Human Lumbar Cerebrospinal Fluid Research Articles

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.

Indole levels in human lumbar and ventricular cerebrospinal fluid and the effect of L-tryptophan administration.

Levels of tryptophan (TRP), 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in human lumbar and ventricular cerebrospinal fluid (CSF) were measured by reversed phase liquid chromatography (HPLC) with electrochemical detection. The levels of TRP ranged from 1593 to 4865 nmol/l in ventricular (VF) and from 1257 to 2557 nmol/l in lumbar CSF. The level of 5-HTP varied from 1.1 to 68.9 nmol/l in VF and from 5.3 to 10.8 nmol/l in lumbar CSF; no previous reports of 5-HTP levels in CSF exist. The serotonin level was 1.9-27.3 nmol/l in VF and 5.7-12.0 nmol/l in lumbar CSF. The levels of 5-HIAA were considerably higher in VF than in lumbar fluid with respective means of 498 +/- 52.4 nmol/l and 112 +/- 15.6 nmol/l (P less than 0.001). An oral dose of 2 g L-tryptophan significantly increased all indole levels except that of 5-HT, both in patients with progressive myoclonus epilepsy and in controls.

Differential protein expression in male and female human lumbar cerebrospinal fluid using iTRAQ reagents after abundant protein depletion

Cerebrospinal fluid (CSF) has become one of the most frequently used biological medium for physiological studies for neurological disorders due to its proximity to the brain and clinical availability; however, before undertaking a rational approach to biomarker discovery or diagnostics, it is crucial to understand the underlying characteristics of CSF proteome in subpopulations. In this study, we examined the differential expression of proteins in pooled male and female CSF utilizing isobaric tags for relative and absolute quantification (iTRAQ) reagents after the depletion of six high abundant proteins using a multiple affinity removal system (MARS). A total of 219 proteins were identified (95% confidence level), and 12 proteins showed difference in expression levels. Eleven out of 12 differentially expressed proteins showed ratios of male/female between 1.15 and 1.29 (duplicate average), indicating a remarkable similarity between male and female CSF. One notable exception was the slightly lower expression level of ceruloplasmin (ferroxidase) in male CSF (0.81), a copper containing protein that catalyzes the conversion of ferrous iron to ferric iron with antioxidant properties. We also examined the levels of ceruloplasmin in each individual patient sample which constituted the pooled CSF using Western blot analysis which confirmed the lower expression levels of ceruloplasmin in male CSF.

Interaction of Phospholipase C-γ1 with Villin Regulates Epithelial Cell Migration

Interaction of Phospholipase C-γ1 with Villin Regulates Epithelial Cell Migration

Human cerebrospinal fluid peptidomics

Cerebrospinal fluid (CSF) has frequently been extensively studied to explore several different central nervous system (CNS) disorders because it contains proteins, enzymes, hormones, neuropeptides and neurotransmitters that play critical regulatory roles in many different physiological processes. Individual neuropeptidergic systems in CSF have been studied. In theory, peptidomics offers a bird's-eye, comprehensive and systems-level approach to analyze all of the peptidergic systems that have been expressed in CSF at any given time. In this study, low molecular mass (M(r) < 5 kDa) peptides were isolated by ultrafiltration. The isolated peptides, with or without trypsin digestion, were preferentially enriched with a solid-phase extraction cartridge, and the peptides were separated with capillary liquid chromatography and analyzed with on-line quadrupole time-of-flight mass spectrometry (MS). In this proof-of-principle study, the 20 representative MS-characterized peptides were shown to be derived from 12 proteins, among which four proteins, amyloid-like protein 1, secretogranin I, granin-like neuroendocrine peptide precursor and neurosecretory protein VGF, have been shown elsewhere to be either associated with CNS disorders or to play a central role in the CNS. The long-term goals of this peptidomics study are to monitor the changes (amount; modifications) of CSF peptides, clarify the aberrant processing of large intact protein precursors, elucidate the molecular mechanisms of CNS disorders and find biomarkers. This analytical method is effective for the analysis of the human lumbar CSF peptidome.

Proteomics analysis of prefractionated human lumbar cerebrospinal fluid

Cerebrospinal fluid (CSF) is produced by the chorioid plexus in the ventricles. It surrounds the brain and bone marrow, and reflects several different disorders of the central nervous system (CNS). Proteomics has been used to analyze CSF in order to discover disease-associated proteins and to elucidate the basic molecular mechanisms that either cause, or result from, CNS disorders. However, some disease-associated proteins are of low-abundance and are difficult to detect. A low total-protein concentration, a high amount of albumin and immunoglobins, and a wide dynamic range (several orders of magnitude) of protein concentration cause several difficulties in the identification of low-abundance CSF proteins. In this study, advantage was taken of the range of different hydrophobic properties of CSF proteins, and a reversed-phase solid-phase extraction (SPE) cartridge was used to prefractionate human lumbar CSF proteins into three separate fractions prior to two-dimensional gel electrophoresis resolution of the proteome. A portion of the high-abundance CSF proteins were removed from two (eluted with 35% and 50% acetonitrile) of the three fractions. Some trace CSF proteins were preferentially enriched in the two fractions, and many proteins were detected in the two-dimensional (2-D) gels of the two fractions. Among the novel proteins identified, sixty-two protein spots that represent forty-two proteins were characterized. Most of the proteins have not been annotated in any previous 2-D map of human CSF, and several have been implicated in CNS diseases. The prefractionation of CSF proteins with SPE, followed by proteomics analysis, provides a new method to explore low-abundance, disease-specific CSF proteins.

Between-gel reproducibility of the human cerebrospinal fluid proteome.

This manuscript describes the between-gel reproducibility of the two-dimensional gel electrophoresis analysis of the human lumbar cerebrospinal fluid (CSF) proteome. This reproducibility study is a necessary component for our long-term research program that uses comparative proteomics to analyze lumbar CSF samples in a study of human idiopathic low back pain. A Protein-Plus Dodeca Cell electrophoresis apparatus and PDQuest software were used to measure the level of between-gel reproducibility of the CSF proteome. One pooled CSF sample was used to evaluate the level of within-sample, between-gel reproducibility, and a set of seven different CSF samples (CSF-1 to 7) was used to test the level of within-group and between-group variability. Differentially expressed proteins (six CSF samples versus the designated control, CSF-3) were characterized with mass spectrometry. The number of spots found in the pooled CSF sample was 490 +/- 30 (n = 10 gels); the percentage of protein spots found in those 10 gels was 92 +/- 6%, with a coefficient of variation of 6%; and a positive coefficient of correlation (r = 0.82) was found. In order to test the proof-of-principle, that set of seven CSF samples served as a test of our ability to perform reproducibility comparative proteomics, and to detect differentially expressed proteins within that set of test samples. One sample (CSF-3) served as the control for the other six to locate the differentially expressed proteins. A comparison of fifteen differentially expressed proteins found in that set of test CSF samples correlated with pathology. Matrix-assisted laser desorption/ionization-time-of-flight and electrospray ionization quadrupole ion trap mass spectrometry were used to characterize thirteen of those fifteen differentially expressed proteins. These results (reproducibility, protein characterization, set of test samples, and proof-of-principle) suggest that the analysis of human CSF two-dimensional gels can achieve a high level of within-sample and between-sample reproducibility, and that PDQuest software can measure the relative protein abundance in the human CSF proteome.

Proteomics Analysis of Phosphotyrosyl-Proteins in Human Lumbar Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a secretion product of several different central nervous system (CNS) structures, including the choroid plexus in the ventricles. Pathological CNS processes are reflected in the protein composition of CSF. To elucidate the molecular events that occur in the homeostatic and pathological processes of the CNS, the high-throughput characterization of differentially expressed proteins, and of post-translationally modified proteins, is needed for proteomics studies of CSF. Among the post-translational modifications of proteins, phosphorylation is the most common and important mechanism for the reversible regulation of protein function. In this study, CSF phosphotyrosyl (p-Tyr)-proteins were detected with antibodies and were analyzed with proteomics methods. Three different combination methods--1D gel electrophoresis and Western blotting, immunoprecipitation and 2D gel electrophoresis, and 2D gel electrophoresis and Western blotting--were used to detect p-Tyr-proteins in human lumbar CSF samples. Six protein spots, representing four proteins on a 2D Western blot, were identified as p-Tyr-proteins with the 2D gel electrophoresis and Western blotting method. Those four p-Tyr-proteins are kallikrein-6 precursor, complement C4 gamma-chain, gelsolin, and ceruloplasmin precursor. Additionally, four other nonphosphorylated CSF proteins--beta-2-glycoprotein I precursor, fibulin-1 precursor, EGF-containing fibulin-like extracellur matrix protein 1 precursor, and angiotensinogen precursor--were characterized for the first time.

Acute changes in Cerebrospinal Fluid 5-HIAA following Oral Paroxetine Challenge in Healthy Humans

A number of studies have reported decreased human lumbar cerebrospinal fluid (CSF) concentrations of the major serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), following chronic administration of selective serotonin reuptake inhibitors (SSRIs). This decrease has been thought to be a consequence of elevated extracellular serotonin and to be mediated through terminal autoreceptor feedback inhibition of serotonin turnover. We wished to study the previously unexamined acute effects of SSRI administration on human CSF 5-HIAA. A serial lumbar puncture (LP) procedure was used to collect CSF samples before and after a single oral 40 mg dose of the SSRI paroxetine (PAR) or matching placebo in eight healthy adult humans in a randomized, double-blind fashion. CSF 5-HIAA concentrations did not change following placebo, but showed a statistically significant 27% mean increase 3 h following PAR. Our findings stand in contrast to the decreases reported for CSF 5-HIAA after chronic SSRI treatment in humans and the decreases seen in brain extracellular 5-HIAA after acute or chronic administration of SSRIs to animals.

Acetylcholinesterase in lumbar and ventricular cerebrospinal fluid

Background: Soluble acetylcholinesterase (AChE, E.C. 3.1.1.7.) is released by neurons, glial and meningeal cells into the CSF. AChE activity in cerebrospinal fluid (CSF) is altered in various disorders of the nervous system. The objects of this study are to define a reference range for CSF AChE activity in human lumbar CSF, to prove that the enzyme activity does not depend on the blood/CSF barrier function, and to provide information about AChE in ventricular CSF. In addition, drugs used in neurosurgical care have been examined for their in vitro effects on CSF AChE activity to exclude interference with the test system. Methods: We tested the AChE activity in 64 lumbar CSF samples collected from a clinically healthy population and in 169 ventricular CSF samples obtained from 90 neurosurgical patients. AChE activity was assayed with our inhibitor-free test procedure. Results: The reference range determined for lumbar CSF AChE activity is 9.2–24.4 nmol/min per ml. Lumbar CSF AChE activity does not correlate with parameters characterising the status of the blood/CSF barrier. Ventricular puncture is only justified for underlying pathology making it impossible to provide reference data for ventricular CSF. Most measurements reveal ventricular enzyme activity below 4 nmol/min per ml. Conclusion: The results of this study suggest the utility of lumbar CSF AChE activity as a measure of specific secretory function in enzyme releasing cells of the nervous system.

Expression of the beta‐trace protein in human pachymeninx as revealed by in situ hybridization and immunocytochemistry

In the pachymeninx (dura mater) from human spinal cord and brain, expression of mRNA for the beta-trace protein (beta-trace) was studied by in situ hybridization with digoxigenin-labeled cRNA probes. The localization of the protein was investigated using monoclonal and polyclonal antibodies, respectively. Very strong hybridization signals were observed in the fibroblasts of the pachymeninx. The results obtained by in situ hybridization were essentially confirmed by immunocytochemistry. By immunoblotting of proteins solubilized from the dura mater, strong immunoreactions were found with a polyclonal beta-trace antibody. Furthermore, beta-trace was quantified in human ventricular and lumbar cerebrospinal fluid (CSF) by use of an immunonephelometric assay. The beta-trace concentration in human lumbar CSF was elevated 11-fold as compared with ventricular CSF. Beta-trace determined in lumbar CSF most probably originates from fibroblasts of the pachymeninx. According to the bi- or probably multifunctional features of beta-trace, various sites of mRNA expression and protein synthesis exist in the human central nervous system (CNS). The major ones are the fibroblasts of the pachymeninx, and as previously shown, the epithelial layer of the choroid plexus. J. Neurosci. Res. 57:730–734, 1999. © 1999 Wiley-Liss, Inc.

Expression of the beta-trace protein in human pachymeninx as revealed by in situ hybridization and immunocytochemistry

In the pachymeninx (dura mater) from human spinal cord and brain, expression of mRNA for the beta-trace protein (beta-trace) was studied by in situ hybridization with digoxigenin-labeled cRNA probes. The localization of the protein was investigated using monoclonal and polyclonal antibodies, respectively. Very strong hybridization signals were observed in the fibroblasts of the pachymeninx. The results obtained by in situ hybridization were essentially confirmed by immunocytochemistry. By immunoblotting of proteins solubilized from the dura mater, strong immunoreactions were found with a polyclonal beta-trace antibody. Furthermore, beta-trace was quantified in human ventricular and lumbar cerebrospinal fluid (CSF) by use of an immunonephelometric assay. The beta-trace concentration in human lumbar CSF was elevated 11-fold as compared with ventricular CSF. Beta-trace determined in lumbar CSF most probably originates from fibroblasts of the pachymeninx. According to the bi- or probably multifunctional features of beta-trace, various sites of mRNA expression and protein synthesis exist in the human central nervous system (CNS). The major ones are the fibroblasts of the pachymeninx, and as previously shown, the epithelial layer of the choroid plexus.

N-acetylaspartic acid (NAA) and N-acetylaspartylglutamic acid (NAAG) in human ventricular, subarachnoid, and lumbar cerebrospinal fluid.

N-Acetylaspartic and N-acetylaspartylglutamic acid concentrations in human ventricular, subarachnoid and lumbar cerebrospinal fluid were measured by combined gas chromatography-mass spectrometry using selected ion monitoring with deuterated internal standards. N-Acetylaspartate concentrations were in the range 55, 9, and 1 microM, respectively; N-acetylaspartylglutamate concentrations in the same fluids were in the range 8, 3 and 4 microM, respectively. There did not appear to be any difference in lumbar fluid concentrations of either compound between control subjects, schizophrenic patients, Alzheimer's disease patients and a pooled group of patients with neurological degeneration. Ventricular concentrations of both compounds were greatly increased in deceased patients suggesting that maintenance of their intracellular concentrations is probably energy dependent. The concentrations of these compounds in lumbar cerebrospinal fluid from living, and ventricular cerebrospinal fluid from deceased subjects were weakly correlated with one another. In lumbar fluid neither compound appeared to be correlated with age. Analysis of serially collected lumbar samples from two subjects showed a weak concentration gradient for both compounds. Neither antipsychotic medication nor the acid transport inhibitor probenecid had any effect on lumbar concentrations of either compound. Attempts to use anion exchange high pressure liquid chromatography with UV detection for measurement of the low concentrations of N-acetylaspartate found in cerebrospinal fluid from living subjects were unsuccessful.

Densities of dextrose-free intrathecal local anesthetics, opioids, and combinations measured at 37 degrees C.

Dextrose-free anesthetic medications are commonly used to provide subarachnoid anesthesia and analgesia. Hypobaricity has been proposed as a mechanism to explain postural effects on the extent of sensory block produced by these drugs. Densities of dextrose-free solutions of local anesthetics and opioids, and commonly used anesthetic/opioid mixtures were determined at 37.00 degrees C for comparison with the density of human cerebrospinal fluid (CSF). Measurements accurate to 0.00001 g/mL were performed using a mechanical oscillation resonance frequency density meter. All undiluted solutions tested are hypobaric relative to human lumbar CSF with the exception of lidocaine 1.5% and 2.0% with epinephrine 1:200,000. All mixtures of local anesthetics and opioids tested are hypobaric. We observed good agreement between measured densities and calculated weighted average densities of anesthetic mixtures. While the influence of baricity on the clinical effects of dextrose-free intrathecal anesthetics remains controversial, attempts to attribute postural effects to the baricity of these drugs requires establishment of accurate density values. These density data may facilitate elucidation of the mechanisms underlying the behavior of dextrose-free intrathecal anesthetics.

Contrast Media Leakage to the Cerebrospinal Fluid After Intravenous Injection

We have tested a method to evaluate leakage of i.v. injected contrast media (CM) through the 3 partitions between blood and cerebrospinal fluid (CSF) in controls and in patients with acute cerebrovascular disease (ACBVD) to detect differences between normal brains and brains with ischemic lesions. High-osmolar (HOCM) and low-osmolar (LOCM) CM were used. In 55 patients and in 41 controls who underwent CT after i.v. contrast administration, lumbar CSF was collected 1 hour after injection and the iodine content in the CSF was measured. The concentration of iodine in CSF was very low, between 0.57 and 11.20 ng/l, and no significant difference could be found between patients and controls or between HOCM and LOCM. We conclude that under the conditions mentioned above, iodine detected in the human lumbar CSF does not reflect the true leakage of contrast agent through the blood-brain barrier.

Contrast Media Leakage to the Cerebrospinal Fluid after Intravenous Injection

We have tested a method to evaluate leakage of i.v. injected contrast media (CM) through the 3 partitions between blood and cerebrospinal fluid (CSF) in controls and in patients with acute cerebrovascular disease (ACBVD) to detect differences between normal brains and brains with ischemic lesions. High-osmolar (HOCM) and low-osmolar (LOCM) CM were used. In 55 patients and in 41 controls who underwent CT after i.v. contrast administration, lumbar CSF was collected 1 hour after injection and the iodine content in the CSF was measured. The concentration of iodine in CSF was very low, between 0.57 and 11.20 ng/l, and no significant difference could be found between patients and controls or between HOCM and LOCM. We conclude that under the conditions mentioned above, iodine detected in the human lumbar CSF does not reflect the true leakage of contrast agent through the blood-brain barrier.

Increased angiotensin-converting enzyme activity in cerebrospinal fluid of treated patients with Parkinson's disease

In the human brain angiotensin converting enzyme (ACE, EC 3.4.15.1) exists in two forms of activity [il. ACE is mainly found in a membrane-fund fraction of cortical epithelial cells, catalysing the conversion of inactive an~otensin I to hypertensive angiotensin II [2}. A soluble ACE form is located neuronally within the corpus striatum and may hydrolyse neuropeptides with a messenger function [3]. ACE activity in normal human lumbar cerebrospinal fluid (CSF), may largely reflect ACE activity in non-cortical regions of the brain, for instance the basal ganglia [4]. It is therefore of interest to evaluate the activity of ACE in neurological disease, such as Parkinson’s disease (PD), with a degeneration of the nigrostriatal dopaminergic neurons and consequent striatal dopamine depletion. Previous studies in PD patients have shown either decreased [5] or normal [6] CSF ACE activity. These authors, however, have not studied untreated and treated PD patients separately. Recently, increased CSF ACE activity in schizophrenic patients

Substance P inactivating enzymes in human cerebrospinal fluid

The K m and V max values were determined for enzymes in human lumbar cerebrospinal fluid (CSF) that inactivate synthetic substance P ( SP = RPKPQQFFGLM-NH 2 ) and produce metabolic products. For the human lumbar CSF samples analyzed in this study, K m = 2.24 ± 0.93 mM and V max = 0.113 ± 0.035 nmol/ml/min ( n = 10; mean ± SEM) for the rate of decrease of SP. HPLC analysis of the incubated synthetic peptide fragments demonstrated that the primary enzymatically produced fragment is SP(3–11), with minor amounts in decreasing order of SP(1–4), SP(1–7), and SP(1–9). Electrospray ionization mass spectrometry (ESI-MS) confirmed the appropriate molecular weights for the four peptides, SP(3–11), SP(1–4), SP(1–7), and SP(1–9). These data demonstrate that the primary enzyme in human lumbar CSF that acts on synthetic SP is a post-proline cleaving enzyme (PPCE).

Assay of Preproenkephalin A Processing Enzymes in Human Lumbar Cerebrospinal Fluid

Vmax and Km measurements have been obtained for endogenous peptidases that are important for methionine enkephalin (ME) homeostasis in humans. Those peptidases in human lumbar cerebrospinal fluid (CSF) act upon several synthetic biologically significant peptides that are also contained within the preproenkephalin Ahuman, 1-267 molecule. The amount of endogenous methionine enkephalin-like immunoreactivity (ME-li) in human lumbar CSF is 74.1 ± 5.7 fmol ME-li/ml CSF (n = 56; x ± SE). The kinetic parameters of the various enzymes that inactivate exogenous, synthetic methionine enkephalin (ME, YGGFM) and that also produce ME from two different portions of the preproenkephalin Ahuman, 1-267 precursor molecule were determined. The enzyme that inactivates synthetic ME to FM, and that correlates to the rate of decrease of ME, has a Vmax = 560 ± 43.3 nmol/ml/min and a Km = 4514 ± 373 μM (n = 56; x ± SE). Preproenkephalin Ahuman, 186-193 (PA = YGGFMRGL) was added to CSF samples to characterize those processing and converting enzymes that produce the ME pentapeptide. Vmax, as measured by the rate of the decrease of PA to produce YGGFMR, was 0.192 ± 0.038 nmol/ml/min and a Km of 513 ± 121 μM (n = 10; x ± SE). Similarly, a bovine analog to preproenkephalin Ahuman, 128-140 (PPEhuman, GSEILAKRYGGFM; PPEbovine, 125-137, GGEVLGKRYGGFM) was used to characterize that enzyme system that produces ME from an N-terminally extended ME peptide. That endopeptidase had a Vmax of 0.120 ± 0.048 nmol/ml/min with a Km of 734 ± 296 μM (n = 10). Those endogenous enzymes in human CSF may relate to the proopiomelanocortin convertase enzymes that contain the subtilisin-like catalytic domain.

Characterization of inositol monophosphatase in human cerebrospinal fluid

Inositol monophosphatase (IMPase) has been identified and characterized in human lumbar cerebrospinal fluid (CSF). The CSF enzyme has a K m for inositol 1-phosphate (Ins(1)P; 0.12 mM), a magnesium dependence (optimum concentration 10 mM) and a sensitivity to inhibition by either the bisphosphonate inhibitor 1-(4-hydroxyphenyloxy)ethane-1,1-bisphosphonic acid (L-690,330) or LiCl (IC 50's: 1.3 μM and 1.6 mM, respectively) similar to native human brain and human recombinant enzymes. In CSF, antiserum raised against purified bovine brain IMPase recognised a protein of 30 kDa, identical to that seen in human brain homogenate. It remains to be determined whether CSF IMPase activity may be a useful in vivo marker of CNS phosphatidyl inositol cycle activity in disorders where this signalling pathway may be altered (e.g. manic depression).