Atsushi Hiraoka

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.

Capillary electrophoretic analyses of β-trace protein and other low molecular weight proteins in cerebrospinal fluid from patients with central nervous system diseases

Ordinary capillary-zone electrophoresis (CZE), as well as CZE in a sodium dodecylsulfate-containing polymer solution (SDS-CZE) and capillary isoelectrofocusing (CIEF), was applied to the analysis of low molecular weight proteins in cerebrospinal fluid (CSF) from patients with various neuropsychiatric disorders. Under the CZE conditions employed, a peaks of beta-trace protein (beta TP), which is the most abundant low MW protein in CSF, was clearly detected on the electropherograms of all the samples examined, and the CSF beta TP level could be tentatively determined using allylamine added at a constant concentration as the internal standard. The results revealed that beta TP in CSF was non-specifically increased in organic disease in the central nervous system (CNS), especially in ones giving severe physical damage to the brain tissues. On the other hand, SDS-CZE allowed us to determine simultaneously the CSF minor low MW proteins other than beta TP, such as beta 2-microglobulin, gamma-trace protein, myelin basic protein, etc., while the CIEF electropherograms suggested that beta TP were separated into several fractions with the different PI values. These capillary electrophoresis systems seem to be powerful as aids in the biochemical examinations of beta TP and other low molecular weight proteins in CSF from patients with CNS diseases.

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.

γ-Radiation-stimulated formation of ortho-quinone from chlorogenic acid in Xanthium occidentale

The effect of γ-irradiation on the conversion of chlorogenate to its o-quinone in aqueous solution as well as in Xanthium occidentale leaves was studied. In aqueous solution, this compound linearly decreased with increased dose. The rate of transformation decreased exponentially with increased concentration, whilst the number of molecules transformed was unrelated to a concentration, suggesting that chlorogenate was mainly attacked by an indirect mechanism. The formation of unstable o-quinone was demonstrated by converting it to a stable derivative, 6-benzosulphonylchlorogenate formed on reacting it with benzenesulphinate. As a result of irradiation, chlorogenate in aqueous solutions was stoichiometrically converted to benzosulphonylchlorogenate in the presence of benzenesulphinate. This was also the case for solutions added with mushroom tyrosinase instead of irradiation treatment. These observations indicated this method to be adequate for estimating the amount of o-quinone. In actively growing leaves of X. occidentale, chlorogenate o-quinone was also detected in non-irradiated tissues; the amount was five-fold higher in the continuously illuminated tissues than in those incubated in the dark. On exposure to γ-rays, o-quinone increased five-fold in tissues which were kept in the dark after irradiation, whilst it was maintained at a constant level in tissues illuminated by ordinary white light. Chlorogenate increased at doses up to 80 Gy in both tissue, probably due to an activation of its biosynthetic system. It is concluded that in normal tissues, a certain amount of o-quinone is constantly present, and when irradiated at moderate doses, there is an additional conversion of chlorogenate to its o-quinone, especially in tissues kept in the dark.

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.

Inhibition of γ-irradiation-induced oxidation of chlorogenate by 1,3-dicaffeoylquinate in Xanthium occidentale

Abstract The endogeneous inhibition of the radiation-induced conversion of chlorogenate into its o -quinone in Xanthium occidentale leaves has been studied. In addition to chlorogenate, this plant contains 1,3-dicaffeoylquinate (cynarin). On exposure to γ-radiation, the concentrations of both substances in aqueous solution were reduced by an indirect mechanism, i.e. a distinct number of molecules were transformed by a given dose of radiation irrespective of concentration. In the presence of cynarin, chlorogenate was protected from radiation-induced oxidation, and the degree of protection increased exponentially with increased cynarin amount; the protection was almost complete at a concentration exceeding ca five times that of chlorogenate. Similarly, chlorogenate reduced the conversion of cynarin, but this protection proceeded only linearly with increased concentration. The high degree of protection afforded by cynarin may be ascribed to the presence in the molecule of two oxidizable (oxidant-scavenging) caffeoyl groups, as compared with chlorogenate which has only one. Since the physiological concentrations of cynarin and chlorogenate were estimated to be 8.7 and 2.1 mM, respectively, it is very probable that in X. occidentale cells only a slight conversion of chlorogenate can occur as a result of irradiation.