Prothrombin in Normal Human Cerebrospinal Fluid Originates from the Blood

In spite of the fact that prothrombin is produced by cells within the central nervous system, its presence in the cerebrospinal fluid (CSF) has not been investigated. We determined the concentration of prothrombin in CSF with reference to the concentration in plasma in paired samples from 18 normal control patients and 4 patients with relapsing-remitting type of multiple sclerosis (MS). The newly developed ELISA was very specific (no cross-reactivity with thrombin) and sensitive (detection limit—0.7 ng/ml) with an imprecision of CV = 8.3% (intraseries) and 7.0% (interassay). The mean prothrombin concentration in normal CSF was 0.55 mg/l (CV ± 33%, range: 0.28–0.93 mg/l), in normal plasma 121.8 mg/l ± 21%, resulting in a mean CSF/plasma concentration quotient (Q_Proth)—4.5 · 10^–3 (CV ± 35%, range: 2.1–8.3 · 10^–3) corresponding to a mean albumin quotient in this group of subjects of Q_Alb = 5.8 · 10^–3. Due to the Q_Proth and the molecular weight of prothrombin (72 kDa)—similar to that of albumin—we conclude that prothrombin in normal human CSF originates predominantly (>95%) from blood. The enzymatic activity in CSF is conserved. Comparable results obtained in MS patients with only few small MRI lesions suggest that local chronic inflammatory disease of the central nervous system does not influence prothrombin concentration in the CSF if the blood-CSF barrier function is normal.     

Establishing the Proteome of Normal Human Cerebrospinal Fluid

Background

Knowledge of the entire protein content, the proteome, of normal human cerebrospinal fluid (CSF) would enable insights into neurologic and psychiatric disorders. Until now technologic hurdles and access to true normal samples hindered attaining this goal.

Methods and Principal Findings

We applied immunoaffinity separation and high sensitivity and resolution liquid chromatography-mass spectrometry to examine CSF from healthy normal individuals. 2630 proteins in CSF from normal subjects were identified, of which 56% were CSF-specific, not found in the much larger set of 3654 proteins we have identified in plasma. We also examined CSF from groups of subjects previously examined by others as surrogates for normals where neurologic symptoms warranted a lumbar puncture but where clinical laboratory were reported as normal. We found statistically significant differences between their CSF proteins and our non-neurological normals. We also examined CSF from 10 volunteer subjects who had lumbar punctures at least 4 weeks apart and found that there was little variability in CSF proteins in an individual as compared to subject to subject.

Conclusions

Our results represent the most comprehensive characterization of true normal CSF to date. This normal CSF proteome establishes a comparative standard and basis for investigations into a variety of diseases with neurological and psychiatric features.     

Quantitative Proteomics and Metabolomics Analysis of Normal Human Cerebrospinal Fluid Samples*

The analysis of cerebrospinal fluid (CSF) is used in biomarker discovery studies for various neurodegenerative central nervous system (CNS) disorders. However, little is known about variation of CSF proteins and metabolites between patients without neurological disorders. A baseline for a large number of CSF compounds appears to be lacking. To analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples of patients undergoing routine, non-neurological surgical procedures, we determined the variation of various proteins and metabolites by multiple analytical platforms. A total of 126 common proteins were assessed for biological variations between individuals by ESI-Orbitrap. A large spread in inter-individual variation was observed (relative standard deviations [RSDs] ranged from 18 to 148%) for proteins with both high abundance and low abundance. Technical variation was between 15 and 30% for all 126 proteins. Metabolomics analysis was performed by means of GC-MS and nuclear magnetic resonance (NMR) imaging and amino acids were specifically analyzed by LC-MS/MS, resulting in the detection of more than 100 metabolites. The variation in the metabolome appears to be much more limited compared with the proteome: the observed RSDs ranged from 12 to 70%. Technical variation was less than 20% for almost all metabolites. Consequently, an understanding of the biological variation of proteins and metabolites in CSF of neurologically normal individuals appears to be essential for reliable interpretation of biomarker discovery studies for CNS disorders because such results may be influenced by natural inter-individual variations. Therefore, proteins and metabolites with high variation between individuals ought to be assessed with caution as candidate biomarkers because at least part of the difference observed between the diseased individuals and the controls will not be caused by the disease, but rather by the natural biological variation between individuals.The analysis of CSF¹ is indispensable in the diagnosis and understanding of various neurodegenerative CNS disorders (1–3). CSF is a fluid that has different functions, such as the protection of the brain from outside forces, transport of biological substances, and excretion of toxic and waste substances. It is in close contact with the extracellular fluid of the brain. Therefore, the composition of CSF can reflect biological processes of the brain (4). By discovering the characterization of the proteome and metabolome of CSF we may gain better insight on the pathogenesis of CNS disorders. This would be significant because, for many of these disorders, the etiology is still unclear.CSF is produced in the ventricles of the brain and in the subarachnoidal spaces. Humans normally produce around 500 mL of CSF each day, and the total volume of CSF at a given time is approximately 150 mL. CSF reflects the composition of blood plasma, although the concentrations of most proteins and metabolites in CSF are lower. However, individual proteins and metabolites can act differently. Active transport from blood and secretion from the brain contribute to the specific composition of CSF. This composition can be disturbed in neurological disorders (5–6). Since CNS-specific proteins and metabolites are typically low in abundance compared with their levels in blood, this change in composition is more likely to be found in CSF because in blood the more abundant plasma proteins can completely mask the signal of the less abundant proteins. Also, if the disease markers do not cross the blood-brain-barrier, then the CSF is the only viable biofluid source. Therefore, CSF might be an excellent source for biomarker discovery for CNS disorders if we follow the hypothesis that neurological diseases induce alterations in CSF protein and metabolite levels.Analysis of metabolites in CSF has been common practice in clinical chemistry for decades to analyze biomarkers for inborn errors of metabolism. The approaches used are either metabolite profiling of CSF using NMR (7), or targeted analysis of one or a few metabolites using specific analytical methods (8). Metabolomics includes the analysis of metabolites in biofluids by NMR or MS-based approaches, i.e. LC-MS or GC-MS. Several metabolite profiling studies were performed on CSF using NMR, some of which were published only recently (9,10). Surprisingly, very few metabolomics studies using MS-based methods have been performed on CSF to date (11,12). One of the reasons is the fact that the human CSF metabolome has not yet been characterized very well. Many CSF metabolites remain unidentified, and for those that have been identified there is not much known about normal concentration ranges. A systematic categorization of the CSF metabolome is necessary and expected to be beneficial for future biomarker discoveries. Recently, Wishart et al. made a good start in exploring the human CSF metabolome with their computer-aided literature survey that resulted in 308 detectable metabolites in human CSF (13).The CSF proteome has been characterized to a much larger extent than the CSF metabolome and is currently the topic of investigations in several research groups worldwide. Recently, studies have been published with numerous identities and quantities of CSF proteins. Pan and co-workers were able to identify 2,594 proteins in well-characterized pooled human CSF samples using strict proteomics criteria with a combination of linear trap quadrupole LTQ-FT (Thermo Fisher Scientific, Bremen, Germany) and MALDI TOF/TOF equipment (14). They were also able to quantify several proteins using a targeted LC MALDI TOF/TOF approach (15). Hu et al. have studied the intra- and inter-individual variation in human CSF and found large variations in protein concentrations in six patients by means of two dimensional–gel electrophoresis (16), focusing mainly on the variations within individuals at two different time-points. Although only a limited number of proteins was analyzed, the variation between the time-points was profound, exceeding 200% for seven proteins.Unique CSF biomarkers may contribute to a deeper understanding of the mechanisms of CNS disorders. However, for this assumption to come true, there are still challenges ahead. Although CSF is not as complex as blood (almost missing the cellular part and the clotting system present in blood), it is expected to consist of thousands of organic- and non-organic salts, sugars, lipids, and proteins. A large part of the CSF consists of a few highly abundant metabolites and proteins, which hamper, if no precautions are undertaken, the identification and quantification of metabolites and proteins that occur in lower amounts. The analysis of the CSF metabolome is complicated because of the diverse chemical nature of metabolites and the lower concentration of metabolites compared with blood. Analytical method development is still required because it is not possible to identify the entire range of CSF metabolites with one single analytical method. Although in proteome research efforts have been made to quantify proteins, metabolomics studies up to now either do not provide quantitative information or they only give information for the most abundant metabolites.Another challenge is the sample amount obtained by lumbar puncture to collect CSF. Lumbar puncture is an invasive method that is not performed as frequently as blood sampling. However, often after the analysis of various clinical parameters, only a limited amount of CSF sample is available for biomarker discovery. Metabolomics studies are hampered by limited CSF sample amount. Therefore, analytical methods are required that are suitable to handle relatively small sample volumes.The main objectives of this study were (1) to analyze the variation in CSF protein and metabolite abundances in a number of well-defined individual samples by multiple analytical platforms; and (2) to integrate metabolomics and proteomics to present biological variations in metabolite and protein abundances and compare these with technical variations with the currently used analytical methods. The results will facilitate and increase the application of CSF for future biomarker discovery studies in the field of neurodegenerative diseases and neuro-oncology.     

Is Pentane a Normal Constituent of Human Breath?

Breath analysis is a non-invasive method for investigation of the volatile compounds produced by humans. Pentane has often been taken as an indicator of lipid peroxidation. Our purpose in this study was to determine its normal concentration in the breath of healthy humans. Using a specific and sensitive gas chromatography-mass spectrometry technique pentane concentrations in breath were lower than 10 pmoles/1. The high levels of pentane found by some authors in healthy humans were probably due to the coelution of pentane with isoprene, a volatile hydrocarbon present in human breath.     

2-Pyrrolidinone in Human Cerebrospinal Fluid: A Major Constituent of Total γ-Aminobutyric Acid

2-Pyrrolidinone, the lactam of gamma-aminobutyric acid (GABA), is identified as the major constituent of total GABA in human CSF. Structural elucidation was done by mass spectrometry. In lumbar CSF of four patients, 2-pyrrolidinone represented about 54% of GABA found after acid hydrolysis, thus accounting for essentially all of the hitherto unknown GABA fraction in CSF.     

3-Methyluridine in Normal Human Urine

Abstract

3-Methyluridine has been identified in normal human urine using gas chromatography mass spectrometry, confirming an earlier report of the presence of this modified nucleoside as a natural component of human biological fluids. Variable recoveries and the lack of quantitative data concerning normal or disease state levels of this compound make selection of 3-methyluridine a poor choice as an internal standard for the quantitation of other nucleosides in biological matrices.     

Anticholinesterase Action of a Bromine Compound Isolated from Human Cerebrospinal Fluid

Abstract: L-l-Methylheptyl-γ-bromoacetoacetate was found to be a competitive inhibitor of the acetylcholines-terases (electric eel, K_i= 17.2 μM; rat brain, K_i= 32.6 μM) and of butyrylcholinesterase (horse serum, K_i= 1.2 μM). The L-isomer was a more effective inhibitor than the D-isomer. The bromine atom at the γ-position of the acidic moiety, the specific length of the carbon chain constituting the secondary alcohol moiety, and the presence of the ketone radical at the acidic moiety of the ester were necessary for the anticholinesterase action. 1-Methyl-heptyl-γ-bromoacetoacetate formed a complex with acetylcholinesterase or butyrylcholinesterase without hydrolysis of its own molecule.     

Characterization of the Serpin, α1-Antichymotrypsin, in Normal Human Cerebrospinal Fluid

Cerebrospinal fluid (CSF) from 20 male patients with nonneurologic disease (age 64.5 +/- 2.8 SEM) was analyzed for the presence of the serpin alpha 1-antichymotrypsin (alpha 1-ACT). A chymotrypsin-specific chromogenic substrate (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) was used to examine the CSF samples. All CSF samples showed inhibitory activity ranging from 45 to 80% inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the samples revealed the presence of a 68-kDa protein migrating identical to authentic human plasma alpha 1-ACT. Complex formation was performed with iodinated bovine chymotrypsin for several representative CSF samples having the highest chymotrypsin inhibitory activity. Comparison was made with complex formation performed with commercially available authentic human plasma alpha 1-ACT. These studies showed the formation of complexes at 37 degrees C, regardless of whether the sample was subsequently boiled or not. In the case of CSF, two complex bands, mass smaller than with plasma alpha 1-ACT, were formed at the lower temperature whereas a single higher Mr band was formed when the samples were boiled. To determine whether cleavage of the serpin occurred, these studies were repeated using human neutrophil cathepsin G as target protease. A complex of approximately 90 kDa was formed with human alpha 1-ACT under these same conditions. alpha 1-ACT has been detected in senile amyloid plaques in brains of Alzheimer's disease patients, the only plasma serine protease inhibitor localized to these structures. Another serpin, protease nexin I, is also found in these plaques, but this inhibitor does not circulate in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Free and Conjugated Amines in Human Lumbar Cerebrospinal Fluid

Significant amounts of acid-hydrolyzable conjugates of 3,4-dihydroxyphenylethylamine, norepinephrine, and 5-hydroxytryptamine were detected in lumbar CSF from 22 awake unpremedicated healthy individuals. In the CSF samples, the amounts of conjugated amines almost always exceeded the amounts of free amines, but were less than the amounts of the acid metabolites 3,4-dihydroxyphenylacetic acid, homovanillic acid, and 5-hydroxyindoleacetic acid.     

Gelsolin in Cerebrospinal Fluid as a Potential Biomarker of Epilepsy

Gelsolin is an actin regulatory protein that generally distributed in a wide variety of body tissues, especially the brain tissues and cerebrospinal fluid. In this study we found that lumbar CSF-gelsolin concentrations markedly decreased in epileptic patients by enzyme linked immunosorbent assay. In order to help judge the result, we determined gelsolin expression in temporal lobe tissues of patients with temporal lobe epilepsy using double-label immunofluorescence to location and using western blot to quantitation. Then we observed that gelsolin was co-expressed with microtubule-associated protein-2 in axons and cytoplasms of neurons and gelsolin protein level was also down-regulated in temporal lobe tissues of epileptic patients. Our findings suggested that CSF-gelsolin level might reflect the alteration of gelsolin in brain tissue of epileptic patients and CSF-gelsolin seems to be a potential biomarker for epilepsy.     

Immunocytochemical Detection of Anti-Hippocampal Antibodies in Alzheimer's Disease and Normal Cerebrospinal Fluid

Immunocytochemical staining was performed to investigate the presence of anti-hippocampal antibodies in cerebrospinal fluid (CSF) from patients with probable Alzheimer's disease (AD) (n = 19), aged normal controls (n = 9), and young normal controls (n = 10). Marked staining of neurons in the granule cell layer of the dentate gyrus and in pyramidal neurons in CA1-3 of the rat hippocampus was observed in 5 AD CSF samples (26%), 1 aged control sample (11%), and 1 young control sample (10%). These differences were not statistically significant. One of the immunoreactive AD CSF specimens also contained high concentrations of C5b-9, the membrane attack complex. The infrequent occurrence of anti-hippocampal antibodies in AD CSF, and the detection of similar immunoreactivity in control CSF specimens, suggest that these antibodies are unlikely to play a role in the neurodegenerative process in most individuals with AD. However, elevated C5b-9 concentration in an AD CSF specimen with marked immunoreactivity to hippocampal neurons suggests the possibility that anti-neuronal antibodies may contribute to complement activation in some AD patients.     

Quantification of Free Fatty Acids in Human Cerebrospinal Fluid

Free fatty acids (FFA) in cerebrospinal fluid (CSF) are well-recognized markers of brain damage in animal studies. Information is limited regarding human CSF in both normal and pathological conditions. Samples of CSF from 73 patients, who had undergone lumbar puncture for medically indicated reasons, came from a core laboratory upon completion of ordered tests. Using high performance liquid chromatography, mean FFA concentrations (g/L ± SEM) were: arachidonic 26.14 ± 3.44; docosahexaenoic 60.74 ± 5.70; linoleic 105.07 ± 10.98; myristic 160.38 ± 16.17; oleic 127.91 ± 10.13; and palmitic 638.34 ± 37.27. No differences in FFA concentrations were seen with gender, race, age, and/or indication for lumbar puncture. This is the first study to document normal human CSF FFA concentrations in a large series. Further characterization of FFA in pathological conditions may provide markers for evaluating clinical treatments and assisting in prognostication of neurological disease.     

Fluorometrical Analysis of Guanidino Compounds in Human Cerebrospinal Fluid

Abstract: Guanidino compounds in CSF of 57 human subjects were determined fluorometrically after reaction with phenanthrenequinone in alkali solution, using HPLC. Creatinine (65.2 ± 13.4 nmol/ml), arginine (24.7 ± 6.4 nmol/ml), and homoarginine (0.7 ± 0.3 nmol/ml) were found in all subjects. Trace amounts of guanidinosuccinic acid and guanidinoacetic acid were detected in some of the subjects. Brain guanidino compounds, taurocyamine, N -acetylarginine, and methylguanidine were not detected in CSF.     

High-Resolution Proton Magnetic Resonance Analysis of Human Cerebrospinal Fluid

High-resolution proton magnetic resonance spectroscopy was used to analyze human cerebrospinal fluid obtained from patients with several neurological problems. The major metabolites measured included glucose, lactate, glutamine, citrate, inositol, acetate, creatine, creatinine, beta-hydroxybutyrate, alanine, and pyruvate. A drug vehicle, propylene glycol, was also measured. Alterations in the cerebrospinal fluid of these metabolites provided information concerning metabolism of the brain. Magnetic resonance spectroscopy offered a simple and rapid means of assessing these and other exogenous and endogenous compounds in diseases affecting the nervous system.     

Increased Interleukin-17 in Peripheral Blood and Cerebrospinal Fluid of Neurosyphilis Patients

BackgroundTreponema pallidum infection evokes vigorous immune responses, resulting in tissue damage. Several studies have demonstrated that IL-17 may be involved in the pathogenesis of syphilis. However, the role of Th17 response in neurosyphilis remains unclear.ConclusionsThese findings indicate that Th17 response may be involved in central nervous system damage and associated with clinical symptoms in neurosyphilis patients. Th17/IL-17 may be used as an alternative surrogate marker for assessing the efficacy of clinical treatment of neurosyphilis patients.     

Gas Chromatographic-Mass Spectrometric Determination of myo-Inositol in Human Cerebrospinal Fluid

The concentration of free myo-inositol in CSF was determined with a gas chromatographic-mass spectrometric method using deuterated myo-inositol as an internal standard after conversion to the hexa-O-acetyl derivative with acetic anhydride and pyridine. Twenty microliters of CSF is sufficient for the analysis which has a coefficient of variation of 9%. Identical analytical results were obtained on two different mass numbers. Schizophrenic patients were compared with healthy control persons. In addition, patients with rheumatoid arthritis or with neurological illnesses were studied. No consistent differences related to the illness could be found. The mean concentration of myo-inositol was about 25 micrograms/ml. Treatment of schizophrenic patients with chlorpromazine or sulpiride had no significant effect on the concentration of myo-inositol in CSF.     

Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in Human Plasma and Cerebrospinal Fluid

The measurement of cholinesterase activities in either plasma or cerebrospinal fluid (CSF) may ultimately prove to be relevant in the diagnosis of neurological and neuropsychiatric disorders. However, studies to date have examined only total enzyme activities. Therefore in the present study we have examined the distribution of the individual molecular forms of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in plasma and CSF using sucrose density gradient centrifugation. Although the total activities of AChE were of the same order of magnitude in plasma and CSF, there was a considerable difference (120-500-fold) between total BChE activity in the CSF and the BChE-rich plasma. The analysis of the individual molecular forms revealed that the predominant molecular species of AChE and BChE in the CSF--both lumbar and ventricular--was the G4 form. The G4 form also constituted the majority of the plasma BChE activity and, on average, over half (56%) of the plasma AChE activity. The significance of the AChE and BChE molecular form compositions of both plasma and CSF and their possible relationship to pathological states are discussed.     

Studies of Specificity and Inhibition of Human Cerebrospinal Fluid Dynorphin Converting Enzyme

Abstract

Dynorphin-converting activity was recently discovered in human cerebrospinal fluid.¹ This enzyme (hCSF-DCE) cleaves dynorphin A, dynorphin B and alpha-neoendorphin to release Leu-enkephalin-Arg⁶. To characterize the enzyme further we used several protease inhibitors, including N-peptidyl-O-acyl hydroxylamines which are known to act as potent irreversible inhibitors of serine and cysteine proteinases.^2-4

No irreversible inactivation occurred but strong, reversible effects on the dynorphin-converting activity by some of the inhibitors tested could be observed. Although, hCSF-DCE binds its substrates (dynorphin A and B) in the μM-mM concentration range, it exhibits high specificity in recognizing and cleaving the linkage between the two basic amino acids in the substrate sequence.     

Cerebrospinal Fluid Biomarker Candidates Associated with Human WNV Neuroinvasive Disease

During the last decade, the epidemiology of WNV in humans has changed in the southern regions of Europe, with high incidence of West Nile fever (WNF) cases, but also of West Nile neuroinvasive disease (WNND). The lack of human vaccine or specific treatment against WNV infection imparts a pressing need to characterize indicators associated with neurological involvement. By its intimacy with central nervous system (CNS) structures, modifications in the cerebrospinal fluid (CSF) composition could accurately reflect CNS pathological process. Until now, few studies investigated the association between imbalance of CSF elements and severity of WNV infection. The aim of the present study was to apply the iTRAQ technology in order to identify the CSF proteins whose abundances are modified in patients with WNND. Forty-seven proteins were found modified in the CSF of WNND patients as compared to control groups, and most of them are reported for the first time in the context of WNND. On the basis of their known biological functions, several of these proteins were associated with inflammatory response. Among them, Defensin-1 alpha (DEFA1), a protein reported with anti-viral effects, presented the highest increasing fold-change (FC>12). The augmentation of DEFA1 abundance in patients with WNND was confirmed at the CSF, but also in serum, compared to the control individual groups. Furthermore, the DEFA1 serum level was significantly elevated in WNND patients compared to subjects diagnosed for WNF. The present study provided the first insight into the potential CSF biomarkers associated with WNV neuroinvasion. Further investigation in larger cohorts with kinetic sampling could determine the usefulness of measuring DEFA1 as diagnostic or prognostic biomarker of detrimental WNND evolution.