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Separation of human cerebrospinal fluid proteins by capillary isoelectric focusing in the absence of denaturing agents.

Conditions of capillary isoelectric focusing (CIEF) to separate human cerebrospinal fluid (CSF) proteins were examined referring to those which we have established for the separation of human plasma/serum proteins. Since the average protein concentration in CSF is about 1/200 of plasma and the salt concentration is at almost the same level as plasma, desalting of CSF samples with minimum dilution was a prerequisite for CIEF analysis of CSF proteins. We constructed an apparatus to dialyze CSF at the level of 20-30 microL, this volume being sufficient for 3-4 repeated analyses of the CSF sample. To trace the process of dialysis, a simple device to measure the conductivity of the dialyzate was also constructed. Most of the CIEF conditions for plasma protein analysis could be applied for CSF protein analysis. However, the addition of N,N,N',N'-tetramethylethylenediamine (TEMED) at a suitable concentration was necessary to improve the resolution of basic proteins (IgG region), since some CSF patterns showed peaks of basic proteins which are not obvious in the serum of the same patient. About 70 peaks and shoulders of CSF proteins could be detected by the established CIEF technique. The results of CIEF analysis of CSF samples suggested that the technique will be useful as a survey method to detect specific proteins in CSF, which might relate to disorders in the central nervous system.

Blood Proteins↗

Cerebrin-50, a human cerebrospinal fluid protein whose mRNA is present in multiple tissues but predominantly expressed in the lymphoblastoid cells and the brain.

A full-length cDNA of 2295 bp coding for a human cerebrospinal fluid protein, designated cerebrin-50, has been isolated from a human brain cDNA expression library. Nucleotide sequence analysis of this cDNA revealed that it codes for a 435 amino acid polypeptide which starts with an ATG initiation codon from the 5'-end and ends with a TGA termination codon with a calculated molecular weight of 51,484 daltons. A 10-amino acid peptide of NH2-SGDLETRYWG based on the deduced amino acid sequence of the cerebrin-50 cDNA was synthesized, conjugated to bovine serum albumin and was used to raise a monospecific polyclonal antibody in a rabbit. Immunoblot analysis using this antibody indicated the presence of a 52 kD protein in the human cerebrospinal fluid, brain cytosol, T lymphoblastoid cell-conditioned medium, and human serum; it is also noted that the concentration of this 52 kD protein is several order of magnitude higher in the cerebrospinal fluid than in other biological fluids. The presence of its mRNA in the brain, T lymphoblastoid cells, spleen, liver, and testis was confirmed by sequential use of reverse-transcription and polymerase chain reaction (RT-PCR). Preliminary analysis by quantitative RT-PCR has noted that the expression of the cerebrin-50 mRNA is higher in the lymphoblastoid cells and brain than the spleen, liver, and testis.

Amino Acid Sequence↗

Electrophoretic pattern of cerebrospinal fluid proteins in non-neoplastic infantile hydrocephalus.

Lumbar cerebrospinal fluid (CSF) from 28 non-neoplastic hydrocephalic children was studied for total protein and electrophoretic protein patterns. These were classified as normal, degenerative, transudative and gamma-globulinic according to Laterre. We found higher total CSF protein mean values than in normal cases with the same age and abnormal electrophoretic patterns in 72% of the cases, of which degenerative was the most common (54%). Gamma-globulinic and transudative patterns were found in 11% and 7% of the cases, respectively. Several factors which may explain the increase in the total CSF protein in infantile hydrocephalus are described: low age, ventricular block in the noncommunicating hydrocephalus, and probable passage of tissue proteins to the CSF from the damaged brain. The predominance of degenerative patterns may be explained by the enrichment of the CSF in tissue proteins resulting from the white matter damage provoked by the abnormal conditions of production, flow and absorption of the CSF in hydrocephalus. Ventricular CSF was studied in four cases, and the results obtained are in agreement with the above-mentioned findings.

Albumins↗

Cerebrospinal fluid protein concentration in pediatric patients: defining clinically relevant reference values.

OBJECTIVES: To define clinically relevant reference ("normal") values for cerebrospinal fluid (CSF) protein concentrations in pediatric patients who were evaluated for meningitis by traditional criteria and by enterovirus-polymerase chain reaction (EV-PCR). DESIGN AND PATIENTS: A cohort of 906 consecutive pediatric patients to receive CSF analysis at St Louis Children's Hospital, St Louis, Mo, from June 1, 1998, to December 31,1998, was studied for clinical and laboratory data. Age-dependent CSF protein concentrations were then derived from a reference group of 225 patients in whom meningitis and other neurologic diseases were excluded by traditional clinical or laboratory criteria (excluding EV-PCR). Available CSF samples from 132 patients of the reference group were subsequently tested for EV-PCR. RESULTS: In the reference group, the CSF protein concentration was highest and most variable in neonates, with a maximum of approximately 1.0 g/L. Cerebrospinal fluid protein concentration decreased rapidly to a nadir by 6 months and remained low throughout childhood, rarely exceeding 0.3 g/L and, finally, increasing in adolescence toward adult values. Enterovirus- polymerase chain reaction was positive in CSF of 11% of the reference group, with EV-PCR-positive patients having significantly higher CSF protein concentrations than EV-PCR-negative patients aged between 4 months and 14 years. CONCLUSIONS: Reference values for CSF protein exhibit a characteristic age dependence in pediatric patients. Continued standard use of adult reference values in the pediatric population is inappropriate. The unexpected finding of a positive EV-PCR in patients not diagnosed with meningitis by traditional criteria further emphasizes the importance of selecting the most clinically relevant reference group for age and other variables when defining normal laboratory values. Arch Pediatr Adolesc Med. 2000;154:827-831

Adolescent↗

Cerebrospinal fluid protein fractions in health and disease.

Samples of lumbar cerebrospinal fluid from 20 healthy subjects and 20 patients with a variety of neurological disorders have been subjected to cellulose acetate electrophoresis and quantitative analyses carried out on the seven separated protein fractions. Normal percentage and absolute concentration limits for each fraction have been determined and compared with previous reports. It is concluded that absolute values are more valuable than percentage values in the discrimination of disease from health, but that the mechanisms governing protein abnormalities are difficult to ascertain from spinal fluid electrophoresis alone.

Cerebrospinal Fluid Proteins↗

[Cerebrospinal fluid protein tau levels in the differential diagnosis of Alzheimer's disease].

Tau protein concentration in cerebrospinal fluid was determined in 55 patients with Alzheimer's disease (AD), 18 patients with vascular dementia (VD), 19 patients with dementia caused by other disorders and 14 patients with major depression. Significantly (p < 0.05) elevated protein tau concentrations were found in AD patients (564.5 +/- 275.5 pg/ml) compared to all other patient groups (VD: 406.5 +/- 263.9 pg/ml; other dementia: 275.0 +/- 135.4 pg/ml; depression: 212.9 +/- 115.6 pg/ml). However, tau levels in AD patients covered a broad range (163.2 pg/ml-1200 pg/ml). AD patients with tau levels below the 25%-percentile of the distribution (among them a high percentage of patients with presenile onset) showed tau levels similar to those of the patients with late life depression. No significant correlations between tau levels and clinical variables such as severity of dementia, age, age of onset, duration of illness, and cerebral changes as assessed by volumetric magnetic resonance imaging could be demonstrated. Similarly, we could not find an influence of either APO-E genotype or psychotropic medication on the tau levels in AD patients. In accordance with other studies our results confirm elevated tau levels in AD compared to elderly not demented control subjects. Comparing groups, this finding applies as well with respect to VD and other dementing disorders. However, elevated tau levels cannot be detected in a subgroup of AD patients. This finding needs to be further investigated in future studies.

Aged↗

[Cerebrospinal fluid proteins: I. Comparative study of concentration methods].

Four protein concentration methods, applicable to cerebrospinal fluid, were compared in order to choose a procedure that not causing protein denaturation, is accurate, precise and easy to perform in routine laboratories. The studied methods were: dialysis against arabic-gum solution, centrifuge-ultrafiltration, absorption of low molecular weight substances by dry polyacrylamide gel and pressure ultrafiltration. Blood serum was diluted, then concentrated by these procedures and analyzed by cellulose acetate electrophoresis; the undiluted serum served as control. It was observed that two methods: pressure ultrafiltration and absorption of low molecular weight substances by polyacrylamide hydrogel, are not suitable for concentrating diluted fluids because they frequently cause protein denaturation. It was concluded that dialysis against arabic-gum is the most suitable, of the methods studied, for application in routine laboratories taking in account that this procedure is precise, relatively accurate, simple and of low cost.

Absorption↗

[Diagnostic significance of the cerebrospinal fluid protein spectrum in bacterial and viral meningitis in children].

Protein spectrum of the cerebrospinal fluid (CSF) was studied by polyacrylamide gel disk electrophoresis in 119 children with bacterial and 17 with viral meningitis of different origin over the course of disease. Liquor proteinograms of patients were compared with those of 37 children without meningitis. Protein spectra of the CSF were shifted in the patients. These shifts can serve as differential diagnostic markers: types 1-2 and 2-2 haptoglobins and beta-lipoprotein appears in the blood during the acute period of disease. More pronounced and stubborn changes in the CSF protein spectrum in pneumococcal and hemophilic meningitis correlated with a more severe clinical course and formation of residual neurological consequences. Progress of dysproteinemic shifts in the protein spectrum of the CSF was characterized by a many-fold increase in the concentrations of high-molecular-weight proteins and decreased relative content of liquor-specific fractions, indicating sharp impairment of the blood-brain barrier permeability and formation of brain edema. Disk electrophoresis method is characterized by high resolution, informative value, requires low amounts of CSF for analysis, and is recommended as an accessory diagnostic test for infectious hospitals.

Cerebrospinal Fluid Proteins↗

Cerebrospinal fluid proteins as indicators of nerve root compression in patients with sciatica caused by disc herniation.

Patients with sciatica caused by lumbar disc herniation were studied to identify biochemical changes in the cerebrospinal fluid related to myelographic findings and clinical observations. One hundred forty-three patients were evaluated by myelography with regard to involvement of the dural sac and the nerve root. A medial group (20 patients) with evidence of dural sac impingement was compared to a lateral group (63 patients) and an extreme lateral group (9 patients) whose condition primarily affected the nerve root. The remaining 51 patients comprised a mixed group with involvement of both the dural sac and the nerve root. The mean cerebrospinal fluid/serum albumin ratio, cerebrospinal fluid/serum immunoglobulin G ratio, and cerebrospinal fluid total proteins showed a significantly increasing trend from the medial through the lateral to the extreme lateral groups. Patients with lateral lumbar disc herniations more often showed neurologic deficits. These results indicate that the elevated cerebrospinal fluid total protein found in the patients with sciatica is due to leaking of plasma proteins primarily from the nerve root into the cerebrospinal fluid. The cerebrospinal fluid proteins may be used as diagnostic parameters of nerve root compression, especially when surgery is a consideration or in patients in whom sciatica is unlikely.

Adolescent↗

[The pattern of cerebrospinal fluid proteins in infants and children. Determination of normal values (author's transl)].

Cerebrospinal fluid (CSF) protein values were measured in 652 children between the ages of 1 day and 17 years, allowing the authors to define the dynamics of the blood-brain barrier under normal conditions and during inflammation of the nervous system. The ratio of CSF albumin/serum albumin (whose upper limit was 0.65 in the study) was the best sign of alteration of blood-brain barrier permeability. The CSF IgG level, whose upper limit was 0.85 (for serum IgG between 10 and 14 g/l) is the most useful criterion for detecting an intra-thecal synthesis of IgG. Six patterns of CSF proteins are defined on the basis of immunochemical and electrophoretic studies. The ratio of CSF albumin/serum albumin and the CSF IgG level must be compared to the electrophoretic pattern of CSF proteins in order to better characterize one aspect of the blood-brain barrier under normal and pathologic conditions of the central nervous system.

Adolescent↗

[Spinal epidural abscess with persistent increase in cerebrospinal fluid protein: a case study].

A 67-year-old man under hemodialysis treatment developed neck stiffness, fever and conscious disturbances. The patient was infected with Methicilin-resistant Staphylococcus aureus (MRSA) sepsis caused by an infection on a dialysis shunt. On admission, he was diagnosed with bacterial meningoencephalitis and underwent a series of antibiotic chemotherapies. The treatment brought cell count in the cerebrospinal fluid to a subnormal level but his clinical status did not improve. The patient continued to have high level of cerebrospinal fluid protein (898 mg/dl). Cervical MRI demonstrated two abscesses deep in the neck as well as in the epidural region of the cervical spinal cord, from C2 to C5 vertebral levels. Based on these findings, spinal epidural abscess (SEA) was diagnosed. Intensive antibiotic chemotherapy especially targeted for MRSA could eradicate abscesses and improve clinical status. However, persistent high protein level in the cerebrospinal fluid could suggest SEA.

Aged↗

[Determination of cerebrospinal fluid proteins using the Cobas Fara centrifugal analyser. Adaptation of the turbidimetric technic to benzethonium chloride].

The authors have adapted a turbidimetric assay on centrifugal Cobas Fara analyser for cerebrospinal fluid proteins determinations. The later method is proposed by SFBC Protein Analysis Committee using benzethonium chloride as monoreagent. This adaptation is more sensitive than manual method. The linearity range is greater (0.08-1.85 g/l instead of 0.2-1.2 g/l). Sample assay and time of analysis were reduced by 90 p. cent.

Benzethonium↗

Cerebrospinal fluid protein patterns in neurodegenerative disease revealed by liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry.

This study demonstrates the power of a novel proteomic approach developed for the detection and identification of biological markers in body fluids. The goal was to observe alterations in the protein patterns of cerebrospinal fluid (CSF) related to amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder with unknown etiology. In the experiments, tryptic digests of CSF from patients and healthy controls were analyzed by on-line capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry. (FT-ICR MS) Typically, around 4000 peptides were detected in one such experiment, and a pattern recognition program was constructed for the data analysis to distinguish mass chromatograms from patients and controls. This strategy was evaluated comparing the peptide patterns of CSF spiked in vitro with a biomarker, with control CSF. The patterns were clearly separated and the tryptic peptides of the biomarker were successfully selected as characteristic peaks. Hence, the method was applied to compare mass chromatograms of CSF from 12 ALS-patients and 10 matched healthy controls. While no biomarker alone could be identified from the characteristic peaks, we were able to assign 4 out of 5 unknown samples correctly (i.e., 80% correctly diagnosed, 20% false-negative), and it would be 100% if we reject a possible outlier believed to be caused by an occlusion in the spinal CSF compartment. These findings are very promising, although the clinical relevance is not fully established due to the low number of unknown samples analyzed. In addition to the diagnostic potential, these results may be important steps towards understanding the neurodegenerative process.

Adult↗

Effect of decreased cerebrospinal fluid proteins on the spread of local anaesthetic drugs in pregnancy.

Cerebrospinal fluid (CSF) and plasma protein concentrations were determined in 60 ASA-I female patients, 30 non-pregnant women, who were to undergo lower abdominal or lower limb surgery (group I, controls) and 30 pregnant women at term, who were posted for lower segment caesarean section (group II). All patients received spinal analgesia. Time of onset of analgesia and level of analgesia achieved were compared in two groups. A significant fall (16.6%) was noted in the plasma proteins in pregnant (6.10 +/- 0.6 g/dl) women as compared to non-pregnant patients (7.30 +/- 0.44 g/dl; P less than 0.01). CSF proteins also showed a significant fall (43.2%) in pregnant (25.80 +/- 5.52 mg/dl), as compared to non-pregnant women (45.43 +/- 7.66 mg/dl; P less than 0.001). Dose of local anaesthetic drug required was significantly less (44%) in pregnant (3.21 +/- 0.29 mg/segment) as compared to non-pregnant women (5.73 +/- 0.74 mg/segment; P less than 0.01). Time of onset of block was significantly less in pregnant than in non-pregnant patients after the injection of drug (2.86 +/- 0.42 sec and 3.41 +/- 0.43 sec respectively; P less than 0.01). No correlation was found between plasma proteins and CSF proteins. CSF protein concentration also did not correlate with dose of local anaesthetic drug, or with time of onset of block. It is suggested that fall in CSF protein concentration may be another contributory factor in the reduced dose requirement of local anaesthetic drug for subarachnoid block during pregnancy.

Adult↗