Human Cerebrospinal Fluid Samples Research Articles

Simultaneous determination of LY3214996, abemaciclib, and M2 and M20 metabolites in human plasma, cerebrospinal fluid, and brain tumor by LC-MS/MS

A sensitive and rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method was established for the quantification of total and unbound concentrations of LY3214996, an extracellular signal-regulated kinase inhibitor; abemaciclib, a cyclin-dependent kinase 4/6 inhibitor; and abemaciclib active metabolites, M2 and M20, in human plasma, brain tumor, and cerebrospinal fluid samples. The method was validated over a concentration range of 0.2–500 nM within a total run time of 3.8 min using isocratic elution on a Kinetex™ F5 column. Detection was performed on a Sciex QTRAP 6500+ mass spectrometer employing multiple reaction monitoring mode under positive electrospray ionization. The intra- and inter-batch accuracy as well as the precision of the method for all matrices was within ±20% and ≤20% at the lower limit of quantification, and within ±15% and ≤15% for other quality control levels for all analytes. The unbound fractions of drugs and metabolites in spiked and patient samples were determined using an optimized equilibrium dialysis. The validated method was successfully applied in a phase 0/2 clinical trial to assess the central nervous system penetration of LY3214996 and abemaciclib.

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.

First Indications of Omsk Haemorrhagic Fever Virus beyond Russia.

Omsk haemorrhagic fever virus (OHFV) is the agent leading to Omsk haemorrhagic fever (OHF), a viral disease currently only known in Western Siberia in Russia. The symptoms include fever, headache, nausea, muscle pain, cough and haemorrhages. The transmission cycle of OHFV is complex. Tick bites or contact with infected small mammals are the main source of infection. The Republic of Kazakhstan is adjacent to the endemic areas of OHFV in Russia and febrile diseases with haemorrhages occur throughout the country—often with unclear aetiology. In this study, we examined human cerebrospinal fluid samples of patients with suspected meningitis or meningoencephalitis with unknown origins for the presence of OHFV RNA. Further, reservoir hosts such as rodents and ticks from four Kazakhstan regions were screened for OHFV RNA to clarify if this virus could be the causative agent for many undiagnosed cases of febrile diseases in humans in Kazakhstan. Out of 130 cerebrospinal fluid samples, two patients (1.53%) originating from Almaty city were positive for OHFV RNA. Screening of tick samples revealed positive pools from different areas in the Akmola region. Of the caught rodents, 1.1% out of 621 were positive for OHFV at four trapping areas from the West Kazakhstan region. In this paper, we present a broad investigation of the spread of OHFV in Kazakhstan in human cerebrospinal fluid samples, rodents and ticks. Our study shows for the first time that OHFV can not only be found in the area of Western Siberia in Russia, but can also be detected up to 1.600 km away in the Almaty region in patients and natural foci.

In Vivo Microdialysis in Mice Captures Changes in Alzheimer's Disease Cerebrospinal Fluid Biomarkers Consistent with Developing Pathology.

Preclinical models of Alzheimer's disease (AD) can provide valuable insights into the onset and progression of the disease, such as changes in concentrations of amyloid-β (Aβ) and tau in cerebrospinal fluid (CSF). However, such models are currently underutilized due to limited advancement in techniques that allow for longitudinal CSF monitoring. An elegant way to understand the biochemical environment in the diseased brain is intracerebral microdialysis, a method that has until now been limited to short-term observations, or snapshots, of the brain microenvironment. Here we draw upon patient-based findings to characterize CSF biomarkers in a commonly used preclinical mouse model for AD. Our modified push-pull microdialysis method was first validated ex vivo with human CSF samples, and then in vivo in an AD mouse model, permitting assessment of dynamic changes of CSF Aβ and tau and allowing for better translational understanding of CSF biomarkers. We demonstrate that CSF biomarker changes in preclinical models capture what is observed in the brain; with a decrease in CSF Aβ observed when plaques are deposited, and an increase in CSF tau once tau pathology is present in the brain parenchyma. We found that a high molecular weight cut-off membrane allowed for simultaneous sampling of Aβ and tau, comparable to CSF collection by lumbar puncture in patients. Our approach can further advance AD and other neurodegenerative research by following evolving neuropathology along the disease cascade via consecutive sampling from the same animal and can additionally be used to administer pharmaceutical compounds and assess their efficacy.

ExoRBase 2.0: an atlas of mRNA, lncRNA and circRNA in extracellular vesicles from human biofluids.

Extracellular vesicles (EVs) are small membranous vesicles that contain an abundant cargo of different RNA species with specialized functions and clinical implications. Here, we introduce an updated online database (http://www.exoRBase.org), exoRBase 2.0, which is a repository of EV long RNAs (termed exLRs) derived from RNA-seq data analyses of diverse human body fluids. In exoRBase 2.0, the number of exLRs has increased to 19 643 messenger RNAs (mRNAs), 15 645 long non-coding RNAs (lncRNAs) and 79 084 circular RNAs (circRNAs) obtained from ∼1000 human blood, urine, cerebrospinal fluid (CSF) and bile samples. Importantly, exoRBase 2.0 not only integrates and compares exLR expression profiles but also visualizes the pathway-level functional changes and the heterogeneity of origins of circulating EVs in the context of different physiological and pathological conditions. Our database provides an attractive platform for the identification of novel exLR signatures from human biofluids that will aid in the discovery of new circulating biomarkers to improve disease diagnosis and therapy.

DDRE-12. DETERMINATION OF TOTAL AND UNBOUND LEVELS OF A POTENT INHIBITOR OF FIBROBLAST GROWTH FACTOR RECEPTOR, INFIGRATINIB, IN HUMAN PLASMA, CEREBROSPINAL FLUID AND BRAIN TUMOR BY A VALIDATED LC-MS/MS ASSAY

Abstract BACKGROUND Infigratinib is an orally available potent inhibitor of fibroblast growth factor receptor (FGFR) under investigation in a Phase 0 clinical trial for recurrent high-grade gliomas harboring oncogenic FGFR mutation or translocation (NCT04424966). Here, we report on our development and validation of an LC-MS/MS method for determination of total and unbound levels of infigratinib in human plasma, cerebrospinal fluid (CSF) and glioblastoma tissue. METHODS Infigratinib was extracted from human plasma, CSF and glioblastoma homogenate samples by protein precipitation with methanol. Unbound fractions of infigratinib in plasma and brain tissues were determined by equilibrium dialysis. Validation was performed using Sciex ExionLC UHPLC system coupled with Sciex QTRAP 6500+ MS/MS detector using positive electrospray ionization mode. The method was validated according to FDA guidelines and CAP/CLIA regulations. RESULTS The method was validated for 0.2-1000 nmol/L concentration range using plasma as matrix for all biospecimens. The analytical separation was optimized on Phenomenex Kinetex™ F5 column with total run time of 3.5 min using isocratic mode. Maximum coefficient of variation for intra- and inter-day precision was 12.7% and the accuracy was within 88.5-115.0% in all matrixes. The analyte is stable in plasma for at least 19 hours at room temperature (RT). Plasma stability at -20°C was demonstrated for at least 119 days. Stability of stock and working solutions was demonstrated for at least 15 hours (RT) and 60 days (2-8°C). The unbound fraction of infigratinib in pooled human plasma and brain were determined to be 0.01. CONCLUSIONS A sensitive and rapid bioanalytical method is successfully developed and validated to quantify total and unbound infigratinib levels in human plasma, CSF and glioblastoma tissue. The method is presently employed to evaluate plasma pharmacokinetics and CNS penetration of infigratinib in recurrent glioblastoma patients in an ongoing Phase 0 clinical trial.

Generation and Release of Neurogranin, Vimentin, and MBP Proteolytic Peptides, Following Traumatic Brain Injury.

Traumatic brain injury (TBI) is a major neurological disorder without FDA-approved therapies. In this study, we have examined the concept that TBI might trigger global brain proteolysis in the acute post-injury phase. Thus, we conducted a systemic proteolytic peptidomics analysis using acute cerebrospinal fluid (CSF) samples from TBI patients and normal control samples. We employed ultrafiltration-based low molecular weight (LMW; < 10kDa) peptide enrichment, coupled with nano-reversed-phase liquid chromatography/tandem mass spectrometry analysis, followed with orthogonal quantitative immunoblotting-based protein degradation analysis. We indeed identified novel patterns of injury-dependent proteolytic peptides derived from neuronal components (pre- and post-synaptic terminal, dendrites, axons), extracellular matrix, oligodendrocytes, microglial cells, and astrocytes. Among these, post-synaptic protein neurogranin was identified for the first time converted to neurogranin peptides including neurogranin peptide (aa 16-64) that is phosphorylated at Ser-36/48 (P-NG-fragment) in acute human TBI CSF samples vs. normal control with a receiver operating characteristic area under the curve of 0.957. We also identified detailed processing of astroglia protein (vimentin) and oligodendrocyte protein (MBP and Golli-MBP) to protein breakdown products (BDPs) and/or LMW proteolytic peptides after TBI. In addition, using MS/MS selected reaction monitoring method, two C-terminally released MBP peptides TQDENPVVHFF and TQDENPVVHF were found to be elevated in acute and subacute TBI CSF samples as compared to their normal control counterparts. These findings imply that future therapeutic strategies might be placed on the suppression of brain proteolysis as a target. The endogenous proteolytic peptides discovered in human TBI biofluid could represent useful diagnostic and monitoring tools for TBI.

Cobalt-decorated 3D hybrid nanozyme: A catalytic amplification platform with intrinsic oxidase-like activity

Research about constructing multi-dimensional carbon nanomaterials is of great significance in electrocatalytic and sensing fields in order to integrate structural merits of each individual unit. Also, nanomaterials with enzyme-like activities are prospective candidates for artificial enzyme design and electrochemical application. Herein, we fabricate Co, N co-doped hierarchical hybrid (Co@NCNTs/NC) nanozyme, which integrates of both N-doped carbon nanotubes (NCNTs) and N-doped carbon sheets (NC). The three-dimensional (3D) porous carbon composite is prepared by thermal treatment of metal-organic framework (MOF) which was synthesized by growing of ZIF-67 on ZIF-L at room temperature. The obtained nanomaterial not only possesses an improved oxidase-like activity that can catalyze 3,3′,5,5′-tetramethylbenzidine (TMB) in the absence of hydrogen peroxide (H2O2), but also constructs a signal amplification platform towards dopamine (DA) due to the synergistic catalysis of Co species and N-doped porous carbon architecture. The electrocatalytic performance for DA detection shows a broad linear range from 30 nM to 710 μM and a detection limit of 9 nM. The Co@NCNTs/NC/GCE is employed to practically detect DA in human serum and artificial cerebrospinal fluid (aCSF) samples with satisfactory results. The present work exhibits a great promising in colorimetric and electrochemical sensing fields and presents a new sight for the fabrication of MOF-derived nanozyme.

Simultaneous Quantification and Speciation of Trace Metals in Paired Serum and CSF Samples by Size Exclusion Chromatography-Inductively Coupled Plasma-Dynamic Reaction Cell-Mass Spectrometry (SEC-DRC-ICP-MS).

Background: Transition metals play a crucial role in brain metabolism: since they exist in different oxidation states they are involved in ROS generation, but they are also co-factors of enzymes in cellular energy metabolism or oxidative defense. Methods: Paired serum and cerebrospinal fluid (CSF) samples were analyzed for iron, zinc, copper and manganese as well as for speciation using SEC-ICP-DRC-MS. Brain extracts from Mn-exposed rats were additionally analyzed with SEC-ICP-DRC-MS. Results: The concentration patterns of transition metal size fractions were correlated between serum and CSF: Total element concentrations were significantly lower in CSF. Fe-ferritin was decreased in CSF whereas a LMW Fe fraction was relatively increased. The 400–600 kDa Zn fraction and the Cu-ceruloplasmin fraction were decreased in CSF, by contrast the 40–80 kDa fraction, containing Cu- and Zn-albumin, relatively increased. For manganese, the α-2-macroglobulin fraction showed significantly lower concentration in CSF, whereas the citrate Mn fraction was enriched. Results from the rat brain extracts supported the findings from human paired serum and CSF samples. Conclusions: Transition metals are strictly controlled at neural barriers (NB) of neurologic healthy patients. High molecular weight species are down-concentrated along NB, however, the Mn-citrate fraction seems to be less controlled, which may be problematic under environmental load.

Design of an Innovative Methodology for Cerebrospinal Fluid Analysis: Preliminary Results.

Cerebrospinal fluid (CSF) analysis supports diagnosis of neurodegenerative diseases (NDs), however a number of issues limits its potentialities in clinical practice. Here, a newly developed technique for fluid voltammetry, relying on a simple sensor (BIOsensor-based multisensorial system for mimicking Nose, Tongue and Eyes, BIONOTE), was used to test the applicability for CSF analysis. BIONOTE was initially calibrated on an artificial CSF-like solution and then applied on human CSF, either immediately after collection or after refrigerated storage. Following optimization, it was used to evaluate 11 CSF samples correlating the electrochemical dataset with CSF routine parameters and biomarkers of neurodegeneration. Multivariate data analysis was performed for model elaboration and calibration using principal component analysis and partial least squares discriminant analysis. BIONOTE presented a high capacity to predict both physiological and pathological constituents of artificial CSF. It differentiated distinct fresh human CSF samples well but lost accuracy after refrigerated storage. The electrochemical analysis-derived data correlated with either CSF routine cytochemical indexes or a biomarker of neurodegeneration. BIONOTE resulted as being a reliable system for electrochemical analysis of CSF. The CSF fingerprint provided by the sensor has shown itself to be sensitive to CSF modification, thus it is potentially representative of CSF alteration. This result opens the way to its testing in further study addressed at assessing the clinical relevance of the methodology. Because of its advantages due to the ease and rapidity of the methodology, a validation study is now required to translate the technique into clinical practice and improve diagnostic workup of NDs.

Characterization of Hevin (SPARCL1) Immunoreactivity in Postmortem Human Brain Homogenates

Hevin is a matricellular glycoprotein that plays important roles in neural developmental processes such as neuronal migration, synaptogenesis and synaptic plasticity. In contrast to other matricellular proteins whose expression decreases when development is complete, hevin remains highly expressed, suggesting its involvement in adult brain function. In vitro studies have shown that hevin can have different post-translational modifications. However, the glycosylation pattern of hevin in the human brain remains unknown, as well as its relative distribution and localization. The present study provides the first thorough characterization of hevin protein expression by Western blot in postmortem adult human brain. Our results demonstrated two major specific immunoreactive bands for hevin: an intense band migrating around 130 kDa, and a band migrating around 100 kDa. Biochemical assays revealed that both hevin bands have a different glycosylation pattern. Subcellular fractionation showed greater expression in membrane-enriched fraction than in cytosolic preparation, and a higher expression in prefrontal cortex (PFC) compared to hippocampus (HIP), caudate nucleus (CAU) and cerebellum (CB). We confirmed that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and matrixmetalloproteinase 3 (MMP-3) proteases digestion led to an intense double band with similar molecular weight to that described as SPARC-like fragment (SLF). Finally, hevin immunoreactivity was also detected in human astrocytoma, meningioma, cerebrospinal fluid and serum samples, but was absent from any blood cell type.

The effect of pre-analytical handling on the stability of fractalkine, monocyte chemoattractant protein 1 (MCP1), interleukin 6 and interleukin 8 in samples of human cerebrospinal fluid

Cytokine networks in cerebrospinal fluid (CSF) are important to our understanding of several neuroinflammatory diseases. Knowledge about optimal handling of samples is limited but important to minimize bias and reduce costs in CSF biomarker studies. The aim of this study was to examine the effect of storage temperature and time delay from CSF sample collection until freezing on the concentration of 11 different cytokines thought to be associated with chronic pain. CSF samples from 21 individuals undergoing hip or knee arthroplasty under spinal anesthesia were divided between two tubes. One tube was stored and centrifuged (within 30 min) at room temperature, and one tube was stored in ice water and centrifuged (within 30 min) at 4 °C. Each tube was split into six vials that were frozen at −80 °C, 0.5, 1, 2, 3, 4, or 5 h after collection. Cytokines were analyzed using a multiplex panel. A random effect panel data regression was conducted for each biomarker including the variables of storage temperature until freezing and time delay. Four cytokines had detectable levels: Fractalkine, monocyte chemoattractant protein 1(MCP-1), interleukine 6 (IL-6), and interleukine 8 (IL-8). There was no significant effect of storage temperature and time delay on MCP-1, IL-6, or IL-8 concentrations. Fractalkine concentration showed no clear trend. No concentration differences were observed between samples kept in ice water and those at room temperature except at the 3-h time point, and there was no overall significant effect of time delay on fractalkine concentration. We found no clear effect of storage temperature and time delay up to five hours from sample collection until freezing on the CSF concentrations of fractalkine, MCP-1, IL-6, or IL-8.

Changes in the metabolic profile of human male postmortem frontal cortex and cerebrospinal fluid samples associated with heavy alcohol use.

Heavy alcohol use is one of the top causes of disease and death in the world. The brain is a key organ affected by heavy alcohol use. Here, our aim was to measure changes caused by heavy alcohol use in the human brain metabolic profile. We analyzed human postmortem frontal cortex and cerebrospinal fluid (CSF) samples from males with a history of heavy alcohol use (n = 74) and controls (n = 74) of the Tampere Sudden Death Series cohort. We used a nontargeted liquid chromatography mass spectrometry-based metabolomics method. We observed differences between the study groups in the metabolite levels of both frontal cortex and CSF samples, for example, in amino acids and derivatives, and acylcarnitines. There were more significant alterations in the metabolites of frontal cortex than in CSF. In the frontal cortex, significant alterations were seen in the levels of neurotransmitters (e.g., decreased levels of GABA and acetylcholine), acylcarnitines (e.g., increased levels of acylcarnitine 4:0), and in some metabolites associated with alcohol metabolizing enzymes (e.g., increased levels of 2-piperidone). Some of these changes were also significant in the CSF samples (e.g., elevated 2-piperidone levels). Overall, these results show the metabolites associated with neurotransmitters, energy metabolism and alcohol metabolism, were altered in human postmortem frontal cortex and CSF samples of persons with a history of heavy alcohol use.

Bank vole prion protein extends the use of RT-QuIC assays to detect prions in a range of inherited prion diseases

The cerebrospinal fluid (CSF) real-time quaking-induced conversion assay (RT-QuIC) is an ultrasensitive prion amyloid seeding assay for diagnosis of sporadic Creutzfeldt–Jakob disease (CJD) but several prion strains remain unexplored or resistant to conversion with commonly used recombinant prion protein (rPrP) substrates. Here, bank vole (BV) rPrP was used to study seeding by a wide range of archived post-mortem human CSF samples from cases of sporadic, acquired and various inherited prion diseases in high throughput 384-well format. BV rPrP substrate yielded positive reactions in 70/79 cases of sporadic CJD [Sensitivity 88.6% (95% CI 79.5–94.7%)], 1/2 variant CJD samples, and 9/20 samples from various inherited prion diseases; 5/57 non-prion disease control CSFs had positive reactions, yielding an overall specificity of 91.2% (95% CI 80.1–97.1%). Despite limitations of using post-mortem samples and our results’ discrepancy with other studies, we demonstrated for the first time that BV rPrP is susceptible to conversion by human CSF samples containing certain prion strains not previously responsive in conventional rPrPs, thus justifying further optimisation for wider diagnostic and prognostic use.

Antibody-Free Determinations of Low-Mass, Soluble Oligomers of Aβ42 and Aβ40 by Planar Bilayer Lipid Membrane-Based Electrochemical Biosensor

Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly. Abnormal aggregates of both β-amyloid peptide (Aβ) subtypes, Aβ42 and Aβ40, are the typical neuropathology hallmarks of AD. However, because of the lack of specific recognition elements such as an antibody and aptamer, it is difficult to differentiate and determine the oligomers of Aβ42 and Aβ40 in clinic. In this paper, we developed a planar bilayer lipid membrane (BLM)-based electrochemical biosensor. According to the dynamic differences on oligomer-induced BLM damage, both low-mass, soluble oligomers of Aβ42 and Aβ40 (L-Aβ42O and L-Aβ40O) were measured in turn by electrochemical impedance spectroscopy. The BLM was supported by a porous 11-mercaptoundecanoic acid layer on a gold electrode, which amplified the impedance signal corresponding to the membrane damage and improved the detection sensitivity. The weakly charged surface of the BLM ensured the low non-specific adsorption of coexisting proteins in cerebrospinal fluid (CSF). Using the electrochemical biosensor, L-Aβ42O was determined within 20 min, with a linear range from 5 to 500 pM and a detection limit of 3 pM. Meanwhile, L-Aβ40O was determined within 60 min, with a linear range from 60 pM to 6.0 nM and a detection limit of 26 pM. The recoveries in oligomer-spiked artificial CSF and human CSF samples confirmed the accuracy and applicability of this proposed method in clinic. This work provides an antibody-free, highly selective, and sensitive method for simultaneous detections of L-Aβ42O and L-Aβ40O in real CSF samples, which is significant for the early diagnosis and prognosis of AD.

In-depth Site-specific Analysis of N-glycoproteome in Human Cerebrospinal Fluid and Glycosylation Landscape Changes in Alzheimer's Disease

As the body fluid that directly interchanges with the extracellular fluid of the central nervous system (CNS), cerebrospinal fluid (CSF) serves as a rich source for CNS-related disease biomarker discovery. Extensive proteome profiling has been conducted for CSF, but studies aimed at unraveling site-specific CSF N-glycoproteome are lacking. Initial efforts into site-specific N-glycoproteomics study in CSF yield limited coverage, hindering further experimental design of glycosylation-based disease biomarker discovery in CSF. In the present study, we have developed an N-glycoproteomic approach that combines enhanced N-glycopeptide sequential enrichment by hydrophilic interaction chromatography (HILIC) and boronic acid enrichment with electron transfer and higher-energy collision dissociation (EThcD) for large-scale intact N-glycopeptide analysis. The application of the developed approach to the analyses of human CSF samples enabled identifications of a total of 2893 intact N-glycopeptides from 511 N-glycosites and 285 N-glycoproteins. To our knowledge, this is the largest site-specific N-glycoproteome dataset reported for CSF to date. Such dataset provides molecular basis for a better understanding of the structure–function relationships of glycoproteins and their roles in CNS-related physiological and pathological processes. As accumulating evidence suggests that defects in glycosylation are involved in Alzheimer's disease (AD) pathogenesis, in the present study, a comparative in-depth N-glycoproteomic analysis was conducted for CSF samples from healthy control and AD patients, which yielded a comparable N-glycoproteome coverage but a distinct expression pattern for different categories of glycoforms, such as decreased fucosylation in AD CSF samples. Altered glycosylation patterns were detected for a number of N-glycoproteins including alpha-1-antichymotrypsin, ephrin-A3 and carnosinase CN1 etc., which serve as potentially interesting targets for further glycosylation-based AD study and may eventually lead to molecular elucidation of the role of glycosylation in AD progression.

High-sensitivity quantification of acetylcholine and choline in human cerebrospinal fluid with a validated LC-MS/MS method

Acetylcholine is the neurotransmitter of the parasympathetic nervous system, synthesized from choline and involved in several neurodegenerative diseases. Exploration of cholinergic neurotransmission in the human central nervous system is limited by the lack of a sensitive and specific method for the determination of acetylcholine and choline expression. We developed an hydrophilic interaction liquid chromatography – mass spectrometry method for the quantification of both molecules in human cerebrospinal fluid samples. An extensive selectivity study towards endogenous interfering compounds, in particular γ-butyrobetain, was performed and the method was validated according to the European Medicine Agency and Food and Drug Administration guidelines for the validation of bioanalytical methods. The performance of the method was excellent with a lower limit of quantification at 5 ng/L (34.2 pmol/L) for acetylcholine and 5 μg/L for choline, a precision in the range 1.3–11.9% and an accuracy between 85.2 and 113.1%. This suitability of the method for the quantification of acetylcholine and choline in clinical samples was demonstrated with the analysis of patient cerebrospinal fluid samples. Altogether, this validated method allows the simultaneous quantitative analysis of acetylcholine and choline in human cerebrospinal fluid with high sensitivity and selectivity. It will allow to better characterize the cholinergic neurotransmission in human pathologies and to study the effects of drugs acting on this system.

1719-P: Novel Mass Spectrometry–Based Selected Reaction Monitoring Proteomics for Analysis of Low Abundant Insulin Levels in Cerebrospinal Fluid

Circulating insulin levels are routinely measured using Enzyme-linked Immunosorbent Assay (ELISA). However, ELISA is not sensitive enough to measure cerebrospinal fluid (CSF) insulin levels which are 10-100 times lower than those of plasma: yet ELISA results are often reported for insulin analysis in CSF where it serves as a surrogate for brain interstitial fluid insulin levels. We developed a novel LC-MS/MS based selected reaction monitoring (SRM) proteomics assay for quantitative analyses targeting predetermined peptides derived from trypsin digests of insulin; each peptide depends on specific SRM transitions. We determined insulin levels using our novel insulin SRM proteomics method in the matching plasma and CSF samples that were from individuals fasting ≥12 hours and individuals after 2 hours of steady state hyperglycemia by clamp technology (2 hr SS HG). All samples were obtained in individuals with normal fasting glucose and HbA1C levels. Statistically significant differences were observed in both plasma and CSF between fasting and 2 hr SS HG by two tailed and unpaired student t test and 2-way matched ANOVA as shown in Table below. Our results confirmed that peripheral endogenous insulin appears in CSF at least 2 hours after steady state hyperglycemia in subjects with normal glucose tolerance. Our SRM insulin tryptic peptide panel reliably quantified insulin peptide levels in human CSF samples. Disclosure Q. Liu: None. M. Zhu: None. C.W. Chia: None. J.F. O’Connell: None. P. Zhang: None. J.M. Egan: None. Funding National Institute on Aging

A Multiplexed Microarray Platform for Targeted Proteomics

A Multiplexed Microarray Platform for Targeted Proteomics

Formation and Detection of Highly Oxidized Hemoglobin Forms in Biological Fluids during Hemolytic Conditions.

Hemolytic diseases are characterized by an accelerated breakdown of red blood cells (RBCs) and the release of hemoglobin (Hb). Following, RBC lysis Hb oxidation occurs with the formation of different redox states of Hb (metHb and ferrylHb) and the release of heme. ferrylHb is unstable and decomposes to metHb with the concomitant formation of globin radicals and eventually covalently crosslinked Hb multimers. The goal of the present study was to determine the concentrations of the different redox states of Hb in biological samples during hemolytic conditions. We used plasma and urine samples of mice with intravascular hemolysis and human cerebrospinal fluid (CSF) samples following intraventricular hemorrhage. Because ferrylHb is highly unstable, we also addressed the fate of this species. metHb and free heme time-dependently accumulate in plasma and CSF samples following intravascular hemolysis and intraventricular hemorrhage, respectively. ferrylHb is hardly detectable in the biological samples during hemolytic conditions. Under in vitro conditions, ferrylHb decomposes quickly to metHb, which process is associated with the formation of covalently crosslinked Hb multimers. We detected these covalently crosslinked Hb multimers in plasma, urine, and CSF samples during hemolytic conditions. Because globin modification is specific for these Hb forms, we propose to call this heterogeneous form of Hb produced during ferrylHb decomposition as globin-modified oxidized Hb (gmoxHb). Understanding the formation and the contribution of gmoxHb species to the pathogenesis of hemolytic conditions could have therapeutic implications in the treatment of hemolytic diseases.