Determination of D- and L-aspartate in cell culturing medium, within cells of MPT1 cell line and in rat blood by a column-switching high-performance liquid chromatogrpahic method.

HPLC fluorometric methods have been used to analyze trace amounts of D-amino acids in biological samples. In this study, we established an expedient column-switching fluorometric HPLC system that would improve the analysis of D-amino acids, in particular D-aspartate (Asp). Our system consists of the fluorogenic derivatization of amino acids with NBD-F and two chromatographic steps, one that separates individual amino acids in reverse phase mode and another that separates the chiral forms of each amino acid in normal-phase mode. The two separation steps are linked through a trapping column by an automated column-switching system. In addition, sample preparation is simplified and improved, where trichloroacetic acid is used for deproteinization, and borate buffer, pH 9.5 is employed for the fluorescent derivatization. The detection limit for D-Asp in culturing medium is 5 nM. The resulting peak heights correlated well with concentrations that ranged from 12.5 to 250 nM for both D- and L-Asp. The present method was applied to determine D- and L-Asp levels in cell culturing medium, and within cells of MPT1 cell line. The detected cellular levels of D- and L-Asp agree with those detected by our previous method. In addition, this method was used to measure D- and L-Asp levels in rat blood samples, and the results are consistent with the reported values.

Localization, transport, and uptake of D-aspartate in the rat adrenal and pituitary glands.

Large amounts of D-aspartate (D-Asp) are present in the rat adrenal and pituitary glands. D-Asp is thought to be synthesized in the mammalian body and also accumulates in various tissues following intraperitoneal or intravenous administration. This report examines the origins of D-Asp in the adrenal and pituitary glands. We administered D-Asp to male rats intraperitoneally and immunolocalized this exogenous D-Asp in adrenal and pituitary tissue, using an anti-D-Asp antiserum which was previously developed in our laboratory. D-Asp levels in the rat adrenal gland have been shown to undergo a transient increase at 3 weeks of age and to decrease rapidly thereafter. We found that in the adrenal gland, exogenous D-Asp administered intraperitoneally was incorporated into the same region of the adrenal cortex in which endogenous D-Asp was present. By Northern and Western blot analysis and immunohistochemistry of glutamate (Glu) transporter, we also found that expression of the Glu transporter (GLAST), which has an affinity for D-Asp, transiently increased at 3 weeks of age and that localization patterns of the Glu transporter within the tissue were almost coincident with those of endogenous D-Asp. These observations suggest that D-Asp in the adrenal cortex of 3-week-old male rats is primarily acquired by uptake from the vascular system. We have previously shown that D-Asp is specifically localized in prolactin (PRL)-containing cells in the anterior lobe of the adult rat pituitary gland. Here we report that in the pituitary gland, exogenous D-Asp accumulated in endothelial cells, but not in PRL-containing cells. Northern and Western blot analysis and immunohistochemistry of Glu transporter revealed that developmental changes in the Glu transporter (GLAST) expression did not correlate with tissue levels of D-Asp and that the Glu transporter was not expressed in PRL-containing cells. These observations suggest that, in contrast to the adrenal gland, most of the D-Asp in the pituitary gland of adult male rats originates inside the gland itself.

Occurrence of D-Amino Acids and a pyridoxal 5'-phosphate-dependent aspartate racemase in the acidothermophilic archaeon, Thermoplasma acidophilum.

Free D-amino acid content in some archaea was investigated and D-forms of several amino acids were found in them. In the acidothermophilic archaeon, Thermoplasma acidophilum, the proportion of D-aspartate (D-Asp) to total Asp was as high as 39.7%. Crude extracts of Thermoplasma acidophilum had Asp-specific racemase activity that was pyridoxal 5'-phosphate (PLP)-dependent. The relative insensitivity to a SH-modifying reagent distinguished this activity from those of the PLP-independent Asp racemases found in other hyperthermophilic archaea (Matsumoto, M., et al., J. Bacteriol. 181, 6560-6563 1999). Thus, high levels of d-Asp should be produced by a new type(s) of Asp-specific racemase in Thermoplasma acidophilum, although the function of d-Asp in this archaeon remains unknown.

d-Aspartate in a prolactin-secreting clonal strain of rat pituitary tumor cells (GH(3)).

d-Aspartate (d-Asp) is found in prolactin (PRL)-containing cells of the rat anterior pituitary gland [Lee et al., Brain Res. 838, 193-199, 1999]. In order to determine whether d-Asp is actually produced by the anterior pituitary gland and whether it plays a physiological role in PRL function, a PRL-secreting clonal strain of rat pituitary tumor cells (GH(3)) was employed in this study. HPLC analysis and immunocytochemical staining detected the presence and synthesis of d-Asp in the cytoplasm of these cells. In addition, thyrotropin-releasing hormone-stimulated PRL secretion was increased in a dose-dependent fashion by d-Asp from these cells. These results suggest that the anterior pituitary gland synthesizes d-Asp and that d-Asp acts as a messenger in this gland.

Investigation of enantioselective separation of quinolonecarboxylic acids by capillary zone electrophoresis using vancomycin as a chiral selector.

When a chiral selector that is a pharmaceutical compound is added to the separation buffer in capillary electrophoresis, the enantioselectivity and the mobility of analytes which interact with that chiral selector may be altered. The changes in enantioselectivity and mobility of the analyte are a function of the strength of the affinity interaction, which depends on the structure of each. The macrocyclic antibiotic vancomycin contains a variety of functionalities that are known to be useful for enantioselective interactions (e.g., hydrogen bonding groups, hydrophobic pockets, aromatic groups, amide linkages). Capillary electrophoresis with vancomycin as a buffer additive was used to separate the enantiomers of different compounds. In this study, the chiral separation of quinolonecarboxylic acids that exhibit marked antibacterial activity and of related compounds was achieved by capillary electrophoresis using vancomycin. The correlations between the separation parameters and analyte structures were investigated. The molecular interaction, which is based on the differences of structure, and the effect of experimental parameters on the enantioselective separation between the quinolonecarboxylic acids and vancomycin are discussed.

Reversed-phase HPLC separation of proteins on chemically bonded silica gel columns.

Reversed-phase high-performance liquid chromatographic (RP-HPLC) separation of proteins on chemically bonded silica gel columns is described. Efficiency of nonporous alkylsilyl bonded silica gel is compared with that of a macroporous gel that has been widely used for the purpose. A comparative study of the separation under conventional and fast separation conditions is also given. The fast separation technique on the nonporous reversed-phase column has the advantage of improving the recovery of late-eluting hydrophobic and large proteins, such as ovalbumin and apoferritin.

Investigation of enantioselective ofloxacin-albumin binding and displacement interactions using capillary affinity zone electrophoresis.

The direct chiral separation of ofloxacin by capillary affinity zone electrophoresis using serum albumins from different animal sources as chiral selector in the supporting electrolyte is described. In addition, the effects of displacers on the mobility and enantioselectivity of ofloxacin were studied. Firstly, the separation behaviour of the enantiomers of the ofloxacin (OFLX) and tryptophan (Trp) was compared. The influence of albumin types, including chemically modified bovine serum albumins (BSAs), and buffer types on the migration behaviour of enantiomers was investigated. The results showed that stereoselectivity of Trp is independent of the type of albumin used. However, chiral separation of OFLX depends on the biological species of albumin. Use of chemically modified BSA led to poorer resolution of enantiomers. Only with acetylated BSA could chiral separation of Trp be achieved. Using Good's buffer solutions (DIPSO and HEPES) as a supporting electrolyte affected the migration times of OFLX enantiomers. Finally, a variety of displacers were added to the buffer along with the protein, and the effects on separation behaviour were observed. The displacers included warfarin, ketoproten, diazepam, propranolol, benzoinphenylbutazone, digitoxin and octanoic acid. From the results obtained, it is concluded that capillary affinity zone electrophoresis using albumin as a chiral selector may allow screening of OFLX-displacer interactions.

Diol-bonded silica gel as a restricted access packing forming a binary-layered phase for direct injection of serum for the determination of drugs.

Direct serum injection for drug determinations can be achieved on a diol-bonded silica gel as a restricted access packing. The diol-bonded phase, 3-(2,3-dihydroxypropoxy)propylsilylsilica, contains two different functions, a hydrophilic function at the tip of the single chemical bond and a hydrophobic function on the inside part of the bond to form a "binary-layered phase" on the support surface. Proteins, as large molecules, contact only the hydrophilic surface of the diol phase, and they are eluted at the solvent front based on size-exclusion chromatography. On the other hand, small molecules such as synthetic drugs are retained on the internal hydrophobic function and separate based on reversed-phase chromatography. Accordingly, the diol-bonded silica gel performs as a restricted access packing for direct serum injection for the determination of relatively hydrophobic drugs.

Chiral separation by capillary affinity zone electrophoresis using an albumin-containing support electrolyte.

Chiral separations of some pharmaceutical compounds were studied by capillary affinity zone electrophoresis. Bovine serum albumin (BSA) was used as a chiral selector and added to electrolyte. For the chiral separation of new quinolone bactericidal reagents, phosphate buffer was more appropriate than borate buffer solution as the support electrolyte. The effects of BSA concentration, albumin type, pH, chiral additive, and voltage on separation were observed. As a result, chiral separations were performed in the pH range 7-8. The migration and stereoselectivities of enantiomers were changed by varying the protein concentration (more than 0.2% w/v) and voltage and by adding amino acids as chiral modifiers. This procedure is easily applicable to other compounds for chiral separation or for studies of protein binding interaction.

[Stabilization of ascorbic acid aqueous solution by protamine].

Protamine was found to stabilize ascorbic acid (AsA) in aqueous solution. The concentration of AsA (50 mumol/l) dissolved in 0.05 M phosphate buffer, pH 7, decreased by 57% after standing for 90 min at 30 degrees C, whereas 91% of AsA remained under the same conditions in the presence of 60 mumol/l protamine. When 0.5 mumol/l of copper ion (II) was added to the above AsA solution (0.05 M phosphate buffer, pH 7) in the absence of protamine, AsA decreased by 5% after standing for 15 min at 30 degrees C. On the other hand, 91% of AsA remained after 15 min and 52% of AsA remained after 90 min under the same conditions in the presence of 60 mumol/l protamine. Furthermore, the effect of protamine on the stability of AsA in various pH solutions (pH 1-10) was examined after standing for 90 min at 30 degrees C. Degradation of AsA was accelerated by increasing the pH from 1 to 10. On the other hand, the effect of protamine (60 mumol/l) on the stabilization of AsA was recognizable above pH 5 and degradation of AsA was controlled at higher pH. When 0.5 mumol/l of copper ion (II) was added to the above solutions in the absence of protamine, degradation of AsA was greatly accelerated above pH 5. When 60 mumol/l of protamine was added to the solutions, the effect of protamine on the stabilization of AsA was observed above pH 5 and degradation of AsA was controlled at higher pH.

Study of chemobiological reactions. 1. Selectivity of aromatic amino compounds and saccharides in glycosylation reactions.

The reactivities between saccharides and primary amino compounds were studied by liquid chromatography on an amino-bonded column and by measurement of the reaction rates of aromatic amines with saccharides. The recoveries from the column and the reaction rates were correlated with their physicochemical properties calculated by the CAChe program. The reactivities between amines and saccharides correlated well with their hydrogen bonding energies calculated by molecular mechanics.

High performance liquid chromatography of nucleic acids and the related compounds on a fluorinated silica gel column.

Separations of nucleic acids and the related compounds were investigated by HPLC on a new fluorinated bonded silica gel column. Polyadenylate (Poly (A)) enzymatic partial hydrolysate sample and the mixture of various polynucleotide samples were sufficiently separated by the reversed-phase mode using a gradient elution with aqueous ammonium acetate/acetonitrile system. Mixed-mode separation on the fluorinated bonded phase coated with a tert-alkylammonium salt was also examined for the separation of the various polynucleotides including tRNAs.

Fast protein separation by reversed-phase high-performance liquid chromatography on octadecylsilyl-bonded nonporous silica gel. II. Improvement in recovery of hydrophobic proteins.

Recovery of hydrophobic proteins from an RP-HPLC column was improved using a fast-separation RP-HPLC system operated at room temperature. Hydrophobic proteins such as ovalbumin could be adequately eluted from a nonporous octadecylsilyl (C18) spherical silica gel with a particle diameter of 20 microns using steep gradient elution with a 0.1% aqueous trifluoroacetic acid-acetonitrile system at a constant flow rate of 4 ml/min. Recoveries improved under fast separation since the protein sample suffered only a slight amount of irreversible denaturation on the hydrophobic surface of the stationary phase. The fast-separation system was also applied to the separation of larger proteins such as apo-ferritin (443 kDa) and thyroglobulin (669 kDa) as well as egg white proteins.

Precolumn derivatization technique for high-performance liquid chromatographic determination of penicillins with fluorescence detection.

A precolumn derivatization method was developed for the high-performance liquid chromatographic (HPLC) determination of penicillins using fluorescence detection. Penicillins were derivatized by a two-step reaction, the beta-lactam ring being opened by hydrolysis in aqueous sodium carbonate solution in the first step to give a secondary amine functionality, and the secondary amino group being reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole in the second step to give a fluorescent derivative. The resulting reaction mixture was injected directly onto a reversed-phase column and analysed by HPLC. At a penicillin concentration of 10 micrograms/ml, the precision (relative standard deviation) ranged from 1.49 to 2.20%. In the concentration range 0.2-100 micrograms/ml, a linear response was observed. The detection limits of this method were 30-85 ng/ml for five different penicillins at a signal-to-noise ratio of 3:1. The proposed method was applied to the determination of penicillins added to serum following pretreatment by deproteinization and removal of compounds containing amino functionalities with a cation-exchange resin.

New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives.

A preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for carboxylic acids is described. 9-Anthraldehyde hydrazone is oxidized with an organic oxidant, N-chlorosuccinimide, in an organic solvent such as ethyl acetate to give ADAM, and then the reaction mixture is directly used as the reagent solution for the derivatization of carboxylic acids. Both the oxidation and the derivatization reaction are carried out at room temperature, and an aliquot of the derivatization mixture is directly injected into a chromatograph. 9-Anthrylmethyl ester derivatives formed from ADAM and various carboxylic acids are sufficiently separated on a reversed-phase column and are sensitively detected fluorometrically. The present method was applied to the high-performance liquid chromatographic determination of long and short chain fatty acids, keto acids, and hydroxy acids.

Determination of carnitine by high-performance liquid chromatography using 9-anthryldiazomethane.

The high-performance liquid chromatographic determination of carnitine chloride was investigated by using 9-anthryldiazomethane (ADAM) as a pre-column derivatization reagent. Carnitine chloride and the internal standard N,N-dimethylglycine reacted with ADAM to give a stable ester derivative in the presence of sodium dodecyl sulphate (SDS) used to mask the basic function. The ADAM derivative of carnitine was separated from decomposition products of the reagent and related compounds such as amino acid derivatives on a silica gel column eluted with methanol-5% aqueous SDS-phosphoric acid (990:10:1). The calibration plot was linear over a sample concentration range from 0.02 to 100 ng per injection. The detection limit for carnitine chloride was about 1 pg per injection (signal to noise ratio = 4), by fluorometric detection.

Postcolumn fluorometric detection system for liquid chromatographic analysis of amino and imino acids using o-phthalaldehyde/N-acetyl-L-cysteine reagent.

A high-performance liquid chromatographic system for the determination of amino acid and imino acid was developed using a two-step reaction with sodium hypochlorite and o-phthalaldehyde/N-acetyl-L-cysteine (OPTA/AcCys) reagent. This reagent improved the sensitivity in the analysis of proline presumably because the fluorophore is more stable to hypochlorite which has been used for the oxidative cleavage of the imino linkage. The use of OPTA/AcCys facilitated the detection of imino acids at the same concentration level as that of amino acids. The detection limit for all the amino and imino acids was a few picomoles. This detection system, together with cation-exchange chromatographic separation, was applied to the determination of amino and imino acids in biological samples.

Amino acid analysis by reversed-phase high-performance liquid chromatography. Automatic pre-column derivatization with activated carbamate reagent.

Succinimido alpha-naphthylcarbamate, an activated carbamate reagent, facilitated the simple and rapid pre-column derivatization of amino acids for fluorimetric detection. The method is based on the formation of naphthylcarbamyl derivatives of amino acids. The carbamylation of amino acids is completed within 1 min at room temperature, and the reaction mixture can be injected directly into a liquid chromatograph equipped with an octadecylsilyl reversed-phase column. The effluent stream is monitored fluorimetrically at 370 nm excited at 290 nm at the sub-picomole level. Naphthylcarbamyl derivatives of common protein amino acids were separated within 30 min by gradient elution with aqueous sodium acetate and acetonitrile. Excess of the reagent does not interfere with the analysis of amino acids, because it is hydrolysed in 2-3 min to give naphthylamine, which is more strongly retained than any amino acid derivatives.

Optical resolution of enantiomeric amino acid derivatives on a naphthylethylurea multiple-bonded chiral stationary phase prepared via an activated carbamate intermediate.

A new chiral stationary phase (CSP) was developed for the direct optical resolution of enantiomeric amino acid derivatives. The CSP was readily prepared by a three-step reaction carried out in a pre-packed aminopropylsilyl silica gel column. In the first step, a solution of disuccinimido carbonate (DSC) was delivered through the pre-packed column to give a succinimido carbamyl aminopropylsilyl-bonded, activated-carbamate type silica gel (ACsil) column. Through the column was then delivered a solution of pentaethylenehexamine to afford a polyamine-bonded column. Finally, a solution of optically active succinimido (S)- or (R)-naphthylethyl carbamate was delivered through the polyamine column, to give a naphthylethylurea multiple-bonded CSP. p-Bromophenylcarbamyl derivatives of enantiomeric protein amino acids were resolved on these CSPs by elution with an aqueous mobile phase. Simultaneous analysis of these amino acid enantiomers by means of gradient elution was also accomplished.