Cerebrospinal Fluid Assays Research Articles

Establishment of beta-hydroxy fatty acids as chemical marker molecules for bacterial endotoxin by gas chromatography-mass spectrometry.

Selected ion-monitoring gas chromatography-mass spectrometry was used for detection of beta-hydroxy fatty acids as an independent assay for the presence or absence of endotoxin in materials claimed to induce nonspecific activation of Limulus amoebocyte lysate. To this end, suspensions of gram-negative and -positive bacteria, one fungal species, cerebrospinal fluid, and hollow-fiber hemodialyzer rinses were assayed for endotoxin by gas chromatography-mass spectrometry and the Limulus amoebocyte lysate assay. Good qualitative agreement was shown for both methods when suspensions of test organisms were assayed. Two false-negative results were obtained by gas chromatography-mass spectrometry assays of cerebrospinal fluid and were shown to be a result of insufficient endotoxin in the cerebrospinal fluid specimens for detection by gas chromatography-mass spectrometry. Hemodialyzer rinses were Limulus assay positive; however, no beta-hydroxy fatty acids were detected by gas chromatography-mass spectrometry. These data were compared with data obtained from USP rabbit pyrogen tests of the rinse materials (nonpyrogenic) and chemical characterization of the Limulus assay-reactive rinses, which showed the rinses to be cellulosic in nature. It is suggested that beta-hydroxy fatty acids, as assayed by selected ion-monitoring gas chromatography-mass spectrometry, be used as chemical marker molecules for the presence or absence of endotoxin in materials reported to cause nonspecific activation of Limulus amoebocyte lysate.

Immunoenzymatic test (ELISA) in the diagnosis of neurocysticercosis: study of various antigenic extracts in the detection of IgG antibodies in serum and cerebrospinal fluid samples

Five different antigens from Cysticercus cellulosae, a vesicular fluid, a saline and an alkaline total extracts, an escolex and membrane, were studied in the ELISA immunoenzymatic assay to demonstrate IgG antibodies in cerebrospinal fluid (CSF) and serum samples. For the 5 antigens the 20 micrograms/ml concentration was selected for polyvinyl plates sensitization for CSF and serum assays. The IgG fraction of a sheep anti-human IgG antiserum was labeled with horseradish peroxidase and revealed with hydrogen peroxide-5-aminosalicilic acid. For positive results 10 ELISA Units for sera and 5 EU for CSF were taken. A total of 182 serum samples and 115 CSF samples were tested. The ELISA sensitivity for sera were 88.6% for vesicular fluid, escolex and membrane; 85.7% for saline extract and 62.8% for alkaline extract. The ELISA sensitivity for CSF was 100% for vesicular fluid and saline total extract, 91.7% for membrane, 89.6% for excolex and 77.1% for alkaline. The ELISA specificity for sera was 100% to the 5 antigens studied; to the CSF was 100% for the alkaline and escolex and 98.5% for the other antigens. An ELISA geometric mean titer of sera and CSF was respectively the 121.1 and 27.3 for vesicular fluid, 111.6 and 31.1 for saline total extract, 30.3 and 5.3 for alkaline total extract, 63.2 and 14.8 for escolex and 69.1 and 18.8 for membrane antigen. ELISA was then compared to immunofluorescence, hemagglutination and complement fixation tests in CSF and sera. ELISA immunoenzymatic assay with saline total extract is recommended for the easy preparation and for the high quantity of antigens obtained, for the high sensitivity and great specificity for sera and CSF; we suggest that this test may be used as a substitute for immunofluorescence, hemagglutination and complement fixation in sera or CSF for the diagnosis of neurocysticercosis.

Increased levels of somatomedins in human lumbar and central cerebrospinal fluid after subarachnoid haemorrhage

The somatomedins, multitargit growth-promoting peptide hormones, were measured with radio receptor assay in cerebrospinal fluid (CSF) after subarachnoid haemorrhage (SAH) in 21 patients and after head injury in 2 patients. In the first group of 10 patients, lumbar (n = 8) or central (n = 2) CSF was collected on days three, six and nine after SAH. 6 of the 8 patients with SAH showed an increase in somatomedin concentrations ranging between 0.52-1.26 U/ml while 2 patients fell within the normal range between 0.19-0.48 U/ml. In the 2 patients with head injury, the somatomedin concentrations were scarcely detectable. In the second group of 13 patients with SAH, CSF was collected preoperatively during surgical clipping of an aneurysm. These patients fell into two groups: 6 patients who had CSF somatomedin levels within the normal range and 7 patients with pathologically increased somatomedin concentrations ranging between 0.38-1.26 U/ml. Neither the neurological condition nor the cerebral vascular diameter correlated with the somatomedin concentrations. It is suggested that the increased somatomedin levels in CSF after SAH could be a compensatory response in order to stimulate cerebral anabolism after injury.

Diagnosis of spirochetal meningitis by enzyme-linked immunosorbent assay and indirect immunofluorescence assay in serum and cerebrospinal fluid.

The antibody response against a spirochetal strain isolated from Swedish Ixodes ricinus ticks was determined by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay of cerebrospinal fluid (CSF) and serum specimens from 45 patients with chronic meningitis. Samples of CSF, serum, or both from patients with various infections of the central nervous system, multiple sclerosis, syphilis, or infectious mononucleosis and from healthy individuals served as control samples. Probable spirochetal etiology could be demonstrated for 41 of 45 (91%) patients with clinical symptoms of chronic meningitis. Approximately 25% of the patients had significantly elevated titers of antibody to the spirochete in CSF but not in serum. The highest diagnostic sensitivity, 91%, was demonstrated by measurement of CSF antibodies and calculation of a spirochetal CSF titer index, which is the ratio of (ELISA titer in CSF/ELISA titer in serum) to (albumin in CSF/albumin in serum) and which also considers the degree of blood-CSF barrier damage. The highest specificity, 98%, was obtained by calculation of a CSF titer index. Patients with short duration of disease were especially prone to be antibody negative in serum but positive in CSF. Significant rise in serum antibody titers was seldom demonstrated in patients treated with antibiotics. It is concluded that measurement of CSF antibodies, especially by ELISA, is a highly sensitive and specific method for the immunological diagnosis of spirochetal meningitis.

Monitoring of gamma-aminobutyric acid in human cerebrospinal fluid: downward revision of previous control values.

gamma-Aminobutyric acid (GABA) levels were measured by a radioreceptor assay in cerebrospinal fluid (CSF) specimens from two groups of subjects, one without evidence of neurological or psychiatric disease and one with a variety of neurological disorders for which GABA involvement is not known. Mean GABA concentrations in CSF for the two groups were 117 +/- 10 and 127 +/- 7.4 pmol/ml (means +/- SE for 17 and 41 individuals, respectively). These values were about half of most previously reported mean CSF GABA control values, a fact which may be attributed, at least in part, to a reduction of artifactual increase in GABA levels during sampling, storing, and thawing of CSF samples and GABA analysis. Age and sex had no significant influence on CSF GABA concentration.

Release of neuron specific enolase (NSE) in cerebrospinal fluid following experimental lesions of the rat brain

Neuron-specific enolase (NSE) levels were measured by sandwich enzymo-immunoassay as well as by enzymatic assay in rat cerebrospinal fluid (CSF), following mechanical lesions of the brain tissue. Significant increases of NSE were observed in CSF, with a peak 2 h following lesions located near the lateral ventricle. Values returned to normal around 48 h later. In another experimental group, lesions were realized further away from the lateral ventricle; the elevation of NSE in CSF reached the maximal value 11 h later. In addition, measurements which were performed following lesions at the same location but of various sizes, indicated that the quantity of NSE released is proportional to the extent of brain damage. The possible factors which govern the time course and amount of NSE release in CSF are discussed. These results suggest that NSE could be a useful and easily detected marker of neuronal damage.

The free fraction of valproic acid in tears, saliva, and cerebrospinal fluid.

Valproic acid (VPA) was determined by EMIT assay in plasma, tears, saliva, and cerebrospinal fluid (CSF) of patients with epilepsy. Closer correlation was shown between tear/plasma and tear/CSF ratios than between saliva/plasma and saliva/CSF ratios. The VPA CSF/serum ratio was in good agreement with data in the literature. Salivary levels were extremely erratic, while those for tears were much more reliable. Determination of VPA in tears is therefore the best method of studying the VPA free fraction in those cases in which investigations of protein binding of the drug are necessary.

Brain uptake of carbamazepine in the cat.

Carbamazepine (CBZ) was determined by EMIT assay in plasma, cerebrospinal fluid (CSF), and brain samples of cats after administration of the drug (40 mg/kg, i.p.). Plasma and CSF levels increase in a parallel manner from time 0 to 90 min after completion of injection, indicating a passive transport from one body fluid to the other. Brain levels reach a steady state at 15 min, with no significant difference being found between the concentrations at 15, 30, 60, and 90 min postinjection; this indicates that substantial early binding occurs in the brain. Since peak CSF concentrations occur at 90 min, concomitant with those for plasma, while brain levels are already high at 15 min, it is likely that CBZ enters the two compartments by independent mechanisms.

Cerebrospinal fluid and blood assays of diagnostic usefulness in multiple sclerosis.

Although the ultimate diagnosis of multiple sclerosis (MS) still remains clinical, confirmatory laboratory aids can be of marked assistance, especially in early and atypical cases. Whereas numerous tests have been described, the only ones to withstand scrutiny in numerous laboratories have been various immunoglobulin assays and myelin basic protein measurements in the cerebrospinal fluid (CSF). Of the newer assays that are commercially available and readily adapted to routine clinical laboratory use, the most discriminating are the production of a CSF IgG:albumin ratio, using an electroimmunodiffusion method, and agarose electrophoresis of concentrated CSF to demonstrate oligoclonal IgG bands. Together, these assays can be performed on less than 3 ml of CSF, and will show relatively specific abnormalities in over 95% of clinically definite MS patients. They both detect abnormalities that frequently occur in the course of disease, and thus add considerable weight to the clinical impression of MS.

A longitudinal study of tryptophan involvement in hepatic coma

Previous cross-sectional studies have demonstrated that hepatic coma is associated with abnormally high levels of plasma free tryptophan. Establishment of a more definitive relationship between this biochemical abnormality and hepatic coma requires an evaluation of biochemical changes in individual patients as they undergo alterations in the function of their central nervous systems. The present report is an evaluation of tryptophan and substances believed to influence its entry into the brain in six patients whose clinical status progressed from hepatic coma to complete recovery. Consistent significant decreases in plasma free tryptophan and free fatty acids were demonstrated to occur as the patients recovered. No consistent changes, however, were found in the plasma levels of the amino acids which have been reported to compete with tryptophan for transport across the blood-brain barrier, except for leucine which was significantly decreased in all six patients upon recovery from coma. Assay of lumbar cerebrospinal fluid for both tryptophan and the serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), showed no consistent changes as the patients recovered from hepatic coma.

Clinical and laboratory studies with amikacin in newborns, infants, and children.

Thirty (86%) of 35 infants and older children with proven gram-negative sepsis had a complete clinical remission after treatment with amikacin. In 27 (82%) of 33 infectious episodes for which bacteriologic results were available before and after treatment, the organism was eradicated. The dosage of amikacin was either 7.5 mg/kg or 15 mg/kg given intramuscularly at 12-hr intervals. No adverse clinical effects or laboratory abnormalities were observed during treatment, which lasted from five to 14 days. All bacteria were sensitive to amikacin when tested by the disk diffusion method, and all but a single strain of Pseudomonas were sensitive when tested by the agar dilution method. Assays of serum and urine demonstrated adequate levels of amikacin after single intramuscular injections of 3.75 or 7.5 mg/kg; simultaneous assays of serum and cerebrospinal fluid in two cases demonstrated comparable concentrations of drug suggestive of a high degree of penetration into the cerebrospinal fluid in two cases demonstrated comparable concentrations of drug suggestive of a high degree of penetration into the cerebrospinal fluid during infection. Serial measurements of amikacin in serum from 0.5 to 12 hr after administration of single doses of 7.5 mg of drug/kg to six newborns revealed no significant differences in the concentrations achieved with intramuscular or intravenous administration of the drug.

Competitive protein binding assay for methotrexate.

A competitive protein binding assay has been developed for methotrexate based on the tight binding of this drug to Lactobacillus casei dihydrofolate reductase (= tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase; EC 1.5.1.3). Free drug may be separated from that bound to reductase by adsorption with dextran--albumin coated charcoal. Scatchard plot analysis of the enzyme--drug interaction confirmed the presence of a single homogeneous class of binding sites with an association constant Ka of 2.1 X 10(8) M-1. This high affinity binding permits detection of methotrexate in the range of 0.3--30 pmol with a coefficient of variation of 15% or less. The predominant circulating folate, 5-methyl tetrahydrofolate, and the clinically useful rescue agent leucovorin (5-formyl tetrahydropteroyl-glutamic acid) do not interfere with the assay, nor does the methotrexate metabolite 4-amino-4-deoxy-10-methylpteroic acid. Assay of clinical samples, including plasma and cerebrospinal fluid, showed close agreement between the previously described enzyme inhibition assay and the more rapid competitive binding method.

Administration of human leukocyte interferon in herpes zoster. I. Safety, circulating, antiviral activity, and host responses to infection.

levels > 100 units/ml were obtained more rapidly and were sustained longer after im administration. When an injection was given im on three successive days, peak daily titers rose progressively, suggesting accumulation in tissues. The rate of rise of varicella-zoster complement-fixing antibodies and the titers of interferon in vesicular fluid and cell counts in the patients treated with interferon did not differ significantly from those of a group of control patients. The only adverse side effect of treatment with interferon was fever of 38 C-40 C after injections. Assays of cerebrospinal fluid and urine specimens generally failed to demonstrate significant titers of interferon during periods when serum levels were known to be >100 units/ml. The average level of circulating interferon attained by either the im or iv route was higher than reported levels in naturally occurring human viral diseases and was approximately 17 times the minimal inhibitory concentration for varicellazoster virus in human cell culture.

Chorionic Gonadotropin In Choriocarcinoma

To the Editor.— Although Stilp et al 1 recommended assay of cerebrospinal fluid for chorionic gonadotropin in metastatic choriocarcinoma of the brain, in none of their four cases are such titers reported. Curiously, their first reference 2 does not mention the use of cerebrospinal fluid titers. I wish to call your attention to an article 3 that describes two patients with markedly elevated titers attributable to brain metastases from choriocarcinoma. We reported cerebrospinal fluid titers in ten patients and suggested that titration of the cerebrospinal fluid for chorionic gonadotropin may be useful in diagnosing intracranial metastatic choriocarcinoma.

Assay of IgG and Other Human Plasma Proteins by Quantitative Inhibition of Passive Hemagglutination

Abstract Using tanned human red cells coated optimally with isolated plasma proteins, specific agglutination in an AutoAnalyzer was quantitatively inhibited by solutions of the coating protein. IgG, IgM, IgA, IgD, IgE, transferrin, fibrinogen and ceruloplasmin were assayed in this manner. Lowest detectable concentrations varied from 0.2 µg IgE/ml and 0.4 µg IgG/ml to 9 µg IgD/ml. The amount of 125I-labeled IgG bound by tanned cells was a linear function of the amount applied when both variables were treated as logarithms, and about 2000 bound molecules per cell were optimal for agglutination. IgG appeared to bind at its Fab region, but less than 5% of the lowest applied concentrations was bound. Assays of cerebrospinal fluid for IgG compared favorably with quantitative precipitin data, but some myeloma proteins were not assayed reliably in reference to normal IgG.

ACETYLCHOLINE AND NEURONAL ACTIVITY: I. CHOLINESTERASE PATTERNS AND ACETYLCHOLINE IN THE CEREBROSPINAL FLUIDS OF PATIENTS WITH CRANIOCEREBRAL TRAUMA

Cerebrospinal fluid acetylcholine and cholinesterase determinations were made on 112 neurological and neurosurgical patients. Results on 15 patients with craniocerebral trauma and six psychiatric patients treated with electric shock convulsant therapy (E.C.T.) are reported in detail. Except for epileptics the only cerebrospinal fluid assays positive for acetylcholine occurred in the traumatic and E.C.T. groups, in which acetylcholine levels were 0.2 to 100 +μgm. %. In contrast to all other patients cerebrospinal fluid cholinesterases of traumatic and E.C.T. groups showed reversals of normal fraction patterns. These consisted of reduction of specific and increase of unspecified cholinesterase fractions, together with decrease of total cholinesterase activity. Acetylcholine levels and extent of cholinesterase fraction reversals paralleled the severity of cerebral damage, judged by clinical and electroencephalographic (E.E.G.) signs. Coma or semiconsciousness and depression of E.E.G. activity were associated with presence of acetylcholine and marked reversals of cholinesterase fractions in cerebrospinal fluids. In less severe cases and during recovery, when patients were disoriented and confused and E.E.G. activity was increased, cerebrospinal fluid acetylcholine decreased and disappeared, and cholinesterase values returned toward normal. Similar cerebrospinal fluid abnormalities were seen in patients after E.C.T. Changes in cerebrospinal fluid cholinesterase fraction patterns seem to be sensitive indications of extent of cerebral injury and progress toward recovery.