Cerebrospinal fluid, brain, and spinal cord levels of L-aspartate signal excitatory neurotransmission abnormalities in multiple sclerosis patients and experimental autoimmune encephalomyelitis mouse model.

The neuroinflammatory process characterizing multiple sclerosis (MS) is associated with changes in excitatory synaptic transmission and altered central concentrations of the primary excitatory amino acid, L-glutamate (L-Glu). Recent findings report that cerebrospinal fluid (CSF) levels of L-Glu positively correlate with pro-inflammatory cytokines in MS patients. However, to date, there is no evidence about the relationship between the other primary excitatory amino acid, L-aspartate (L-Asp), its derivative D-enantiomer, D-aspartate, and the levels of pro-inflammatory and anti-inflammatory cytokines in the CSF of MS. In the present study, we measured by HPLC the levels of these amino acids in the cortex, hippocampus, cerebellum, and spinal cord of mice affected by experimental autoimmune encephalomyelitis (EAE). Interestingly, in support of glutamatergic neurotransmission abnormalities in neuroinflammatory conditions, we showed reduced L-Asp levels in the cortex and spinal cord of EAE mice and increased D-aspartate/total aspartate ratio within the cerebellum and spinal cord of these animals. Additionally, we found significantly decreased CSF levels of L-Asp in both relapsing-remitting (n = 157) MS (RR-MS) and secondary progressive/primary progressive (n = 22) (SP/PP-MS) patients, compared to control subjects with other neurological diseases (n = 40). Importantly, in RR-MS patients, L-Asp levels were correlated with the CSF concentrations of the inflammatory biomarkers G-CSF, IL-1ra, MIP-1ß, and Eotaxin, indicating that the central content of this excitatory amino acid, as previously reported for L-Glu, reflects a neuroinflammatory environment in MS. In keeping with this, we revealed that CSF L-Asp levels were positively correlated with those of L-Glu, highlighting the convergent variation of these two excitatory amino acids under inflammatory synaptopathy occurring in MS.

Cerebrospinal fluid and serum d-serine concentrations are unaltered across the whole clinical spectrum of Alzheimer's disease.

The diagnosis of Alzheimer's disease (AD) relies on the presence of amyloidosis and tauopathy, as reflected in cerebrospinal fluid (CSF), independently from the clinical stage. Recently, CSF d-serine has been proposed as a possible new AD biomarker, reflecting dysfunctional activation of neuronal glutamatergic N-methyl-d-aspartate receptor (NMDAR). In this study, we measured blood serum and CSF concentration of two NMDAR modulators, such as d-serine and d-aspartate, in a cohort of drug-free subjects encompassing the whole AD clinical spectrum. In addition, we also analyzed d-serine levels in a cohort of post-mortem AD and control cortex samples. We reported unaltered serum and CSF concentrations of d-serine and d-aspartate in AD patients both during the AD progression and compared to non-demented controls. Accordingly, no correlation was detected between serum or CSF d-serine content and mini-mental state examination or Clinical Dementia Rating. Similarly, cortical d-serine levels were also unaltered in post-mortem samples of AD patients. Overall, our results failed to confirm previous findings indicating the CSF d-serine as a novel biomarker for AD.

Bioanalytical methodology for determination of glutamate and aspartate for use in pharmacological sciences with application of Integrated Calibration Method.

A new calibration approach based on the adaptation of Integrated Calibration Method (ICM) to consecutive two components analysis in separation science is presented. Consecutive ICM method (C-ICM) was conceptually developed and applied to determination of two excitatory amino acids - glutamate and aspartate in cerebrospinal fluids collected by the use of brain microdialysis from freely-moving animals. Both analytes as a neurotransmitters play an important role in formation of the memory trace, and thus the processes of learning and memory. Due to their low concentration and presence of interferences, considered analytical system - animal brain - was a big challenge. High-performance liquid chromatography (HPLC) with electrochemical detection (ECD) was used in all experimental work. The most important feature of proposed method is integration of interpolative and extrapolative ways to calculate analyte concentration in single calibration procedure, which consequently leads to obtain series of six estimations of analytical result. Comparison of individual estimations with each other allows for a more in-depth analysis of systematic errors. It was proved that C-ICM approach enables diagnosis and compensation of systematic errors induced by occurrence of interference effects and improvement of accuracy of analytical results. Most of all, it was demonstrated that application of this method is efficient and useful analytical tool in analysis of complicated biological samples in pharmacology and neuroscience.

Elevated cerebrospinal fluid concentrations of N-acetylaspartate correlate with poor outcome in a pilot study of severe brain trauma.

Primary objective: Examine the correlation between acute cerebrospinal fluid (CSF) levels of N-acetylaspartate (NAA) and injury severity upon admission in addition to long-term functional outcomes of severe traumatic brain injury (TBI). Design and rationale: This exploratory study assessed CSF NAA levels in the first four days after severe TBI, and correlated these findings with Glasgow Coma Scale (GCS) score and long-term outcomes at 3, 6, 12, and 24 months post-injury. Methods: CSF was collected after passive drainage via an indwelling ventriculostomy placed as standard of care in a total of 28 people with severe TBI. NAA levels were assayed using triple quadrupole mass spectrometry. Functional outcomes were assessed using the Glasgow Outcomes Scale (GOS) and Disability Rating Scale (DRS). Results: In this pilot study, better functional outcomes, assessed using the GOS and DRS, were found in individuals with lower acute CSF NAA levels after TBI. Key findings were that average NAA level was associated with GCS (p = .02), and GOS at 3 (p = .01), 6 (p = .04), 12 (p = .007), and 24 months (p = .002). Implications: The results of this study add to a growing body of neuroimaging evidence that raw NAA values are reduced and variable after TBI, potentially impacting patient outcomes, warranting additional exploration into this finding. This line of inquiry could lead to improved diagnosis and prognosis in patients with TBI.

Evaluation of MALDI-TOF/TOF Mass Spectrometry Approach for Quantitative Determination of Aspartate Residue Isomerization in the Amyloid-ß Peptide.

Immunoprecipitation (IP) combined with MALDI-TOF mass spectrometry is a powerful instrument for peptide and protein identification in biological samples. In this study, the analytical capabilities of MALDI-TOF/TOF mass spectrometry for relative quantitation of isoAsp7 in Aß(1-42) and Aß(1-16) were investigated. The possibility of quantitative determination of isoAsp7 in Aß(1-42) with the detection limit as low as 2 pmol has been demonstrated. The same approach was applied for a shorter peptide Aß(1-16) and resulted in enhanced accuracy (± 3.2%), and lower detection limit (50 fmol). Pilot experiments with artificial cerebrospinal fluid and mouse brain tissue were performed and showed that the proposed IP-MALDI-TOF/TOF approach could be applied for measuring isoAß content in biological fluids and tissues. Additionally, it was shown that 6E10 anti-amyloid antibodies might affect the accuracy of the amyloid-ß quantitation in the presence of the isomerized peptide.

SLC13A3 variants cause acute reversible leukoencephalopathy and α-ketoglutarate accumulation.

OBJECTIVE: SLC13A3 encodes the plasma membrane Na+ /dicarboxylate cotransporter 3, which imports inside the cell 4 to 6 carbon dicarboxylates as well as N-acetylaspartate (NAA). SLC13A3 is mainly expressed in kidney, in astrocytes, and in the choroid plexus. We describe two unrelated patients presenting with acute, reversible (and recurrent in one) neurological deterioration during a febrile illness. Both patients exhibited a reversible leukoencephalopathy and a urinary excretion of α-ketoglutarate (αKG) that was markedly increased and persisted over time. In one patient, increased concentrations of cerebrospinal fluid NAA and dicarboxylates (including αKG) were observed. Extensive workup was unsuccessful, and a genetic cause was suspected. METHODS: Whole exome sequencing (WES) was performed. Our teams were connected through GeneMatcher. RESULTS: WES analysis revealed variants in SLC13A3. A homozygous missense mutation (p.Ala254Asp) was found in the first patient. The second patient was heterozygous for another missense mutation (p.Gly548Ser) and an intronic mutation affecting splicing as demonstrated by reverse transcriptase polymerase chain reaction performed in muscle tissue (c.1016 + 3A > G). Mutations and segregation were confirmed by Sanger sequencing. Functional studies performed on HEK293T cells transiently transfected with wild-type and mutant SLC13A3 indicated that the missense mutations caused a marked reduction in the capacity to transport αKG, succinate, and NAA. INTERPRETATION: SLC13A3 deficiency causes acute and reversible leukoencephalopathy with marked accumulation of αKG. Urine organic acids (especially αKG and NAA) and SLC13A3 mutations should be screened in patients presenting with unexplained reversible leukoencephalopathy, for which SLC13A3 deficiency is a novel differential diagnosis. ANN NEUROL 2019;85:385-395.

Derivatization reagent-assisted enantioseparation of 3-hydroxyaspartate with two chiral centers in rat cerebrospinal fluid by capillary electrophoresis-mass spectrometry.

A new analytical method based on capillary zone electrophoresis-mass spectrometry (CZE-MS) was proposed and validated for the simultaneous determination of four stereoisomers of 3-hydroxyaspartate with two chiral centers in rat cerebrospinal fluid (CSF) in absence of optically pure single enantiomer standards. The derivatization reagent 9-fluorenylmethyl chloroformate (FMOC-Cl) was found to assist chiral separation and the derivatized enantiomers of 3-hydroxyaspartate can achieve enantioseparation with a lower concentration (6 mM) of ß-cyclodextrin (ß-CD), while underivatized 3-hydroxyaspartate cannot be separated. The enhanced interactions between derivatized analytes and ß-CD were demonstrated by proton nuclear magnetic resonance (1H NMR). The four stereoisomers of FMOC-3-hydroxyaspartate were identified successfully using a new method based on experimental and calculated electronic circular dichroism (ECD) spectra combined with the comparison of CE peak areas. Large volume sample stacking with polarity switching (LVSS-PS) was used to increase sensitivity and the detection limit of 356 nM was achieved for L-THA, which was around 10-fold improvement compared to the normal CE-MS analysis. The composition of the background electrolyte (BGE) was optimized by response surface methodology (RSM). Under the optimal conditions, satisfactory results of L-THA were obtained in terms of linearity over the range of 2-80 µM (R2 > 0.99) and precision (RSD below 1.43% and 2.56% for migration time and peak area, respectively). The recoveries for all four stereoisomers in spiked rat CSF ranged from 91.2% to 99.5%. The method has been successfully applied to rat CSF analysis and D-erythro-3-hydroxyaspartate (D-EHA) was detected.

Striatal neurometabolite levels in patients with schizophrenia undergoing long-term antipsychotic treatment: A proton magnetic resonance spectroscopy and reliability study.

Previous proton magnetic resonance spectroscopy (1H-MRS) studies have reported disrupted levels of various neurometabolites in patients with schizophrenia. An area of particular interest within this patient population is the striatum, which is highly implicated in the pathophysiology of schizophrenia. The present study examined neurometabolite levels in the striatum of 12 patients with schizophrenia receiving antipsychotic treatment for at least 1 year and 11 healthy controls using 3-Tesla 1H-MRS (PRESS, TE = 35 ms). Glutamate, glutamate+glutamine (Glx), myo-inositol, choline, N-acetylaspartate, and creatine levels were estimated using LCModel, and corrected for fraction of cerebrospinal fluid in the 1H-MRS voxel. Striatal neurometabolite levels were compared between groups. Multiple study visits permitted a reliability assessment for neurometabolite levels (days between paired 1H-MRS acquisitions: average = 90.33; range = 7-306). Striatal neurometabolite levels did not differ between groups. Within the whole sample, intraclass correlation coefficients for glutamate, Glx, myo-inositol, choline, and N-acetylaspartate were fair to excellent (0.576-0.847). The similarity in striatal neurometabolite levels between groups implies a marked difference from the antipsychotic-naïve first-episode state, especially in terms of glutamatergic neurometabolites, and might provide insight regarding illness progression and the influence of antipsychotic medication.

Blood-Brain Barrier Disruption Is Initiated During Primary HIV Infection and Not Rapidly Altered by Antiretroviral Therapy.

Background: We explored the establishment of abnormal blood-brain barrier (BBB) permeability and its relationship to neuropathogenesis during primary human immunodeficiency virus (HIV) infection by evaluating the cerebrospinal fluid (CSF) to serum albumin quotient (QAlb) in patients with primary HIV infection. We also analyzed effects of initiating combination antiretroviral therapy (cART). Methods: The QAlb was measured in longitudinal observational studies of primary HIV infection. We analyzed trajectories of the QAlb before and after cART initiation, using mixed-effects models, and associations between the QAlb and the CSF level of neurofilament light chain (NFL), the ratio of N-acetylaspartate to creatinine levels (a magnetic resonance spectroscopy neuronal integrity biomarker), and neuropsychological performance. Results: The baseline age-adjusted QAlb was elevated in 106 patients with primary HIV infection (median time of measurement, 91 days after infection), compared with that in 64 controls (P = .02). Before cART initiation, the QAlb increased over time in 84 participants with a normal baseline QAlb (P = .006) and decreased in 22 with a high baseline QAlb (P = .011). The QAlb did not change after a median cART duration of 398 days, initiated at a median interval of 225 days after infection (P = .174). The QAlb correlated with the NFL level at baseline (r = 0.497 and P < .001) and longitudinally (r = 0.555 and P < .001) and with the ratio of N-acetylaspartate to creatinine levels in parietal gray matter (r = -0.352 and P < .001 at baseline and r = -0.387 and P = .008 longitudinally) but not with neuropsychological performance. Conclusion: The QAlb rises during primary HIV infection, associates with neuronal injury, and does not significantly improve over a year of treatment. BBB-associated neuropathogenesis in HIV-infected patients may initiate during primary infection.

[Protective effects of Ginkgo Terpene Lactones Meglumine Injection on focal cerebral ischemia in rats].

To investigate the protective effects of ginkgo diterpene lactone meglumine injection (GDLMI) on cerebral focal ischemia reperfusion injury induced by middle cerebral artery occlusion (MCAO) in rats, and explore its possible mechanism. One hundred and forty male SD rats were randomly divided into sham operation group, model group, ginkgo biloba extract injection (Ginaton, 1.0 mL•kg⁻¹) group, nimodipine (0.4 mg•kg⁻¹) group, and GDLMI (5.2, 2.6, 1.3 mg•kg⁻¹) groups; All of rats received corresponding drugs by tail vein injection 4 days before operation (normal saline in model group and sham operation group). Except the sham operation group, the cerebral ischemic stroke model was established by MCAO method in right brain of the other rats. After 3 h of ischemia, all the animals received intravenous administration again. The neurobehavioral scores of rats after ischemia-reperfusion were evaluated and the infarct rate of brain tissue was observed by TTC staining. The super oxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and lactic acid (LA) contents in brain tissue homogenate and the concentration of Ca2+, glutamate (Glu) and aspartate (Asp), creatine phosphate kinase (CK-BB) and lactate dehydrogenase (LDH) content changes in cerebrospinal fluid were measured. As compared with the sham operation group, the cerebral infarction rate was increased significantly in the model group; the content of MDA and LA in the homogenate of brain tissue was increased, and the content of GSH and SOD was decreased; in cerebrospinal fluid, Ca2+ concentration was decreased, and the content of Glu and Asp, CK-BB and LDH increased significantly. As compared with the model group, the high and medium dose GDLMI groups can significantly reduce the cerebral infarction rate and improve the symptoms of neurological impairment; increase SOD and GSH activity, reduce MDA and LA content in serum; increase Ca2+ concentration in cerebrospinal fluid and decrease the content of neurotransmitter Glu and Asp as well as CK-BB and LDH. GDLMI could obviously improve neurologic impairment in model rats, and the mechanism may be related to recovering the blood brain barrier, scavenging free radicals, decreasing free Ca2+ inflow into the cells and the content of excitatory amino acid in cerebrospinal fluid to improve its protective effect on cerebral ischemia.

Increased ventricular cerebrospinal fluid lactate in depressed adolescents.

BACKGROUND: Mitochondrial dysfunction has been increasingly examined as a potential pathogenic event in psychiatric disorders, although its role early in the course of major depressive disorder (MDD) is unclear. Therefore, the purpose of this study was to investigate mitochondrial dysfunction in medication-free adolescents with MDD through in vivo measurements of neurometabolites using high-spatial resolution multislice/multivoxel proton magnetic resonance spectroscopy. METHODS: Twenty-three adolescents with MDD and 29 healthy controls, ages 12-20, were scanned at 3T and concentrations of ventricular cerebrospinal fluid lactate, as well as N-acetyl-aspartate (NAA), total creatine (tCr), and total choline (tCho) in the bilateral caudate, putamen, and thalamus were reported. RESULTS: Adolescents with MDD exhibited increased ventricular lactate compared to healthy controls [F(1,41)=6.98, P=0.01]. However, there were no group differences in the other neurometabolites. Dimensional analyses in the depressed group showed no relation between any of the neurometabolites and symptomatology, including anhedonia and fatigue. CONCLUSIONS: Increased ventricular lactate in depressed adolescents suggests mitochondrial dysfunction may be present early in the course of MDD; however it is still not known whether the presence of mitochondrial dysfunction is a trait vulnerability of individuals predisposed to psychopathology or a state feature of the disorder. Therefore, there is a need for larger multimodal studies to clarify these chemical findings in the context of network function.

N-acetylaspartate and neurofilaments as biomarkers of axonal damage in patients with progressive forms of multiple sclerosis.

Primary and secondary progressive forms of multiple sclerosis (PPMS and SPMS) have different pathological characteristics. However, it is unknown whether neurodegenerative mechanisms are shared. We measured cerebrospinal fluid (CSF) levels of neurofilament (Nf) light and heavy isoforms and N-acetylaspartic acid (NAA) in 21 PP, 10 SPMS patients and 15 non-inflammatory neurological disease controls (NINDC). Biomarkers were related to Expanded Disability Status Scale (EDSS) and Multiple Sclerosis Severity Score (MSSS) over a long period of follow-up [median (interquartile range) 9 (5.5-12.5) years] in 19 PPMS and 4 SPMS patients, and to T2 lesion load, T1 lesion load, and brain parenchymal fraction at the time of lumbar puncture. Nf light was higher in PPMS (p < 0.005) and Nf heavy was increased in both SPMS and PPMS (p < 0.05 and p < 0.01) compared to NINDC, but were comparable between the two MS subtypes. Nf heavy was a predictor of the ongoing disability measured by MSSS (R(2) = 0.17, ß = 0.413; p < 0.05). Conversely, Nf light was the only predictor of the EDSS annual increase (R(2) = 0.195, ß = 0.441; p < 0.05). The frequency of abnormal biomarkers did not differ between the two MS progressive subtypes. Our data suggest that PP and SPMS likely share similar mechanisms of axonal damage. Moreover, Nf heavy can be a biomarker of ongoing axonal damage. Conversely, Nf light can be used as a prognostic marker for accumulating disability suggesting it as a good tool for possible treatment monitoring in the progressive MS forms.

Effect of Gua Lou Gui Zhi decoction on focal cerebral ischemia-reperfusion injury through regulating the expression of excitatory amino acids and their receptors.

Gua Lou Gui Zhi decotion (GLGZD) has been reported to be an effective treatment for post­apoplectic limb spasm in the clinic. The present study aimed to investigate whether GLGZD had an affect on cerebral injuries induced by middle cerebral artery occlusion (MCAO) in rats and its possible mechanism. High­performance liquid chromatography was performed to analyze GLGZD. Furthermore, a model was established to assess the efficacy of GLGZD. Neurological defect scores and screen tests were analyzed. Brain ischemic infarct volume was measured using 2,3,5­triphenyl tetrazolium chloride staining and glutamic acid (Glu), aspartic acid (Asp) and glycine (Gly) levels in the cerebrospinal fluid were measured using the Hitachi automatic amino acid analyzer. Immunohistochemistry was performed to determine the expression of the α­amino­3­hydroxy­5­methyl­4­isoxazole­propionic acid (AMPA) and N­methyl­D­aspartic acid (NMDA) glutamate receptors, and to analyze histopathological change. GLGZD was found to improve neurological performance and reduce infarct volumes in MCAO rats. In addition, GLGZD was observed to enhance motor performance, which was assessed using the screen test. Furthermore, GLGZD was found to reduce Glu, Asp and Gly levels in the cerebrospinal fluid and downregulate the protein expression of the AMPA and NMDA glutamate receptors. Thus, it was demonstrated that GLGZD may exert neuroprotective effects through the modulation of excitatory amino acids, and AMPA and NMDA receptor expression.

Molecularly imprinted polymer-matrix nanocomposite for enantioselective electrochemical sensing of D- and L-aspartic acid.

A new molecularly imprinted polymer-matrix (titanium dioxide nanoparticle/multiwalled carbon nanotubes) nanocomposite was developed for the modification of pencil graphite electrode as an enantioselective sensing probe for aspartic acid isomers, prevalent at ultra trace level in aqueous and real samples. The nanocomposite having many shape complementary cavities was synthesized adopting surface initiated-activators regenerated by electron transfer for atom transfer radical polymerization. The proposed sensor has high stability, nanocomposite uniformity, good reproducibility, and enhanced electrocatalytic activity to respond oxidative peak current of L-aspartic acid quantitatively by differential pulse anodic stripping voltammetry, without any cross-reactivity in real samples. Under the optimized operating conditions, the L-aspartic acid imprinted modified electrode showed a wide linear response for L-aspartic acid within the concentration range 9.98-532.72 ng mL(-1), with the minimum detection limit of 1.73-1.79 ng mL(-1) (S/N=3) in aqueous and real samples. Almost similar stringent limit (1.79 ng mL(-1)) was obtained with cerebrospinal fluid which is typical for the primitive diagnosis of neurological disorders, caused by an acute depletion of L-aspartic acid biomarker, in clinical settings.

Performance on Paced Auditory Serial Addition Test and cerebral blood flow in multiple sclerosis.

BACKGROUND: To assess the relationship between performance on the Paced Auditory Serial Addition Test (PASAT) and both cerebral blood flow (CBF) and axonal metabolic integrity in normal appearing white matter (NAWM) of the centrum semiovale in patients with multiple sclerosis (MS). METHODS: Normal appearing white matter of the centrum semiovale was investigated with magnetic resonance (MR) imaging in 28 non-depressed individuals (18 patients with MS and 10 healthy controls). CBF was assessed with pseudo-continuous arterial spin labeling. N-acetylacetate/creatine (NAA/Cr) ratios (a metabolic axonal marker) were measured using (1) H-MR spectroscopy. CBF was also measured in frontoparietal cortices and cerebellar hemispheres. RESULTS: In subjects with MS, we found a positive correlation between performance on the PASAT and CBF to the left centrum semiovale (P = 0.008), but not with the NAA/Cr ratio. There were no correlations between PASAT scores and CBF to the right centrum semiovale, frontoparietal cortices, and cerebellar hemispheres. There was no correlation between PASAT scores and NAA/Cr ratios. CONCLUSIONS: Our preliminary results suggest that performance on the PASAT in subjects with MS correlates with CBF to the left centrum semiovale, which contains left frontoparietal white matter association tracts involved in information processing speed and working memory.

Highly sensitive and selective hyphenated technique (molecularly imprinted polymer solid-phase microextraction-molecularly imprinted polymer sensor) for ultra trace analysis of aspartic acid enantiomers.

The present work is related to combination of molecularly imprinted solid-phase microextraction and complementary molecularly imprinted polymer-sensor. The molecularly imprinted polymer grafted on titanium dioxide modified silica fiber was used for microextraction, while the same polymer immobilized on multiwalled carbon nanotubes/titanium dioxide modified pencil graphite electrode served as a detection tool. In both cases, the surface initiated polymerization was found to be advantageous to obtain a nanometer thin imprinted film. The modified silica fiber exhibited high adsorption capacity and enantioselective diffusion of aspartic acid isomers into respective molecular cavities. This combination enabled double preconcentrations of d- and l-aspartic acid that helped sensing both isomers in real samples, without any cross-selectivity and matrix complications. Taking into account 6×10(4)-fold dilution of serum and 2×10(3)-fold dilution of cerebrospinal fluid required by the proposed method, the limit of detection for l-aspartic acid is 0.031ngmL(-1). Also, taking into account 50-fold dilution required by the proposed method, the limit of detection for d-aspartic acid is 0.031ngmL(-1) in cerebrospinal fluid.

CSF neurofilament and N-acetylaspartate related brain changes in clinically isolated syndrome.

BACKGROUND: Axonal damage is considered a major cause of disability in multiple sclerosis (MS) and may start early in the disease. Specific biomarkers for this process are of great interest. OBJECTIVE: To study if cerebrospinal fluid (CSF) biomarkers for axonal damage reflect and predict disease progression already in the earliest stages of the disease, that is, in clinically isolated syndrome (CIS). METHODS: We assessed CSF levels of neurofilament heavy (NFH), neurofilament light (NFL) and N-acetylaspartate (NAA) in 67 patients with CIS and 18 controls with neuropsychiatric diseases of non-inflammatory aetiology (NC). Patients with CIS underwent baseline magnetic resonance imaging (MRI) at 3T, and a follow-up MRI after 1 year was obtained in 28 of them. RESULTS: Compared with NC, patients with CIS had higher NFH (p=0.05) and NFL (p<0.001) levels. No significant group differences were found for NAA. Patients' NFH levels correlated with physical disability (r=0.304, p<0.05) and with change in brain volume over 1 year of follow-up (r=-0.518, p<0.01) but not with change in T2 lesion load. CONCLUSION: Our results confirm increased neurofilament levels already in CIS being related to the level of physical disability. The association of NFH levels with brain volume but not lesion volume changes supports the association of these markers with axonal damage.

Serum and CSF N-acetyl aspartate levels differ in multiple sclerosis and neuromyelitis optica.

BACKGROUND: The identification of biomarkers able to improve the differential diagnosis between multiple sclerosis (MS) and neuromyelitis optica (NMO) is challenging because of a different prognosis and response to treatment. Growing evidence indicates that brain and CSF N-acetyl aspartate (NAA) concentration is a useful marker for characterising different phases of axonal pathology in demyelinating diseases, and preliminary studies suggest that increased serum NAA levels may be a telltale sign of acute neuronal damage or defective NAA metabolism in oligodendrocytes. OBJECTIVE: To evaluate whether serum and CSF NAA concentration differs in patients with MS and NMO. DESIGN: Observational, multicentre, prospective, cross sectional study. METHODS: Serum samples were collected from 48 relapsing-remitting MS, 32 NMO and 76 age matched healthy controls. Coeval CSF samples were available for all MS and for 8/32 NMO patients. NAA was measured in serum and CSF by liquid chromatography-mass spectrometry. RESULTS: MS patients showed higher serum and CSF NAA levels than NMO patients, and higher serum NAA levels than healthy controls (p<0.001). High serum NAA values, exceeding the 95th percentile of serum NAA values in healthy controls, were found in 100% of patients with MS and in no patient with NMO. No differences in serum NAA levels were found between NMO and healthy controls. In MS, serum and CSF NAA levels correlated with disability score. CONCLUSIONS: Determination of serum and CSF NAA levels may represent a suitable tool in the diagnostic laboratory workup to differentiate MS and NMO.

Lipoic acid alters amino acid neurotransmitters content in rat hippocampus after pilocarpine-induced seizures.

This study was aimed at investigating the anticonvulsant activity of lipoic acid (LA) against pilocarpine-induced seizures as well as the effects of this metabolic antioxidant on the hippocampal extracellular concentrations of amino acid neurotransmitters glutamate, aspartate, glycine and glutamate and γ-aminobutyric acid (GABA). In vivo microdialysis demonstrated that an intraperitoneal administration of pilocarpine induced a pronounced increment of hippocampal glutamate and aspartate concentrations, whereas no significant change was observed in the levels of glycine or GABA. LA (10, 20 or 30 mg/kg) pretreatment completely blocked pilocarpine-evoked increases in extracellular glutamate and aspartate concentrations. Significant reductions in hippocampal GABA and glycine concentrations were also observed although not as pronounced as those shown by glutamate and aspartate. Based on the finding that LA protected rats against pilocarpine-induced seizures, it could be suggested that the reduction in inhibitory amino acid neurotransmitters levels was comparatively minor and offset by a more pronounced reduction in glutamate and aspartate extracellular concentrations. Therefore, the fact that LA could drastically reduce pilocarpine-induced increases in glutamate and aspartate should account, at least partly, for its anticonvulsant activity observed in pilocarpine-induced seizure in rats.

Stacking and separation of aspartic acid enantiomers under discontinuous system by capillary electrophoresis with light-emitting diode-induced fluorescence detection.

We describe the stacking and separation of d- and l-aspartic acid (Asp) by capillary electrophoresis (CE) with light-emitting diode-induced fluorescence detection (LEDIF). In the presence of cyanide, d- and l-Asp were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to form fluorescent derivatives prior to CE-LEDIF. The separation of NDA-derivatized d- and l-Asp was accomplished using a discontinuous system - buffer vials contained a solution of 0.6% poly(ethylene oxide) (PEO), 150 mM sodium dodecyl sulfate (SDS), and 60mM hydroxypropyl-ß-cyclodextrin (Hp-ß-CD), while a capillary was filled with a solution of 150 mM SDS and 60mM Hp-ß-CD. The role of PEO, Hp-ß-CD, and SDS is to act as a concentrating media, as a chiral selector, and as a pseudostationary phase, respectively. This discontinuous system could be employed for the stacking of 600 nL of NDA-derivatized d- and l-Asp without the loss of chiral resolution. The stacking mechanism is mainly based on the difference in viscosity between sample zone and PEO as well as SDS sweeping. The limits of detection at signal-to-noise of 3 for d- and l-Asp were down to 2.4 and 2.5 × 10(-10)M, respectively. Compared to normal sample injection volume (25 nL), this stacking approach provided a 100- and 110-fold improvement in the sensitivity of d- and l-Asp, respectively. This method was further applied for determining d- and l-Asp in cerebrospinal fluid, soymilk, and beer.