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Journal of Alzheimer’s Disease xx (20xx) x–xx DOI 10.3233/JAD-142046 IOS Press
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Fatty Acid Profiles in Demented Patients: Identification of Hexacosanoic Acid (C26:0) as a Blood Lipid Biomarker of Dementia
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Amira Zarrouka,b,1 , Jean-Marc Riedingerc , Samia Hadj Ahmeda , Sonia Hammamia , Wafa Chaabaned , Meryam Debbabia , Sofiene Ben Ammoue , Olivier Rouaudf , Mahbouba Frihd , G´erard Lizardb,1,∗ and Mohamed Hammamia
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a Laboratoire
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b Equipe
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Nutrition, Aliments Fonctionnels et Sant´e Vasculaire, UR12ES05 Universit´e de Monastir, Tunisia Biochimie du Peroxysome, Inflammation et M´etabolisme Lipidique EA, 7270/Universit´e de Bourgogne/INSERM, Dijon, France c Centre de Lutte Contre le Cancer GF Leclerc, Dijon, France d Service Neurologie, CHU Fattouma Bourguiba, Monastir, Tunisia e Service Neurologie, CHU Sahloul, Sousse, Tunisia f Service Neurologie, CHU de Dijon, Dijon, France
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Accepted 3 November 2014
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Keywords: Dementia, fatty acid profiles, hexacosanoic acid (C26:0), lipid biomarkers, plasma, red blood cells
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INTRODUCTION
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Abstract. Background: Several lipid metabolism alterations have been described in the brain and plasma of Alzheimer’s disease (AD) patients, suggesting a relation between lipid metabolism alteration and dementia. Objective: We attempted to identify blood fatty acids as biomarkers of dementia. Methods: Fatty acid profiles were established using gas chromatography with or without mass spectrometry on matched plasma and red blood cells (RBCs) of demented patients diagnosed with AD, vascular dementia, or other dementia, and compared with a control group of elderly individuals. The severity of dementia was evaluated with the Mini-Mental State Examination test. Results: Fatty acid analysis showed significant variations of fatty acid levels in demented patients including AD patients. The highest plasma and RBC accumulation was found with hexacosanoic acid (C26:0). These data suggest that alterations of desaturase and elongase activity may contribute to cognitive dysfunction. Conclusion: The variations of fatty acid levels and the accumulation of C26:0 in the plasma and RBCs highlight an alteration of fatty acid metabolism in demented patients and point toward possible peroxisomal dysfunction. It is suggested that C26:0 may constitute a convenient blood biomarker of dementia that could be useful in routine medical practice.
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Alzheimer’s disease (AD; OMIM#104300) is the most common cause of dementia. Therefore, the 1 These
authors contributed equally to this work. ∗ Correspondence to: Dr. G´ erard Lizard, Laboratoire BIOperoxIL–EA 7270/INSERM, Facult´e des Sciences Gabriel, 6 Bd Gabriel, 21000 Dijon, France. Tel.: +33 380396256; Fax: +33 380396250; E-mail: [email protected].
etiology of the disease needs to be detailed more precisely to predict its occurrence and progression. Currently, the participation of lipid metabolism in AD, especially fatty acid (FA) metabolism, is not well known, although several lipid metabolism disorders have been described [1]. Isoform 4 of Apolipoprotein E is known to increase the risk of AD [2]. The oxysterol, 24(S)-hydroxycholesterol, is the predominant metabolite of brain cholesterol, and the plasma level of
ISSN 1387-2877/15/$27.50 © 2015 – IOS Press and the authors. All rights reserved
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Among FA disorders, which could contribute to the development of AD, it is also widely suspected that PUFAs and monounsaturated fatty acids (MUFAs) play a significant role. It is well established that a deficit in PUFAs is involved in several neurological disorders: schizophrenia, hyperactivity, depression, and AD [24]. The metabolism of MUFAs and PUFAs could be altered in AD. Consequently, abnormal levels of MUFAs and PUFAs could contribute to modifying membrane fluidity and neuronal activity. Indeed, arachidonic acid (AA), eicosapentaenoic acid (EPA), and DHA are powerful modulators of membrane fluidity [25]. This fluidity is modulated by PUFAs, and it depends on the rate of unsaturation of FAs incorporated into membrane phospholipids. Interestingly, hippocampal membranes of AD patients showed significantly lower fluidity compared with membranes from elderly nondemented controls [26]. Membrane fluidity in AD patients was also correlated with abnormal amyloid- protein precursor processing and cognitive decline [27]. These different data suggest substantial dysfunction of FA metabolism in AD and support the notion that FAs play a major role in the physiopathology of AD. It is therefore tempting to speculate that some FAs could constitute reliable biomarkers of dementia. In the present study, a lipidomic approach was used to identify peripheral lipid biomarkers of dementia, especially AD. With gas chromatography (GC) and gas chromatography coupled with mass spectrometry set to the selected-ion monitoring mode (GC/MS-SIM), we determined FA profiles (MUFA and PUFA profiles) on plasma and membranes of red blood cells (RBCs) from patients with AD, vascular dementia, and other forms of dementia compared to healthy controls within the same age range. The desaturation and elongation index were calculated (C16:1/C16:0 and C18:1/C18:0: 9 desaturation index; C18:3/C18:2: 6 desaturation index; C20:4/C20:3: 5 desaturation index; C24:1/C18:1 and C18:0/C16:0 elongation index). Since modifications of peroxisomal metabolism are suspected of contributing to the development of AD, several markers of peroxisomal metabolism were studied: VLCFAs (C22:0, C24:0, C26:0), DHA, phytanic acid, and plasmalogen-C16:0. Altogether, these data provide evidence of altered FA profiles and FA metabolism in demented patients. They demonstrate an accumulation of several FAs in plasma and RBCs, most particularly C26:0, which suggests possible peroxisomal dysfunction, possibly a convenient blood biomarker of dementia that may be useful in routine medical practice.
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this oxysterol may be higher in the early stages of cognitive impairment and lower in more advanced stages of AD when compared to cognitively normal controls [3]. Ceramides and phospholipids, which are ubiquitous components of cell membranes and play major roles in signal transduction, could also be indicators of neurodegeneration. A positive association was found between the concentrations of cerebrospinal fluid C18 ceramide, total sphingomyelins, and amyloid- and tau levels in a cohort of cognitively normal individuals with a confirmed parental history of AD [4]. Serum sphingomyelin and ceramides may be good preclinical predictors of memory impairment [5]. Plasma phospholipids could also be of interest to predict phenoconversion to either amnestic mild cognitive impairment or AD [6]. Postmortem analyses of brain samples have also revealed multiple lipid abnormalities in AD, including changes in ceramides, n-3 polyunsaturated fatty acids (PUFAs) and PUFAderived signaling lipids [7, 8]. It was reported that docosahexaenoic acid (DHA) content in the brain is often decreased in several neurodegenerative diseases, including AD [9]. DHA is an essential constituent of nervous tissue and is present in the brain under its esterified form with phospholipids [10]. In addition to its structural role in membrane lipids, DHA is required for brain development, and it participates in neurotransmission and synaptic plasticity [11, 12]. Moreover, decreased levels of DHA have been shown in phosphatidylcholine and phosphatidylethanolamine fractions from various regions of the brain (frontal grey, frontal white, hippocampus, and pons) in AD [13, 14]. Given that DHA is a substrate of peroxisomal oxidation [15], it is assumed that alteration of its plasma and tissue levels could be the consequence of peroxisomal dysfunction [9]. Indeed, the main functions of the peroxisome involve lipid metabolism with the -oxidation of various compounds including verylong-chain fatty acids (VLCFAs: C≥22, which are abundant in myelin) [16] and the precursor of DHA (C24:6 n-3), the ␣-oxidation of phytanic acid, and the first two steps of plasmalogen synthesis [17–19]. In vitro results support the hypothesis that a peroxisomal dysfunction is involved in AD: peroxisomal proliferation protects from amyloid- neurodegeneration [20]. In animal models, morphological and biochemical peroxisomal modifications were found in the brain of transgenic mouse models of AD [21, 22]. The potential role of peroxisome in AD was strengthened by results obtained on brain lesions of AD patients containing increased levels of VLCFAs (C22:0; C24:0; C26:0) [23].
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Blood sample preparation Blood was collected in EDTA tubes after overnight fasting. Plasma and RBCs were separated by centrifugation. Conventional biochemical characteristics of the patients including glycemia and lipid profiles were determined as described previously [28].
Statistical analyses Statistical analyses were performed by the Centre de Lutte Contre le Cancer G.F. Leclerc (Dr. J.M. Riedinger, Dijon, France), an independent organization. Data were analyzed using the Statistical Package for Social Sciences (SPSS 11.0 for Windows). Demographic and clinical characteristics of controls and demented patients were compared with a Student’s ttest. Since biological variable distributions were not normally distributed, the Kruskal-Wallis and MannWhitney nonparametric tests were used. To reduce the chance of obtaining false-positive results, Bonferroni correction was applied for the 35 tests in Tables 1 and 2, as well as for the seven tests in Table 3, and the 0.001 significance level was retained. Receiver operating characteristic curves (ROC) were carried out with Winstat for Microsoft Excel (version 2012.1) to identify discriminant biological variables and optimal threshold. The Spearman correlation test was also used to evaluate the relationships between various parameters, and for this statistical test the 0.05 significance level was retained.
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Extraction of fatty acids and fatty acid profile analysis Fatty acids were analyzed as fatty acid methyl esters (FAMEs) by GC analysis. Total lipids were extracted as described by Folch et al. [29]. Aliquots of total lipids were converted into methyl esters using 14% methanolboron trifluoride (BF3 ) at 50◦ C for 30 min. FAMEs were analyzed in duplicate and 1 L of each sample was injected into the GC system (Hewlett Packard, Palo Alto, CA, USA) equipped with a flame ionization detector and a polar fused silica capillary column HP-Innowax with cross-linked PEG. Carbowax 20 M (30 m × 0.25 mm ID and 0.25 m film thickness). The FA composition was reported as a relative percentage of the total peak area using a HP Chemstation integrator. VLCFAs (C22:0; C24:0; C26:0), phytanic acid, and plasmalogen-C16:0 were analyzed according to the method described by Takemoto et al. [30]. In a glass tube, 100 L of plasma or RBCs and 100 L of
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All assessments were carried out after obtaining written or verbal informed consent from patients and healthy subjects. A total of 64 subjects (28 women, 36 men; age: mean ± SD: 65 ± 10 years; median/range: 63/50–93 years) were studied, all from Central Tunisia, who consulted at Fattouma Bourguiba University Hospital (Monastir, Tunisia) between January and July 2011. The control group contained 128 subjects (60 women, 68 men; age: mean ± SD: 72 ± 8 years; median/range: 72/58–91 years) with no memory complaints or other cognitive impairment symptoms. These control subjects were recruited from a nursing home for the elderly in Central Tunisia (Sousse, Madhia, and Monastir). All participants underwent a complete clinical investigation; including medical history, neurological and neuropsychological examination, Mini-Mental State Examination (MMSE), screening laboratory tests, and neuroimaging consisting of magnetic resonance imaging. Patient characteristics are presented in Supplementary Tables 1 and 2.
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C17:0 (1 mg/mL) used as an internal standard were mixed with 2 mL of methanolic/5% hydrochloric acid and tightly capped. After shaking, the mixture was incubated for 2 h at 100◦ C. After cooling to room temperature, the methyl derivatives were extracted twice with 2 mL of n-hexane, dried under a stream of nitrogen, and dissolved in 1 mL of n-hexane. Methylated samples were subjected to a GC/MS system (Series II Gas Chromatograph Hewlett Packard 5890/Hewlett Packard 5972 MS) equipped with HP Chemstation program for instrument management and data reduction and with an HP-Innovax column (internal diameter: 250 m, length: 30 m, film thickness: 0.25 m; Hewlett-Packard). The initial oven temperature was 100◦ C, increased to 240◦ C (15◦ C/min) and maintained for 10 min. One microliter of sample was injected into the GC/MS system in the splitless mode. Electron impact ionization was applied at 70 eV. Mass spectrometry acquisition of each substance was set to the selected-ion monitoring mode (GC/MS-SIM).
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RESULTS
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Clinical and cognitive data
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The clinical features and biochemical parameters of healthy subjects and demented patients are summarized (Supplementary Tables 1 and 2). No significant
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49.85 ± 11.65 24.23 ± 6.25 17.57 ± 8.04 0.45 ± 0.29 2.56 ± 0.52 1.78 ± 0.83 3.26 ± 1.03∗∗ 25.34 ± 8.09∗∗ 0.52 ± 0.82 0.52 ± 0.32∗∗ 13.09 ± 5.43∗∗ 1.75 ± 0.57 2.19 ± 1.50∗∗ 1.55 ± 0.52 5.72 ± 3.25∗∗ 24.81 ± 1.54∗∗ 6.10 ± 2.44 0.29 ± 0.31 1.17 ± 0.21 5.65 ± 1.36∗∗ 1.91 ± 0.69 7.78 ± 4.35∗∗ 1.37 ± 0.98 0.53 ± 0.39 0.02 ± 0.01 0.76 ± 0.35 0.43 ± 0.21∗∗ 0.04 ± 0.32 1.37 ± 1.58 0.70 ± 0.29 4.98 ± 1.12 19.82 ± 1.09∗∗ 0.25 ± 0.20 0.51 ± 0.21 7.12 ± 2.46
Vascular Dementia 53.67 ± 11.24 26.87 ± 6.35 19.10 ± 8.13 0.64 ± 0.25 2.11 ± 0.90 1.92 ± 1.38 3.04 ± 0.85∗∗ 22.04 ± 1.16∗∗ 0.34 ±0.40 0.37 ± 0.25∗∗ 12.47 ± 7.46∗∗ 1.96 ± 1.05 1.69 ± 1.66 1.16 ± 1.04 4.05 ± 2.78∗∗ 24.28 ± 7.24∗∗ 5.92 ± 4.19 0.41 ± 0.25 1.15 ± 0.11 6.61 ± 1.39∗∗ 2.54 ± 0.64 4.63 ± 1.21 2.55 ± 0.94 0.47 ± 0.32 0.01 ± 0.01 0.65 ± 0.31 0.32 ± 0.79∗∗ 0.07 ± 0.04 0.70± 1.23 0.71 ± 0.30 6.71 ± 1.31 19.1± 1.14 0.35 ± 0.27 0.41 ± 0.29 7.07 ± 2.52
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50.18 ± 11.89 25.09 ± 6.30 18.27 ± 7.90 0.46 ± 0.23 2.34 ± 0.38 1.88 ± 1.64 2.53 ± 0.66∗∗ 23.64 ± 8.02∗∗ 0.52 ± 0.75 0.46 ± 0.27∗∗ 12.77 ± 5.87∗∗ 1.87 ± 1.14 1.88 ± 0.49∗∗ 1.36 ± 0.61 4.78 ± 3.11∗∗ 26.18 ± 2.03∗∗ 7.62 ± 3.62 0.81 ± 0.51 0.91 ± 0.20 5.24 ± 4.02 2.15 ± 1.32 7.22 ± 4.71∗∗ 1.87 ± 0.97 0.36 ± 0.17 0.02 ± 0.01 0.69 ± 0.54 0.40 ± 0.12∗∗ 0.38 ± 0.22∗∗ 1.37 ± 1.38 0.76 ± 0.29 5.29 ± 1.93 20.89 ± 2.71∗∗ 0.38 ± 0.50 0.47 ± 0.22 6.86 ± 3.26
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50.56 ± 6.82 30.05 ± 4.71 16.51 ± 3.41 0.62 ± 0.27 1.71 ± 0.62 0.79 ± 0.29 0.88 ± 0.06 30.71 ± 5.13 1.52 ± 1.23 1.53 ± 0.97 22.01 ± 4.62 2.41 ± 0.68 0.49 ± 0.29 1.33 ± 1.05 1.42 ± 1.08 18.73 ± 4.97 9.49 ± 3.31 0.39 ± 0.45 0.47 ± 0.47 2.01 ± 0.85 1.71 ± 0.98 3.32 ± 2.06 0.61 ± 0.41 0.73 ± 0.07 0.05 ± 0.04 1.38 ± 0.53 0.06 ± 0.1 0.04 ± 0.03 1.65 ± 1.25 0.55 ± 0.10 3.52 ± 1.88 15.21 ± 2.44 0.23 ± 0.23 0.60 ± 0.15 3.38 ± 1.87
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SFA C16:0 C18:0 C20:0 C22:0 C24:0 C26:0 MUFA C14:1 n-5 C16:1 n-5 C18:1 n-9 (OA) C18:1 n-7 C20:1 n-9 C22:1 n-9 C24:1 n-9 PUFA C18:2 n-6 (LA) C18:3 n-6 C18:3 n-3 (ALA) C20:3 n-6 C20:3 n-3 C20:4 n-6 (AA) C20:5 n-3 (EPA) C22:6 n-3 (DHA) C16:1/C16:0 C18:1/C18:0 C24:1/C18:1 C18:3/C18:2 C20:4/C20:3 C18:0/C16:0 PUFA n-3 PUFA n-6 PUFA n-3/ PUFA n-6 MUFA/ SFA VLCFA
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Other Dementia 47.71 ± 1.89 24.09 ± 5.80 18.51 ± 7.59 1.34 ± 0.23 1.09 ± 0.64 1.34 ± 0.49 1.34 ± 0.74 23.43 ± 8.14∗∗ 0.92 ± 0.59 0.52 ± 0.20∗∗ 12.66 ± 5.34∗∗ 1.96 ± 0.59 1.37 ± 1.22 0.81 ± 0.14 5.19 ± 3.13∗∗ 28.86 ± 1.35∗∗ 7.56 ± 3.29 2.00 ± 0.58 0.55 ± 0.38 5.93 ± 1.27∗∗ 1.54 ± 0.54 8.32 ± 3.92#∗∗ 2.58 ± 1.03 0.38 ± 0.26 0.02 ± 0.01 0.68 ± 0.34 0.40 ± 0.24 0.26 ± 0.13∗∗ 1.40 ±0.28 0.73 ± 0.25 4.80 ± 1.75 23.56 ± 1.65∗∗ 0.20 ± 0.10 0.49 ± 0.21 3.77 ± 1.13
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Data obtained by GC are expressed as relative values (%): mean % of total FA ± SD. SFAs, sum of saturated fatty acids; MUFAs, sum of monounsaturated fatty acids; PUFAs, sum of polyunsaturated fatty acids. OA, oleic acid; LA, linoleic acid, ALA, alpha linolenic acid; AA, arachidonic acid, EPA, eicosapentaenoic acid, DHA, docosahexaenoic acid. A significant difference between controls and demented patients is indicated by ∗∗ p < 0.001. Data with ∗∗ p < 0.001 are underlined in dark grey. C16:1/C16:0 and C18:1/C18:0: 9 desaturation index; C18:3/C18:2 : 6 desaturation index; C20:4/C20:3 : 5 desaturation index; C24:1/C18:1 and C18:0/C16:0: elongation index. Data with ∗∗ p < 0.001 are underlined in dark grey. The Mann-Whitney test with Bonferroni correction was used.
differences in glucose, cholesterol, triglycerides, and lipoproteins were observed between demented patients and controls. Evaluation of fatty acid profile in red blood cells and plasma The FA profile of RBC membranes is given in Table 1. Analysis of FA composition of RBCs in demented patients showed a slight nonsignificant decrease of SFA in the group with AD or other dementia. Whereas a nonsignificant increase of C18:0 and C22:0 was found, a significantly higher level of
C26:0 was revealed in all groups of demented patients, excepted in other dementia. MUFA was significantly lower in all groups of demented patients. It was associated with a significantly lower level of C16:1 n-5 and C18:1 n-9 (oleic acid). Nervonic acid (C24:1 n-9) in RBCs was almost four times higher in AD patients than in controls. PUFA was significantly higher in all groups. It was associated with higher C20:3 n-6 and C20:4 n-6 (AA) proportions. The highest proportion of PUFAs was mainly represented by PUFA n-6. It is noteworthy that a lower fatty acid 9-desaturase index (C18:1/C18:0), although not significant, was found in all patient groups. Major differences between
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Table 2 Fatty acid profiles in the plasma of controls and demented patients determined by gas chromatography Controls
Other Dementia
35.71 ± 5.12 22.78 ± 4.54 8.64 ± 2.94 0.30 ± 0.27 0.57 ± 0.33 1.16 ± 0.78 2.26 ± 1.07∗∗ 26.48 ± 7.33 0.31 ± 0.40 2.01 ± 0.95 20.09 ± 7.25 2.29 ± 0.68 1.29 ± 0.27 0.16 ± 0.10 0.33 ± 0.13 37.81 ± 7.34 25.05 ± 9.45 0.51 ± 0.42 0.24 ± 0.18 2.93 ± 1.10 2.87 ± 1.05 5.41 ± 2.62 0.42 ± 0.38 0.38 ± 0.14 0.08 ± 0.03 2.32 ± 1.13 1.88 ± 1.51∗∗ 0.02 ± 0.02 0.01 ± 0.01 0.38 ± 0.13 3.53 ± 2.01 28.87 ± 3.75 0.74 ± 0.24 0.12 ± 0.09 3.99 ± 1.55∗∗
34.64 ±4.32 22.8 ± 3.24 8.84 ±2.21 0.28 ± 0.18 0.40 ± 0.23 0.22 ± 0.12 2.10 ± 0.88∗∗ 26.54 ± 8.78 0.21 ± 0.38 1.61 ± 0.32 20.77 ± 6.87 2.51 ± 1.15 1.03 ± 0.49 0.10 ± 0.09 0.31 ± 0.15 38.83 ± 3.56 27.34 ± 3.56 0.55 ±0.29 0.31 ± 0.30 1.53 ± 0.48 2.05 ± 0.90 5.88 ± 2.58 0.83 ± 0.51 0.34 ± 0.13 0.07 ± 0.02 2.35 ± 1.18 3.84 ± 1.65∗∗ 0.02 ± 0.01 0.01 ± 0.02 0.38 ± 0.10 3.35 ± 2.44 35.37 ± 3.27 0.76 ± 0.34 0.10 ± 0.01 2.72 ± 1.01∗∗
35.86 ± 7.24 24.5 ± 4.33 8.18 ± 2.28 0.45 ± 0.28 0.52 ± 0.37 0.15 ± 0.10 2.06 ± 1.33∗∗ 29.84 ± 8.64 0.38 ± 0.66 2.00 ± 0.57 24.16 ± 5.36∗∗ 2.15 ± 0.60 0.60 ± 0.51 0.19 ± 0.28 0.36 ± 0.19 34.3 ± 3.02 22.94 ± 2.22∗∗ 0.62 ± 0.20 0.38 ± 0.10 1.81 ± 0.29 2.07 ±0.59 6.01 ± 2.02 0.19 ± 0.20 0.28 ± 0.21 0.08 ± 0.03 2.95 ± 0.64 3.32 ± 1.63∗∗ 0.03 ± 0.02 0.01 ± 0.01 0.33 ± 0.11 2.92 ± 0.71 31.38 ± 3.46 0.83 ± 0.24 0.09 ± 0.01 2.73 ± 1.45∗∗
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36.23 ± 5.98 23.61 ± 4.43 8.93 ± 2.70 0.34 ± 0.26 0.51 ± 0.32 0.63 ± 0.10 2.21 ± 1.12∗∗ 27.43 ± 7.49 0.32 ± 0.49 1.91 ± 0.71 21.30 ± 6.91 2.41 ± 0.81 1.02 ± 0.16 0.11 ± 0.18 0.36 ± 0.16 36.34 ± 7.86 24.23 ± 9.23 0.56 ± 0.45 0.32 ± 0.32 2.18 ± 1.45 2.40 ± 1.46 5.83 ± 2.48 0.46 ± 0.31 0.35 ± 0.16 0.08 ± 0.02 2.38 ± 1.49 2.42 ± 1.48∗∗ 0.02 ± 0.01 0.01 ± 0.01 0.38 ± 0.11 3.07 ± 1.58 32.80 ± 2.39 0.75 ± 0.30 0.09 ± 0.29 3.35 ± 1.24∗∗
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34.22 ± 3.26 26.24 ± 2.75 7.16 ± 1.52 0.25 ± 0.35 0.30 ± 0.12 0.17 ± 0.05 0.10 ± 0.05 28.20 ± 3.48 1.32 ± 1.21 2.53 ± 0.90 21.01 ± 3.56 1.93 ± 0.48 0.64 ± 0.13 0.43 ± 0.16 0.34 ± 0.12 37.58 ± 4.67 27.78 ± 5.26 0.59 ± 0.30 0.71 ± 0.23 0.57 ± 0.37 1.75 ± 0.48 4.45 ± 1.24 0.30 ± 0.31 1.43 ± 0.27 0.09 ± 0.03 2.93 ± 3.21 7.80 ± 2.19 0.02 ± 0.02 0.01 ± 0.02 0.27 ± 0.06 3.89 ± 2.36 33.39 ± 3.44 0.82 ± 0.21 0.12 ± 0.23 0.57 ± 0.37
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SFA C16:0 C18:0 C20:0 C22:0 C24:0 C26:0 MUFA C14:1 n-5 C16:1 n-5 C18:1 n-9 (OA) C18:1 n-7 C20:1 n-9 C22:1 n-9 C24:1 n-9 PUFA C18:2 n-6 (LA) C18:3 n-6 C18:3 n-3 (ALA) C20:3 n-6 C20:3 n-3 C20:4 n-6 (AA) C20:5 n-3(EPA) C22:6 n-3 (DHA) C16:1/C16:0 C18:1/C18:0 C20:4/C20:3 C18:3/C18:2 C24:1/C18:1 C18:0/C16:0 PUFA n-3 PUFA n-6 MUFA/ SFA PUFA n-3/ PUFA n-6 VLCFA
Demented Patients All Dementia
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patients and controls were also observed with the elongase index (C24:1/C18:1). The 5-desaturation activity index (C20:4/C20:3) was similar in these groups. Moreover, the 6-desaturation activity index (C18:3/C18:2) was significantly higher in patients with all or other dementia (Table 1). The FA composition of the plasma is given in Table 2. A significantly higher level of C26:0 was revealed in all groups of demented patients, and VLCFA was significantly higher in these groups A lower 5-desaturation activity index (C20:4/C20:3) was found in all patient groups. The elongase activity index (C18:0/C16:0) and the 6-desaturation activity index (18:3/18:2) were not modified.
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Data obtained by GC are expressed as relative values (%): mean % of total FA ± SD. SFA, sum of saturated fatty acids; MUFA, sum of monounsaturated fatty acids; PUFA, sum of polyunsaturated fatty acids; OA, oleic acid; LA, linoleic acid; ALA, alpha linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaeno¨ic acid. A significant difference between controls and demented patients is indicated by ∗∗ p < 0.001. Data with ∗∗ p < 0.001 are underlined in dark grey. The Mann-Whitney test with Bonferroni correction was used. C16:1/C16:0 and C18:1/C18:0 : 9 desaturation index; C18:3/C18:2 : 6 desaturation index; C20:4/C20:3 : 5 desaturation index; C24:1/C18:1 and C18:0/C16:0 : elongation index.
Quantification of lipids linked with peroxisomal metabolism Quantification of VLCFAs (C22:0; C24:0; C26:0), phytanic acid, and plasmalogen-C16:0 levels (linked with peroxisomal metabolism) [9], in plasma and RBCs was conducted by GC/MS-SIM (Table 3). Comparing VLCFAs between controls and demented patients showed a significantly higher (p < 0.001) level of C26:0 in the plasma and RBCs of demented patients. In RBCs, the C26:0 concentration, which was 1.92 ± 2.07 M in the control group, was 16.78 ± 4.37 M, 16.35 ± 4.91 M, 17.66 ± 3.85 M, and 16.59 ± 4.15 M in all dementia,
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Table 3 Levels of very long chain fatty acids, phytanic acid, and plasmalogen-C16:0 analyzed by GC/MS-SIM in plasma and red blood cells of controls and demented patients Controls
All Dementia
Phytanic acid Plasmalogen-C16:0 C26:0 C24:0 C22:0 C22:6 n-3 (DHA) Desaturation index C24:1/C24:0
RBCs
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16.39 ± 6.67 55.85 ± 7.17 1.92 ± 2.07 31.72 ± 9.52 21.57 ± 8.32 79.88 ± 49.75 2.78 ± 2.64
16.90 ± 3.16 22.36 ± 2.67 1.08 ± 1.48 24.68 ± 11.71 23.59 ± 7.09 71.47 ± 55.11 2.92 ± 0.59
RBCs
18.61 ± 7.35 48.02 ± 8.29 16.78 ± 4.37∗∗ 24.32 ± 5.47 24.96 ± 5.83 52.19 ± 4.21 7.22 ± 1.78
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25.20 ± 17.32 17.41 ± 3.62 12.86 ± 5.42∗∗ 28.53 ± 7.00 26.50 ± 7.86 45.65 ± 14.06 2.52 ±0.97
Demented patients
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RBCs
Plasma
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20.15 ± 5.07 49.06 ± 4.79 16.35 ± 4.91∗∗ 24.26 ± 5.70 25.69 ± 4.11 43.85 ± 2.57∗∗ 10.80 ± 2.57
24.33 ± 5.64 12.86 ± 6.66 12.64 ± 6.11∗∗ 29.82 ± 6.4 24.77 ± 7.45 45.69 ± 8.25 1.90 ± 0.98
Vascular Dementia RBCs 16.04 ± 8.70 53.31 ± 6.54 17.66 ± 3.85∗∗ 24.29 ± 6.16 25.54 ± 6.32 59.05 ± 30.67 5.02 ± 1.19
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Others Dementia
Plasma 29.43 ± 9.19 17.55 ± 11.94 14.00 ± 5.11∗∗ 29.92 ± 7.93 28.70 ± 8.36 40.39 ± 14.22 1.61 ± 0.80
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RBCs
Plasma
18.87 ± 1.49 42.49 ± 3.37 16.59 ± 4.15∗∗ 24.41 ± 4.85 23.73 ± 6.99 55.99 ± 26.60 4.29 ± 1.58
23.16 ± 8.15 23.21 ± 8.99 12.21 ± 4.76∗∗ 25.74 ± 6.46 26.98 ± 7.86 49.80 ± 18.54 4.05 ± 1.90
RBCs: red blood cells (measurements were performed on membrane RBCs), Gas chromatography/mass spectrometry acquisition of each substance was set to the selected-ion monitoring mode (GC/MS–SIM). Data are expressed as mean concentrations ± SD expressed in M. A significant difference between controls and demented patients is indicated by ∗∗ p < 0.001 (Mann Whitney test with Bonferroni correction). Data with ∗∗ (p < 0.001) are underlined in dark grey.
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Test 1, A = 0.99
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Fig. 1. Distribution and receiver operating characteristic (ROC) curves established on controls and demented patients with C26:0 concentrations measured in RBCs. ROC curve: AUC = 0.999 ± 0.001 [95% CI, 0.998–1.001], threshold = 4.16 M, sensitivity = 97.9%, specificity = 98.7%.
Fig. 2. Distribution and receiver operating characteristic (ROC) curves established on controls and demented patients with C26:0 concentrations measured in the plasma. ROC curve: AUC = 0.993 ± 0.006 [95% CI, 0.982–1.005], threshold = 2.80 M, sensitivity = 98.8%, specificity = 98.4%.
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AD patients, vascular dementia, and other dementia, respectively. In demented patients, the analysis of the ROC curve, corresponding to C26:0 concentrations in RBCs, showed an area under the ROC curves (AUC) of 0.999 (95% confidence interval (CI), 0.998–1.001) and a predictive threshold of 4.16 M. The AUC was significantly different from 0.5 (p < 10–4 ). Sensitivity and specificity of the level found were 97.9% and 98.7%, respectively (Fig. 1). The measurement of C26:0 in RBCs made it possible to classify 98.3% of the AD patients correctly. In plasma, the concentration of C26:0 was significantly higher in demented patients: 1.08 ± 1.48 M in the control group and 12.86 ± 5.42 M in demented patients (Table 3). In the
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plasma of AD patients, C26:0 concentration was 12fold higher than in control subjects (12.64 ± 6.11 M versus 1.08 ± 1.48 M). This plasma accumulation of C26:0 was also observed in vascular dementia and in other dementia groups. In AD patients, the analysis of the ROC curve corresponding to plasma C26:0 showed an AUC of 0.993 (95% CI, 0.982–1.005) and a predictive threshold of 2.80 M. The AUC differed significantly from 0.5 (p < 10–4 ). The sensitivity of the level was 98.8%, its specificity was 98.4%, and the level of C26:0 in plasma accurately classified 99.1% of AD patients (Fig. 2). In AD, vascular dementia and other dementia patients (in agreement with data shown in Table 3), similar characteristics of ROC curves
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Correlations between plasma and membrane red blood cell fatty acid composition
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DISCUSSION
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Significant correlations were seen between C22:0 and AA, EPA, and C26:0, EPA and DHA in RBCs (r = –0.65, p < 0.001; r = –0.449, p < 0.05; r = 0.529, p < 0.05; respectively) and between plasma EPA and AA (r = –0.403, p < 0.05). We found that the desaturation index (MUFA/SFA) in plasma displayed i) a negative correlation with the MMSE test, which assesses global cognition (r = –0.411; p < 0.05) and ii) a positive correlation between these two parameters in RBCs (r = 0.437, p = 0.05) (Supplementary Figure 2). A correlation was shown between MMSE and SFA in the plasma of demented patients (r = 0.295; p < 0.05).
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results reporting a lower level of these FAs, in particular oleic acid [31]. Moreover, in agreement with previous investigations, we observed a notably higher level of nervonic acid (C24:1 n-9) in the RBCs of demented patients, which was also found at elevated levels in brain samples from AD patients and which was strongly correlated with cognitive impairment [7]. The increase of C18:0, although not significant, supports the notion that enhanced levels of this fatty acid could be associated with an increased risk of cognitive decline [32]. The total PUFA proportion (PUFA) was also higher in the RBCs of all groups due to the significantly higher level of total n-6 fatty acids (PUFA n-6) as well as AA and C20:3 n-6. The accumulation of PUFA n-3 in RBCs may explain, at least in part, the lower MUFA levels, as suggested by several studies. Indeed, it has been reported that PUFA repress stearoyl-CoA desaturase (9 desaturase) mRNA expression [33]. Although not statistically significant, the lower level of DHA in demented patients found in the present study reinforces the results of other investigations that described lower levels of DHA in dementia [9]. In addition, we report a negative correlation between the MMSE and the desaturation index (MUFA/SFA) in the plasma of AD patients. This finding suggests the existence of a functional link between lower MUFA levels in plasma and deterioration of mental functions in subjects with AD, and is in accordance with data obtained by Astarita et al. [7], who showed that the desaturation index measured in brains of AD patients was correlated with the MMSE score. It is known that modification of FA proportions and alteration of 5 and 6 desaturase activity may result from genetic variants of these enzymes encoded by the FADS1 and FADS2 genes. However, the effect of the polymorphism of these genes on PUFA n-6 has not been thoroughly investigated [34]. Furthermore, it was reported that the activity of 5 and 6 desaturase is modulated by dietary fatty acid intake and the level of FAs in the tissues [34]. Whereas the desaturation index depends on several intrinsic and extrinsic factors, 5 and 6 desaturases may be valuable from several points of view in following the outcome of demented patients. To identify potential peripheral biomarkers in demented patients, we used a quantitative lipidomic strategy based on GC/MS-SIM quantifying VLCFAs, plasmalogen-C16:0, and phytanic acid. Noteworthy, an accumulation of VLCFAs was observed in RBCs and plasma from AD patients and those with other forms of dementia. This increase of VLCFAs observed
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as those observed in all dementia were found (our data not shown). It should be noted that the C26:0 concentration in RBCs was significantly correlated to its concentration in plasma (r = 0.787; p < 0.001) (Supplementary Figure 1). Comparison of the C24:0 mean values between the control group and demented patients demonstrated slightly higher but nonsignificant plasma C24:0 in the group of demented patients. In fact, the C24:0 concentration was 24.68 ± 11.71 M in controls and 28.53 ± 7.00 M in demented patients. In AD, the plasma C24:0 level was 29.82 ± 6.40 M. Although not significant, this accumulation was also observed in the plasma of patients with vascular dementia (29.92 ± 7.93 M) or in other dementia (25.74 ± 6.46 M). With plasmalogen-C16:0, a nonsignificant lower level was observed in RBCs and plasma of all demented patients and AD patients. Comparison of phytanic acid levels in plasma and RBCs between controls and demented patients also showed a higher level (although nonsignificant) of this branched FA in all demented patients (Table 3).
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These data obtained by GC and GC/MS-SIM on the plasma and RBCs of patients with AD, vascular dementia, and other types of dementia demonstrate important modifications of FA profiles: i) significant variations of several SFAs, including VLCFAs; ii) a higher elongation index and modifications of the 5, 6, and 9 desaturation index, supporting modifications in FA metabolism. This study also establishes that C26:0 could constitute a convenient peripheral blood biomarker of dementia, including AD. The MUFA level was lower in demented patients than in controls in the RBCs, in agreement with the
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which reflects peroxisomal ␣-oxidation [15, 38], also supports peroxisome dysfunctions and points toward involvement of this organelle in the development of dementia. Complementary studies are required on a large number of demented patients from various origins to determine the value of a fatty acid profile in the diagnosis of dementia. It cannot be excluded that fatty acid metabolism may vary with ethnic origin. In addition, dietary habits can also influence the fatty acid profile. It would also be useful to correlate the fatty acid profile, especially the RBCs and plasma level of C26:0, with conventional biomarkers of AD (amyloid and tau) present in the cerebrospinal fluid to establish clearly whether C26:0 can be considered as an alternative biomarker of dementia. We must also determine whether increased plasma levels of VLCFAs, known to occur in peroxisomopathies, can also be observed in other forms of neurological diseases. From a practical point of view, the quantification of C26:0, which only requires a blood sample (easier to take than collecting cerebrospinal fluid to quantify conventional biomarkers of AD and requiring specialized care units), could make the diagnosis of dementia available in routine medical practice. Easy-to-measure biomarkers could be applied to a high number of patients. This would contribute to rapidly obtaining a great deal of data for better knowledge of the physiopathology of dementia in order to develop appropriate and effective treatments that are not yet available. Moreover, since the plasma levels of VLCFAs are approximately 12 times higher in demented patients than in controls, the risk of false-positive results will be avoided. In conclusion, our data underline important differences in the FA profile of plasma and RBCs in demented patients, suggesting pronounced alterations of lipid metabolism in dementia. In addition, the substantial accumulation of VLCFAs (especially C26:0) in dementia favors the hypothesis of possible peroxisomal dysfunctions, which are assumed to play critical roles in cell senescence and aging [39, 40].
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in demented patients is in agreement with FA analyses of cortical regions of brains of AD patients with Braak classification stages V–VI, which revealed accumulation of VLCFAs compared with those modestly affected (stages I–II) [23]. In these patients, this accumulation of C22:0, C24:0, and C26:0 was significantly correlated with the appearance of neurofibrillary tangles [23], and the accumulation of C24:0 and C26:0 was assumed to reveal peroxisomal dysfunctions in the cascade of events contributing to the peroxisomal beta-oxidation of C24:0 and C26:0 [9, 23]. In brain cells, VLCFA incorporation into complex lipids could destabilize cellular membranes and change their physiological properties, including those of lipid rafts, which serve as platforms for cell signaling and membrane trafficking [35]. The accumulation of VLCFAs could also result, at least in part, from enhanced FA elongation, whereas the enhanced elongase index observed in the present study was not significant. Therefore, peroxisomal dysfunctions and enhanced elongase activities could explain the marked accumulation of C26:0, in the plasma and RBCs of demented patients. It is noteworthy that the plasmatic levels of C26:0 found in patients with AD (12.64 ± 6.11 M), vascular dementia (14.00 ± 5.11 M), and other dementia (12.21 ± 4.76 M) are within the range of those observed in patients with peroxisomal disorders (X-linked adrenoleukodystrophy (X-ALD): 2.87 ± 1.46 M; Zellweger syndrome: 18.25 ± 8.11 M; D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacylCoA dehydrogenase deficiency (D-BP): 18.02 M; and acylCoA oxidase deficiency (AOX): 4.31 M) [30]. In control subjects, the plasma level of C26:0 (measured in infants, school-aged children, and young adults) [30, 36] was within the range of 0.10–0.40 M. The reduced amounts of plasmalogen-C16:0, requiring enzymes located both in the peroxisome and the endoplasmic reticulum [17, 18], suggest dysfunctions of these organelles, which could have important consequences on brain functions. A pronounced decrease of plasmalogen content has been reported in the white matter, at a very early stage of dementia [36]. A reduction of ethanolamine plasmalogen, in the grey matter of AD patients, has also been described, and it was correlated with a synaptic loss and brain degeneration [37]. In addition, it was reported that reduction of ethanolamine plasmalogen and choline plasmalogen in the brain and RBCs of AD patients was correlated with the severity of dementia [37]. Although not statistically significant, in AD patients and in patients with vascular dementia, the plasma accumulation of phytanic acid, which can be based on dietary intake, and
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ACKNOWLEDGMENTS This work was supported by grants from the Minist`ere de l’Enseignement Sup´erieur et de la Recherche Scientifique and the Direction G´en´erale de la Recherche Scientifique et Technologique (Tunisia). We thank Dr. Imed Cheraif for his helpful technical assistance. We also acknowledge INSERM, the Universit´e de Bourgogne, the Department of Neurology
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(CHU de Dijon, France), and ABASIM (Dijon, France) for their financial support. The authors are indebted to Mrs. Linda Northrup for reviewing the English version of the manuscript. Authors’ disclosures available online (http://j-alz. com/manuscript-disclosures/14-2046r2). The data presented are included in two patents from the Universit´e de Bourgogne: French patent deposit (2013 09 05) and international patent deposit (2014 05 13).
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Journal of Alzheimer’s Disease xx (20xx) x–xx DOI 10.3233/JAD-142046 IOS Press
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Fatty Acid Profiles in Demented Patients: Identification of Hexacosanoic Acid (C26:0) as a Blood Lipid Biomarker of Dementia
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Amira Zarrouka,b,1 , Jean-Marc Riedingerc , Samia Hadj Ahmeda , Sonia Hammamia , Wafa Chaabaned , Meryam Debbabia , Sofiene Ben Ammoue , Olivier Rouaudf , Mahbouba Frihd , G´erard Lizardb,1,∗ and Mohamed Hammamia
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Nutrition, Aliments Fonctionnels et Sant´e Vasculaire, UR12ES05 Universit´e de Monastir, Tunisia Biochimie du Peroxysome, Inflammation et M´etabolisme Lipidique EA, 7270/Universit´e de Bourgogne/INSERM, Dijon, France c Centre de Lutte Contre le Cancer GF Leclerc, Dijon, France d Service Neurologie, CHU Fattouma Bourguiba, Monastir, Tunisia e Service Neurologie, CHU Sahloul, Sousse, Tunisia f Service Neurologie, CHU de Dijon, Dijon, France
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Accepted 3 November 2014
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Keywords: Dementia, fatty acid profiles, hexacosanoic acid (C26:0), lipid biomarkers, plasma, red blood cells
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INTRODUCTION
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Abstract. Background: Several lipid metabolism alterations have been described in the brain and plasma of Alzheimer’s disease (AD) patients, suggesting a relation between lipid metabolism alteration and dementia. Objective: We attempted to identify blood fatty acids as biomarkers of dementia. Methods: Fatty acid profiles were established using gas chromatography with or without mass spectrometry on matched plasma and red blood cells (RBCs) of demented patients diagnosed with AD, vascular dementia, or other dementia, and compared with a control group of elderly individuals. The severity of dementia was evaluated with the Mini-Mental State Examination test. Results: Fatty acid analysis showed significant variations of fatty acid levels in demented patients including AD patients. The highest plasma and RBC accumulation was found with hexacosanoic acid (C26:0). These data suggest that alterations of desaturase and elongase activity may contribute to cognitive dysfunction. Conclusion: The variations of fatty acid levels and the accumulation of C26:0 in the plasma and RBCs highlight an alteration of fatty acid metabolism in demented patients and point toward possible peroxisomal dysfunction. It is suggested that C26:0 may constitute a convenient blood biomarker of dementia that could be useful in routine medical practice.
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Alzheimer’s disease (AD; OMIM#104300) is the most common cause of dementia. Therefore, the 1 These
authors contributed equally to this work. ∗ Correspondence to: Dr. G´ erard Lizard, Laboratoire BIOperoxIL–EA 7270/INSERM, Facult´e des Sciences Gabriel, 6 Bd Gabriel, 21000 Dijon, France. Tel.: +33 380396256; Fax: +33 380396250; E-mail: [email protected].
etiology of the disease needs to be detailed more precisely to predict its occurrence and progression. Currently, the participation of lipid metabolism in AD, especially fatty acid (FA) metabolism, is not well known, although several lipid metabolism disorders have been described [1]. Isoform 4 of Apolipoprotein E is known to increase the risk of AD [2]. The oxysterol, 24(S)-hydroxycholesterol, is the predominant metabolite of brain cholesterol, and the plasma level of
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Among FA disorders, which could contribute to the development of AD, it is also widely suspected that PUFAs and monounsaturated fatty acids (MUFAs) play a significant role. It is well established that a deficit in PUFAs is involved in several neurological disorders: schizophrenia, hyperactivity, depression, and AD [24]. The metabolism of MUFAs and PUFAs could be altered in AD. Consequently, abnormal levels of MUFAs and PUFAs could contribute to modifying membrane fluidity and neuronal activity. Indeed, arachidonic acid (AA), eicosapentaenoic acid (EPA), and DHA are powerful modulators of membrane fluidity [25]. This fluidity is modulated by PUFAs, and it depends on the rate of unsaturation of FAs incorporated into membrane phospholipids. Interestingly, hippocampal membranes of AD patients showed significantly lower fluidity compared with membranes from elderly nondemented controls [26]. Membrane fluidity in AD patients was also correlated with abnormal amyloid- protein precursor processing and cognitive decline [27]. These different data suggest substantial dysfunction of FA metabolism in AD and support the notion that FAs play a major role in the physiopathology of AD. It is therefore tempting to speculate that some FAs could constitute reliable biomarkers of dementia. In the present study, a lipidomic approach was used to identify peripheral lipid biomarkers of dementia, especially AD. With gas chromatography (GC) and gas chromatography coupled with mass spectrometry set to the selected-ion monitoring mode (GC/MS-SIM), we determined FA profiles (MUFA and PUFA profiles) on plasma and membranes of red blood cells (RBCs) from patients with AD, vascular dementia, and other forms of dementia compared to healthy controls within the same age range. The desaturation and elongation index were calculated (C16:1/C16:0 and C18:1/C18:0: 9 desaturation index; C18:3/C18:2: 6 desaturation index; C20:4/C20:3: 5 desaturation index; C24:1/C18:1 and C18:0/C16:0 elongation index). Since modifications of peroxisomal metabolism are suspected of contributing to the development of AD, several markers of peroxisomal metabolism were studied: VLCFAs (C22:0, C24:0, C26:0), DHA, phytanic acid, and plasmalogen-C16:0. Altogether, these data provide evidence of altered FA profiles and FA metabolism in demented patients. They demonstrate an accumulation of several FAs in plasma and RBCs, most particularly C26:0, which suggests possible peroxisomal dysfunction, possibly a convenient blood biomarker of dementia that may be useful in routine medical practice.
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this oxysterol may be higher in the early stages of cognitive impairment and lower in more advanced stages of AD when compared to cognitively normal controls [3]. Ceramides and phospholipids, which are ubiquitous components of cell membranes and play major roles in signal transduction, could also be indicators of neurodegeneration. A positive association was found between the concentrations of cerebrospinal fluid C18 ceramide, total sphingomyelins, and amyloid- and tau levels in a cohort of cognitively normal individuals with a confirmed parental history of AD [4]. Serum sphingomyelin and ceramides may be good preclinical predictors of memory impairment [5]. Plasma phospholipids could also be of interest to predict phenoconversion to either amnestic mild cognitive impairment or AD [6]. Postmortem analyses of brain samples have also revealed multiple lipid abnormalities in AD, including changes in ceramides, n-3 polyunsaturated fatty acids (PUFAs) and PUFAderived signaling lipids [7, 8]. It was reported that docosahexaenoic acid (DHA) content in the brain is often decreased in several neurodegenerative diseases, including AD [9]. DHA is an essential constituent of nervous tissue and is present in the brain under its esterified form with phospholipids [10]. In addition to its structural role in membrane lipids, DHA is required for brain development, and it participates in neurotransmission and synaptic plasticity [11, 12]. Moreover, decreased levels of DHA have been shown in phosphatidylcholine and phosphatidylethanolamine fractions from various regions of the brain (frontal grey, frontal white, hippocampus, and pons) in AD [13, 14]. Given that DHA is a substrate of peroxisomal oxidation [15], it is assumed that alteration of its plasma and tissue levels could be the consequence of peroxisomal dysfunction [9]. Indeed, the main functions of the peroxisome involve lipid metabolism with the -oxidation of various compounds including verylong-chain fatty acids (VLCFAs: C≥22, which are abundant in myelin) [16] and the precursor of DHA (C24:6 n-3), the ␣-oxidation of phytanic acid, and the first two steps of plasmalogen synthesis [17–19]. In vitro results support the hypothesis that a peroxisomal dysfunction is involved in AD: peroxisomal proliferation protects from amyloid- neurodegeneration [20]. In animal models, morphological and biochemical peroxisomal modifications were found in the brain of transgenic mouse models of AD [21, 22]. The potential role of peroxisome in AD was strengthened by results obtained on brain lesions of AD patients containing increased levels of VLCFAs (C22:0; C24:0; C26:0) [23].
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Blood sample preparation Blood was collected in EDTA tubes after overnight fasting. Plasma and RBCs were separated by centrifugation. Conventional biochemical characteristics of the patients including glycemia and lipid profiles were determined as described previously [28].
Statistical analyses Statistical analyses were performed by the Centre de Lutte Contre le Cancer G.F. Leclerc (Dr. J.M. Riedinger, Dijon, France), an independent organization. Data were analyzed using the Statistical Package for Social Sciences (SPSS 11.0 for Windows). Demographic and clinical characteristics of controls and demented patients were compared with a Student’s ttest. Since biological variable distributions were not normally distributed, the Kruskal-Wallis and MannWhitney nonparametric tests were used. To reduce the chance of obtaining false-positive results, Bonferroni correction was applied for the 35 tests in Tables 1 and 2, as well as for the seven tests in Table 3, and the 0.001 significance level was retained. Receiver operating characteristic curves (ROC) were carried out with Winstat for Microsoft Excel (version 2012.1) to identify discriminant biological variables and optimal threshold. The Spearman correlation test was also used to evaluate the relationships between various parameters, and for this statistical test the 0.05 significance level was retained.
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Extraction of fatty acids and fatty acid profile analysis Fatty acids were analyzed as fatty acid methyl esters (FAMEs) by GC analysis. Total lipids were extracted as described by Folch et al. [29]. Aliquots of total lipids were converted into methyl esters using 14% methanolboron trifluoride (BF3 ) at 50◦ C for 30 min. FAMEs were analyzed in duplicate and 1 L of each sample was injected into the GC system (Hewlett Packard, Palo Alto, CA, USA) equipped with a flame ionization detector and a polar fused silica capillary column HP-Innowax with cross-linked PEG. Carbowax 20 M (30 m × 0.25 mm ID and 0.25 m film thickness). The FA composition was reported as a relative percentage of the total peak area using a HP Chemstation integrator. VLCFAs (C22:0; C24:0; C26:0), phytanic acid, and plasmalogen-C16:0 were analyzed according to the method described by Takemoto et al. [30]. In a glass tube, 100 L of plasma or RBCs and 100 L of
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All assessments were carried out after obtaining written or verbal informed consent from patients and healthy subjects. A total of 64 subjects (28 women, 36 men; age: mean ± SD: 65 ± 10 years; median/range: 63/50–93 years) were studied, all from Central Tunisia, who consulted at Fattouma Bourguiba University Hospital (Monastir, Tunisia) between January and July 2011. The control group contained 128 subjects (60 women, 68 men; age: mean ± SD: 72 ± 8 years; median/range: 72/58–91 years) with no memory complaints or other cognitive impairment symptoms. These control subjects were recruited from a nursing home for the elderly in Central Tunisia (Sousse, Madhia, and Monastir). All participants underwent a complete clinical investigation; including medical history, neurological and neuropsychological examination, Mini-Mental State Examination (MMSE), screening laboratory tests, and neuroimaging consisting of magnetic resonance imaging. Patient characteristics are presented in Supplementary Tables 1 and 2.
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C17:0 (1 mg/mL) used as an internal standard were mixed with 2 mL of methanolic/5% hydrochloric acid and tightly capped. After shaking, the mixture was incubated for 2 h at 100◦ C. After cooling to room temperature, the methyl derivatives were extracted twice with 2 mL of n-hexane, dried under a stream of nitrogen, and dissolved in 1 mL of n-hexane. Methylated samples were subjected to a GC/MS system (Series II Gas Chromatograph Hewlett Packard 5890/Hewlett Packard 5972 MS) equipped with HP Chemstation program for instrument management and data reduction and with an HP-Innovax column (internal diameter: 250 m, length: 30 m, film thickness: 0.25 m; Hewlett-Packard). The initial oven temperature was 100◦ C, increased to 240◦ C (15◦ C/min) and maintained for 10 min. One microliter of sample was injected into the GC/MS system in the splitless mode. Electron impact ionization was applied at 70 eV. Mass spectrometry acquisition of each substance was set to the selected-ion monitoring mode (GC/MS-SIM).
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RESULTS
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Clinical and cognitive data
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The clinical features and biochemical parameters of healthy subjects and demented patients are summarized (Supplementary Tables 1 and 2). No significant
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A. Zarrouk et al. / Fatty Acids, Lipid Biomarkers, and Dementia Table 1 Fatty acid profiles of red blood cells in controls and demented patients determined by gas chromatography
49.85 ± 11.65 24.23 ± 6.25 17.57 ± 8.04 0.45 ± 0.29 2.56 ± 0.52 1.78 ± 0.83 3.26 ± 1.03∗∗ 25.34 ± 8.09∗∗ 0.52 ± 0.82 0.52 ± 0.32∗∗ 13.09 ± 5.43∗∗ 1.75 ± 0.57 2.19 ± 1.50∗∗ 1.55 ± 0.52 5.72 ± 3.25∗∗ 24.81 ± 1.54∗∗ 6.10 ± 2.44 0.29 ± 0.31 1.17 ± 0.21 5.65 ± 1.36∗∗ 1.91 ± 0.69 7.78 ± 4.35∗∗ 1.37 ± 0.98 0.53 ± 0.39 0.02 ± 0.01 0.76 ± 0.35 0.43 ± 0.21∗∗ 0.04 ± 0.32 1.37 ± 1.58 0.70 ± 0.29 4.98 ± 1.12 19.82 ± 1.09∗∗ 0.25 ± 0.20 0.51 ± 0.21 7.12 ± 2.46
Vascular Dementia 53.67 ± 11.24 26.87 ± 6.35 19.10 ± 8.13 0.64 ± 0.25 2.11 ± 0.90 1.92 ± 1.38 3.04 ± 0.85∗∗ 22.04 ± 1.16∗∗ 0.34 ±0.40 0.37 ± 0.25∗∗ 12.47 ± 7.46∗∗ 1.96 ± 1.05 1.69 ± 1.66 1.16 ± 1.04 4.05 ± 2.78∗∗ 24.28 ± 7.24∗∗ 5.92 ± 4.19 0.41 ± 0.25 1.15 ± 0.11 6.61 ± 1.39∗∗ 2.54 ± 0.64 4.63 ± 1.21 2.55 ± 0.94 0.47 ± 0.32 0.01 ± 0.01 0.65 ± 0.31 0.32 ± 0.79∗∗ 0.07 ± 0.04 0.70± 1.23 0.71 ± 0.30 6.71 ± 1.31 19.1± 1.14 0.35 ± 0.27 0.41 ± 0.29 7.07 ± 2.52
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50.18 ± 11.89 25.09 ± 6.30 18.27 ± 7.90 0.46 ± 0.23 2.34 ± 0.38 1.88 ± 1.64 2.53 ± 0.66∗∗ 23.64 ± 8.02∗∗ 0.52 ± 0.75 0.46 ± 0.27∗∗ 12.77 ± 5.87∗∗ 1.87 ± 1.14 1.88 ± 0.49∗∗ 1.36 ± 0.61 4.78 ± 3.11∗∗ 26.18 ± 2.03∗∗ 7.62 ± 3.62 0.81 ± 0.51 0.91 ± 0.20 5.24 ± 4.02 2.15 ± 1.32 7.22 ± 4.71∗∗ 1.87 ± 0.97 0.36 ± 0.17 0.02 ± 0.01 0.69 ± 0.54 0.40 ± 0.12∗∗ 0.38 ± 0.22∗∗ 1.37 ± 1.38 0.76 ± 0.29 5.29 ± 1.93 20.89 ± 2.71∗∗ 0.38 ± 0.50 0.47 ± 0.22 6.86 ± 3.26
cte
50.56 ± 6.82 30.05 ± 4.71 16.51 ± 3.41 0.62 ± 0.27 1.71 ± 0.62 0.79 ± 0.29 0.88 ± 0.06 30.71 ± 5.13 1.52 ± 1.23 1.53 ± 0.97 22.01 ± 4.62 2.41 ± 0.68 0.49 ± 0.29 1.33 ± 1.05 1.42 ± 1.08 18.73 ± 4.97 9.49 ± 3.31 0.39 ± 0.45 0.47 ± 0.47 2.01 ± 0.85 1.71 ± 0.98 3.32 ± 2.06 0.61 ± 0.41 0.73 ± 0.07 0.05 ± 0.04 1.38 ± 0.53 0.06 ± 0.1 0.04 ± 0.03 1.65 ± 1.25 0.55 ± 0.10 3.52 ± 1.88 15.21 ± 2.44 0.23 ± 0.23 0.60 ± 0.15 3.38 ± 1.87
Alzheimer’s Disease
rre
SFA C16:0 C18:0 C20:0 C22:0 C24:0 C26:0 MUFA C14:1 n-5 C16:1 n-5 C18:1 n-9 (OA) C18:1 n-7 C20:1 n-9 C22:1 n-9 C24:1 n-9 PUFA C18:2 n-6 (LA) C18:3 n-6 C18:3 n-3 (ALA) C20:3 n-6 C20:3 n-3 C20:4 n-6 (AA) C20:5 n-3 (EPA) C22:6 n-3 (DHA) C16:1/C16:0 C18:1/C18:0 C24:1/C18:1 C18:3/C18:2 C20:4/C20:3 C18:0/C16:0 PUFA n-3 PUFA n-6 PUFA n-3/ PUFA n-6 MUFA/ SFA VLCFA
Demented patients All dementia
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Other Dementia 47.71 ± 1.89 24.09 ± 5.80 18.51 ± 7.59 1.34 ± 0.23 1.09 ± 0.64 1.34 ± 0.49 1.34 ± 0.74 23.43 ± 8.14∗∗ 0.92 ± 0.59 0.52 ± 0.20∗∗ 12.66 ± 5.34∗∗ 1.96 ± 0.59 1.37 ± 1.22 0.81 ± 0.14 5.19 ± 3.13∗∗ 28.86 ± 1.35∗∗ 7.56 ± 3.29 2.00 ± 0.58 0.55 ± 0.38 5.93 ± 1.27∗∗ 1.54 ± 0.54 8.32 ± 3.92#∗∗ 2.58 ± 1.03 0.38 ± 0.26 0.02 ± 0.01 0.68 ± 0.34 0.40 ± 0.24 0.26 ± 0.13∗∗ 1.40 ±0.28 0.73 ± 0.25 4.80 ± 1.75 23.56 ± 1.65∗∗ 0.20 ± 0.10 0.49 ± 0.21 3.77 ± 1.13
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Data obtained by GC are expressed as relative values (%): mean % of total FA ± SD. SFAs, sum of saturated fatty acids; MUFAs, sum of monounsaturated fatty acids; PUFAs, sum of polyunsaturated fatty acids. OA, oleic acid; LA, linoleic acid, ALA, alpha linolenic acid; AA, arachidonic acid, EPA, eicosapentaenoic acid, DHA, docosahexaenoic acid. A significant difference between controls and demented patients is indicated by ∗∗ p < 0.001. Data with ∗∗ p < 0.001 are underlined in dark grey. C16:1/C16:0 and C18:1/C18:0: 9 desaturation index; C18:3/C18:2 : 6 desaturation index; C20:4/C20:3 : 5 desaturation index; C24:1/C18:1 and C18:0/C16:0: elongation index. Data with ∗∗ p < 0.001 are underlined in dark grey. The Mann-Whitney test with Bonferroni correction was used.
differences in glucose, cholesterol, triglycerides, and lipoproteins were observed between demented patients and controls. Evaluation of fatty acid profile in red blood cells and plasma The FA profile of RBC membranes is given in Table 1. Analysis of FA composition of RBCs in demented patients showed a slight nonsignificant decrease of SFA in the group with AD or other dementia. Whereas a nonsignificant increase of C18:0 and C22:0 was found, a significantly higher level of
C26:0 was revealed in all groups of demented patients, excepted in other dementia. MUFA was significantly lower in all groups of demented patients. It was associated with a significantly lower level of C16:1 n-5 and C18:1 n-9 (oleic acid). Nervonic acid (C24:1 n-9) in RBCs was almost four times higher in AD patients than in controls. PUFA was significantly higher in all groups. It was associated with higher C20:3 n-6 and C20:4 n-6 (AA) proportions. The highest proportion of PUFAs was mainly represented by PUFA n-6. It is noteworthy that a lower fatty acid 9-desaturase index (C18:1/C18:0), although not significant, was found in all patient groups. Major differences between
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Table 2 Fatty acid profiles in the plasma of controls and demented patients determined by gas chromatography Controls
Other Dementia
35.71 ± 5.12 22.78 ± 4.54 8.64 ± 2.94 0.30 ± 0.27 0.57 ± 0.33 1.16 ± 0.78 2.26 ± 1.07∗∗ 26.48 ± 7.33 0.31 ± 0.40 2.01 ± 0.95 20.09 ± 7.25 2.29 ± 0.68 1.29 ± 0.27 0.16 ± 0.10 0.33 ± 0.13 37.81 ± 7.34 25.05 ± 9.45 0.51 ± 0.42 0.24 ± 0.18 2.93 ± 1.10 2.87 ± 1.05 5.41 ± 2.62 0.42 ± 0.38 0.38 ± 0.14 0.08 ± 0.03 2.32 ± 1.13 1.88 ± 1.51∗∗ 0.02 ± 0.02 0.01 ± 0.01 0.38 ± 0.13 3.53 ± 2.01 28.87 ± 3.75 0.74 ± 0.24 0.12 ± 0.09 3.99 ± 1.55∗∗
34.64 ±4.32 22.8 ± 3.24 8.84 ±2.21 0.28 ± 0.18 0.40 ± 0.23 0.22 ± 0.12 2.10 ± 0.88∗∗ 26.54 ± 8.78 0.21 ± 0.38 1.61 ± 0.32 20.77 ± 6.87 2.51 ± 1.15 1.03 ± 0.49 0.10 ± 0.09 0.31 ± 0.15 38.83 ± 3.56 27.34 ± 3.56 0.55 ±0.29 0.31 ± 0.30 1.53 ± 0.48 2.05 ± 0.90 5.88 ± 2.58 0.83 ± 0.51 0.34 ± 0.13 0.07 ± 0.02 2.35 ± 1.18 3.84 ± 1.65∗∗ 0.02 ± 0.01 0.01 ± 0.02 0.38 ± 0.10 3.35 ± 2.44 35.37 ± 3.27 0.76 ± 0.34 0.10 ± 0.01 2.72 ± 1.01∗∗
35.86 ± 7.24 24.5 ± 4.33 8.18 ± 2.28 0.45 ± 0.28 0.52 ± 0.37 0.15 ± 0.10 2.06 ± 1.33∗∗ 29.84 ± 8.64 0.38 ± 0.66 2.00 ± 0.57 24.16 ± 5.36∗∗ 2.15 ± 0.60 0.60 ± 0.51 0.19 ± 0.28 0.36 ± 0.19 34.3 ± 3.02 22.94 ± 2.22∗∗ 0.62 ± 0.20 0.38 ± 0.10 1.81 ± 0.29 2.07 ±0.59 6.01 ± 2.02 0.19 ± 0.20 0.28 ± 0.21 0.08 ± 0.03 2.95 ± 0.64 3.32 ± 1.63∗∗ 0.03 ± 0.02 0.01 ± 0.01 0.33 ± 0.11 2.92 ± 0.71 31.38 ± 3.46 0.83 ± 0.24 0.09 ± 0.01 2.73 ± 1.45∗∗
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Vascular Dementia
36.23 ± 5.98 23.61 ± 4.43 8.93 ± 2.70 0.34 ± 0.26 0.51 ± 0.32 0.63 ± 0.10 2.21 ± 1.12∗∗ 27.43 ± 7.49 0.32 ± 0.49 1.91 ± 0.71 21.30 ± 6.91 2.41 ± 0.81 1.02 ± 0.16 0.11 ± 0.18 0.36 ± 0.16 36.34 ± 7.86 24.23 ± 9.23 0.56 ± 0.45 0.32 ± 0.32 2.18 ± 1.45 2.40 ± 1.46 5.83 ± 2.48 0.46 ± 0.31 0.35 ± 0.16 0.08 ± 0.02 2.38 ± 1.49 2.42 ± 1.48∗∗ 0.02 ± 0.01 0.01 ± 0.01 0.38 ± 0.11 3.07 ± 1.58 32.80 ± 2.39 0.75 ± 0.30 0.09 ± 0.29 3.35 ± 1.24∗∗
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34.22 ± 3.26 26.24 ± 2.75 7.16 ± 1.52 0.25 ± 0.35 0.30 ± 0.12 0.17 ± 0.05 0.10 ± 0.05 28.20 ± 3.48 1.32 ± 1.21 2.53 ± 0.90 21.01 ± 3.56 1.93 ± 0.48 0.64 ± 0.13 0.43 ± 0.16 0.34 ± 0.12 37.58 ± 4.67 27.78 ± 5.26 0.59 ± 0.30 0.71 ± 0.23 0.57 ± 0.37 1.75 ± 0.48 4.45 ± 1.24 0.30 ± 0.31 1.43 ± 0.27 0.09 ± 0.03 2.93 ± 3.21 7.80 ± 2.19 0.02 ± 0.02 0.01 ± 0.02 0.27 ± 0.06 3.89 ± 2.36 33.39 ± 3.44 0.82 ± 0.21 0.12 ± 0.23 0.57 ± 0.37
Alzheimer’s Disease
rre
SFA C16:0 C18:0 C20:0 C22:0 C24:0 C26:0 MUFA C14:1 n-5 C16:1 n-5 C18:1 n-9 (OA) C18:1 n-7 C20:1 n-9 C22:1 n-9 C24:1 n-9 PUFA C18:2 n-6 (LA) C18:3 n-6 C18:3 n-3 (ALA) C20:3 n-6 C20:3 n-3 C20:4 n-6 (AA) C20:5 n-3(EPA) C22:6 n-3 (DHA) C16:1/C16:0 C18:1/C18:0 C20:4/C20:3 C18:3/C18:2 C24:1/C18:1 C18:0/C16:0 PUFA n-3 PUFA n-6 MUFA/ SFA PUFA n-3/ PUFA n-6 VLCFA
Demented Patients All Dementia
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patients and controls were also observed with the elongase index (C24:1/C18:1). The 5-desaturation activity index (C20:4/C20:3) was similar in these groups. Moreover, the 6-desaturation activity index (C18:3/C18:2) was significantly higher in patients with all or other dementia (Table 1). The FA composition of the plasma is given in Table 2. A significantly higher level of C26:0 was revealed in all groups of demented patients, and VLCFA was significantly higher in these groups A lower 5-desaturation activity index (C20:4/C20:3) was found in all patient groups. The elongase activity index (C18:0/C16:0) and the 6-desaturation activity index (18:3/18:2) were not modified.
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Data obtained by GC are expressed as relative values (%): mean % of total FA ± SD. SFA, sum of saturated fatty acids; MUFA, sum of monounsaturated fatty acids; PUFA, sum of polyunsaturated fatty acids; OA, oleic acid; LA, linoleic acid; ALA, alpha linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaeno¨ic acid. A significant difference between controls and demented patients is indicated by ∗∗ p < 0.001. Data with ∗∗ p < 0.001 are underlined in dark grey. The Mann-Whitney test with Bonferroni correction was used. C16:1/C16:0 and C18:1/C18:0 : 9 desaturation index; C18:3/C18:2 : 6 desaturation index; C20:4/C20:3 : 5 desaturation index; C24:1/C18:1 and C18:0/C16:0 : elongation index.
Quantification of lipids linked with peroxisomal metabolism Quantification of VLCFAs (C22:0; C24:0; C26:0), phytanic acid, and plasmalogen-C16:0 levels (linked with peroxisomal metabolism) [9], in plasma and RBCs was conducted by GC/MS-SIM (Table 3). Comparing VLCFAs between controls and demented patients showed a significantly higher (p < 0.001) level of C26:0 in the plasma and RBCs of demented patients. In RBCs, the C26:0 concentration, which was 1.92 ± 2.07 M in the control group, was 16.78 ± 4.37 M, 16.35 ± 4.91 M, 17.66 ± 3.85 M, and 16.59 ± 4.15 M in all dementia,
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Table 3 Levels of very long chain fatty acids, phytanic acid, and plasmalogen-C16:0 analyzed by GC/MS-SIM in plasma and red blood cells of controls and demented patients Controls
All Dementia
Phytanic acid Plasmalogen-C16:0 C26:0 C24:0 C22:0 C22:6 n-3 (DHA) Desaturation index C24:1/C24:0
RBCs
Plasma
16.39 ± 6.67 55.85 ± 7.17 1.92 ± 2.07 31.72 ± 9.52 21.57 ± 8.32 79.88 ± 49.75 2.78 ± 2.64
16.90 ± 3.16 22.36 ± 2.67 1.08 ± 1.48 24.68 ± 11.71 23.59 ± 7.09 71.47 ± 55.11 2.92 ± 0.59
RBCs
18.61 ± 7.35 48.02 ± 8.29 16.78 ± 4.37∗∗ 24.32 ± 5.47 24.96 ± 5.83 52.19 ± 4.21 7.22 ± 1.78
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Plasma
25.20 ± 17.32 17.41 ± 3.62 12.86 ± 5.42∗∗ 28.53 ± 7.00 26.50 ± 7.86 45.65 ± 14.06 2.52 ±0.97
Demented patients
Alzheimer’s Disease
RBCs
Plasma
dA
20.15 ± 5.07 49.06 ± 4.79 16.35 ± 4.91∗∗ 24.26 ± 5.70 25.69 ± 4.11 43.85 ± 2.57∗∗ 10.80 ± 2.57
24.33 ± 5.64 12.86 ± 6.66 12.64 ± 6.11∗∗ 29.82 ± 6.4 24.77 ± 7.45 45.69 ± 8.25 1.90 ± 0.98
Vascular Dementia RBCs 16.04 ± 8.70 53.31 ± 6.54 17.66 ± 3.85∗∗ 24.29 ± 6.16 25.54 ± 6.32 59.05 ± 30.67 5.02 ± 1.19
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Plasma 29.43 ± 9.19 17.55 ± 11.94 14.00 ± 5.11∗∗ 29.92 ± 7.93 28.70 ± 8.36 40.39 ± 14.22 1.61 ± 0.80
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RBCs
Plasma
18.87 ± 1.49 42.49 ± 3.37 16.59 ± 4.15∗∗ 24.41 ± 4.85 23.73 ± 6.99 55.99 ± 26.60 4.29 ± 1.58
23.16 ± 8.15 23.21 ± 8.99 12.21 ± 4.76∗∗ 25.74 ± 6.46 26.98 ± 7.86 49.80 ± 18.54 4.05 ± 1.90
RBCs: red blood cells (measurements were performed on membrane RBCs), Gas chromatography/mass spectrometry acquisition of each substance was set to the selected-ion monitoring mode (GC/MS–SIM). Data are expressed as mean concentrations ± SD expressed in M. A significant difference between controls and demented patients is indicated by ∗∗ p < 0.001 (Mann Whitney test with Bonferroni correction). Data with ∗∗ (p < 0.001) are underlined in dark grey.
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Test 1, A = 0.99
Test 1, A = 0.99
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Fig. 1. Distribution and receiver operating characteristic (ROC) curves established on controls and demented patients with C26:0 concentrations measured in RBCs. ROC curve: AUC = 0.999 ± 0.001 [95% CI, 0.998–1.001], threshold = 4.16 M, sensitivity = 97.9%, specificity = 98.7%.
Fig. 2. Distribution and receiver operating characteristic (ROC) curves established on controls and demented patients with C26:0 concentrations measured in the plasma. ROC curve: AUC = 0.993 ± 0.006 [95% CI, 0.982–1.005], threshold = 2.80 M, sensitivity = 98.8%, specificity = 98.4%.
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AD patients, vascular dementia, and other dementia, respectively. In demented patients, the analysis of the ROC curve, corresponding to C26:0 concentrations in RBCs, showed an area under the ROC curves (AUC) of 0.999 (95% confidence interval (CI), 0.998–1.001) and a predictive threshold of 4.16 M. The AUC was significantly different from 0.5 (p < 10–4 ). Sensitivity and specificity of the level found were 97.9% and 98.7%, respectively (Fig. 1). The measurement of C26:0 in RBCs made it possible to classify 98.3% of the AD patients correctly. In plasma, the concentration of C26:0 was significantly higher in demented patients: 1.08 ± 1.48 M in the control group and 12.86 ± 5.42 M in demented patients (Table 3). In the
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plasma of AD patients, C26:0 concentration was 12fold higher than in control subjects (12.64 ± 6.11 M versus 1.08 ± 1.48 M). This plasma accumulation of C26:0 was also observed in vascular dementia and in other dementia groups. In AD patients, the analysis of the ROC curve corresponding to plasma C26:0 showed an AUC of 0.993 (95% CI, 0.982–1.005) and a predictive threshold of 2.80 M. The AUC differed significantly from 0.5 (p < 10–4 ). The sensitivity of the level was 98.8%, its specificity was 98.4%, and the level of C26:0 in plasma accurately classified 99.1% of AD patients (Fig. 2). In AD, vascular dementia and other dementia patients (in agreement with data shown in Table 3), similar characteristics of ROC curves
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Correlations between plasma and membrane red blood cell fatty acid composition
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Significant correlations were seen between C22:0 and AA, EPA, and C26:0, EPA and DHA in RBCs (r = –0.65, p < 0.001; r = –0.449, p < 0.05; r = 0.529, p < 0.05; respectively) and between plasma EPA and AA (r = –0.403, p < 0.05). We found that the desaturation index (MUFA/SFA) in plasma displayed i) a negative correlation with the MMSE test, which assesses global cognition (r = –0.411; p < 0.05) and ii) a positive correlation between these two parameters in RBCs (r = 0.437, p = 0.05) (Supplementary Figure 2). A correlation was shown between MMSE and SFA in the plasma of demented patients (r = 0.295; p < 0.05).
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results reporting a lower level of these FAs, in particular oleic acid [31]. Moreover, in agreement with previous investigations, we observed a notably higher level of nervonic acid (C24:1 n-9) in the RBCs of demented patients, which was also found at elevated levels in brain samples from AD patients and which was strongly correlated with cognitive impairment [7]. The increase of C18:0, although not significant, supports the notion that enhanced levels of this fatty acid could be associated with an increased risk of cognitive decline [32]. The total PUFA proportion (PUFA) was also higher in the RBCs of all groups due to the significantly higher level of total n-6 fatty acids (PUFA n-6) as well as AA and C20:3 n-6. The accumulation of PUFA n-3 in RBCs may explain, at least in part, the lower MUFA levels, as suggested by several studies. Indeed, it has been reported that PUFA repress stearoyl-CoA desaturase (9 desaturase) mRNA expression [33]. Although not statistically significant, the lower level of DHA in demented patients found in the present study reinforces the results of other investigations that described lower levels of DHA in dementia [9]. In addition, we report a negative correlation between the MMSE and the desaturation index (MUFA/SFA) in the plasma of AD patients. This finding suggests the existence of a functional link between lower MUFA levels in plasma and deterioration of mental functions in subjects with AD, and is in accordance with data obtained by Astarita et al. [7], who showed that the desaturation index measured in brains of AD patients was correlated with the MMSE score. It is known that modification of FA proportions and alteration of 5 and 6 desaturase activity may result from genetic variants of these enzymes encoded by the FADS1 and FADS2 genes. However, the effect of the polymorphism of these genes on PUFA n-6 has not been thoroughly investigated [34]. Furthermore, it was reported that the activity of 5 and 6 desaturase is modulated by dietary fatty acid intake and the level of FAs in the tissues [34]. Whereas the desaturation index depends on several intrinsic and extrinsic factors, 5 and 6 desaturases may be valuable from several points of view in following the outcome of demented patients. To identify potential peripheral biomarkers in demented patients, we used a quantitative lipidomic strategy based on GC/MS-SIM quantifying VLCFAs, plasmalogen-C16:0, and phytanic acid. Noteworthy, an accumulation of VLCFAs was observed in RBCs and plasma from AD patients and those with other forms of dementia. This increase of VLCFAs observed
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as those observed in all dementia were found (our data not shown). It should be noted that the C26:0 concentration in RBCs was significantly correlated to its concentration in plasma (r = 0.787; p < 0.001) (Supplementary Figure 1). Comparison of the C24:0 mean values between the control group and demented patients demonstrated slightly higher but nonsignificant plasma C24:0 in the group of demented patients. In fact, the C24:0 concentration was 24.68 ± 11.71 M in controls and 28.53 ± 7.00 M in demented patients. In AD, the plasma C24:0 level was 29.82 ± 6.40 M. Although not significant, this accumulation was also observed in the plasma of patients with vascular dementia (29.92 ± 7.93 M) or in other dementia (25.74 ± 6.46 M). With plasmalogen-C16:0, a nonsignificant lower level was observed in RBCs and plasma of all demented patients and AD patients. Comparison of phytanic acid levels in plasma and RBCs between controls and demented patients also showed a higher level (although nonsignificant) of this branched FA in all demented patients (Table 3).
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These data obtained by GC and GC/MS-SIM on the plasma and RBCs of patients with AD, vascular dementia, and other types of dementia demonstrate important modifications of FA profiles: i) significant variations of several SFAs, including VLCFAs; ii) a higher elongation index and modifications of the 5, 6, and 9 desaturation index, supporting modifications in FA metabolism. This study also establishes that C26:0 could constitute a convenient peripheral blood biomarker of dementia, including AD. The MUFA level was lower in demented patients than in controls in the RBCs, in agreement with the
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which reflects peroxisomal ␣-oxidation [15, 38], also supports peroxisome dysfunctions and points toward involvement of this organelle in the development of dementia. Complementary studies are required on a large number of demented patients from various origins to determine the value of a fatty acid profile in the diagnosis of dementia. It cannot be excluded that fatty acid metabolism may vary with ethnic origin. In addition, dietary habits can also influence the fatty acid profile. It would also be useful to correlate the fatty acid profile, especially the RBCs and plasma level of C26:0, with conventional biomarkers of AD (amyloid and tau) present in the cerebrospinal fluid to establish clearly whether C26:0 can be considered as an alternative biomarker of dementia. We must also determine whether increased plasma levels of VLCFAs, known to occur in peroxisomopathies, can also be observed in other forms of neurological diseases. From a practical point of view, the quantification of C26:0, which only requires a blood sample (easier to take than collecting cerebrospinal fluid to quantify conventional biomarkers of AD and requiring specialized care units), could make the diagnosis of dementia available in routine medical practice. Easy-to-measure biomarkers could be applied to a high number of patients. This would contribute to rapidly obtaining a great deal of data for better knowledge of the physiopathology of dementia in order to develop appropriate and effective treatments that are not yet available. Moreover, since the plasma levels of VLCFAs are approximately 12 times higher in demented patients than in controls, the risk of false-positive results will be avoided. In conclusion, our data underline important differences in the FA profile of plasma and RBCs in demented patients, suggesting pronounced alterations of lipid metabolism in dementia. In addition, the substantial accumulation of VLCFAs (especially C26:0) in dementia favors the hypothesis of possible peroxisomal dysfunctions, which are assumed to play critical roles in cell senescence and aging [39, 40].
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in demented patients is in agreement with FA analyses of cortical regions of brains of AD patients with Braak classification stages V–VI, which revealed accumulation of VLCFAs compared with those modestly affected (stages I–II) [23]. In these patients, this accumulation of C22:0, C24:0, and C26:0 was significantly correlated with the appearance of neurofibrillary tangles [23], and the accumulation of C24:0 and C26:0 was assumed to reveal peroxisomal dysfunctions in the cascade of events contributing to the peroxisomal beta-oxidation of C24:0 and C26:0 [9, 23]. In brain cells, VLCFA incorporation into complex lipids could destabilize cellular membranes and change their physiological properties, including those of lipid rafts, which serve as platforms for cell signaling and membrane trafficking [35]. The accumulation of VLCFAs could also result, at least in part, from enhanced FA elongation, whereas the enhanced elongase index observed in the present study was not significant. Therefore, peroxisomal dysfunctions and enhanced elongase activities could explain the marked accumulation of C26:0, in the plasma and RBCs of demented patients. It is noteworthy that the plasmatic levels of C26:0 found in patients with AD (12.64 ± 6.11 M), vascular dementia (14.00 ± 5.11 M), and other dementia (12.21 ± 4.76 M) are within the range of those observed in patients with peroxisomal disorders (X-linked adrenoleukodystrophy (X-ALD): 2.87 ± 1.46 M; Zellweger syndrome: 18.25 ± 8.11 M; D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacylCoA dehydrogenase deficiency (D-BP): 18.02 M; and acylCoA oxidase deficiency (AOX): 4.31 M) [30]. In control subjects, the plasma level of C26:0 (measured in infants, school-aged children, and young adults) [30, 36] was within the range of 0.10–0.40 M. The reduced amounts of plasmalogen-C16:0, requiring enzymes located both in the peroxisome and the endoplasmic reticulum [17, 18], suggest dysfunctions of these organelles, which could have important consequences on brain functions. A pronounced decrease of plasmalogen content has been reported in the white matter, at a very early stage of dementia [36]. A reduction of ethanolamine plasmalogen, in the grey matter of AD patients, has also been described, and it was correlated with a synaptic loss and brain degeneration [37]. In addition, it was reported that reduction of ethanolamine plasmalogen and choline plasmalogen in the brain and RBCs of AD patients was correlated with the severity of dementia [37]. Although not statistically significant, in AD patients and in patients with vascular dementia, the plasma accumulation of phytanic acid, which can be based on dietary intake, and
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ACKNOWLEDGMENTS This work was supported by grants from the Minist`ere de l’Enseignement Sup´erieur et de la Recherche Scientifique and the Direction G´en´erale de la Recherche Scientifique et Technologique (Tunisia). We thank Dr. Imed Cheraif for his helpful technical assistance. We also acknowledge INSERM, the Universit´e de Bourgogne, the Department of Neurology
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