22]. Genotyping was repeated for all Ala12
homozygotes and Pro12Ala heterozygotes; reproducibil-
ity was 100%.
Statistical analysis
Continuous variables were expressed as mean (SD) or
median (i nterquartile range) when necessary. The distri-
bution of 24S-OH-Cholesterol values was skewed; con-
sequently, data were expressed as median values
(interquartile range). Means were compared by ANOVA
using the Fishers least significant difference (LSD) test
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for post-hoc analysis. 24S-OH-Cholesterol values were
LOG-tranformed before entering ANOVA. Bivariate
correlations were tested by the Pearsons test. Prevalence
was compared by the c
2
test. Multivariate linear regres-
sion analysis (method stepwise forward) was used to test
the association between the 24S-OH-Chol/TC ratio and
other variables previously selected by univariate analysis.
Dicotomous variables were included as dummy variables
(0: absent; 1: present).
SPSS for Windows, version 7.0 (SP SS, Inc, Chicago,
IL)statistical packages were used.
Results
In Table 1 are reported the general characteristics and
the plasma levels of 24S-OH-Chol in patients with
LOAD, VD, CIND, and in C. The prevalence of female
gender was h igher in LOAD and lower in VD compared
with the other groups. Mean age was lower, while MMSE
score was higher in C compared with the other groups.
The Barthel index score was higher in C and CIND com-
pared with LOAD and VD. Brain atrop hy on C T scan
was more frequently reported in LOAD patients, while
ischemic lesions (both lacunar and cortical infarcts) were
more frequent in VD (Chi square: all p: 0.001).
Compared with C, plasma 24S-OH-Chol levels were
higher in LOAD (LSD post-hoc test p: 0. 01) and lower
in VD (LSD p: 0.05), while no differences were observed
as regards the CIND group (model ANOVA p < 0.001).
The distribution of 24S-OH-Chol levels (boxplots) in
Controls and patients affected by VD, LOAD or CIND
is reported in Figure 1.
In Table 2 are reported the correlation s between the
24S-OH-Chol and other varia bles observed in the whole
sample (n. 160 subjects). Since 24S-OH-Chol and TC
plasma levels are known to be correlated [4] (in our
sample r: 0.28, p: 0.005) as they are both transported by
the low-density lipoproteins, 24S-OH-Chol values were
adjusted for TC levels by calculating the 24S-OH-Chol/
TC ratio (ng/mg). Indeed, it has been su ggested that the
24S-OH-Chol/TC ratio may better reflect brain choles-
terol homeostasis than 24S-OH-Chol absolute level [4].
The 24S-OH-Chol/TC ratio was significantly corre-
lated with serum albumin (r: -020; p: 0.03), and hs.CPR
levels (r: 0.33; p: 0.001); no significant correlations
emerged with age, Barthel index, and creatinine levels.
Interestingly, the 24S-OH-Chol/TC ratio correlate d
negatively with the Babcock test score (both immediat e
and delayed recall), and positively with the Frontal
Assessment Battery (FAB) score. In both cases, the
higher the 24S-OH-Chol/TC ratio, the worse the perfor-
mance obtained in neuropsychological tests.
As regards TC scan findings, 24S-OH-Chol/TC ratio
was positively related to the presence of brain atrophy.
By multivariate linear regression analysis we demon-
strated that the 24S-O H-Chol/TC ratio was significantly
correlated with hs.CRP independent of age, albumin
Table 1 General characteristics and plasma levels of 24S-OH-Chol in patients with Late Onset Alzheimers Disease
(LOAD), Vascular Dementia (VD), Cognitive Impairment No Dementia (CIND), and in older controls.
Parameter LOAD VD CIND Controls
(n. 60) (n. 35) (n. 25) (n. 40)
Age (years)* 78 ± 5.5 77 ± 7.6 74 ± 8.2 73 ± 8.7
Female gender ^ 78,00% 53,00% 60,00% 68,00%
MMSE score (mean/SD)* 21.3 ± 3.8 21.7 ± 3.9 25 ± 3.1 28 ± 2
Barthel index § 82.5/100 79/100 96/100 96/100
GDS 5.7/15 5.3/15 6.3/15 6.2/15
Tot. Chol. (mg/dL) 210 ± 42 204 ± 32 207 ± 43 215 ± 28
Albumin (g/dL) 4.14 ± 0.38 4.11 ± 0.37 4.08 ± 0.34 4.22 ± 0.31
Hypertension 67,00% 83,00% 63,00% 67,00%
Diabetes 15,00% 34.5% 18,00% 19,00%
CHD ^ 16,00% 34.5% 12,00% 9.5%
24S-OH-Chol (ng/ml)° 51.0 (42.0-64.7) 39.1 (36.0-46.5) 47.0 (42.6-56.3) 46.3 (37.5-52.7)
Brain atrophy ^^ 84% 53% 51% 47%
Single lacune ^^ 5% 22% 7% 6.5%
Multiple lacunes ^^ 0% 55% 20% 10%
Cortical infarcts ^^ 0% 23% 11% 6%
* ANOVA (model P < 0.001) Fishers least significant difference (LSD) post-hoc test: C vs LOAD p < 0.01; C vs VD p < 0.05; VD vs LOAD and CIND p < 0.005
§ ANOVA (model P < 0.001) LSD p < 0.05 C and CIND vs LOAD and VD
^Chi square test p < 0.05; ^^Chi square tes t p < 0.001
° ANOVA (model P < 0.001) LSD p < 0.01 LOAD vs C; p < 0.05 VD vs C and CIND; p < 0.01 for LOAD vs VD
MMSE: min i mental state examination; GDS: geriatric depression scale; CHD: coronary heart disease. For 24S-OH-Chol: median (interquartile range)
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levels, brain atrophy, Babcock test, and FAB score
(ANOVA: p:0.04; r
2
: 0.104).
In Table 3 are reported the results of PPARgamma
Pro12Ala polymorphism genotyping in patients with
LOAD, VD, or CIND, and in 144 older contr ols. Th e
distribution of the two alleles was in Hardy-Weinberg
equilibrium. No differences emerged by comparing their
distribution in the four groups of su bjects. Furthermore,
no differences in median/mean plasma 2 4S-OH-Chol
levels were found when individuals bearing the Ala allele
(heterozygous + homozygous) were compared with Pro/
Pro homozygote subjects (data not shown).
Discussion
We evaluated the plasma levels of 24S-OH-Chol in a
sample of aging patient s affe cted by two different forms
Figure 1 24 Shydroxycholesterol p lasma levels ( boxplots) i n older normal indiv iduals (C ontrols) and in older s ubjects affected by
vascular dementia (VD), late onset Alzheimers disease (LOAD) or cognitive impairment-no dementia (CIND). The dashed line represents
the median value of plasma 24Shydroxycholesterol in controls (46.3 ng/ml).
Table 2 Pearsons correlations between the 24S-OH-Chol/
TC ratio and other variables in the whole sample (160
individuals).
Variable R P
Age 0.11 0.18
Barthel index -0.06 0.70
Serum Albumin -0.20 0.03
Serum Creatinine 0.04 0.96
Hs.CRP 0.33 0.001
MMSE -0.07 0.69
Rey test (short) -0.14 0.17
Rey test (long) -0.15 0.14
Token test -0.06 0.71
Verbal fluency (letter) -0.02 0.79
Verbal fluency (category) -0.11 0.27
Babcock (immediate) -0.29 0.01
Babcock (delayed) -0.22 0.03
FAB 0.26 0.04
Trail making A 0.18 0.31
Trail making B 0.03 0.89
CT SCAN IMAGING
- Atrophy 0.17 0.05
- Cortical infarction -0.04 0.70
- Single lacune 0.01 0.84
- Multiple lacunes -0.16 0.06
MMSE: min i mental state examination; FAB: frontal assessment battery
Table 3 PPARgamma Pro12Ala polymorphism in older
patients with LOAD, VD, CIND, and in older controls.
LOAD VD CIND Controls
(n. 60) (n. 35) (n. 25) (n. 144)
Pro allele 0.92 0.91 0.89 0.92
Ala allele 0.08 0.09 0.11 0.08
Homo Pro/Pro 48 29 19 126
Etero Pro/Ala 10 6 6 16
Homo Ala/Ala 200 2
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of deme ntia, LOAD and VD, an d in individual s with
CIND. The first interesting data emerging from our
study is the finding of higher circulating levels of 24S-
OH-Chol in patients with LOAD compared with Con-
trols. Our results seem to confirm the unique observa-
tion by Lutjohann et al. [12], while are in contrast with
fol lowing studies reporting decreased levels of 24S-OH-
Chol in LOAD patients [13-15]. Unlike that study [12],
we were not able to demonstrate in LOAD patients a
significant correlation between 24S-OH-Chol levels and
the global cog nitive function (MMSE) or functional
impairment (Barthel inde x), perhaps because our sub-
jects were not in the very-early stage of the disease
(Global Deterioration Scale: stage 3 to 5). Our data sup-
port the hypothesis of a trend toward higher levels of
plasma 24S-OH-Chol in the early stages of LOAD,
when the rate of neurodegeneration is higher than nor-
mal, but t he amount of cell loss and resulting brain
atrophy is still small. The precise mechanisms leading to
increased plasma 24S-OH-Chol levels in the early stages
of LOAD are not known. It is possible that, in these
specific conditions, cholesterol turnover might be
increased i n CNS (due to neuronal degradation) and/or
that the rate of conversion of brain cholesterol to 24S-
OH-Chol might be higher compared to normal.
Interestingly, it has been observed by Brown et al. that
24S-OH-Chol is an efficient inhibitor of beta-amyloid
formationinvitro[23];ifthisistruealsounderinvivo
conditions, the increase of 24S-OH-Chol levels in CNS
(and consequently in the circulation) might be consid-
ered as an early attempt t o counteract beta-amyloid
deposition. On the contrary, in the advanced stages of
the disease low levels of 24S-OH-Chol in CNS (and
consequently in the circulation), would even accelerate
beta-amyloid deposition and the progression of LOAD.
In part, increased 24S-OH-Chol levels might also
result from a defect in blood-brain barrier, which seems
to be a frequent finding in different neurological dis-
eases including LOAD [24].
Unlike the study of Lutjohann et al. [12], but in agree-
ment with ot her following clinical observations [13-15],
we found that plasma 24S-OH-Chol levels were lower in
VD patients compared with controls. The difference we
observed between LOAD and VD is somehow unex-
pected, and might be related to different mechanisms.
In particular, it has to be underlined that, on avera ge,
our VD patients were in a more advanced stage of the
disease (GDS: stage 4-6) compared with LOAD (GDS:
stage 3-5). Actually, not only it has been reported that a
decrease in plasma levels of 24S-OH-Chol would be
typical of dementia [25,8], but it has been also shown
that plasma 24S-OH-Chol levels progressively decrease
with the worsening of the disease [4]. Thus, the differ-
ences between LOAD and VD might be se condary to a
different stage of disease. A lternatively, the differences
in levels of 24S-OH-Chol between LOAD and VD might
reflect the different pathogenetic mechanisms and/or
evolution of these two forms of dementia.
We also evaluated the possible relationship between
24S-OH-Chol and other available variables. Interestingly,
by multivariate regression analysis we found that the
24S-OH-Chol/TC ratio independently correlated with
hs.CRP levels, and this explained about 10% of its total
variability. Hs.CRP is a sensitive marker of sy stemic
inflammation. The assoc iation between the 24S-OH-
Chol and inflammation has been already reported in
vitro [26]; indeed, Alexandrov found that in primary co-
culture of h uman neurons and glia, 24S-OH-Chol is
able to induce the expression of several pro-inflamma-
torygenes.Thepresenceofalow-gradesystemic
inflamma tion has been reported both in LOAD and VD
by several Authors, including our group [20]. The find-
ing of a significant a ssociation between 24S-OH-Chol
and CRP levels suggest a possible link between the
degree of neurodegeneration (plasma 24S-OH-Chol
would be a marker of it) and the degree of peripheral
system ic inflammation. As a matter of fact, the relation-
ship between 24S-OH-Chol/TC and CRP was strong in
LOAD (r: 0.39), was still present in C IND (r: 0.20 ), but
it was pratically absent in VD patients (r: 0.08). The spe-
cifici ty of the relationship between LOAD and increa sed
24S-OH-Chol plasma concentrations might be indirectly
supported by the result of univariate analysis (Table 2)
showing a significant correlation of 24S-OH-Chol with
memory tests impairment and brain atrophy on CT
scan, both typical characteristics of LOAD but not of
VD.
Finally,weevaluatedthepossibleeffectofthePPAR-
gamma Pro12Ala poly morphism on the risk of being
affected by dementia or CIND, as well as on 24S-OH-
Chol plasma levels. Infact, it has been consistently
reported that PPARgamma plays an import ant role in
glucose and lipid met abolism[27],whichinturnhave
been associated with LOAD [28,29]. We found no as so-
ciation between the PPARgamma poly morphism and
LOAD, VD or CIND. Furt hermore, unlike Sauder [17]
we didn t find any increase in 24S-OH-Chol/TC ratio
among the carriers of the Ala allele both in the whole
sample and in the three groups of patients.
The principal limitations of the study must be also
acknowledged. First, br ain morphology was evaluated by
qualitative CT scan assessment and not by quantitativ e
MRI analysis, which is t he best validated method and
would give important information. Second, CSF biomar-
kers were not ava ilable for LOAD, VD and CIND
patients enrolled into this study, and this might signifi-
cantly influence sensibility and specificity of our diag-
noses. Third, we did not systematically evaluated Apo E
Zuliani et al. BMC Neurology 2011, 11:121
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Page 6 of 8
polymorphism in our sample, and it is probable that apo
E phenotype m ight inf luenc e plas ma levels of 24S-OH-
Chol. Nevertheless, we evaluated apo E genotype in
DNA from 70% of patients (84/120); we found no differ-
ences in mean/median 24S-OH-Chol levels by compar-
ing patients bearing or not the ε4 allele (ANOVA -
Mann-Whitney test p: 0.86 and 0.96, respectively).
Fourth, although we investigated plasma 24S-OH-Chol
concentrations in a much larger sample of subjects com-
pared with previous studies, sample size might be small
when investigat ing the po ssible effect of genetics. For
this reason our negative data on PPARgamma poly-
morphism need to be replicated in larger groups of
individuals.
Conclusions
In conclusion, the results of the present study suggest
that 24S-OH-Chol plasma levels might be i ncreased in
thefirststagesofLOAD,andmaybecorrelatedwith
systemic inflammation.
The finding of lower 24S-OH-Chol concentrations in
VD might be related with a more advanced stage of VD
compared with LOAD in our sample, and/or to different
pathogenetic mechanisms and evolution of these two
forms of dementia.
Author details
1
Department of Clinical and Experimental Medicine, Section of Internal
Medicine, Gerontology and Clinical Nutrition; Azienda Ospedaliero-
Universitaria Arcispedale S. Anna, Ferrara, Italy.
2
Associazione Alzheimer-
Perusini, Ferrara, Italy.
3
Department of Clinical Biochemistry and Molecular
Biology, University of Rome 2, Tor Vergata, Italy.
4
Geriatrics Division; Azienda
Ospedaliero-Universitaria Arcispedale S. Anna, Ferrara, Italy.
Authors contributions
GZ contributed to conception, design, and acquisition of data and
manuscript drafting; MPD participated to analysis and interpre tation of data
and revised the manuscript critically; CB contributed to acquisition and
interpretation of data and revised the manuscript critically; AP participated
to analysis and interpretation of data and manuscript drafting; EDN
contributed to conception and interpretation of data and revised the
manuscript critically; AZ participated to conception and interpretation of
data, and revised the manuscript critically; FB contributed to conception of
data and revised the manuscript critically; AFM participated to analysis and
interpretation of data and revised the manuscript critically; CC contributed
to conception, analysis, and interpretation of data and revised the
manuscript critically. All the Authors gave the final approval to the version
to be published.
Competing interests
The authors declare that they have no competing interests.
Received: 9 March 2011 Accepted: 4 October 2011
Published: 4 October 2011
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Cite this article as: Zuliani et al.: Plasma 24S-hydroxycholesterol levels in
elderly subjects with late onset Alzheimers disease or vascular
dementia: a case-control study. BMC Neurology 2011 11:121.
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... A negative correlation between plasma 24-OHC levels and AD severity was found in another investigation, although in this case 24-OHC levels were higher in AD patients than in healthy controls [78]. A subsequent study partially confirmed this evidence, reporting increased plasma levels of 24-OHC in the early stage of late-onset AD patients compared to controls [79]. In contrast, plasma 24-OHC content was found to be lower in probable AD patients compared to controls, but this reduction did not reflect the clinical severity of the disease [80]. ...
... Post-mortem human MCI brain (frontal cortex) with aging [56] MCI, AD versus control subjects [67] MCI, AD versus control subjects [66,67] AD versus control subjects [66,78,79] AD versus control subjects [82][83][84][85][86][87] Post-mortem human AD brain (frontal and occipital cortex, basal ganglia, pons) versus control subjects [27] AD versus control subjects [68,76,77,80] AD subjects genotyping for RXRα polymorphism versus control subjects [90] Post-mortem human AD brain (frontal cortex) with aging [56] AD subjects genotyping for RXRα polymorphism versus control subjects [90] AD subjects genotyping for CYP46A1 polymorphism versus control subjects [91] ...
... Data of 24OHC, 26OHC and 7-oxycholesterol levels were also inconsistent in literature. Out of ten studies analysed for 24OHC, six studies reported high circulating levels , Iuliano et al. 2010, Zuliani et al. 2011, Hughes et al. 2012, Popp et al. 2013, Zarrouk et al. 2020) and four studies reported low circulating levels (Qureischie et al. 2008, Mateos et al. 2011, Costa et al. 2018, Roy et al. 2019 in AD compared to control. For 26OHC, three studies reported high circulating levels (Iuliano et al. 2010b, Popp et al. 2013, Zarrouk et al. 2020, two studies reported low levels (Mateos et al. 2011, Hughes et al. 2012) and one study reported no change (Costa et al. 2018) compared to control. ...
... . WhileHughes et al. (2012) andPopp et al. (2012) reported increased levels of circulating 24OHC in MCI,Mateos et al. (2011) reported decreased levels. For 26OHC,Hughes et al. (2012) andPopp et al. (2012) reported decreased circulating levels in MCI butLiu et al. (2016) andZuliani et al. (2011) reported 26OHC is higher in MCI plasma. ...
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Despite decades of research, the cause and series of events underlying the advancement of Alzheimer’s disease (AD) has not yet been established. Lipids and especially cholesterol levels have been proposed to be implicated in AD. Several studies have been undertaken and many ongoing in different directions looking at the importance of circulating cholesterols and oxidised cholesterols in Alzheimer’s disease with inconsistent methods and results. This meta-analysis aims to systematically analyse available data describing the involvement of oxidised cholesterols in Mild cognitive impairment (MCI) and Alzheimer’s disease (AD). We conducted a systematic literature search of 6 databases MEDLINE (PubMed), BIOSIS (Web of Science), EMBASE (Elsevier), PsycNET, Scopus and Cochrane library for studies measuring oxysterols (24-hydroxycholesterol (24OHC); 26-hydroxycholesterol (26OHC) and 7-oxycholesterols) in serum or plasma from MCI / AD patients compared to age and gender matched cognitively normal controls. Data was analysed using the inverse variance and standard mean difference with random effect analysis model at 95% confidence interval for association between oxysterols and MCI / AD in Review Manager (RevMan) software version 5.4.1. 175 studies between January 2000 and April 2022 were identified by 2 independent researchers out of which 14 met the inclusion criteria and were analysed with a total of 957 controls, 469 MCI cases and 509 AD cases. The standard mean differences between MCI / AD participants and controls did not show any difference in the oxysterol levels except for 26OHC level which were higher in AD but not statistically significant.
... A negative correlation between plasma 24-OHC levels and AD severity was found in another investigation, although in this case 24-OHC levels were higher in AD patients than in healthy controls [78]. A subsequent study partially confirmed this evidence, reporting increased plasma levels of 24-OHC in the early stage of late-onset AD patients compared to controls [79]. In contrast, plasma 24-OHC content was found to be lower in probable AD patients compared to controls, but this reduction did not reflect the clinical severity of the disease [80]. ...
... Post-mortem human MCI brain (frontal cortex) with aging [56] MCI, AD versus control subjects [67] MCI, AD versus control subjects [66,67] AD versus control subjects [66,78,79] AD versus control subjects [82][83][84][85][86][87] Post-mortem human AD brain (frontal and occipital cortex, basal ganglia, pons) versus control subjects [27] AD versus control subjects [68,76,77,80] AD subjects genotyping for RXRα polymorphism versus control subjects [90] Post-mortem human AD brain (frontal cortex) with aging [56] AD subjects genotyping for RXRα polymorphism versus control subjects [90] AD subjects genotyping for CYP46A1 polymorphism versus control subjects [91] ...
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The development of Alzheimer’s disease (AD) is influenced by several events, among which the dysregulation of cholesterol metabolism in the brain plays a major role. Maintenance of brain cholesterol homeostasis is essential for neuronal functioning and brain development. To maintain the steady-state level, excess brain cholesterol is converted into the more hydrophilic metabolite 24-S-hydroxycholesterol (24-OHC), also called cerebrosterol, by the neuron-specific enzyme CYP46A1. A growing bulk of evidence suggests that cholesterol oxidation products, named oxysterols, are the link connecting altered cholesterol metabolism to AD. It has been shown that the levels of some oxysterols, including 27-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol, significantly increase in AD brains contributing to disease progression. In contrast, 24-OHC levels decrease, likely due to neuronal loss. Among the different brain oxysterols, 24-OHC is certainly the one whose role is most controversial. It is the dominant oxysterol in the brain and evidence shows that it represents a signaling molecule of great importance for brain function. However, numerous studies highlighted the potential role of 24-OHC in favoring AD development, since it promotes neuroinflammation, amyloid β (Aβ) peptide production, oxidative stress and cell death. In parallel, 24-OHC has been shown to exert several beneficial effects against AD progression, such as preventing tau hyperphosphorylation and Aβ production. In this review we focus on the current knowledge of the controversial role of 24-OHC in AD pathogenesis, reporting a detailed overview of the findings about its levels in different AD biological samples and its noxious or neuroprotective effects in the brain. Given the relevant role of 24-OHC in AD pathophysiology, its targeting could be useful for disease prevention or slowing down its progression.
... Further research delivered contradictory outcomes, reporting normal (Schönknecht et al., 2002) or lowered (Bretillon et al., 2000;Kölsch et al., 2004;Solomon et al., 2009) levels of 24(S)-OHC in demented vs. cognitively normal individuals. Some studies indicate that plasma concentrations of 24(S)-OHC may be higher in initial AD and Vascular dementia (VaD), possibly due to cholesterol turnover elevation associated with neuronal degradation or a defect in the BBB, present in neurodegenerative disorders, including AD (Zuliani et al., 2011). BBB defects, the occurrence of inflammation, or elevated cholesterol turnover can all counterbalance this notion, causing an increase in, or-occasionally-modification of 24(S)-OHC plasma concentrations. ...
... Therefore, possibly the plasma concentrations of this parameter could be a candidate biochemical marker of altered homeostasis of cholesterol in CNS . Studies on plasma 24(S)-OHC levels in the context of dementia have brought conflicting results throughout (Bretillon et al., 2000;Lütjohann et al., 2000;Schönknecht et al., 2002;Kölsch et al., 2004;Solomon et al., 2009) with an interesting hypothesis of higher concentrations of brain cholesterol when neurodegeneration markers are elevated, but neuron loss and brain atrophy degree remains small (Zuliani et al., 2011). This is following other clinical observations Solomon et al., 2009), also indicating that lowered plasma 24(S)-OHC levels are typical for dementia (Liu et al., 1998;Leoni and Caccia, 2013), but they lower gradually with disease progression (Björkhem and Meaney, 2004). ...
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... In AD, an increase, decrease or no change in plasma 24HC levels were documented in affected patients (Bretillon et al., 2000;Lutjohann et al., 2000;Papassotiropoulos et al., 2002;Schonknecht et al., 2002;Heverin et al., 2004;Kolsch et al., 2004;Zuliani et al., 2011;Popp et al., 2012;Leoni and Caccia, 2013a;Li et al., 2018). This inconsistency was explained by the disease stage, as different criteria were used for the disease stage determination and patient enrollment. ...
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... There was neither a significant difference in distributions of the CYP46A1 genotype between the MCI group and control group nor in plasma 24-OHC concentration among the three genotype subgroups. Previous studies reported elevated plasma 24-OHC concentration levels in patients with MCI and AD Zuliani et al., 2011), which was consistent with our results. This conclusion supports that the plasma level of the 24-OHC concentration is proportional to the degree of brain atrophy and the loss of active gray matter (Liu et al., 2016). ...
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... Previous studies have found increased plasma levels of 24-OHC in patients with Alzheimer's disease 9,10 , though other studies have found decreased levels 11,12 ; in part, these mixed findings could be due to evidence that 24-OHC is elevated in early or mild Alzheimer's but decreased in more advanced illness 13,14 . Plasma levels of 24-OHC are also decreased in Huntington's disease 15,16 . ...
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... In a comparison between patients with late onset AD, VD, cognitive impairment, and cognitively normal controls (Zuliani et al., 2011), plasma 24S-HC levels were higher in late onset AD and lower in VD. ...
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The adult brain exhibit a characteristic cholesterol homeostasis, with low synthesis rate and active catabolism. Brain cholesterol turnover is possible thanks to the action of the enzyme Cytochrome P450 46A1 (CYP46A1) or 24‐cholesterol hydroxylase, that transforms cholesterol into 24S‐hydroxycholesterol (24S‐HC). But before crossing the blood‐brain barrier (BBB), this oxysterol that is the most abundant in the brain can act locally, affecting the functioning of neurons, astrocytes, oligodendrocytes, and vascular cells. The first part of this review addresses different aspects of 24S‐HC production and elimination from the brain. The second part concentrates in the effects of 24S‐HC at the cellular level, describing how this oxysterol affects cell viability, amyloid β production, neurotransmission, and transcriptional activity. Finally, the role of 24S‐HC in Alzheimer, Huntington and Parkinson diseases, multiple sclerosis and amyotrophic lateral sclerosis, as well as the possibility of using this oxysterol as predictive and/or evolution biomarker in different brain disorders is discussed.
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Alterations in brain cholesterol homeostasis have been broadly implicated in neurological disorders. Notwithstanding the complexity by which cholesterol biology is governed in the mammalian brain, excess neuronal cholesterol is primarily eliminated by metabolic clearance via cytochrome P450 46A1 (CYP46A1). No methods are currently available for visualizing cholesterol metabolism in the living human brain; therefore, a noninvasive technology that quantitatively measures the extent of brain cholesterol metabolism via CYP46A1 could broadly affect disease diagnosis and treatment options using targeted therapies. Here, we describe the development and testing of a CYP46A1-targeted positron emission tomography (PET) tracer, ¹⁸ F-CHL-2205 ( ¹⁸ F-Cholestify). Our data show that PET imaging readouts correlate with CYP46A1 protein expression and with the extent to which cholesterol is metabolized in the brain, as assessed by cross-species postmortem analyses of specimens from rodents, nonhuman primates, and humans. Proof of concept of in vivo efficacy is provided in the well-established 3xTg-AD murine model of Alzheimer’s disease (AD), where we show that the probe is sensitive to differences in brain cholesterol metabolism between 3xTg-AD mice and control animals. Furthermore, our clinical observations point toward a considerably higher baseline brain cholesterol clearance via CYP46A1 in women, as compared to age-matched men. These findings illustrate the vast potential of assessing brain cholesterol metabolism using PET and establish PET as a sensitive tool for noninvasive assessment of brain cholesterol homeostasis in the clinic.
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We thank our colleagues Beth Duncan, Jay Horton, Axel Nohturfft, Jih-tung Pai, Juro Sakai, Jin Shimano, and Iichiro Shimomura for helpful comments in the preparation of this review. We thank Ravi Pathak for the immunofluorescence micrograph (Figure 3Figure 3). Our research is supported by grants from the National Institutes of Health (HL20948) and the Perot Family Foundation.
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