Biomedical subjects

J Haginaka

Publications and source records attributed to J Haginaka.

At least 37 records · Page 2Linked to original sources

PubMed1999

Chiral resolution of basic drugs by capillary electrophoresis with new glycosaminoglycans.

New glycosaminoglycans, fucose-containing glycosaminoglycan (FGAG) and depolymerized holothurian glycosaminoglycan (DHG), were investigated as chiral additives for the separation of drug enantiomers by capillary electrophoresis. The average molecular masses of FGAG and DHG were estimated to be about 59,000 and 14,000, respectively. A variety of basic drug enantiomers were resolved using 10 mM phosphate buffer, pH 5.0, containing 3% FGAG or DHG. Since chiral recognition properties of FGAG and DHG are different, some drug enantiomers were only separated by using FGAG or DHG. With regard to comparison of chiral recognition abilities of FGAG and DHG with other chiral selectors, tolperisone and eperisone enantiomers were not separated with alpha- or beta-cyclodextrin, or heparin as the chiral additives, but were separated with FGAG and DHG. The results obtained reveal that FGAG and DHG are useful as the chiral selectors for separations of drug enantiomers by CE, and that they could be complementarily used with other chiral additives.

Buffers↗

PubMed1999

Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein. VII. Comparison of chiral recognition ability of ovoglycoprotein from chicken and Japanese quail egg whites.

Ovoglycoproteins from chicken and Japanese quail egg whites (OGCHI and OGJPQ, respectively) were isolated, and characterized by isoelectric focusing, high-performance capillary electrophoresis, reversed-phase HPLC and matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS. The isoelectric point (pI) of natural OGCHI was 4.37-4.51 by isoelectric focusing, while natural OGJPQ showed two discrete bands at pI 4.68 and 4.77. The average molecular masses of natural OGCHI and OGJPQ were estimated to be about 30000 and 27400 by MALDI-TOF-MS. Both natural OGCHI and OGJPQ were either fully or partially glycosylated with a ratio of ca. 3:1. Next, natural OGCHI and OGJPQ were bound to aminopropyl-silica gels activated with N,N'-disuccinimidylcarbonate to compare retentive and enantioselective properties of the two columns. The OGCHI column is suitable for chiral resolution of basic compounds, while the OGJPQ column is suitable for that of acidic compounds. With regard to chiral resolution of neutral compounds, it is dependent on a compound resolved which column could be suitable. Differences in the retentivity and enantioselectivity between OGCHI and OGJPQ columns are due to differences in the enantioselective binding properties. The results obtained reveal that chiral recognition of various solutes could be efficiently attained by using both columns complementarily.

Animals↗

PubMed1999

Uniform-sized molecularly imprinted polymer for (S)-ibuprofen retention properties in aqueous mobile phases.

A uniform-sized molecularly imprinted polymer for (S)-ibuprofen has been prepared by a multi-step swelling and thermal polymerization method using 4-vinylpyridine (4-VPY) and ethylene glycol dimethacrylate (EDMA) as a host functional monomer and cross-linker, respectively. The obtained (S)-ibuprofen imprinted 4-VPY-EDMA materials were evaluated using aqueous eluents by HPLC. Hydrophobic and hydrogen bonding interactions between ibuprofen enantiomers and 4-VPY-EDMA materials could play an important role in the retentivity and enantioselectivity. Further, partial resolution of the enantiomers of ibuprofen metabolites, 2-hydroxy- and 2-carboxyibuprofen, was attained with the (S)-ibuprofen imprinted 4-VPY-EDMA materials.

Chromatography, High Pressure Liquid↗

PubMed1999

High-performance liquid chromatographic assay of hydroperoxide levels in oxidatively modified lipoproteins.

An anion-exchange HPLC method has been developed for the chemiluminescence (CL) assay of hydroperoxide (HPO) levels in native and oxidized low density lipoproteins (N- and Ox-LDLs, respectively) of Watanabe heritable hyperlipidemic (WHHL) rabbits. The method involves anion-exchange HPLC separation in N- and Ox-LDLs using a DEAE-glucomannan gel, and direct CL detection of HPOs in them without extraction of the lipids following postcolumn reaction with isoluminol, microperoxidase and Triton X-100. Addition of Triton X-100, which could solubilize lipids, were essential for the detection of HPOs in N- and Ox-LDLs. With an increase in the degree of oxidation, Ox-LDL was more retained on the DEAE-glucomannan gel with a concomitant increase in the CL intensity. The proposed method could analyze the HPO levels in N- and Ox-LDLs of WHHL rabbits without extraction of the lipids.

Animals↗

PubMed1999

Uniform-sized molecularly imprinted polymer for (S)-naproxen selectively modified with hydrophilic external layer.

A uniform-sized molecularly imprinted polymer (MIP) for (S)-naproxen selectively modified with hydrophilic external layer has been prepared. First, the molecularly imprinted polymer for (S)-naproxen was prepared using 4-vinylpyridine and ethylene glycol dimethacrylate (EDMA) as a functional monomer and cross-linker, respectively, by a multi-step swelling and thermal polymerization method. Next, a 1:1 mixture of glycerol monomethacrylate (GMMA) and glycerol dimethacrylate (GDMA) was used for hydrophilic surface modification, and it was added directly to the molecularly imprinted polymer for (S)-naproxen 4 h after the start of molecular imprinting. The retention factors of all solutes tested were decreased with the surface modified molecularly imprinted polymer, compared with the unmodified molecularly imprinted polymer. However, chiral recognition of racemic naproxen was attained with the surface modified molecularly imprinted polymer as well as the unmodified molecularly imprinted polymer. Further, bovine serum albumin was completely recovered from the surface modified molecularly imprinted polymer. These results revealed that the chiral recognition sites of (S)-naproxen remained unchanged with hydrophilic surface modification, and that the molecularly imprinted polymer for (S)-naproxen was selectively modified with hydrophilic external layer. Preliminary results reveal that the surface modified molecularly imprinted polymer could be applicable to direct serum injection assays of (S)-naproxen.

Chromatography, High Pressure Liquid↗

PubMed1999

Separation and sensing based on molecular recognition using molecularly imprinted polymers.

Molecular recognition-based separation and sensing systems have received much attention in various fields because of their high selectivity for target molecules. Molecular imprinting has been recognized as a promising technique for the development of such systems, where the molecule to be recognized is added to a reaction mixture of a cross-linker(s), a solvent(s), and a functional monomer(s) that possesses a functional groups(s) capable of interacting with the target molecule. Binding sites in the resultant polymers involve functional groups originating from the added functional monomer(s), which can be constructed according to the shape and chemical properties of the target molecules. After removal of the target molecules, these molecularly imprinted complementary binding sites exhibit high selectivity and affinity for the template molecule. In this article, recent developments in molecularly imprinted polymers are described with their applications as separation media in liquid chromatography, capillary electrophoresis, solid-phase extraction, and membranes. Examples of binding assays and sensing systems using molecularly imprinted polymers are also presented.

Chemistry Techniques, Analytical↗

PubMed1999

Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein. V. Influence of immobilization method on chiral resolution.

Ovoglycoprotein from chicken egg whites (OGCHI) was bound to aminopropyl-silica gels via an amino or carboxyl group(s) of OGCHI. In the former case, OGCHI was bound to N,N'-disuccinimidyl carbonate-activated aminopropyl-silica gels, while in the latter case OGCHI activated by a water-soluble carbodiimide and N-hydroxysulfosuccinimide was bound to aminopropyl-silica gels. The obtained OGCHI materials were compared with regard to the bound amounts, retentivity and enantioselectivity. The OGCHI materials prepared via a carboxyl group(s) of OGCHI are suitable for chiral resolution of acidic solutes, and those via an amino group(s) of OGCHI are suitable for chiral resolution of basic solutes. It is suggested that the electrostatic interaction between an amino or carboxyl group of OGCHI and a charged solute should play an important role in chiral recognition of the solute.

Benzoin↗

PubMed1999

Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein. III. Effect of aggregation of ovoglycoprotein on chiral resolution.

Ovoglycoprotein from chicken egg whites (OGCHI) subjected to 80 degrees C has produced the associated OGCHI, which is a heat-induced aggregate of OGCHI. The molecular weight of the aggregate was estimated to be ca. 5.7 million daltons by a low-angle laser light-scattering detection. The heat-induced aggregate of OGCHI was found to dissociate reversibly to the OGCHI monomer. The OGCHI aggregate has little chiral recognition ability, or has much lower chiral recognition ability than the native OGCHI. Further, the OGCHI monomer from the reversibly dissociated OGCHI aggregate has chiral recognition ability comparable to that of the native OGCHI.

Chromatography, Gel↗

PubMed1998

Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins of rabbits, rats and mice.

A fast, accurate and precise high-performance liquid chromatographic assay method has been developed for plasma lipoproteins of experimental animals, rabbits, rats and mice. The method includes complete separation of high, low and very low density lipoproteins from one another within 20 min by a DEAE-glucomannan gel using stepwise elution, and determination by postcolumn reaction with an enzymatic cholesterol reagent as the total cholesterol level. The relative standard deviation of each lipoprotein assay was highly reproducible, being less than 2.0 and 2.4% for repeatability and intermediate precision, respectively. The method was successfully applied to the assays of plasma lipoproteins in three species of normolipidemic and hyperlipidemic animals.

Animals↗

PubMed1998

Determination of ATP and its metabolites released from rat caudal artery by isocratic ion-pair reversed-phase high-performance liquid chromatography.

A sensitive and selective assay method for adenine compounds (purines) using high-performance liquid chromatography with fluorescent detection was developed. The 1,N6-ethenoderivatives of adenine, adenosine, AMP, ADP, and ATP formed by reaction with chloroacetaldehyde at 80 degreesC were separated by ion-pair reversed-phase chromatography within 15 min under isocratic conditions. alpha,beta-Methylene adenosine 5'-diphosphate could be used as an internal standard for the determination of purines. The calibration graphs constructed with peak area ratios against amounts were linear between 0.1 and 10.0 pmol of each purine. The repeatability and intermediate precision were less than 6% (RSD, n = 5) and 8% (RSD, n = 3), respectively, at 0.5 pmol of each purine. A method for calculation of each purine amount which considers hydrolysis by derivatization is described. The optimized method was applied to determine the purines released from the rat caudal artery stimulated by noradrenaline.

Acetaldehyde↗

PubMed1998

Evidence of modified lipoprotein in the plasma of Watanabe heritable hyperlipidemic rabbits by anion-exchange high-performance liquid chromatographic assay.

We searched for evidence for the presence of oxidatively modified low density lipoproteins (Ox-LDL) in the plasma of Watanabe heritable hyperlipidemic (WHHL) rabbits using an anion-exchange high-performance liquid chromatographic (HPLC) method newly developed for the assay of artificially modified lipoproteins. Various Ox-LDLs were prepared by incubation of native LDL (N-LDL) from rabbit plasma with 5 microM CuCl2 for 0, 1, 3 and 24 h, and separated by anion-exchange HPLC method using a DEAE-glucomannan gel column. With an increase in the incubation time, LDL was further oxidized and retained on the DEAE-glucomannan gel. This oxidized LDL migrated faster in the anodic direction in agarose gel electrophoresis, accompanied by an increase in lipid peroxide levels, estimated as thiobarbituric acid reactive substances (TBARS). There was a good agreement in the chromatographic and electrophoretic behaviors of the various Ox-LDLs. LDL from WHHL rabbits showed increases in TBARS levels, electrophoretic mobility on agarose gel electrophoresis and cholesteryl ester accumulation in mouse peritoneal macrophages compared to that from normolipidemic Japanese white (JW) rabbits. When LDLs from both animals were compared by the anion-exchange HPLC method using linear gradient or stepwise elution, LDL from WHHL rabbits had a longer retention time and was eluted by a higher concentration of sodium chloride. These results suggest that LDL in WHHL rabbit plasma may undergo mild oxidative modification.

Animals↗

PubMed1997

Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein. II. Comparison of chiral recognition properties with crude ovomucoid.

Chiral stationary phases based on ovoglycoprotein from chicken egg whites (OGCHI) and crude ovomucoid from chicken egg whites (OMCHI) were compared with regard to the bound amounts of OGCHI and chiral recognition abilities. Crude OMCHI included 11% OGCHI, by weight. Since pure OMCHI had no appreciable chiral recognition ability, the chiral recognition ability of crude OMCHI originated from OGCHI, which was present in crude OMCHI preparations as an impurity. However, a chiral stationary phase based on crude OMCHI showed good chiral recognition ability, despite the 11% OGCHI content in crude OMCHI preparations. When crude OMCHI was reacted with N,N'-disuccinimidylcarbonate (DSC)-activated aminopropyl-silica gels, the ratio of bound OGCHI to that of totally bound protein was 0.23. These results reveal that the good chiral recognition ability of a stationary phase based on crude OMCHI is due to OGCHI being preferentially bound to DSC-activated aminopropyl-silica gels rather than the OMCHI. In addition, OMCHI did not contribute to the enantioselectivity of the solute at all and made little contribution to the retention characteristics.

Animals↗

PubMed1997

Separation of enantiomers on a chiral stationary phase based on ovoglycoprotein. I. Influences of the pore size of base silica materials and bound protein amounts on chiral resolution.

The influences of the pore size of base silica gels and the bound amounts of ovoglycoprotein (OGCHI) on the chiral resolution of racemates have been investigated. To the 12-, 20- and 30-nm pore size silica gels, aminopropyl groups were introduced, activated with N,N'-disuccinimidyl carbonate and then reacted with OGCHI followed by blocking with D-glucosamine. For the OGCHI material prepared with the same pore size silica gel, a linear correlation was obtained between the capacity factor or each enantiomer and the amount of bound OGCHI. Enantioselectivity and resolution obtained with the reaction of 40 mg OGCHI per 1 g silica gel were lower than those obtained with the reaction of 80, 160 and 320 mg OGCHI, when the same pore size silica gel was used. This is due to the superfluous achiral interaction with base silica gels and/or spacers because of low protein coverage. With regard to comparison of the pore sizes of silica gel, the OGCHI materials prepared with the 12-nm pore size silica gel gave the largest capacity factor, and the highest enantioselectivity and/or resolution for the racemates tested, when the same amount of OGCHI was reacted. Thus, the OGCHI materials were prepared with the reaction of 80 mg OGCHI per 1 g silica gel having a 12-nm pore size followed by blocking with D-glucosamine. By diminishing the superfluous achiral interaction with base silica materials and/or spacers, much more efficient OGCHI materials should be obtainable.

Chemical Phenomena↗

PubMed1997

Enantioselectivity of bovine serum albumin-bonded columns produced with isolated protein fragments. II. Characterization of protein fragments and chiral binding sites.

Enantioselectivity of bovine serum albumin (BSA)-bonded columns produced with isolated protein fragments has been investigated. The BSA fragment, BSA-FG75, was isolated by size exclusion chromatography following peptic digest of BSA. The isolated BSA-FG75 was further fractionated to two fractions, BSA-F1 and BSA-F2, by anion-exchange chromatography. BSA-F1 and BSA-F2 had molecular mass of about 35000 daltons, estimated by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, BSA-F1 has amino acid residues 1-307 estimated by electrospray ionization (ESI) mass spectrometry, while BSA-F2 is an N-terminal half BSA fragment. The BSA, BSA-FG75, BSA-F1 and BSA-F2 proteins were bound to aminopropyl-silica gels activated by N,N'-disuccinimidyl carbonate. The bound amounts of the BSA fragments were 2.2-2.7 times more than that of the intact BSA. Chiral recognition of 2-arylpropionic acid derivatives, benzodiazepines, warfarin and benzoin was obtained with the BSA fragment-bonded columns. The non-enantioselective interactions of benzoin and benzodiazepines except for clorazepate with BSA fragments were increased with protein surface coverages, while those of 2-arylpropionic acid, clorazepate and warfarin were decreased. The BSA fragment columns gave higher enantioselectivity for lorazepam and benzoin, and lower enantioselectivity for other compounds tested, compared with the BSA column. These results might be due to changes in the globular structure of the BSA fragment and/or changes in the local environment around the binding sites.

Benzodiazepines↗

PubMed1996

[Development of HPLC packing materials for drug analysis and their applications].

High-performance liquid chromatography (HPLC) has been used for the assays of pharmaceuticals and their impurities, and drugs and their metabolites in biological fluids. However, HPLC packing materials so far developed are not necessarily suitable for the above mentioned purposes. During the past decade, we developed of new HPLC packing materials for direct serum injection analysis of drugs, and for chiral resolution of drug enantiomers. A sample preparation step such as deproteinization and/or extraction is required for HPLC assays of drugs and their metabolites in biological fluids. Direct serum injection analysis of drugs can be achieved by the use of the packing materials having properties as follows: large molecules such as proteins are eluted in the void volume without destructive accumulation, but small molecules such as drugs and their metabolites can reach the hydrophobic sites and be separated. Such a material is called restricted access stationary phase (RASP). We developed two RASPs, an improved internal-surface reversed-phase (ISRP) material and a mixed functional phase (MFP) material. The preparation methods of the ISRP and MFP materials and their applications to the assays of drugs in the serum are described. HPLC chiral stationary phases based on chiral small molecules, and macromolecules such as polysaccharides and proteins have been used for separations of drug enantiomers. Disadvantages of protein-bonded stationary phases include low capacity, lack of column ruggedness and limited understanding of the chiral recognition mechanism. We tried to overcome the disadvantages of protein-bonded stationary phases by modification of side-chain amino acid and preparation of protein-domain or -fragment column. We isolated each ovomucoid domain and examined chiral recognition ability of each domain. The ovomucoid third domain exhibited chiral recognition for only a few benzodiazepines and 2-arylpropionic acid derivatives. The chiral binding site and mechanism on the ovomucoid third domain was investigated by proton NMR measurements and molecular modeling of the ligand-protein complex. Further, new protein, ovoglycoprotein, was isolated from crude ovomucoid preparations, characterized and bound to silica gels. It was found that the chiral recognition ability of the ovomucoid reported previously came from the ovoglycoprotein, which is present in crude ovomucoid as an impurity.

Amino Acid Sequence↗

PubMed1995

Anion-exchange high-performance liquid chromatographic assay of plasma lipoproteins.

A fast, accurate, and precise high-performance liquid chromatographic (HPLC) assay method for human plasma lipoproteins has been developed. The method includes complete separation of high-, low-, and very-low-density lipoproteins within 20 min using a DEAE-glucomannan gel with stepwise elution, enzymatic oxidation of cholesterol (following ester hydrolysis) followed by a two-step reaction to form a fluorogenic product, and fluorometric detection as the total cholesterol (TC) level of each lipoprotein. The calibration range is 5-1000 micrograms/ml for the TC level of 20-microliters plasma samples and the relative standard deviation (n = 5) of the precision at 5 micrograms/ml is less than 10%. Each lipoprotein level determined by this optimized HPLC method agrees well with levels determined by ultracentrifugation or precipitation methods. This new method was successfully applied to assays of plasma lipoproteins in various forms of hyperlipidemia.

Calibration↗

PubMed1995

The absence of chiral recognition ability in ovomucoid: ovoglycoprotein-bonded HPLC stationary phases for chiral recognition.

Commercial chicken ovomucoid (OMCHI) and OMCHI, isolated by precipitation of egg whites with organic solvents, both of which as crude products, were fractionated by anion- and cation-exchange chromatography. The obtained four fractions were characterized by reversed-phase chromatography, N-terminal sequencing, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, determination of sugar contents, and trypsin-inhibitory activities. Three fractions were OMCHI variants differing in carbohydrate composition, especially in sialic acid content, and the other fraction was tentatively termed ovoglycoprotein (OGCHI). The OMCHI and OGCHI are different in physicochemical and biochemical properties: average molecular weight 26000-27700 for OMCHI variants and 29700 for OGCHI; N-terminal amino acid, Ala for OMCHI and Thr for OGCHI; and trypsin-inhibitory activity, positive for OMCHI and negative for OGCHI. These OMCHI variants and OGCHI were bound to aminopropyl silica gels to evaluate chiral recognition ability. OMCHI is reported to have chiral recognition ability (Miwa, T.; et al. Chem. Pharm. Bull. 1987, 35, 682-686). However, neither OMCHI variant had appreciable chiral recognition ability, while the OGCHI had excellent chiral recognition properties as compared to those of the OMCHI reported previously. This reveals that the chiral recognition ability of the OMCHI reported previously comes from the OGCHI, which is present in crude OMCHI as an impurity.

Amino Acid Sequence↗

PubMed1995

Protein binding chiral discrimination of HPLC stationary phases made with whole, fragmented, and third domain turkey ovomucoid.

Individual protein domains and two domains in combination were prepared by enzymatic and chemical cleavage of turkey ovomucoid followed by isolation and purification by size-exclusion and ion-exchange chromatography. Silica bonded-phase HPLC columns were made from either whole or isolated domains of turkey ovomucoid. The protein columns were tested for chiral recognition by their abilities to resolve enantiomers among a wide range of racemates. The columns made from whole turkey ovomucoid displayed chiral activity toward many racemates, where as a combination of the first and second domain resolved only a selected number of aromatic weak bases. The first and second domains independently gave no appreciable chiral activity. The turkey ovomucoid third domain exhibited enantioselective protein binding for fused-ring aromatic weak acids. Glycosylation of the third domain did not affect chiral recognition. Titration of the third domain with model compounds in conjunction with NMR measurements enabled the identification of the amino acids responsible for binding. Molecular modeling of the ligand-protein complexation provided insights into the ability of a protein surface to discriminate enantiomers on the basis of multiple intermolecular interactions.

Amino Acid Sequence↗