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Decreased levels of serum nitric oxide in different forms of dementia
Neuroscience Letters 420 (2007) 263–267
Decreased levels of serum nitric oxide in different forms of dementia Lola Corzo a,∗ , Raquel Zas a , Susana Rodr´ıguez a , Luc´ıa Fern´andez-Novoa b , Ram´on Cacabelos c a
b
Department of Clinical Biochemistry, EuroEspes Biomedical Research Center, Santa Marta de Bab´ıo s/n, 15166 Bergondo, La Coru˜na, Spain Department of Molecular Genetics, EBIOTEC, Pol´ıgono Industrial de Bergondo, Calle Parroquia de Gu´ısamo parcela A-6, 15165 Bergondo, A Coru˜na, Spain c Department of Clinical Neuroscience, EuroEspes Biomedical Research Center, Santa Marta de Bab´ıo s/n, 15166 Bergondo, La Coru˜ na, 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 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 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 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. 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 neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) [24]. Tetrahydrobiopterin (BH4) is a necessary cofactor for NO synthesis [32]. NO is involved in normal physiological functions and can also damage tissues due in part to its free radical nature (oxidative stress). This process refers to the cytopathologic consequences of a mismatch between antioxidant defenses and free radical production leading to cellular death [23]. Oxidative stress and vascular dysfunction have been recognized as contributing factors in the pathogenesis of Alzheimer disease (AD) and vascular dementia (VD) [10,30]. Reactive oxygen species (ROS) are involved in cellular damage in most tissues including neurons and glia [29]. Amyloid-beta (A) deposition and Apolipoprotein E (ApoE) can induce ROS generation in the CNS (central nervous system) [3,35]. The APOE gene (19q13.2) encodes apolipoprotein E (ApoE), a
∗
Corresponding author. Tel.: +34 981 780505; fax: +34 981 780511. E-mail address: [email protected] (L. Corzo).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.05.008
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 genotype of an intronic polymorphism located at 3 to exon 8 of the PSEN1 gene was also associated with an approximately two-fold risk of developing AD. The most common allele has an A at nucleotide position 16 (allele 1) in this intron, while the variant allele has a C at this position (allele 2). Neither the 1/2 nor the 2/2 genotype was associated with increased risk of AD [38]. Perry et al. [30] suggested that many pathogenic theories potentially implicated in AD are directly related to oxidative processes. However, it is unclear whether or not oxidative stress is a primary pathogenic event in AD or the consequence of AD pathology. In the vascular system, NO is generated by eNOS and has an important role in vascular tone. NO causes vasodilation, reduces the aggregation and activation of platelets [19], attenuates adhesion of leukocytes to the endothelium [27] and inhibits proliferation and migration of vascular smooth muscle cells [18]. In general, NO is associated with an atheroprotective effect [36];
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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
Age (years) Gender (%) MMSE scores ADAS-cog scores Total-cholesterol HDL-cholesterol LDL-cholesterol Tryglicerides ApoE -Amyloid(1–42) Total nitric oxide
Control (n = 55)
Dementia (n = 99)
Alzheimer (n = 42)
Vascular (n = 19)
Mixed (n = 29)
66.7 ± 6.9 45% females 28.1 ± 1.5 1.8 ± 1.8 221 ± 37 54 ± 14 144 ± 33 111 ± 76 5.1 ± 1.07 18.0 ± 10.5 53.9 ± 25.3
73.6 ± 8.1 54% females 14.6 ± 8.3a 21.5 ± 13.6b 218 ± 43 51 ± 15 143 ± 36 116 ± 61 4.9 ± 1.06 18.5 ± 9.7 44.8 ± 21.1e
69.8 ± 7.2 62% females 13.0 ± 7.8a 24.6 ± 12.6b 231 ± 35c 52 ± 13 156 ± 31d 116 ± 74 5.0 ± 1.17 17.9 ± 7.4 42.5 ± 17.0f
75.6 ± 8.3 42% females 14.1 ± 9.3a 19.6 ± 14.0b 200 ± 38 47 ± 14 128 ± 34 124 ± 46 4.9 ± 0.88 23.1 ± 15.7 43.2 ± 19.6g
76.6 ± 8.1 48% females 17.9 ± 7.8a,z 16.5 ± 13.4b,z 213 ± 56 53 ± 17 136 ± 44 107 ± 54 4.6 ± 1.14 17.0 ± 8.1 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 developing dementia [4,13]. Previous studies have reported changes in 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 HDLcholesterol, demonstrated by several authors [22] might be a mechanism of action. The aim of this study was to investigate oxidative and vascular factors in patients with dementia and correlate these factors 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)] 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, 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 subjects, and serum and plasma were removed after centrifuging at 3000 rpm for 10 min. These specimens were frozen at −40 ◦ C until analysis of NOx , ApoE and A levels. Lipids, vitamin B12 and folate levels were measured the same day of venipuncture. Commercial vacutubes containing tripotassic-EDTA 15% were used for genetic analysis. For accurate assay of the total nitric oxide generated, we monitored both nitrate and nitrite (NOx ) levels by a colorimetric 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 convert the nitrate to nitrite prior to quantitation using the Griess
Reagent, thus providing accurate determination of the total NO produced [28]. To avoid possible interferences with proteins, we removed excess proteins from serum samples by boiling during 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 procedure 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 triglycerides measurements. LDL-cholesterol levels were calculated by the Friedewald formula. The serum levels of vitamin B12 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. 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 assessed by Pearson’s method. Our data showed a significant decrease of serum NOx levels in dementia (44.8 ± 21.1 M), especially in probable AD and VD patients, as compared with controls (53.9 ± 25.3 M; p < 0.02) with no differences between probable AD and VD. A and ApoE levels were similar in controls and dementia (Table 1). Although serum ApoE levels decrease according to APOE genotype (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 concentration with A levels. No correlation was observed in dementia; however, in the control group, we detected a significant correlation 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
21.9 ± 11.0 (n = 11) 17.6 ± 6.7 (n = 44) 16.9 ± 9.4 (n = 31) 17.7 ± 8.4 (n = 45) Results are expressed as mean ± S.D. a P < 0.001 vs. APOE genotype 3–3. b P < 0.01 vs. absence of APOE allele 4.
18.9 ± 7.9 (n = 9) 16.9 ± 8.5 (n = 34) 18.4 ± 9.8 (n = 44)
19.42 ± 11.1 (n = 48)
5.1 ± 1.0 (n = 12) 4.8 ± 1.2 (n = 45) 4.7 ± 1.2b (n = 46) 4.0 ± 0.5a (n = 9) 4.7 ± 1.2 (n = 35) 4.9 ± 0.8 (n = 45)
5.0 ± 0.9 (n = 49)
4.8 ± 0.9 (n = 31)
47.5 ± 27.6 (n = 12) 48.0 ± 21.5 (n = 45) 39.8 ± 18.7 (n = 31) 45.5 ± 18.6 (n = 46)
Total nitric oxide (M) Apolipoprotein E (mg/dL) -Amyloid(1–42) (pg/mL)
43.8 ± 17.0 (n = 9) 44.3 ± 17.8 (n = 35) 42.5 ± 23.1 (n = 45)
43.9 ± 23.6 (n = 49)
1–1 Absence 3–4 3–3
4–4
APOE allele 4 APOE genotype
Table 2 Plasma -amyloid(1–42) and serum nitric oxide and apolipoprotein E levels according to AD-related genes in dementia
Presence
PSEN1 polymorphism
1–2
2–2
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levels of total and LDL-cholesterol in AD but not in dementia 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, 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). Folate levels were decreased in dementia as compared to controls (controls: 6.76 ± 2.4 ng/mL; dementia: 5.62 ± 2.5 ng/mL; 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 scores: r = −.2123; p < 0.05 and ADAS-cog scores: r = .2235; p < 0.05) and NOx levels has been detected (Table 1). In a preliminary study [4], an increase of serum NOx levels 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 number of subjects included in the control sample. The present data show significant decreased concentrations of total NOx in the serum of AD and vascular dementia patients. This evidence has been recently documented by Selley et al. [33] who found an association between decreased plasma NO and increased homocysteine 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 AD patients and controls; however Kuiper et al. [16] reported decreased CSF nitrate levels in AD that were associated with decreased tetrahydrobiopterin (BH4) levels [14]. Since folate and vitamin B12 appear to be required for the biosynthesis of 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 implicate A in oxidative stress, ROS generation and in decreasing endothelial nitric oxide production [3,31]. We did not find any correlation between serum A and NOx levels in demented patients. Likewise, we could not find any association between AD-related genetic polymorphisms in the APOE and PSEN 1 genes and NOx levels. In contrast, basic studies in mice reported an oxidative damage induced by ApoE deficiency, or associated with APOE4 genotype and PSEN1 mutations [35,6,17]. The correlation between HDL-cholesterol and NOx levels in dementia and elevated total and LDL-cholesterol concentrations in AD observed in our study might support the hypothesis that the reduced levels of NOx could be implicated in AD and VD pathology by a vascular mechanism related to HDL-cholesterol [11] or lipid metabolism. Increased total-cholesterol levels were associated with NO levels in AD patients [5] and an association between reduced levels of VLDL + LDL cholesterol and elevated eNOS activity in mice has been reported [36]. Circulating levels of HDL-cholesterol are inversely related to the risk of atherosclerosis and it has been demonstrated that HDL causes potent stimulation of eNOS activity, probably, through binding to scavenger receptor class B, member I (SR-B1), which is expressed in endothelium [39]. The HDL-induced increase in NO production may be critical to the atheroprotective features of HDL. However, the mechanism by which HDL activates
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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 correlation 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 markers [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 an HDL cholesterol-related atheroprotective effect in dementia. The mechanism by which decreased NOx levels affect the pathogenesis of dementia remains unclear and requires further elucidation.
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Decreased levels of serum nitric oxide in different forms of dementia Lola Corzo a,∗ , Raquel Zas a , Susana Rodr´ıguez a , Luc´ıa Fern´andez-Novoa b , Ram´on Cacabelos c a
b
Department of Clinical Biochemistry, EuroEspes Biomedical Research Center, Santa Marta de Bab´ıo s/n, 15166 Bergondo, La Coru˜na, Spain Department of Molecular Genetics, EBIOTEC, Pol´ıgono Industrial de Bergondo, Calle Parroquia de Gu´ısamo parcela A-6, 15165 Bergondo, A Coru˜na, Spain c Department of Clinical Neuroscience, EuroEspes Biomedical Research Center, Santa Marta de Bab´ıo s/n, 15166 Bergondo, La Coru˜ na, 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 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 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 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. 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 neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS) [24]. Tetrahydrobiopterin (BH4) is a necessary cofactor for NO synthesis [32]. NO is involved in normal physiological functions and can also damage tissues due in part to its free radical nature (oxidative stress). This process refers to the cytopathologic consequences of a mismatch between antioxidant defenses and free radical production leading to cellular death [23]. Oxidative stress and vascular dysfunction have been recognized as contributing factors in the pathogenesis of Alzheimer disease (AD) and vascular dementia (VD) [10,30]. Reactive oxygen species (ROS) are involved in cellular damage in most tissues including neurons and glia [29]. Amyloid-beta (A) deposition and Apolipoprotein E (ApoE) can induce ROS generation in the CNS (central nervous system) [3,35]. The APOE gene (19q13.2) encodes apolipoprotein E (ApoE), a
∗
Corresponding author. Tel.: +34 981 780505; fax: +34 981 780511. E-mail address: [email protected] (L. Corzo).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.05.008
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 genotype of an intronic polymorphism located at 3 to exon 8 of the PSEN1 gene was also associated with an approximately two-fold risk of developing AD. The most common allele has an A at nucleotide position 16 (allele 1) in this intron, while the variant allele has a C at this position (allele 2). Neither the 1/2 nor the 2/2 genotype was associated with increased risk of AD [38]. Perry et al. [30] suggested that many pathogenic theories potentially implicated in AD are directly related to oxidative processes. However, it is unclear whether or not oxidative stress is a primary pathogenic event in AD or the consequence of AD pathology. In the vascular system, NO is generated by eNOS and has an important role in vascular tone. NO causes vasodilation, reduces the aggregation and activation of platelets [19], attenuates adhesion of leukocytes to the endothelium [27] and inhibits proliferation and migration of vascular smooth muscle cells [18]. In general, NO is associated with an atheroprotective effect [36];
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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
Age (years) Gender (%) MMSE scores ADAS-cog scores Total-cholesterol HDL-cholesterol LDL-cholesterol Tryglicerides ApoE -Amyloid(1–42) Total nitric oxide
Control (n = 55)
Dementia (n = 99)
Alzheimer (n = 42)
Vascular (n = 19)
Mixed (n = 29)
66.7 ± 6.9 45% females 28.1 ± 1.5 1.8 ± 1.8 221 ± 37 54 ± 14 144 ± 33 111 ± 76 5.1 ± 1.07 18.0 ± 10.5 53.9 ± 25.3
73.6 ± 8.1 54% females 14.6 ± 8.3a 21.5 ± 13.6b 218 ± 43 51 ± 15 143 ± 36 116 ± 61 4.9 ± 1.06 18.5 ± 9.7 44.8 ± 21.1e
69.8 ± 7.2 62% females 13.0 ± 7.8a 24.6 ± 12.6b 231 ± 35c 52 ± 13 156 ± 31d 116 ± 74 5.0 ± 1.17 17.9 ± 7.4 42.5 ± 17.0f
75.6 ± 8.3 42% females 14.1 ± 9.3a 19.6 ± 14.0b 200 ± 38 47 ± 14 128 ± 34 124 ± 46 4.9 ± 0.88 23.1 ± 15.7 43.2 ± 19.6g
76.6 ± 8.1 48% females 17.9 ± 7.8a,z 16.5 ± 13.4b,z 213 ± 56 53 ± 17 136 ± 44 107 ± 54 4.6 ± 1.14 17.0 ± 8.1 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 developing dementia [4,13]. Previous studies have reported changes in 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 HDLcholesterol, demonstrated by several authors [22] might be a mechanism of action. The aim of this study was to investigate oxidative and vascular factors in patients with dementia and correlate these factors 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)] 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, 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 subjects, and serum and plasma were removed after centrifuging at 3000 rpm for 10 min. These specimens were frozen at −40 ◦ C until analysis of NOx , ApoE and A levels. Lipids, vitamin B12 and folate levels were measured the same day of venipuncture. Commercial vacutubes containing tripotassic-EDTA 15% were used for genetic analysis. For accurate assay of the total nitric oxide generated, we monitored both nitrate and nitrite (NOx ) levels by a colorimetric 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 convert the nitrate to nitrite prior to quantitation using the Griess
Reagent, thus providing accurate determination of the total NO produced [28]. To avoid possible interferences with proteins, we removed excess proteins from serum samples by boiling during 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 procedure 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 triglycerides measurements. LDL-cholesterol levels were calculated by the Friedewald formula. The serum levels of vitamin B12 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. 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 assessed by Pearson’s method. Our data showed a significant decrease of serum NOx levels in dementia (44.8 ± 21.1 M), especially in probable AD and VD patients, as compared with controls (53.9 ± 25.3 M; p < 0.02) with no differences between probable AD and VD. A and ApoE levels were similar in controls and dementia (Table 1). Although serum ApoE levels decrease according to APOE genotype (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 concentration with A levels. No correlation was observed in dementia; however, in the control group, we detected a significant correlation 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
21.9 ± 11.0 (n = 11) 17.6 ± 6.7 (n = 44) 16.9 ± 9.4 (n = 31) 17.7 ± 8.4 (n = 45) Results are expressed as mean ± S.D. a P < 0.001 vs. APOE genotype 3–3. b P < 0.01 vs. absence of APOE allele 4.
18.9 ± 7.9 (n = 9) 16.9 ± 8.5 (n = 34) 18.4 ± 9.8 (n = 44)
19.42 ± 11.1 (n = 48)
5.1 ± 1.0 (n = 12) 4.8 ± 1.2 (n = 45) 4.7 ± 1.2b (n = 46) 4.0 ± 0.5a (n = 9) 4.7 ± 1.2 (n = 35) 4.9 ± 0.8 (n = 45)
5.0 ± 0.9 (n = 49)
4.8 ± 0.9 (n = 31)
47.5 ± 27.6 (n = 12) 48.0 ± 21.5 (n = 45) 39.8 ± 18.7 (n = 31) 45.5 ± 18.6 (n = 46)
Total nitric oxide (M) Apolipoprotein E (mg/dL) -Amyloid(1–42) (pg/mL)
43.8 ± 17.0 (n = 9) 44.3 ± 17.8 (n = 35) 42.5 ± 23.1 (n = 45)
43.9 ± 23.6 (n = 49)
1–1 Absence 3–4 3–3
4–4
APOE allele 4 APOE genotype
Table 2 Plasma -amyloid(1–42) and serum nitric oxide and apolipoprotein E levels according to AD-related genes in dementia
Presence
PSEN1 polymorphism
1–2
2–2
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levels of total and LDL-cholesterol in AD but not in dementia 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, 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). Folate levels were decreased in dementia as compared to controls (controls: 6.76 ± 2.4 ng/mL; dementia: 5.62 ± 2.5 ng/mL; 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 scores: r = −.2123; p < 0.05 and ADAS-cog scores: r = .2235; p < 0.05) and NOx levels has been detected (Table 1). In a preliminary study [4], an increase of serum NOx levels 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 number of subjects included in the control sample. The present data show significant decreased concentrations of total NOx in the serum of AD and vascular dementia patients. This evidence has been recently documented by Selley et al. [33] who found an association between decreased plasma NO and increased homocysteine 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 AD patients and controls; however Kuiper et al. [16] reported decreased CSF nitrate levels in AD that were associated with decreased tetrahydrobiopterin (BH4) levels [14]. Since folate and vitamin B12 appear to be required for the biosynthesis of 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 implicate A in oxidative stress, ROS generation and in decreasing endothelial nitric oxide production [3,31]. We did not find any correlation between serum A and NOx levels in demented patients. Likewise, we could not find any association between AD-related genetic polymorphisms in the APOE and PSEN 1 genes and NOx levels. In contrast, basic studies in mice reported an oxidative damage induced by ApoE deficiency, or associated with APOE4 genotype and PSEN1 mutations [35,6,17]. The correlation between HDL-cholesterol and NOx levels in dementia and elevated total and LDL-cholesterol concentrations in AD observed in our study might support the hypothesis that the reduced levels of NOx could be implicated in AD and VD pathology by a vascular mechanism related to HDL-cholesterol [11] or lipid metabolism. Increased total-cholesterol levels were associated with NO levels in AD patients [5] and an association between reduced levels of VLDL + LDL cholesterol and elevated eNOS activity in mice has been reported [36]. Circulating levels of HDL-cholesterol are inversely related to the risk of atherosclerosis and it has been demonstrated that HDL causes potent stimulation of eNOS activity, probably, through binding to scavenger receptor class B, member I (SR-B1), which is expressed in endothelium [39]. The HDL-induced increase in NO production may be critical to the atheroprotective features of HDL. However, the mechanism by which HDL activates
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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 correlation 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 markers [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 an HDL cholesterol-related atheroprotective effect in dementia. The mechanism by which decreased NOx levels affect the pathogenesis of dementia remains unclear and requires further elucidation.
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