Human Cerebrospinal Fluid Concentrations Research Articles

An immuno-enrichment free, validated quantification of tau protein in human CSF by LC-MS/MS.

Tau protein is a key target of interest in developing therapeutics for neurodegenerative diseases. Here, we sought to develop a method that quantifies extracellular tau protein concentrations in human cerebrospinal fluid (CSF) without antibody-based enrichment strategies. We demonstrate that the fit-for-purpose validated method in Alzheimer’s Disease CSF is limited to quasi quantitative measures of tau surrogate peptides. We also provide evidence that CSF total Tau measures by LC-MS are feasible in the presence of monoclonal therapeutic antibodies in human CSF. Our Tau LC-MS/MS method is a translational bioanalytical tool for assaying target engagement and pharmacodynamics for anti-tau antibody drug development campaigns.

Quantification of endocannabinoids in human cerebrospinal fluid using a novel micro-flow liquid chromatography-mass spectrometry method

The endocannabinoid system (ECS) is implicated in various brain disorders. Changes in the composition of the cerebrospinal fluid (CSF) may be associated with ECS-related pathologies. Endocannabinoids (eCBs) and their analogues are present at low concentrations in human CSF, which hampered the investigation of the ECS in this body fluid. In this study, we developed a highly sensitive and selective micro-flow liquid chromatography-tandem mass spectrometry (micro-LC-MS/MS) method for the analysis of eCBs and eCB analogues in human CSF. The developed method allowed for the quantitative analysis of 16 eCBs and their analogues in human CSF. Micro-LC-MS/MS analyses were performed at a flow-rate of 4 μL min−1 with a 0.3-mm inner diameter column. A minor modification of a novel spray needle was carried out to improve the robustness of our method. By using an injection volume of 3 μL, our method reached limits of detection in the range from 0.6 to 1293.4 pM and limits of quantification in range from 2.0 to 4311.3 pM while intra- and interday precisions were below 13.7%. The developed workflow was successfully used for the determination of eCBs in 288 human CSF samples. It is anticipated that the proposed approach will contribute to a deeper understanding of the role of ECS in various brain disorders.

Cladribine modifies functional properties of microglia.

SummaryCladribine (CdA), an oral prodrug approved for the treatment of relapsing multiple sclerosis, selectively depletes lymphocytes. CdA passes the blood–brain barrier, suggesting a potential effect on central nervous system (CNS) resident cells. We examined if CdA modifies the phenotype and function of naive and activated primary mouse microglia, when applied in the concentrations 0·1–1 μM that putatively overlap human cerebrospinal fluid (CSF) concentrations. Primary microglia cultures without stimulation or in the presence of proinflammatory lipopolysaccharide (LPS) or anti‐inflammatory interleukin (IL)‐4 were treated with different concentrations of CdA for 24 h. Viability was assessed by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] assay. Phagocytotic ability and morphology were examined by flow cytometry and random migration using IncuCyte Zoom and TrackMate. Change in gene expression was examined by quantitative polymerase chain reaction (qPCR) and protein secretion by Meso Scale Discovery. We found that LPS and IL‐4 up‐regulated deoxycytidine kinase (DCK) expression. Only activated microglia were affected by CdA, and this was unrelated to viability. CdA 0·1–1 μM significantly reduced granularity, phagocytotic ability and random migration of activated microglia. CdA 10 μM increased the IL‐4‐induced gene expression of arginase 1 (Arg1) and LPS‐induced expression of IL‐1β, tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS) and Arg1, but protein secretion remained unaffected. CdA 10 μM potentiated the increased expression of anti‐inflammatory TNF receptor 2 (TNF‐R2) but not TNF‐R1 induced by LPS. This suggests that microglia acquire a less activated phenotype when treated with 0·1–1 μM CdA that putatively overlaps human CSF concentrations. This may be related to the up‐regulated gene expression of DCK upon activation, and suggests a potential alternative mechanism of CdA with direct effect on CNS resident cells.

HIV influences microtubule associated protein-2: potential marker of HIV-associated neurocognitive disorders.

Postmortem brains of patients diagnosed with HIV-1-associated neurocognitive disorders (HAND) exhibit loss of dendrites. However, the mechanisms by which synapses are damaged are not fully understood. Dendrite length and remodeling occurs via microtubules, the dynamics of which are regulated by microtubule-binding proteins, including microtubule-associated protein 2 (MAP2). The HIV protein gp120 is neurotoxic and interferes with neuronal microtubules. We measured MAP2 concentrations in human cerebrospinal fluid (CSF) and MAP2 immunoreactivity in rat cortical neurons exposed to HIV and gp120. First, we examined whether HIV affects MAP2 levels by analyzing the CSF of 27 persons living with HIV (PLH) whose neurocognitive performance had been characterized. We then used rat cortical neurons to study the mechanisms of HIV-mediated dendritic loss. PLH who had HAND had greater MAP2 concentrations within the CSF than cognitive normal PLH. In cortical neurons, the deleterious effect of HIV on MAP2-positive dendrites occurred through a gp120-mediated mechanism. The neurotoxic effect of HIV was blocked by a CCR5 antagonist and prevented by Helix-A, a peptide that displaces gp120 from binding to microtubules, conjugated to a nanolipoprotein particle delivery platform. Our findings support that HIV at least partially effects its neurotoxicity via neuronal cytoskeleton modifications and provide evidence of a new therapeutic compound that could be used to prevent the HIV-associated neuropathology.

Characterization of the Prion Protein Binding Properties of Antisense Oligonucleotides.

Antisense oligonucleotides (ASOs) designed to lower prion protein (PrP) expression in the brain through RNase H1-mediated degradation of PrP RNA are in development as prion disease therapeutics. ASOs were previously reported to sequence-independently interact with PrP and inhibit prion accumulation in cell culture, yet in vivo studies using a new generation of ASOs found that only PrP-lowering sequences were effective at extending survival. Cerebrospinal fluid (CSF) PrP has been proposed as a pharmacodynamic biomarker for trials of such ASOs, but is only interpretable if PrP lowering is indeed the relevant mechanism of action in vivo and if measurement of PrP is unconfounded by any PrP–ASO interaction. Here, we examine the PrP-binding and antiprion properties of ASOs in vitro and in cell culture. Binding parameters determined by isothermal titration calorimetry were similar across all ASOs tested, indicating that ASOs of various chemistries bind full-length recombinant PrP with low- to mid-nanomolar affinity in a sequence-independent manner. Nuclear magnetic resonance, dynamic light scattering, and visual inspection of ASO–PrP mixtures suggested, however, that this interaction is characterized by the formation of large aggregates, a conclusion further supported by the salt dependence of the affinity measured by isothermal titration calorimetry. Sequence-independent inhibition of prion accumulation in cell culture was observed. The inefficacy of non-PrP-lowering ASOs against prion disease in vivo may be because their apparent activity in vitro is an artifact of aggregation, or because the concentration of ASOs in relevant compartments within the central nervous system (CNS) quickly drops below the effective concentration for sequence-independent antiprion activity after bolus dosing into CSF. Measurements of PrP concentration in human CSF were not impacted by the addition of ASO. These findings support the further development of PrP-lowering ASOs and of CSF PrP as a pharmacodynamic biomarker.

Determination of ceftriaxone concentration in human cerebrospinal fluid by high-performance liquid chromatography with UV detection

Determination of ceftriaxone (CTRX) concentration in human cerebrospinal fluid (CSF) is required to clarify whether a high concentration of CTRX in CSF is associated with CTRX-induced encephalopathy (CIE). In our study, in order to perform an accurate analysis of CSF sample from CIE patient, we proposed HPLC with UV detection (HPLC-UV) using an octadecylsilica (ODS) column, a methanol and 10 mM phosphoric acid (25:75, v/v) mixture solution as a mobile phase, and a detection wavelength at 280 nm. The linear range was from 0.1 to 100 μg/mL (r = 0.999) in the present HPLC-UV. In the recovery tests using blank samples of human CSF and control serum spiked with CTRX, the recoveries of CTRX were >95.3%, and the RSD (n = 3) was <5.8%. We applied the proposed HPLC-UV system to determine CTRX in the CSF and serum samples obtained from a patient diagnosed as having CIE, and it was revealed that the CTRX concentrations in the CSF sample and the serum were 2.61 and 37.35 μg/mL, respectively. To the best of our knowledge, this is the first report describing the determination of CTRX concentration in a CSF sample obtained from a peritoneal dialysis patient diagnosed as having CIE.

Kinetic Insights into Zn2+-Induced Amyloid β-Protein Aggregation Revealed by Stopped-Flow Fluorescence Spectroscopy.

Zn2+ has remarkable impacts on amyloid β-protein (Aβ) aggregation, which is crucial in the pathology of Alzheimer's disease. However, the Zn2+ concentration in human cerebrospinal fluid is commonly too low for interaction with Aβ, and only during neurotransmission is there a transient release of a high concentration of Zn2+. It is difficult to explore the details of the interaction between Zn2+ and Aβ within such a short time scale by using ordinary analytical methods. Herein, stopped-flow fluorescence spectroscopy was used to study the fast aggregation kinetics of Aβ42 in the presence of Zn2+ in the time scale from 1 ms to seconds. It was found that Zn2+ bound to Aβ42 within 1 ms; caused immediate conformational transition around Tyr10, which led to the enhancement of Aβ42 hydrophobicity; and then promoted fast aggregation of Aβ42 through enhanced hydrophobic interactions. Among the two Zn2+-binding sites on Aβ42 (KD = 107 nM and 5.2 μM), the first one of higher affinity had a greater impact on the aggregation of Aβ42. Three kinetic phases were observed in the Zn2+-induced fast aggregation of Aβ42, and the fast phase was extremely accelerated by Zn2+, indicating that accelerated aggregation mainly occurred in the fast phase. The reactions occurring in this phase were closely related to the association of Zn2+ and Aβ42. Moreover, Zn2+ largely broadened the pH range of Aβ42 fast aggregation from pH 5.2-6.2 without Zn2+ to pH 5.2-7.8 in the presence of Zn2+. Besides, the promoting effect of Zn2+ on Aβ42 fast aggregation peaked at pH 6.8-7.8, which includes the pH values of the cerebrospinal fluid (pH 7.3) and hippocampus (pH 7.15-7.35). The findings demonstrate the significant effect of Zn2+ on Aβ aggregation and provide new insight into its mechanisms.

Bioanalytical method development and validation for the determination of glycine in human cerebrospinal fluid by ion-pair reversed-phase liquid chromatography–tandem mass spectrometry

A LC–MS/MS method has been developed and validated for the determination of glycine in human cerebrospinal fluid (CSF). The validated method used artificial cerebrospinal fluid as a surrogate matrix for calibration standards. The calibration curve range for the assay was 100–10,000ng/mL and 13C2, 15N-glycine was used as an internal standard (IS). Pre-validation experiments were performed to demonstrate parallelism with surrogate matrix and standard addition methods. The mean endogenous glycine concentration in a pooled human CSF determined on three days by using artificial CSF as a surrogate matrix and the method of standard addition was found to be 748±30.6 and 768±18.1ng/mL, respectively. A percentage difference of −2.6% indicated that artificial CSF could be used as a surrogate calibration matrix for the determination of glycine in human CSF. Quality control (QC) samples, except the lower limit of quantitation (LLOQ) QC and low QC samples, were prepared by spiking glycine into aliquots of pooled human CSF sample. The low QC sample was prepared from a separate pooled human CSF sample containing low endogenous glycine concentrations, while the LLOQ QC sample was prepared in artificial CSF. Standard addition was used extensively to evaluate matrix effects during validation. The validated method was used to determine the endogenous glycine concentrations in human CSF samples. Incurred sample reanalysis demonstrated reproducibility of the method.

The Influence of Preanalytical Conditions on the DJ-1 Concentration in Human Cerebrospinal Fluid

The purpose of this study was to establish the influence of centrifugation and protease activity on the cerebrospinal fluid (CSF) concentrations of DJ-1 and hemoglobin. The concentrations of DJ-1 and hemoglobin were determined in 12 (DJ-1) and six (hemoglobin) pairs of CSF samples, with one sample being stored without centrifugation and the other being centrifuged at 2000 × g before storage. The DJ-1 concentration was also determined in centrifuged and uncentrifuged CSF containing protease inhibitors and compared with values determined in centrifuged and uncentrifuged CSF samples without protease inhibitors. Furthermore, specific protein concentrations were determined in CSF from two groups, each comprising 23 patients with Parkinson's disease. In one group the CSF was centrifuged at 1300-1800 × g, 4°C, 10 min, and in the other at 2000 × g, 4°C, 10 min. Centrifugation at 2000 × g resulted in significantly lower CSF DJ-1 concentrations compared with no centrifugation and centrifugation at a lower g-force. There was a significant difference in the hemoglobin concentration between centrifuged and uncentrifuged CSF. In all centrifuged samples the hemoglobin concentration was <200 ng/ml including blood contaminated samples centrifuged at 2000 × g. When a protease inhibitor cocktail was added to the CSF prior to centrifugation, the DJ-1 concentration was significantly higher. Preanalytical factors such as centrifugation and protease inhibition must be carefully controlled when handling CSF for analysis of DJ-1 and other biomarkers, as DJ-1 was influenced by blood contamination, centrifugation and protease activity.

An alternative derivatization method for the analysis of amino acids in cerebrospinal fluid by gas chromatography–mass spectrometry

The determination of the concentrations of l-amino acids in cerebrospinal fluid (CSF) has been used to gain biochemical insight into central nervous system disorders. This paper describes a microwave-assisted derivatization (MAD) method using N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA) as a derivatizing agent for determining the concentrations of l-amino acids in human CSF by gas chromatography with mass spectrometry (GC/MS). The experimental design used to optimize the conditions showed that the optimal derivatization time was 3min with a microwave power of 210W. The method showed good performance for the validation parameters. The sensitivity was very good, with limits of detection (LODs) ranging from 0.01μmolL(-1) to 4.24μmolL(-1) and limits of quantification (LOQs) ranging from 0.02 to 7.07μmolL(-1). The precision, measured using the relative standard deviation (RSD), ranged from 4.12 to 15.59% for intra-day analyses and from 6.36 to 18.71% for inter-day analyses. The coefficients of determination (R(2)) were above 0.990 for all amino acids. The optimized and validated method was applied to the determination of amino acid concentrations in human CSF.

The impact of P-gp functionality on non-steady state relationships between CSF and brain extracellular fluid

In the development of central nervous system (CNS)-targeted drugs, the prediction of human CNS target exposure is a big challenge. Cerebrospinal fluid (CSF) concentrations have often been suggested as a ‘good enough’ surrogate for brain extracellular fluid (brainECF, brain target site) concentrations in humans. However, brain anatomy and physiology indicates prudence. We have applied a multiple microdialysis probe approach in rats, for continuous measurement and direct comparison of quinidine kinetics in brainECF, CSF, and plasma. The data obtained indicated important differences between brainECF and CSF kinetics, with brainECF kinetics being most sensitive to P-gp inhibition. To describe the data we developed a systems-based pharmacokinetic model. Our findings indicated that: (1) brainECF- and CSF-to-unbound plasma AUC0–360 ratios were all over 100 %; (2) P-gp also restricts brain intracellular exposure; (3) a direct transport route of quinidine from plasma to brain cells exists; (4) P-gp-mediated efflux of quinidine at the blood–brain barrier seems to result of combined efflux enhancement and influx hindrance; (5) P-gp at the blood–CSF barrier either functions as an efflux transporter or is not functioning at all. It is concluded that in parallel obtained data on unbound brainECF, CSF and plasma concentrations, under dynamic conditions, is a complex but most valid approach to reveal the mechanisms underlying the relationship between brainECF and CSF concentrations. This relationship is significantly influenced by activity of P-gp. Therefore, information on functionality of P-gp is required for the prediction of human brain target site concentrations of P-gp substrates on the basis of human CSF concentrations.Electronic supplementary materialThe online version of this article (doi:10.1007/s10928-013-9314-4) contains supplementary material, which is available to authorized users.

Utility of CSF in translational neuroscience

Human cerebrospinal fluid (CSF) sampling is of high value as the only general applicable methodology to obtain information on free drug concentrations in individual human brain. As the ultimate interest is in the free drug concentration at the CNS target site, the question is what CSF concentrations may tell us in that respect. Studies have been performed in rats and other animals for which concentrations in brain extracellular fluid (brain ECF) as a target site for many drugs, have been compared to (cisterna magna) CSF concentrations, at presumed steady state conditions,. The data indicated that CSF drug concentrations provided a rather good indication of, but not a reliable measure for predicting brain ECF concentrations. Furthermore, comparing rat with human CSF concentrations, human CSF concentrations tend to be higher and display much more variability. However, this comparison of CSF concentrations cannot be a direct one, as humans probably had a disease for which CSF was collected in the first place, while the rats were healthy. In order to be able to more accurately predict human brain ECF concentrations, understanding of the complexity of the CNS in terms of intrabrain pharmacokinetic relationships and the influence of CNS disorders on brain pharmacokinetics needs to be increased. This can be achieved by expanding a currently existing preclinically derived physiologically based pharmacokinetic model for brain distribution. This model has been shown to successfully predict data obtained for human lumbar CSF concentrations of acetaminophen which renders trust in the model prediction of human brain ECF concentrations. This model should further evolute by inclusion of influences of drug properties, fluid flows, transporter functionalities and different disease conditions. Finally the model should include measures of target site engagement and CNS effects, to ultimately learn about concentrations that best predict particular target site concentrations, via human CSF concentrations.

N-truncated Aβ peptides in complex fluids unraveled by new specific immunoassays

Previous studies have highlighted the potential physiopathological and diagnostic role of N- and C-terminally truncated amyloid-β (Aβ) peptides in Alzheimer's disease. However, our knowledge about their production remains incomplete, in part due to the lack of very specific and sensitive tools for their detection. We thus developed specific monoclonal antibodies that target either Aβ11-x or Aβ17-x species, which result from the combined cleavages by β/γ- or α/γ-secretases, respectively. The presence of Aβ peptides truncated at residue 11 and 17 peptides was qualitatively and quantitatively assessed, using surface enhanced laser desorption ionization-time of flight mass spectrometry and xMAP (Multi-Analyte Profiling) immunoassays, in the supernatant of HEK293 cells that overexpress wild type or mutant Aβ protein precursor or in which α- and β-secretase activities had been modulated. Our results show a differential secretion of Aβ11–40 and Aβ17–40 species by these HEK293 cell lines. Finally, Aβ11–40 concentration in human cerebrospinal fluid (measured with the new xMAP immunoassays) from a first pilot study was higher in cerebrospinal fluid samples from patients with Alzheimer's disease than in samples from patients with other types of dementia.

Physiologically Based Pharmacokinetic Modeling to Investigate Regional Brain Distribution Kinetics in Rats

One of the major challenges in the development of central nervous system (CNS)-targeted drugs is predicting CNS exposure in human from preclinical data. In this study, we present a methodology to investigate brain disposition in rats using a physiologically based modeling approach aiming at improving the prediction of human brain exposure. We specifically focused on quantifying regional diffusion and fluid flow processes within the brain. Acetaminophen was used as a test compound as it is not subjected to active transport processes. Microdialysis probes were implanted in striatum, for sampling brain extracellular fluid (ECF) concentrations, and in lateral ventricle (LV) and cisterna magna (CM), for sampling cerebrospinal fluid (CSF) concentrations. Serial blood samples were taken in parallel. These data, in addition to physiological parameters from literature, were used to develop a physiologically based model to describe the regional brain pharmacokinetics of acetaminophen. The concentration-time profiles of brain ECF, CSF(LV), and CSF(CM) indicate a rapid equilibrium with plasma. However, brain ECF concentrations are on average fourfold higher than CSF concentrations, with average brain-to-plasma AUC(0-240) ratios of 121%, 28%, and 35% for brain ECF, CSF(LV), and CSF(CM), respectively. It is concluded that for acetaminophen, a model compound for passive transport into, within, and out of the brain, differences exist between the brain ECF and the CSF pharmacokinetics. The physiologically based pharmacokinetic modeling approach is important, as it allowed the prediction of human brain ECF exposure on the basis of human CSF concentrations.

A Common Variant in ERBB4 Regulates GABA Concentrations in Human Cerebrospinal Fluid

The neuregulin 1 (NRG1) receptor ErbB4 is involved in the development of cortical inhibitory GABAergic circuits and NRG1-ErbB4 signaling has been implicated in schizophrenia (SCZ). A magnetic resonance spectroscopy ((1)H-MRS) study has demonstrated that a single-nucleotide polymorphism in ERBB4, rs7598440, influences human cortical GABA concentrations. Other work has highlighted the significant impact of this genetic variant on expression of ERBB4 in the hippocampus and dorsolateral prefrontal cortex in human post mortem tissue. Our aim was to examine the association of rs7598440 with cerebrospinal fluid (CSF) GABA levels in healthy volunteers (n=155). We detected a significant dose-dependent association of the rs7598440 genotype with CSF GABA levels (G-allele standardized β=-0.23; 95% CIs: -0.39 to -0.07; P=0.0066). GABA concentrations were highest in A homozygous, intermediate in heterozygous, and lowest in G homozygous subjects. When excluding subjects on psychotropic medication (three subjects using antidepressants), the results did not change (G-allele standardized β=-0.23; 95% CIs: -0.40 to -0.07; P=0.0051). The explained variance in CSF GABA by rs7598440 in our model is 5.2% (P=0.004). The directionality of our findings agrees with the aforementioned (1)H-MRS and gene expression studies. Our observation therefore strengthens the evidence that the A-allele of rs7598440 in ERBB4 is associated with increased GABA concentrations in the human central nervous system (CNS). To our knowledge, our finding constitutes the first confirmation that CSF can be used to study genotype-phenotype correlations of GABA levels in the CNS. Such quantitative genetic analyses may be extrapolated to other CSF constituents relevant to SCZ in future studies.

The impact of decreased bead count to determine concentrations of amyloid beta1‐42, total‐tau, and phosphorylated‐tau181 in human cerebrospinal fluid using xMAP technology

Alzheimer's disease is the leading cause of human dementia. The lack of diagnostic tests and limited therapeutic options has driven the search for endogenous biomarkers. The INNO‐BIA AlzBio3 assay is a multiplex flow‐based immunoassay measuring Aβ42, tau, and p‐tau in cerebrospinal fluid (CSF). This study assesses assays performance under varying bead count (BC) parameters. Original method validation parameters at 100 BC were acceptable. Reanalyses performed at 3, 10, 25, and 50 BCs were compared to 100 BC data by ANOVA, Bland–Altman analysis, evaluation of concordance correlation coefficients, and frequency distribution of coefficient of variation (CV) ranges. Method validation characteristics were acceptable with 100 BCs. Equivalency for 25 and 50 versus 100 BCs was demonstrated, but not for 3 and 10 BCs. A general trend of decreasing agreement between decreasing BCs and the 100 BC reference resulted in decreases in concordance coefficients ρc. The frequency of CV values greater than 20% increased with decreasing BCs, and CV values of 5% or less decreased with decreased BCs. Statistical analyses demonstrate that BCs of 3 and 10 are not equivalent with the reference and should not be used as a basis for determination of Aβ42, tau, and p‐tau concentration in human CSF. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4655–4663, 2011

LC Method for the Determination of Meropenem in Cerebrospinal Fluid: Application to Therapeutic Drug Monitoring

A simple, rapid and accurate liquid chromatography method using ultrafiltration to pretreat cerebrospinal fluid (CSF) samples was developed to determine meropenem concentrations in human CSF in clinical settings. Meropenem in CSF samples was stabilized by mixing with 1 mol L−1 3-morpholinopropanesulfonic acid buffer (pH 7.0) (1:1). The mixture was transferred to a Nanosep 10 K centrifugal filter device; after centrifugation, the filtrate was subjected to reversed-phase LC and the eluate was monitored at 300 nm. The retention time for meropenem was 5.8 min. The calibration curve of meropenem in human CSF was linear over 0.05–50 μg mL−1. The intra-day and inter-day precision was 0.27–5.66 % and accuracy was 99.0–109.5 %. The limit of detection was 0.01 μg mL−1. This method was successfully applied to neurosurgical patients, showing that it is applicable for therapeutic drug monitoring in patients receiving meropenem.

Age and circadian influences on picolinic acid concentrations in human cerebrospinal fluid

It has been suggested that picolinic acid (PIC), an endogenous metabolite of l-tryptophan, possesses neuro-protective and anti-proliferative effects within the CNS. However, the literature surrounding PIC is limited, and its exact endogenous function is not known. Picolinic acid is produced via the kynurenine pathway which has been implicated in the pathogenesis of a range of neuro-inflammatory diseases. Although not extensively studied, there have been reports of altered PIC production alongside other kynurenine metabolites in inflammatory disorders. In order to investigate whether PIC concentrations are altered with disease in the CNS, we analysed PIC levels in the CSF of 241 patients who underwent lumbar puncture as part of their standard clinical evaluation. In patients with no apparent CNS disease, CSF PIC levels were 10-fold higher in samples taken between 20:00 and 16:00 h compared with those collected between 04:00 and 12:00 h. This result suggests a diurnal variation in PIC synthesis within the CNS. In addition, we observed a direct correlation between a patient's age and their PIC concentration. No significant correlations were observed between CSF PIC levels and any specific disease state.

Use of Peptide Analogue Diversity Library Beads for Increased Depth of Proteomic Analysis: Application to Cerebrospinal Fluid

Biological samples can contain proteins with concentrations that span more than 10 orders of magnitude. Given the limited dynamic range of analysis methods, observation of proteins present at the lower concentrations requires depletion of high-abundance proteins, or other means of reducing the dynamic range of concentrations. Hexapeptide diversity library beads have been used to bind proteins in a complex sample up to a given saturation limit, effectively truncating the maximum concentration of proteins at a desired level. To avoid the potential problem of susceptibility of the hexapeptides to cleavage by proteases in the sample and/or bacterial degradation, peptide analogues that exhibit similar binding characteristics to peptides can be used in place of peptides. We report here the use of hexameric peptoid diversity library beads to reduce the dynamic range of protein concentrations in human cerebrospinal fluid (CSF). Using this method in conjunction with 2D LC/MS/MS analyses, we identified 200 unique proteins, about twice the number identified in untreated CSF.

Elevation of Gas6 protein concentration in cerebrospinal fluid of patients with chronic inflammatory demyelinating polyneuropathy (CIDP)

Gas6 enhances survival of Schwann cells and neurons in vitro and participates in autoimmunity in animal models. Since its concentration in human cerebrospinal fluid (CSF) is unknown, we measured it in samples from patients with non-inflammatory/non-autoimmune neurological diseases (NINAD) and autoimmune polyneuropathies. Samples collected after informed consent during diagnostic lumbar puncture in the period 1999-2006 were stored at -30 degrees C. We considered subjects with NINAD (stroke, ALS, headache, psychiatric conditions simulating neurological diseases, otologic dizziness) or with Guillain-Barré syndrome (GBS) or CIDP. CSF and plasma total protein and age were obtained from clinical records. Gas6 was measured with an ELISA developed and validated in our laboratory (inter-, intra-assay CVs <10%, recovery 96%). Variance, Tukey's post-hoc test, regression were calculated with a statistical software (Statsoft). Mean Gas6 concentration in patients with NINAD was 6.5+/-2.4 ng/ml, 7.2+/-2.6 ng/ml in GBS and significantly higher (11.5+/-1.7 ng/ml) in CIDP than in the other conditions (post-hoc, p<0.005). It was not related to age, CSF total proteins or to CSF/plasma ratio of total proteins (regression, p>0.1). Gas6 is detectable in CSF and may be involved in chronic autoimmune demyelination or myelin repair.