Analysis of chiral amino acids in cerebrospinal fluid samples linked to different stages of Alzheimer disease

El pdf del artículo es la versión de autor.-- et al.Chiral micellar electrokinetic chromatography with laser-induced fluorescence detection (chiral-MEKC-LIF) was used to investigate D- and L-amino acid contents in cerebrospinal fluid (CSF) samples related to different Alzheimer disease (AD) stages. CSF samples were taken from (i) control subjects (S1 pool), (ii) subjects showing a mild cognitive impairment who remained stable (S2 pool), (iii) subjects showing an mild cognitive impairment that progressed to AD (S3 pool) and (iv) subjects diagnosed with AD (S4 pool). The optimized procedure only needed 10μL of CSF and it included sample cleaning, derivatization with FITC and chiral-MEKC-LIF separation. Eighteen standard amino acids were baseline separated with efficiencies up to 703000plates/m, high sensitivity (LODs in the nM range) and good resolution (values ranging from 2.6 to 9.5). Using this method, L-Arg, L-Leu, L-Gln, γ-aminobutyric acid, L-Ser, D-Ser, L-Ala, Gly, L-Lys, L-Glu and L-Asp were detected in all the CSF samples. S3 and S4 samples (i.e. AD subjects) showed significant lower amounts of L-Arg L-Lys, L-Glu and L-Asp compared to the non-AD S1 and S2 samples, showing in the S4 group the lowest amounts of L-Arg L-Lys, L-Glu and L-Asp. Moreover, γ-aminobutyric acid was significantly higher in AD subjects with the highest amount also found for S4. No significant differences were observed for the rest of amino acids including D-Ser. Based on the obtained chiral-MEKC-LIF data, it was possible to correctly classify all the samples into the four groups. These results demonstrate that the use of enantioselective procedures as the one developed in this work can provide some new light on the investigations of AD, including the discovery of new biomarkers related to different stages of AD. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.S.S. thanks Shota Rustaveli Georgia National Science Foundation for her Young Scientists Grant (Project 04/03). C.I. thanks the Ministerio de Ciencia e Innovación for her FPI predoctoral fellowship. This work was supported by: Projects AGL2008-05108-C03-01 and CONSOLIDER INGENIO 2010 CSD2007-00063 FUN-C-FOOD (Ministerio de Educación y Ciencia), Gun och Bertil Stohnes Stiftelse, Karolinska Institutets fund for geriatric research, Stiftelsen Gamla Tjanarinnor, Stiftelsen Dementia, Swedish Alzheimer Foundation, Swedish Brain Foundation, Ramon Areces Foundation and the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and the Karolinska Institute.Peer Reviewe

Chiral separation and determination of excitatory amino acids in brain samples by CE-LIF using dual cyclodextrin system.

Chiral capillary electrophoresis method has been developed to separate aspartate and glutamate enantiomers to investigate the putative neuromodulator function of D-Asp in the central nervous system. To achieve appropriate detection sensitivity fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and laser-induced fluorescence detection was applied. Although, simultaneous baseline separation of the two enantiomer pairs could be achieved by using 3 mM 6-monodeoxy-6-mono(3-hydroxy)propylamino-beta-cyclodextrin (HPA-beta-CD), further improvement of the chemical selectivity was required because of the high excess of L-enantiomers in real samples to be analyzed. The system selectivity was fine-tuned by combination of 8 mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin and 5 mM HPA-beta-CD in order to increase the resolution between aspartate and glutamate enantiomers. The method was validated for biological application. The limits of detection for D-Asp and D-Glu were 17 and 9 nM, respectively, while the limit of quantification for both analytes was 50 nM. This is the lowest quantification limit reported so far for NBD-tagged D-Asp and D-Glu obtained by validated capillary electrophoresis laser-induced fluorescence method. The applicability of the method was demonstrated by analyzing brain samples of 1-day-old chickens. In all the studied brain areas, the D-enantiomer contributed 1-2 % of the total aspartate content, corresponding to 17-45 nmol/g wet tissue