Biomedical subjects

Kazuhiro Imai

Publications and source records attributed to Kazuhiro Imai.

At least 55 records · Page 3Linked to original sources

PubMed2003

Capillary electrophoresis with laser induced-fluorescence detection of profens derivatized with the water-soluble fluorogenic reagent 4-N-(4-N'-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole.

Profens, including pranoprofen, fenoprofen, flurbiprofen, ketoprofen and ibuprofen (Ib), were derivatized by a water-soluble benzofurazan fluorescent reagent, 4-N-(4-N'-aminoethyl)piperazino-7-nitro-2,1,3-benzoxadiazole and then were run on capillary electrophoresis in a NH4Ac-HAc buffer of pH 3.1 containing 2.4 mM beta-cyclodextrin. At room temperature, the derivatization reaction was catalyzed by triphenyl phosphine and diphenyl disulfide in acetonitrile medium, and the derivatives fluoresce around 530 nm when excited at 488 nm. With the CE running on a 50 cm x 50 microm I.D. length fused-silica capillary of by using Ar+ laser induced-fluorescence detection, the detection limits attained were in the range of 0.16 to 0.3 fmol.

4-Chloro-7-nitrobenzofurazan↗

PubMed2003

An identification method for altered proteins in tissues utilizing fluorescence derivatization, liquid chromatography, tandem mass spectrometry, and a database-searching algorithm.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is now widely used as a tool for proteomic studies. For the sensitive determination of proteins in 2D-PAGE, fluorescence derivatization of primary amino moieties of proteins with cyanine dyes was recently developed. However, precipitation of the proteins could occur if completely derivatized because of the lower solubility of the resultant derivatives owing to the hydrophobicity of the reagents and the loss of the hydrophilic primary amino moieties. Thus, in this paper, a water-soluble and thiol-specific fluorogenic reagent, ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate, was adopted for the derivatization of proteins in tissues either with and without stimulation. Then, the method follows a separation of the derivatives by liquid chromatography with fluorescence detection, an isolation of only the altered proteins, an enzymatic digestion of the isolated proteins, and an identification of the proteins by liquid chromatography/MS/MS with the database-searching algorithm. By using this method, we identified the altered expressions of five increased proteins (e.g., pancreatic polypeptide) as well as three decreased proteins (e.g., insulin 2) in the islets of Langerhans in Wistar rats 2 days after they were subcutaneously administered with dexamethasone.

Algorithms↗

PubMed2003

Determination of serum D-lactic and L-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization.

d-Lactic and l-lactic acids were simultaneously determined by means of a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. As a fluorescence reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was employed for the fluorescence derivatization of lactic acid. The proposed HPLC system adopted both octylsilica (Cadenza CD-C8) and amylose-based chiral columns (CHIRALPAK AD-RH), which proved to give a sufficient enantiomeric separation of the lactic acid derivatives with a separation factor ( alpha) of 1.32 and a resolution ( R(s)) of 1.98. Moreover, the features of the first elution of d-lactic acid peak in the proposed HPLC were convenient for the determination of trace amount of serum d-lactic acid, which is known to increase under diabetes. Intra-day and inter-day accuracies were in the range of 90.5-101.2 and 89.0-100.7%, and the intra-day and inter-day precisions were 0.3-1.2 and 0.4-4.8%, respectively. The proposed method was applied to determine d-lactic and l-lactic acids in human serum of normal subjects and diabetic patients, showing that both d-lactic and l-lactic acid concentrations were significantly increased in the serum of diabetic patients ( n=31) as compared with normal subjects ( n=21). This fact was found for the first time owing to the development of the proposed HPLC method which is able to determine d-lactic and l-lactic acid simultaneously. Finally, serum d-lactic acid concentrations determined by the proposed HPLC method were compared with those from a reported enzymatic assay, and the smaller p value between normal subjects and diabetic patients was shown by the proposed HPLC method.

Adult↗

PubMed2003

High-performance liquid chromatography-fluorescent assay of catechol-O-methyltransferase activity in rat brain.

A method to measure catechol- O-methyltransferase (COMT) activity using high performance liquid chromatography-fluorescence detection with norepinephrine (NE) as a natural substrate was optimized for both soluble (S-) and membrane-bound (MB-) COMT activities in rat brain areas, cerebral cortex, cerebellum, hippocampus, brain stem, hypophysis, and hypothalamus. The highest S-COMT activity in Sprague-Dawley rat brain was found in hippocampus. MB-COMT activities in all brain areas were about 3-8 times lower than S-COMT activities. However, considering Vmax/ Km values, specificity constants for NE to S- and MB-COMT contributes mainly to the metabolism of NE in cerebral cortex and cerebellum.

Animals↗

PubMed2003

First fluorescent photoinduced electron transfer (PET) reagent for hydroperoxides.

A novel fluorescent reagent for hydroperoxides, 4-(2-diphenylphosphinoethylamino)-7-nitro-2,1,3-benzoxadiazole (1), was developed on the basis of the method for designing photoinduced electron transfer (PET) reagents having a benzofurazan skeleton. Compound 1 was quantitatively reacted with hydroperoxides to give its fluorescent derivative, 2. In acetonitrile, the Phi value (0.44) of 2 was 31 times greater than that of 1. The long excitation (458 nm) and emission (520 nm) wavelengths of 2 are suitable for the determination of hydroperoxides, especially in biosamples. [structure: see text]

Fluorescence Resonance Energy Transfer↗

PubMed2003

Fluorescence on-off switching mechanism of benzofurazans.

Many fluorescent reagents with a benzofurazan (2,1,3-benzoxadiazole) skeleton have been developed and widely used in bio-analyses. In this study, we try to elucidate the fluorescence on-off switching mechanism of three fluorogenic reagents and their derivatives. Ten 4,7-disubstituted benzofurazans were used for this purpose and the measurements of their fluorescence, phosphorescence, photolysis, and time-resolved thermal lensing signal in acetonitrile were obtained in order to understand the relaxation processes of these compounds. These results indicate that the competition of fluorescence with a fast intersystem crossing or fast photoreaction plays a key role in the fluorescence on-off switching. Semi-empirical molecular orbital calculations show that the existence of the triplet n pi* state is responsible for the fast intersystem crossing while the proximity of the reactive second single pi pi* state to the first singlet pi pi* state contributes to the fast photoreaction in the excited states.

Journal Article↗

PubMed2003

Recent progress in derivatization methods for LC and CE analysis.

The derivatization procedure with a suitable fluorescence or chemiluminescence reagent is performed for the purpose of increasing the detection sensitivity and selectivity, in high-performance liquid chromatography (HPLC) and/or capillary electrophoresis (CE). In this article, recent derivatization methods and their applications to biosamples are described. In HPLC, femto mol order of mass detection limits are obtained by derivatization. Regarding the fluorescence reagents, the use of water-soluble reagents has been effective to avoid an undesired adsorption in the process of determination of peptides. In CE, the advantages of having extremely low mass detection limits (ranging from atto to yocto mol level) and requiring only a very short analysis time (less than a few minutes) are made possible by using laser-induced fluorescence or near infra-red detections.

Chromatography, High Pressure Liquid↗

PubMed2003

Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation.

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).

Adenosine Diphosphate↗

PubMed2003

Decreased serum levels of D-serine in patients with schizophrenia: evidence in support of the N-methyl-D-aspartate receptor hypofunction hypothesis of schizophrenia.

BACKGROUND: The hypofunction of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors has been implicated in the pathophysiology of schizophrenia. Several lines of evidence suggest that D-serine may function as an endogenous agonist of the glycine site of the NMDA receptor. The aim of this study was to examine whether serum levels of D- and L-serine in patients with schizophrenia are different from those of healthy controls. METHODS: Forty-two patients with schizophrenia and 42 age- and sex-matched healthy controls were enrolled in this study. Symptoms were assessed using the Brief Psychiatric Rating Scale. Serum levels of total serine and D- and L-serine were measured by high-performance liquid chromatography. RESULTS: Serum levels of D-serine in the patients with schizophrenia were significantly (z = -3.30, P =.001) lower than those of healthy controls. In contrast, serum levels of total (D and L) serine (z = -2.40, P =.02) and L-serine (z = -2.49, P =.01) in the schizophrenic patients were significantly higher than those of controls. In addition, the percentage of D-serine in the total serine in the schizophrenic patients was significantly (z = -4.78, P<.001) lower than that of controls, suggesting that the activity of serine racemase, an enzyme catalyzing the formation of D-serine from L-serine, may have been reduced in the schizophrenic patients. CONCLUSIONS: Reduced levels of D-serine may play a role in the pathophysiology of schizophrenia, and serum D- and L-serine levels might provide a measurable biological marker for schizophrenia.

Adolescent↗

PubMed2003

Determination of fluoxetine and norfluoxetine in rat plasma by HPLC with pre-column derivatization and fluorescence detection.

Both fluoxetine (FLX) and its N-demethylated metabolite, norfluoxetine (NFLX), have been reported to be potent serotonin-reuptake inhibitors. A sensitive and reliable method that allows simultaneous quantification of their plasma levels would be valuable and was developed in this work. The procedure included extraction of FLX and NFLX from plasma, fluorescence derivatization with 4-(N-chloroformylmethyl-N-methyl) amino-7-nitro-2,1,3-benzoxadiazole (NBD-COCl), separation of the derivatives on an octadecylsilica column with acetonitrile-water (55:45,v/v) as mobile phase and fluorescence detection with emission at 537 nm and excitation at 478 nm. The calibration curves were linear for FLX and NFLX concentration over the range of 10-1000 nM (r = 0.9992 and r = 0.9997) and the limits of quantitation were 10 nM in 100 micro L of plasma. Precision of intra- and inter-day RSD of less than 12% and accuracy of intra- and inter-day RE within -6.0-13% were achieved. The method described was applied to analysis of the plasma samples from rats treated with FLX hydrochloride and to the pharmacokinetic study.

Animals↗

PubMed2003

Altered expression of insulins I and II and their mRNAs in the islets of Langerhans in dexamethasone-induced diabetic rats.

Rats have two isomeric insulins (insulins I and II). There have been no reports on the expression of the isomeric insulins in glucocorticoid-induced diabetic rats. To clarify the relation of the expression of each insulin and its mRNAs in dexamethasone-induced diabetic rats, the amounts of the isomeric insulins and mRNAs in the islets of Langerhans were determined in vivo and in vitro. A sensitive and selective HPLC-fluorescence determination method for the isomeric insulins and a newly developed real-time quantitative RT-PCR method for their mRNAs were used. There was a greater reduction of insulin II than insulin I in the islets of Langerhans in dexamethasone-induced diabetic rats. This alteration may be caused by a disproportionate expression of the respective mRNA for the isomeric insulins that resulted from the direct effect of dexamethasone. In addition, continuous hyperglycemia may also suppress the expression of the insulin II mRNA. The overall effects of dexamethasone and hyperglycemia may cause a greater reduction of insulin II than insulin I in the dexamethasone-induced diabetic rat. Conversely, an elevated ratio of insulin I to II in the islets could suggest a diabetic condition.

Animals↗

PubMed2003

A study of the relationship between the chemical structures and the fluorescence quantum yields of coumarins, quinoxalinones and benzoxazinones for the development of sensitive fluorescent derivatization reagents.

To develop new fluorescent derivatization reagents, we investigated the relationship between the chemical structures and the fluorescence quantum yields (phi(f)) of coumarins, quinoxalinones and benzoxadinones. Forty-six compounds were synthesized and their fluorescence spectra were measured in n-hexane, ethyl acetate, methanol and water. The energy levels of these compounds were calculated by combination of the semi-empirical AM1 and INDO/S (CI = all) methods. The deltaE(Tn(n,pi*), S1(pi,pi*)) (the energy gap between the Tn(n,pi*) and S1(pi,pi*) states) values were well correlated with the phi(f) values, which enables us to predict the phi(f) values from their chemical structures. Based on this relationship, 3-phenyl-7-N-piperazinoquinoxalin-2(1H)-one (PQ-Pz) and 7-(3-(S)-aminopyrrolidin-1-yl)-3-phenylquinoxalin-2-(1H)-one (PQ-APy) were developed as fluorescent derivatization reagents for carboxylic acids. The derivatives of the carboxylic acids with PQ-Pz and PQ-APy showed large phi(f) values even in polar solvents, suggesting that these reagents are suitable for the microanalysis of biologically important carboxylic acids by reversed phase HPLC.

Benzoxazoles↗

PubMed2003

Study on interactions of endocrine disruptors with estrogen receptor using fluorescence polarization.

In this study, we examined the affinities of many (21) compounds such as hormones, pharmaceuticals, industrial chemicals, and phytoestrogens to the estrogen receptor (ER) by ER binding assay using fluorescence polarization (FP). This method is based on the competitive binding assay using fluorescein-labeled estradiol (F-E2), in which the polarization values decreased with the addition of the compounds (competitors). The obtained sigmoidal inhibition curves were transformed into the pseudo-Hill plots, and the concentrations at 50% inhibition (IC50) and Hill coefficients were obtained from the regression equations. We examined the relationship between the chemical structures and estrogenic activities, and finally classified the tested compounds into three categories, agonists, partial agonists and antagonists based on their Hill coefficients.

Binding, Competitive↗

PubMed2003

[Precision of internal standard method in HPLC analysis].

The internal standard methods are known to compensate for the errors from sample preparation and injection into an analytical instrument. However, recent HPLC apparatuses have injectors of excellent repeatability and it is dubious whether the cancellation of injection error can lead to substantial improvement in the precision of analysis. This paper answers the above question experimentally and theoretically. The HPLC analysis of butylscopolamine bromide is taken as an example. The relative standard deviations (RSD) of measurements in the internal standard method and absolute calibration curve method are compared and the advantages of these methods are discussed. The measurement RSD is shown to be well estimated by the (function of mutual information) (FUMI) theory without repeating measurements. This report also demonstrates simple equations for calculating the measurement RSD at an arbitrary concentration of analyte and for selecting the better method between the internal standard method and absolute calibration curve method under specific experimental conditions.

Calibration↗

PubMed2003

[Analytical chemical studies on high-performance recognition and detection of bio-molecules in life].

In order to understand the mechanism for maintaining life of animals based on the search of dynamics of biomolecules, I have developed several sensitive and selective methods for their quantification. Using the methods of derivatization with the developed benzofurazan fluorogenic reagents (4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), ammonium 7-fluoro-2,1,3-benzoxadiazole 4-sulfonate (SBD-F) and etc.) followed by high-performance liquid chromatography (HPLC)--fluorescence detection, a certain kind of biological and clinical importance was demonstrated of chiral bio-molecules (D-amino acids, D-lactic acid and so on), peptides and proteins. The proposed method (derivatization with SBD-F, isolation of the fluorescent proteins by two-dimensional HPLC, enzymatic digestion and identification of the altered proteins by HPLC-mass spectrometry (MS)/MS with database-searching algorithm) for proteomics studies revealed the changed proteins in the islets of Langerhans of the dexamethazone-induced diabetic rats. An importance of catecholamine metabolism on the blood pressure regulation was also suggested by the method of HPLC-chemiluminescence detection of catecholamines and their 3-O-methylmetabolites. A new field of Analytical Chemistry, i.e., Bio-Analytical Chemistry, was also proposed.

Amino Acids↗

PubMed2003

Reduced membrane-bound catechol-O-methyltransferase in the liver of spontaneously hypertensive rats.

We have previously reported that methylation of catecholamines by catechol-O-methyltransferase (COMT) was attenuated in spontaneously hypertensive rats (SHR) with acute hypotension as compared with that of Wistar-Kyoto (WKY) rats. Here we examined the soluble (S-) and membrane-bound (MB-) COMT activities and COMT protein in the liver, kidney, and erythrocytes in both strains. Both the activities and the amounts of MB-COMT in the liver were lower in SHR than in WKY rats, but no such trend was found in the kidney or erythrocytes. Nor was such a trend observed in any of these three tissues for S-COMT. These results indicate that liver MB-COMT may be a relevant factor in blood pressure regulation in rats.

Aging↗

PubMed2003

Study on interactions of endocrine disruptors with estrogen receptor-beta using fluorescence polarization.

In this study, we developed a rapid, simple and homogeneous human recombinant estrogen receptor-beta (hrER-beta) binding assay method using fluorescence polarization (FP) by applying a fluorescent ligand, fluorescein-labeled estradiol (F-E2). A Scatchard plot and a Hill plot analysis of the saturation binding assay using F-E2 and hrER-beta were studied. F-E2 showed a high affinity for hrER-beta, the dissociation constant was 5.53 nM, indicating that F-E2 is applicable to the hrER-beta binding assay. Competitive binding assays using F-E2, in which the FP values decreased upon the addition of compounds (competitors) were carried out to evaluate the binding characteristics of compounds with and without biological activities to hrER-beta. Twenty-one compounds, such as hormones, pharmaceuticals, industrial chemicals and phytoestrogens, were examined. The obtained sigmoidal inhibition curves were transformed into pseudo-Hill plots and the concentrations at 50% inhibition (IC50) and Hill coefficients, the degree of cooperativity in ER-ligand binding, were obtained from the regression equations. Antagonists exhibited larger Hill coefficients than agonists, showing the correlation between the Hill coefficients and the estrogenic/antiestrogenic activities.

Binding, Competitive↗

PubMed2002

Development of hydrophilic fluorogenic derivatization reagents for thiols: 4-(N-acetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole and 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole.

Sensitive, reactive, and hydrophilic fluorogenic reagents for thiols with the benzofurazan skeleton, 4-(N-acetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (AcABD-F) and 4-(N-trichloroacetylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (TCAcABD-F) have been developed. These reagents reacted with thiols within 10 min at 60 degrees C. AcABD-F and TCAcABD-F themselves do not fluoresce but are strongly fluorescent after the reaction with thiol compounds. The generated derivatives were highly water-soluble, since they dissociated a proton and ionized in the neutral pH region. The derivatives with four biologically important thiol compounds were separated on a reversed-phase HPLC column and detected fluorometrically at 504 nm with excitation at 388 nm. The detection limit attained for homocysteine with AcABD-F was 25 fmol on column (11 nM) (signal-to-noise ratio = 3), and that for glutathione with TCAcABD-F was 45 fmol on column (20 nM).

Benzoxazoles↗