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 Neuroscience Letters 420 (2007) 263–267

Decreased levels of serum nitric oxide in different forms of dementia

py
Lola Corzo a,∗ , Raquel Zas a , Susana Rodrı́guez a , Lucı́a Fernández-Novoa b , Ramón Cacabelos c
a Department of Clinical Biochemistry, EuroEspes Biomedical Research Center, Santa Marta de Babı́o s/n, 15166 Bergondo, La Coruña, Spain
b Department of Molecular Genetics, EBIOTEC, Polı́gono Industrial de Bergondo, Calle Parroquia de Guı́samo parcela A-6, 15165 Bergondo, A Coruña, Spain

co
c Department of Clinical Neuroscience, EuroEspes Biomedical Research Center, Santa Marta de Babı́o s/n, 15166 Bergondo, La Coruña, Spain

Received 9 March 2007; received in revised form 27 April 2007; accepted 3 May 2007

Abstract
Nitric oxide is involved in normal physiological functions and also in pathological processes leading to tissue damage due, in part, to its free
radical nature (oxidative stress). Oxidative stress and vascular dysfunction have been recognized as contributing factors in the pathogenesis of

al
Alzheimer disease (AD) and vascular dementia (VD). In order to study the possible links between these processes and dementia, we have analysed
plasma amyloid-beta(1–42) levels (A␤) and total nitric oxide (NOx ), apolipoprotein E (ApoE), lipids, vitamin B12, and folate concentrations in the
serum of 99 patients with dementia and 55 age-matched non-demented controls. Both nitrate and nitrite levels were measured by a colorimetric
on
method using Griess Reagent and plasma A␤ levels were analysed by a hypersensitive ELISA method. Our data showed a significant decrease of
serum NOx levels in dementia, especially in probable AD and VD patients, as compared with controls. We observed a weak correlation between
serum NOx levels and cognitive deterioration in dementia; however, NOx levels were not associated with ApoE and A␤ levels. In dementia and
controls, a similar correlation pattern between HDL-cholesterol versus NOx was found. No apparent association between NOx , A␤ and AD-related
rs

genes [APOE (apolipoprotein E), PSEN1 (Presenilin 1)] was observed. Our data suggest that NOx may contribute to the pathogenesis of dementia
through a process mediated by HDL-cholesterol.
© 2007 Elsevier Ireland Ltd. All rights reserved.
pe

Keywords: Nitric oxide; Amyloid-beta; Apolipoprotein E; HDL-cholesterol; Alzheimer disease; Vascular dementia

Nitric oxide (NO) is a free radical produced by a family of
nitric oxide synthases (NOS), which includes constitutive neu-
ronal NOS (nNOS), endothelial NOS (eNOS) and inducible
r's
glycosylated protein associated with lipoproteins. Three major
isoforms of ApoE are products of three apoE alleles: ␧2, ␧3 and
␧4. Data has shown an association between LOAD (load onset
Alzheimer disease) or sporadic AD with apoE ␧4 [7]. The 1/1

NOS (iNOS) [24]. Tetrahydrobiopterin (BH4) is a necessary

cofactor for NO synthesis [32]. NO is involved in normal physi- genotype of an intronic polymorphism located at 3 to exon 8
ological functions and can also damage tissues due in part to of the PSEN1 gene was also associated with an approximately
its free radical nature (oxidative stress). This process refers two-fold risk of developing AD. The most common allele has
o

to the cytopathologic consequences of a mismatch between an A at nucleotide position 16 (allele 1) in this intron, while the
antioxidant defenses and free radical production leading to cel- variant allele has a C at this position (allele 2). Neither the 1/2
lular death [23]. Oxidative stress and vascular dysfunction have nor the 2/2 genotype was associated with increased risk of AD
th

been recognized as contributing factors in the pathogenesis of [38]. Perry et al. [30] suggested that many pathogenic theories
Alzheimer disease (AD) and vascular dementia (VD) [10,30]. potentially implicated in AD are directly related to oxidative
Reactive oxygen species (ROS) are involved in cellular damage processes. However, it is unclear whether or not oxidative stress
Au

in most tissues including neurons and glia [29]. Amyloid-beta is a primary pathogenic event in AD or the consequence of AD
(A␤) deposition and Apolipoprotein E (ApoE) can induce ROS pathology.
generation in the CNS (central nervous system) [3,35]. The In the vascular system, NO is generated by eNOS and has
APOE gene (19q13.2) encodes apolipoprotein E (ApoE), a an important role in vascular tone. NO causes vasodilation,
reduces the aggregation and activation of platelets [19], attenu-
ates adhesion of leukocytes to the endothelium [27] and inhibits
∗ proliferation and migration of vascular smooth muscle cells [18].
Corresponding author. Tel.: +34 981 780505; fax: +34 981 780511.
E-mail address: analisis@euroespes.com (L. Corzo). In general, NO is associated with an atheroprotective effect [36];

0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.neulet.2007.05.008
264 L. Corzo et al. / Neuroscience Letters 420 (2007) 263–267

Table 1
Demographic data, cognitive scores, plasma ␤-amyloid(1–42) levels (pg/mL) and total nitric oxide (␮M), apolipoprotein E (mg/dL) and total-cholesterol (mg/dL) and
HDL-cholesterol (mg/dL), LDL-cholesterol (mg/dL) and tryglicerides (mg/dL) concentration in serum of demented and control patients
Control (n = 55) Dementia (n = 99) Alzheimer (n = 42) Vascular (n = 19) Mixed (n = 29)

Age (years) 66.7 ± 6.9 73.6 ± 8.1 69.8 ± 7.2 75.6 ± 8.3 76.6 ± 8.1
Gender (%) 45% females 54% females 62% females 42% females 48% females
MMSE scores 28.1 ± 1.5 14.6 ± 8.3a 13.0 ± 7.8a 14.1 ± 9.3a 17.9 ± 7.8a,z
ADAS-cog scores 1.8 ± 1.8 21.5 ± 13.6b 24.6 ± 12.6b 19.6 ± 14.0b 16.5 ± 13.4b,z
Total-cholesterol 221 ± 37 218 ± 43 231 ± 35c 200 ± 38 213 ± 56

py
HDL-cholesterol 54 ± 14 51 ± 15 52 ± 13 47 ± 14 53 ± 17
LDL-cholesterol 144 ± 33 143 ± 36 156 ± 31d 128 ± 34 136 ± 44
Tryglicerides 111 ± 76 116 ± 61 116 ± 74 124 ± 46 107 ± 54
ApoE 5.1 ± 1.07 4.9 ± 1.06 5.0 ± 1.17 4.9 ± 0.88 4.6 ± 1.14
␤-Amyloid(1–42) 18.0 ± 10.5 18.5 ± 9.7 17.9 ± 7.4 23.1 ± 15.7 17.0 ± 8.1

co
Total nitric oxide 53.9 ± 25.3 44.8 ± 21.1e 42.5 ± 17.0f 43.2 ± 19.6g 47.9 ± 27.4

Results are expressed as mean ± S.D. a,b P < 0.00 vs. control group. z P < 0.00 vs. Alzheimer disease. c P < 0.03 vs. vascular and mixed dementia. d P < 0.02 vs. vascular
and mixed dementia. e P < 0.02 vs. controls. f P < 0.02 vs. controls. g P < 0.05 vs. controls.

and vascular risk factors increase the vulnerability of develop- Reagent, thus providing accurate determination of the total NO
ing dementia [4,13]. Previous studies have reported changes in produced [28]. To avoid possible interferences with proteins, we

CSF (cerebrospinal fluid) [16] and plasma nitrate levels [33,9],
but the hypothesis of a relationship between NO and dementia
has not yet been demonstrated. The eNOS stimulation by HDL-
cholesterol, demonstrated by several authors [22] might be a
on
mechanism of action.
The aim of this study was to investigate oxidative and vascu-
lar factors in patients with dementia and correlate these factors
rs
with genetic, clinical and biochemical features involved in the
pathogenesis of dementia.
Ninety-nine patients with dementia [19 VD, 42 probable AD,
29 mixed dementia (possible AD with cerebrovascular disease)]
pe
al
removed excess proteins from serum samples by boiling dur-
ing 5 min and centrifuging at 13,000 rpm during 10 min, prior
to performing the assay. The concentration of ApoE in serum
was measured by an immunoturbidimetric assay using reagents
ApoE-HA from Wako (Japan). Characteristics of this proce-
dure have been described by Kostner [15]. Lipid levels were
measured by an automated spectrophotometric analyser (MIRA
PLUS, ABX Diagnostics) using conventional enzymatic direct
methods for total-cholesterol, HDL-cholesterol and triglyc-
erides measurements. LDL-cholesterol levels were calculated
by the Friedewald formula. The serum levels of vitamin B12

and 9 with other types of dementia), diagnosed according to
DSM-IV and NINCDS-ADRDA criteria, were included in this
study. The cognitive state of demented patients was assessed
with the Mini-Mental State Examination (MMSE) and the
Alzheimer Disease Assessment Scale-cognitive (ADAS-cog).
Patients were not taking vitamin B12, folate or lipid-lowering
drugs and none had a previous history of cancer, alcoholism,
r's
and folate were measured by an automatic quimioluminiscent
method (ACCESS 2, Beckman-Coulter). We measured amyloid-
␤(1–42) in plasma of demented patients and controls with a High
Sensitive INNOTEST ␤-amyloid(1–42) ELISA kit (Innogenetics,
Belgium) modified for measuring low concentration of amyloid-
␤(1–42) [37]. Genomic DNA was extracted from peripheral
blood by a conventional method without phenol–chloroform.

chronic liver dysfunction, chronic renal failure or atypical
dietary habits. The control group included 55 healthy subjects
without clinical signs or symptoms of dementia (Table 1).
Venous blood samples were taken from overnight fasting sub-
o
The APOE and PSEN1 genotypes were carried out in blind
conditions by procedures previously reported [2].
Data were statistically analysed by using the non-parametric
Mann–Whitney U and Kruskal–Wallis test. Correlations were

jects, and serum and plasma were removed after centrifuging at assessed by Pearson’s method.
3000 rpm for 10 min. These specimens were frozen at −40 ◦ C Our data showed a significant decrease of serum NOx lev-
th

until analysis of NOx , ApoE and A␤ levels. Lipids, vitamin B12 els in dementia (44.8 ± 21.1 ␮M), especially in probable AD
and folate levels were measured the same day of venipuncture. and VD patients, as compared with controls (53.9 ± 25.3 ␮M;
Commercial vacutubes containing tripotassic-EDTA 15% were p < 0.02) with no differences between probable AD and VD. A␤
Au

used for genetic analysis.
For accurate assay of the total nitric oxide generated, we
monitored both nitrate and nitrite (NOx ) levels by a colori-
metric method using Griess Reagent in a microtiter format
(Calbiochem, La Jolla, CA). The transient and volatile nature of
NO makes it unsuitable for most convenient detection methods.
However, NO is oxidized to two stable breakdown products,
nitrite and nitrate. Spectrophotometric quantitation of nitrite
using the Griess Reagent is a straightforward method and the
NADH-dependent enzyme nitrate reductase was used to con-
vert the nitrate to nitrite prior to quantitation using the Griess
and ApoE levels were similar in controls and dementia (Table 1).
Although serum ApoE levels decrease according to APOE geno-
type (3/3 > 3/4 > 4/4) [8], we did not observe changes in NOx and
A␤ levels in patients with dementia. No PSEN1-related changes
have been found (Table 2). In order to study the possible link
between A␤ and oxidative stress we correlated NOx concentra-
tion with A␤ levels. No correlation was observed in dementia;
however, in the control group, we detected a significant corre-
lation between serum ApoE and NOx that was not present in
dementia. No differences in A␤ and NOx concentrations were
observed according to both gender and age. We found increased
 L. Corzo et al. / Neuroscience Letters 420 (2007) 263–267 265

levels of total and LDL-cholesterol in AD but not in demen-

47.5 ± 27.6 (n = 12)

21.9 ± 11.0 (n = 11)

5.1 ± 1.0 (n = 12)
tia group. Total-cholesterol, LDL-cholesterol, HDL-cholesterol
and triglyceride levels were correlated with serum NOx levels to
study the NOx -associated atheroprotective effect. A moderate,
2–2
positive and significant correlation between HDL-cholesterol
and NOx concentration was found in the two groups (controls:
r = .5238; p = 0.000; dementia: r = .2435; p = 0.019).
48.0 ± 21.5 (n = 45)

4.8 ± 1.2 (n = 45)

17.6 ± 6.7 (n = 44)

Folate levels were decreased in dementia as compared to con-
trols (controls: 6.76 ± 2.4 ng/mL; dementia: 5.62 ± 2.5 ng/mL;

py
p = 0.009); however, neither folate nor vitamin B12 correlated
with NO levels in serum. With regard to clinical features of
dementia, a weak association between cognitive decline (MMSE
1–2

scores: r = −.2123; p < 0.05 and ADAS-cog scores: r = .2235;

co
PSEN1 polymorphism

39.8 ± 18.7 (n = 31)

p < 0.05) and NOx levels has been detected (Table 1).
4.8 ± 0.9 (n = 31)

16.9 ± 9.4 (n = 31)

In a preliminary study [4], an increase of serum NOx lev-

els in AD patients (n = 30) as compared with controls (n = 9)
had been reported. The differences observed in our preliminary
study with regard to the present one can be attributed to the num-
1–1

ber of subjects included in the control sample. The present data

al
show significant decreased concentrations of total NOx in the
45.5 ± 18.6 (n = 46)

4.7 ± 1.2b (n = 46)

17.7 ± 8.4 (n = 45)

serum of AD and vascular dementia patients. This evidence has

been recently documented by Selley et al. [33] who found an
on
association between decreased plasma NO and increased homo-
Presence

cysteine and asymmetric dimethylarginine concentration in AD.

Previously, Navarro et al. [26] and Milstien et al. [21] did not find
significant differences in CSF and plasma nitrate levels between
Plasma ␤-amyloid(1–42) and serum nitric oxide and apolipoprotein E levels according to AD-related genes in dementia

43.9 ± 23.6 (n = 49)

19.42 ± 11.1 (n = 48)

rs

AD patients and controls; however Kuiper et al. [16] reported

5.0 ± 0.9 (n = 49)

decreased CSF nitrate levels in AD that were associated with

APOE allele 4

decreased tetrahydrobiopterin (BH4) levels [14]. Since folate

and vitamin B12 appear to be required for the biosynthesis of
pe
Absence

BH4 [12] and BH4 is a cofactor in NO synthesis, according to

our studies it appears that the role of both folate and vitamin B12
are not relevant in NO-related pathogenesis in AD. Some reports
43.8 ± 17.0 (n = 9)

4.0 ± 0.5a (n = 9)

18.9 ± 7.9 (n = 9)

implicate A␤ in oxidative stress, ROS generation and in decreas-

ing endothelial nitric oxide production [3,31]. We did not find
any correlation between serum A␤ and NOx levels in demented
r's

patients. Likewise, we could not find any association between

4–4

AD-related genetic polymorphisms in the APOE and PSEN 1

genes and NOx levels. In contrast, basic studies in mice reported
44.3 ± 17.8 (n = 35)

4.7 ± 1.2 (n = 35)

16.9 ± 8.5 (n = 34)

an oxidative damage induced by ApoE deficiency, or associ-

ated with APOE4 genotype and PSEN1 mutations [35,6,17].
o

The correlation between HDL-cholesterol and NOx levels in

dementia and elevated total and LDL-cholesterol concentrations
th

in AD observed in our study might support the hypothesis that

3–4

the reduced levels of NOx could be implicated in AD and VD

42.5 ± 23.1 (n = 45)

pathology by a vascular mechanism related to HDL-cholesterol

b P < 0.01 vs. absence of APOE allele 4.
Au
4.9 ± 0.8 (n = 45)

18.4 ± 9.8 (n = 44)

[11] or lipid metabolism. Increased total-cholesterol levels were

APOE genotype

a P < 0.001 vs. APOE genotype 3–3.

Results are expressed as mean ± S.D.

associated with NO levels in AD patients [5] and an associ-

ation between reduced levels of VLDL + LDL cholesterol and
elevated eNOS activity in mice has been reported [36]. Circulat-
3–3

ing levels of HDL-cholesterol are inversely related to the risk of

atherosclerosis and it has been demonstrated that HDL causes
Apolipoprotein E

potent stimulation of eNOS activity, probably, through bind-

Total nitric oxide

␤-Amyloid(1–42)

ing to scavenger receptor class B, member I (SR-B1), which

(mg/dL)

(pg/mL)

is expressed in endothelium [39]. The HDL-induced increase

(␮M)
Table 2

in NO production may be critical to the atheroprotective fea-

tures of HDL. However, the mechanism by which HDL activates
266 L. Corzo et al. / Neuroscience Letters 420 (2007) 263–267
eNOS and its pathological implication are yet to be clarified.
The HDL stimulation of multiple kinase cascades and calcium
mobilization is other of the main hypothesis [34]. Nanetti et
al. [25] demonstrated in vitro that lipoproteins (HDL and LDL)
can induce the formation of reactive astrocytes, inducing iNOS.
Astrocytes provide structural, trophic and metabolic support to
neurons and modulate synaptic activity. Considering that the
cerebral blood flow dysfunction and synaptic disruption are
well-established features in dementia, the reduced NO levels
may contribute to accelerate AD-related neurodegeneration [4].
Further studies are necessary to validate this theory. The correla-
tion of NOx levels with cognitive deterioration scales is too weak
to get conclusions. It would be useful to confirm the present
results in a larger sample of patients to allow an appropriate
control of the variables. Previous studies have demonstrated an
association between cognitive decline and oxidative stress mark-
ers [20,1]; however, none of these studies evaluated MMSE,
ADAS-cog, and serum NOx levels in patients with dementia.
In conclusion, decreased serum levels of total NO are present
in dementia, either probable AD and VD, and this seems to be
unrelated to genetic risk factors, or serum A␤ and ApoE levels;
however, progressive NOx decline seems to be associated with
on
an HDL cholesterol-related atheroprotective effect in demen-
tia. The mechanism by which decreased NOx levels affect the
pathogenesis of dementia remains unclear and requires further
elucidation.
rs
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