Yoshiko Nagata

d‐Aspartate stimulation of testosterone synthesis in rat Leydig cells

d‐Aspartate increases human chorionic gonadotropin‐induced testosterone production in purified rat Leydig cells. l‐Aspartate, d‐,l‐glutamate or d‐,l‐asparagine could not substitute for d‐aspartate and this effect was independent of glutamate receptor activation. Testosterone production was enhanced only in cells cultured with d‐aspartate for more than 3 h. The increased production of testosterone was well correlated with the amounts of d‐aspartate incorporated into the Leydig cells, and l‐cysteine sulfinic acid, an inhibitor of d‐aspartate uptake, suppressed both testosterone production and intracellular d‐aspartate levels. d‐Aspartate therefore is presumably taken up into cells to increase steroidogenesis. Intracellular d‐aspartate probably acts on cholesterol translocation into the inner mitochondrial membrane, the rate‐limiting process in steroidogenesis.

Stimulation of steroidogenic acute regulatory protein (StAR) gene expression by D-aspartate in rat Leydig cells.

D-aspartate and human chorionic gonadotropin act synergistically to increase testosterone production in purified rat Leydig cells, and D-aspartate stimulates testosterone synthesis even in the absence of human chorionic gonadotropin stimulation. In addition, D-aspartate enhances steady-state cellular mRNA and protein levels of steroidogenic acute regulatory protein, which is a key regulatory factor in gonadal and adrenal steroidogenesis. D-aspartate therefore appears to increase testosterone production in rat Leydig cells by stimulating steroidogenic acute regulatory protein gene expression. To our knowledge, this is the first report demonstrating a direct effect of D-aspartate on gene expression in mammalian cells.

Aqueous chromatographic system for separation of biomolecules using thermoresponsive polymer modified stationary phase

We have investigated a new method for HPLC using packing materials modified with a functional polymer, such as thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm). PNIPAAm-modified silica exhibits temperature-controlled hydrophilic-hydrophobic surface property changes in aqueous systems. Temperature-responsive chromatography is performed with an aqueous mobile phase without using an organic solvent. We designed ternary copolymers of NIPAAm introduced 2-(dimethyl-amino) ethyl methacrylate (DMAEMA) as a cationic monomer and butyl methacrylate (BMA) as a hydrophobic monomer. A cationic thermoresponsive hydrogel grafted surface would produce an alterable stationary phase with both thermally regulated hydrophobicity and charge density for separation of bioactive compounds. In this study, we achieved successful separation of lysozyme without the loss of bioactivity by temperature-responsive chromatography. The electrostatic and hydrophobic interactions could be modulated simultaneously with the temperature in an aqueous mobile phase, thus the separation system would have potential applications in the separation of biomolecules.

Liquid chromatography-mass spectrometry for the determination of medetomidine and other anaesthetics in plasma

A liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric method is presented for the simultaneous determination of medetomidine and other anaesthetic drugs in solutions and dog plasma. The drugs examined were flumazenil, butorphanol, atropine, ketamine, xylazine, medetomidine, atipamezole and midazolam. The separation was carried out on a reversed-phase column using methanol-0.1 M ammonium acetate (3:2) as eluent.

Simultaneous determination of ginsenosides and saikosaponins by high-performance liquid chromatography

Octadecyl porous glass was used as the packing for reversed-phase high-performance liquid chromatography. A mixture of ginsenosides and saikosaponins (saponins of ginseng and bupleurum root, respectively) were analysed with detection at 203 nm. A well resolved chromatogram of ginsenoside Rb1, Rc, Rb2 and Rd and saikosaponin a, b2 and c was obtained with acetonitrile-water (25.5:74.5) as the mobile phase. The whole separation was achieved in 20 min with a flow-rate of 1.5 ml/min. Calibration graphs for ginsenoside Rb1, Rc, Rb2 and Rd and saikosaponin a and c were linear up to 5 micrograms. Rapid and accurate simultaneous determinations of the saponins are possible by the described method.

High-performance liquid chromatographic determination of catecholamine metabolites and 5-hydroxyindoleacetic acid in human urine using a mixed-mode column and an eight-channel electrode electrochemical detector

An HPLC system for the simultaneous determination of acidic catecholamine metabolites, related compounds and 5-hydroxyindoleacetic acid (5-HIAA) in human urine was developed. A mixed-mode (C18/anion-exchange) column with isocratic elution using citrate buffer and an eight-channel electrochemical detector were used. Vanilmandelic acid (VMA), 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxyphenyllactic acid (vanillactic acid, VLA), homovanillic acid (HVA), vanillic acid (VA) and 5-HIAA in urine were determined simultaneously. Detection limits and inter (n = 5) and intra-assay (n = 5) coefficients of variation were satisfactory. The mean of analytical recoveries (n = 3, +/- C.V. (%)) were between 97 +/- 3.2 (VMA) and 105 +/- 4.8 (VA). Correlations between the analytical results for VMA, HVA and 5-HIAA obtained by an established method and the present method were satisfactory. The mean +/- 2 S.D. of the excretion rates of VMA, DOPAC, VLA, HVA, 5-HIAA and VA in urine from healthy adult volunteers were 0.61-4.36, 0.13-1.02, 0-0.35, 0.67-6.55, 0.50-5.14 and 0-0.55 mg/g creatinine, respectively.

Stereospecific analysis of loxoprofen in plasma by chiral column liquid chromatography with a circular dichroism-based detector

The chiral separation of loxoprofen was achieved on a chiral column with UV and circular dichroism (CD) detection. The good resolution of four loxoprofen stereoisomers was obtained. The column used for the chiral separation was Chiralcel OJ column (250 x 4.6 mm) using hexane-2-propanol-trifluoroacetic acid (95:5:0.1), as an eluent. The flow-rate was 1.0 ml/min and the detection was at 225 nm. In addition, CD and UV spectra were obtained by stopped flow scanning. The method allows the determination of the stereoisomers of loxoprofen in human plasma after the administration of therapeutic dose of the racemic drug, thus HPLC with CD detector is useful for the stereospecific determination of loxoprofen products in biological samples.

Determination of acidic saponins in crude drugs by high- performance liquid chromatography on octadecylsilyl porous glass

High-performance liquid chromatographic (HPLC) analysis on octadecylsilyl porous glass was investigated for acidic saponins in ginseng, bupleurum root and senega. The acidic saponins, malonyl-ginsenosides, malonyl-saikosaponins and senegins, as well as neutral saponins in the crude drugs were separated rapidly by HPLC on this column with aqueous acetonitrile containing KH2PO4 as the mobile phase at room temperature.

Comparison of columns of chemically modified porous glass and silica in reversed-phase high-performance liquid chromatography of ginsenosides

Abstract Reversed-phase high-performance liquid chromatograms of an extract of ginseng and mixtures of ginsenosides (ginseng saponins) on a number of columns of chemically modified porous glass (MPG, pore size 550 A) and silica (pore size 80 and 300 A) were compared. Although the retention behaviour of ginsenosides was similar on the columns examined, the capacity factors of ten ginsenosides on an octadecylsilyl-MPG (MPG-ODS) column were smaller than those on silica columns. It is concluded that the MPG-ODS column has a number of advantages over conventional silica-ODS columns for the chromatography of ginsenosides. These properties are attributable to the optimum pore size for the molecular size of the saponins on the one hand and to the narrow distribution range of the pore size on the other.

High performance liquid chromatography of anti-pyretics on chemically modified porous glass

An octadecylsilyl porous glass was prepared and used as the packing for reversed-phase high-performance liquid chromatography. Five antipyretic drugs (aspirin, caffeine, guaiacol glycerol ether, 3-hydroxy-p-butyrophenetidine, and phenacetin) were separated in 2 min with a mobile phase of 20% acetonitrile at a flow-rate of 3.0 ml/min. A pharmaceutical preparation, containing aspirin, phenacetin, caffeine and chlorpheniramine maleate was analysed in 2 min with a mobile phase of acetonitrile-water-acetic acid (20:79:1). The packing seems promising for the rapid analysis of pharmaceuticals and biomedical compounds.