Itaru Tominaga

Capillary zone electrophoretic determination of organic acids in cerebrospinal fluid from patients with central nervous system diseases

Organic acids in cerebrospinal fluid (CSF) from patients with various central nervous system (CNS) diseases were determined by capillary zone electrophoresis (CZE). Under one of the two sets of conditions employed, several anionic components of CSF were separated into corresponding peaks on the electropherograms and determined. The other conditions employed were also useful in measurement of the lactate contents in CSF. The CSF levels of lactate and pyruvate and the ratios of lactate to pyruvate were elevated in patients with cerebral infarction and bacterial meningitis, whereas CSF ascorbate was reduced mainly in inflammatory disorders of the CNS. The results showed that CZE can become a powerful tool in the biochemical diagnosis of CNS diseases.

Sodium dodecyl sulfate-capillary gel electrophoretic analysis of molecular mass microheterogeneity of β-trace protein in cerebrospinal fluid from patients with central nervous system diseases

Molecular mass (M(r)) microheterogeneity of beta-trace protein (beta TP) in cerebrospinal fluid (CSF) from patients with various neurological disorders was analyzed by sodium dodecyl sulfate capillary gel electrophoresis. Under the conditions employed, beta TP with a M(r) distribution of 23,000-30,000 was roughly separated into two subfractions containing the major peaks with M(r) of 26,000 and 28,500, respectively. The peak area ratios of the two subfractions of the electropherograms varied among the samples examined, and elevation in the total beta TP level in the CSF from patients with organic diseases in the central nervous system (CNS) was often accompanied by changes in the ratios of the subfractions. The quantitative changes in the subfraction level in CSF beta TP are considered to reflect the pathological alterations in the CNS.

Analysis of low-molecular-mass proteins in cerebrospinal fluid by sodium dodecyl sulfate capillary gel electrophoresis

Proteins of low molecular (M(r)) in human cerebrospinal fluid (CSF) were analyzed by capillary electrophoresis in a sodium dodecyl sulfate-containing polymer solution. Under the conditions employed, peaks of beta 2-microglobulin (beta MG) (M(r): 11,700), gamma-trace protein (12,300), myelin basic protein (18,000), beta-trace protein (beta TP) (23,000 to 30,000) and alpha 1-acid glycoprotein (42,000) were detected on the electropherograms. The concentrations of beta MG and beta TP were determined based on the peak area relative to that of an internal standard, Orange G, which was added at a constant amount as the front marker. It was demonstrated that their levels in CSF change under various pathological conditions in the central nervous system.

One-step capillary isoelectric focusing of the proteins in cerebrospinal fluid and serum of patients with neurological disorders

One-step capillary isoelectric focusing (cIEF), which uses reduced but non-zero electroosmosis flow to mobilize the focused proteins, was applied to the analysis of proteins in cerebrospinal fluid (CSF) and serum of patients with various neurological disorders. Under the conditions employed, pathological changes in the CSF proteins were clearly detected on the electropherograms within 25 min, although the serum proteins did not vary significantly between samples. The present one-step cIEF system seems to be useful in routine laboratory examinations of a large number of CSF samples as an aid in neurological diagnosis.

Charge microheterogeneity of the β‐trace proteins (lipocalin‐type prostaglandin D synthase) in the cerebrospinal fluid of patients with neurological disorders analyzed by capillary isoelectrofocusing

Charge microheterogeneity of the β‐trace protein (β‐TP = lipocalin‐type prostaglandin D synthase) in the cerebrospinal fluid (CSF) of patients with various neurological disorders was analyzed by capillary isoelectric focusing (CIEF). Under the conditions employed, β‐TP in the low‐molecular‐weight protein fraction of CSF was separated into at least four isoforms with different pI values. An isoform with the pI value of 4.6–4.8 was usually the most abundant. The total β‐TP level in the CSF was determined by enzyme‐linked immunosorbent assay (ELISA) to be elevated in patients recovering from organic damage to the CNS and those with pathological brain atrophy. Changes in the total β‐TP level in the CSF were occasionally accompanied by those in its charge microheterogeneity, as revealed by CIEF. Such quantitative and qualitative changes in β‐TP in human CSF indicated changes in its pathophysiological roles in association with various neurological disorders.

Sodium dodecylsulfate capillary gel electrophoretic measurement of the concentration ratios of albumin and α2-macroglobulin in cerebrospinal fluid and serum of patients with neurological disorders

Sodium dodecylsulfate capillary gel electrophoresis (SDS-CGE) was applied to measure the concentration ratios of albumin (Alb) and alpha2-macroglobulin (alphaMG) in the cerebrospinal fluid (CSF) and concurrent serum samples from patients with various neurological disorders. The values of the alphaMG index in individual patients were calculated on the basis of the peak area ratios of Alb and an alphaMG subunit on the CSF and serum electropherograms. The alphaMG index value thus obtained was most prominently raised in patients with inflammatory diseases of the brain and/or meninges, suggesting that the function of the blood-brain barrier (BBB) was disturbed under the pathological conditions in the central nervous system. The measurement of the concentration ratios of Alb and alphaMG in CSF and the concurrent serum samples by the present SDS-CGE system seems to be useful as an aid in the biochemical examination of the BBB function in patients with neurological disorders.

Capillary-isotachophoretic determination of glutamine in cerebrospinal fluid of various neurological disorders

A rapid and microscale technique to detect and determine L-glutamine (Gln), which is the most abundant free amino acid in human cerebrospinal fluid (Sf), was established on the basis of capillary-isotachophoresis. This system was applied to the measurement of Sf Gln concentration in various neurological disorders. The results showed that Sf Gln was increased under some pathological conditions in the central nervous system, especially those accompanying pleocytosis in Sf.

A silver staining method for protein bands or immunoprecipitates in electrophoresis or immunoelectrophoretic treatment of unconcentrated cerebrospinal fluid proteins using the commercial agarose plates (Corning Universal System)

A highly sensitive silver staining method for protein bands or immunoprecipitates in electrophoresis or immunoelectrophoresis of unconcentrated cerebrospinal fluid using the commercial agarose plates (Corning Universal System) has been established. The new method is based on the reaction between protein-potassium ferrocyanide complex and silver nitrate as is the conventional silver staining in agar gel electrophoresis. However, some modifications were introduced into the procedures in order to obtain the densitometric values of fractionated proteins coincident with those by the usual amidoblack 10B staining for the same concentrated samples. Furthermore, in immunoelectrophoresis, several minor component proteins which could not be found by the usual staining have been detected in the cerebrospinal fluid samples.