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T Toyo'oka

Publications and source records attributed to T Toyo'oka.

At least 19 recordsLinked to original sources
PubMed2001

Determination of triazolam involving its hydroxy metabolites in hair shaft and hair root by reversed-phase liquid chromatography with electrospray ionization mass spectrometry and application to human hair analysis.

A sensitive method using reversed-phase liquid chromatography coupled with electrospray ionization mass spectrometry has been developed for simultaneous determination of triazolam and its hydroxy metabolites in hair. After the addition of deuterium-labeled 1-hydroxymethyltriazolam as an internal standard, the analytes in hair shaft and hair root samples were extracted with a basic medium, CH(2)Cl(2):MeOH:28% NH(4)OH (20:80:2) at room temperature overnight. The chromatographic separation of the analytes was achieved using a semimicro HPLC column (3-microm particle size; 100 x 2.0-mm i.d.) by gradient elution with acetonitrile in water containing 1% acetic acid as eluent. The mass spectrometer was operated in selected-ion monitoring mode at quasi-molecular ions [M+H](+) of triazolam and its metabolites. The method has been applied to determine the incorporation of triazolam and its metabolites into the hair shafts and hair roots of Dark Agouti rats administered 3 or 6 mg/kg triazolam intraperitoneally twice a day for 5 days. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were incorporated into the hair shafts and the hair roots. The concentration of 4-hydroxytriazolam was the highest of all compounds detected. An unknown substance considered to be 1,4-dihydroxytriazolam also appeared in the hair samples. The structural elucidation was performed with online HPLC-MS after acetylation of the substance with acetic anhydride and pyridine. The time course studies of triazolam and the metabolites in both rat hair roots and plasma were carried out after single intraperitoneal administration of triazolam. The concentrations of triazolam and the metabolites in the hair roots reflected those in the plasma. The proposed method using selected-reaction monitoring was applied to the determination of triazolam and the metabolites in human hairs of a triazolam addict. Triazolam, 1-hydroxymethyltriazolam, and 4-hydroxytriazolam were identified in the black hair shafts, whereas only triazolam was detected in the hair roots and the white hair shafts. This is the first report on the detection of triazolam and its metabolites in human hairs.

Aged↗
PubMed2001

Determination of rat hepatocellular glutathione by reversed-phase liquid chromatography with fluorescence detection and cytotoxicity evaluation of environmental pollutants based on the concentration change.

Three methods for the determination of rat hepatocellular thiols by high-performance liquid chromatography (HPLC) with fluorescence (FL) detection have been developed. The thiols in the cells were tagged with three fluorogenic reagents, SBD-F, ABD-F and DBD-F. These reagents could permeate into cells and effectively reacted with thiols to produce highly fluorescent derivatives. These derivatives fluoresced in the long wavelength region at around 530 nm (excitation at around 380 nm). The five biological thiols tagged were perfectly separated by reversed-phase liquid chromatography and were sensitively and selectively detected without any interference from endogenous substanaces. The main thiol in the cells was reduced GSH and the concentration was at the mM level. The proposed procedures were applied to the determination of hepatocellular GSH after treatment of environmental pollutants such as volatile organic compounds (VOC) and endocrine disrupting chemicals (EDC). From the comparison of intracellular GSH concentration, the test compounds were classified into four groups: compounds of strong depletion (eg triphenyltin chloride, hexachlorocyclohexene, nonylphenol, bromoacetic acid, 4-chlorobenzyl chloride and 1,3-dichloropropene), slight decrease (eg bisphenol A, benzo[a]pylene, carbon tetrachloride and benzene), slight increase (eg bromoform and toluene), and no effect (eg 1,1,1-trichloroethane, 1,1,2-trichloroethane and 1,2-dichloroethane). Although the decrease of GSH concentration does not reflect the cytotoxicity of chemicals, the proposed procedure utilizing isolated rat hepatpcytes seems to be useful for investigating the bioactivation of VOC, and EDC, etc.

Animals↗
PubMed2001

R(-)-4-(3-Isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole, a fluorescent chiral tagging reagent: sensitive resolution of chiral amines and amino acids by reversed-phase liquid chromatography.

The usefulness of R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole [R(-)-DBD-PyNCS], a fluorescent chiral tagging reagent, for the determination of racemic amines and amino acids, was studied. The reagent reacted with beta-blockers selected as representative secondary amines to produce corresponding fluorescent diastereomers (excitation at 460 nm and emission at 550 nm). The yields of the derivatization reaction were dependent on the stereostructure arround the NH group in beta-blockers. The resulting diastereomers were completely separated with single chromatographic run using linear gradient elutions by reversed-phase chromatography. R(-)-DBD-PyNCS was also applied to the determination of DL-amino acid, considered to be one of the primary amines, in human urine and foodstuffs. DL-amino acids tested equally reacted with the reagent, and the thiocarbamoyl derivatives were separated with an ODS column. The epimerization during the derivatization reaction was negligible judging from the resolution of opposite diastereomers on the chromatogram. The occurence of D-amino acids (D-Ala, D-Ser, D-Asp and/or D-Glu) was identified in the samples tested. The structures and the purities were elucidated with on-line HPLC-MS. The chiral reagent possessing an isothiocyanate group (-NCS) in the structure seems to be applicable to continuous sequential analysis of peptides containing D-amino acids. The thiocarbamoyl derivatives obtained from the reaction with DL-amino acids were converted to thiohydantoins via thiazolinones in acidic medium. The thiohydantoins produced from acidic, basic, neutral, hydroxyl and aromatic amino acids were completely separated with isocratic elutions using acidic mobile phase containing 0.1% TFA. The separations were sufficient for the identification of DL-amino acid in peptide sequences. Although the epimerization during the conversion reaction to thiohydantoins was not avoidable, the descrimination of D- and L-configuration was demonstrated with some commercially available peptides such as beta-lipotropin and [D-Ala2]-deltorphin II. The Edman degaradation method using R(-)-DBD-PyNCS was also adopted to autoanlaysis by gas-phase sequencer. The separation and the detection (UV 254 nm) conditions of the derivatives were used without any change from those for the Edman degradation method using PITC as the tagging reagent. The three DL-amino acid residues (Tyr, Ala and Gly) in [L-Ala2]-leucine-enkephalin and [D-Ala2]-leucine-enkephalin were perfectly identidied with the autoanalysis.

Amines↗
PubMed2001

Simultaneous determination of morphine and its glucuronides in rat hair and rat plasma by reversed-phase liquid chromatography with electrospray ionization mass spectrometry.

The simultaneous determination of morphine and the glucuronide metabolites [morphine-3-beta-D-glucuronide (M3G) and morphine-6-beta-D-glucuronide (M6G)] in rat hair and rat plasma was carried out using reversed-phase high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS). The chromatographic separation of the analytes was achieved using a semi-micro-HPLC column (3 microm particle size; 100 x 2.0 mm id) by gradient elution with 50 mM ammonium acetate and acetonitrile as eluents. After separation, morphine and the glucuronides were determined by selected ion monitoring (SIM) of ESI-MS using the quasi-molecular ions [M + H]+ at m/z = 286 and 462, respectively. The calibration curves were linear between the concentration of the analytes and the deuterium-labelled morphine (M-d3) selected as internal standard. The method was applied for the determination of the incorporation of morphine and the glucuronides into the hair shafts and hair roots of Dark Agouti rats after single intraperitoneal administration of morphine hydrochloride. Plasma concentrations of morphine and glucuronides were simultaneously determined after administration. Morphine and M3G were detected in the hair shafts and the hair roots. The concentrations of M3G in the hair root were lower than those of morphine in all sampling periods. In contrast, M3G concentrations in plasma were relatively higher at each sampling time. Small quantities of M6G were also identified in the plasma up to 4 h after administration. The concentration difference between the hair root and plasma seems to be due to the incorporation ratio of morphine and glucuronide into hair. As M3G was also identified in the hair shaft 1 week after administration, the incorporation of glucuronide metabolites into hair is obvious. This is the first report of the identification of morphine glucuronide in hair samples without the use of acid hydrolysis or enzyme digestion.

Animals↗
PubMed1999

Separation of 17 DL-amino acids and chiral sequential analysis of peptides by reversed-phase liquid chromatography after labeling with R(-)-4- (3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole.

Seventeen DL-amino acids labeled with a fluorescent chiral labeling reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazole (R(-)-DBD-PyNCS), were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). The reagent reacted with amino functional group in dl-amino acids under basic medium. The thiocarbamoyl derivatives were converted to thiohydantoin via thiazolinone in trifluoroacetic acid (TFA) solution. The epimerization ratios during the reaction of the cyclization were less than 37% in all dl-amino acids tested. The resulting thiohydantoin derivatives of individual dl-amino acids were completely separated with isocratic elutions using acidic mobile phase involving 0.1% TFA. The separations of the thiohydantoins yielded from acidic, basic, neutral, hydroxyl, and aromatic amino acids were good enough for the identification of dl-amino acid. The method using the reagent was adopted to identification of dl-amino acid sequences in eight peptides. The separation and identification of the thiohydantoin derivatives liberated from the peptides labeled were performed by the isocratic elutions. The applicability of the proposed procedure to sequential analysis of peptide was demonstrated with [D-Ala(2)]-leucine enkephalin, [D-Ala(2)]-deltorphin II, d-Phe-Met-Arg-Phe-amide, and Phe-D-Met-Arg-Phe-amide. D-Ala, D-Phe, and D-Met in the peptides were positively identified with the proposed procedures. [L-Ala(2)]-leucine enkephalin, beta-lipotropin, Asp-Ser-Asp-Pro-Arg, and Pro-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-amide were also analyzed as the references without D-amino acid.

Amino Acid Sequence↗
PubMed1999

Determination of D-amino acids labeled with fluorescent chiral reagents, R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles, in biological and food samples by liquid chromatography.

D-Amino acids in food and biological samples labeled with R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles (DBD-PyNCS) were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). DL-Amino acids were efficiently labeled at 55 degrees C for 20 min in basic medium. The resulting thiocarbamoyl-amino acids were resolved by an isocratic elution using water:30% methanol in acetonitrile (72:28) containing 0.1% trifluoracetic acid as mobile phase for hydrophilic amino acids and gradient elutions using sodium acetate buffer (pH 5. 2)/acetonitrile as gradient solvent mixture for hydrophobic amino acids, respectively. The detection limits (S/N = 3) of DL-amino acids tested were in the range of 0.16-0.75 pmol. The proposed method was applied to determine the D-amino acid(s) in milk, cream, fermented dairy products (yogurt and yakult), tomato products (juice, puree, and catchup), fermented beverages (beer and red wine), and human urine. The existence of D-amino acid(s) was demonstrated in all the samples tested. Furthermore, the identification of the D-amino acid(s) was performed using both isomers of DBD-PyNCS and by on-line HPLC-electrospray ionization-MS.

Amino Acids↗
PubMed1999

Ion exchange chromatography of aluminum using 3-carboxy-2-naphthylamine-N,N-diacetic acid as a fluorescent post-column chelating reagent.

Ion exchange chromatography of aluminum ion using 3-carboxy-2-naphthylamine-N,N-diacetic acid (CNDA) as a fluorescent post-column chelating reagent was studied. The solution containing ammonium chloride and hydrochloric acid was used for the eluent, and acetate buffer solution containing CNDA was used for the post column chelating reagent. The peak of aluminum was separated from that of calcium, magnesium and zinc, and the chromatogram was not affected by copper(II) and iron(III). The calibration curve gave linear plots with a range of 0.0027-0.54 ppm aluminum, the regression coefficient of correlation (r2) was 1.000, and the detection limit (S/N = 3) was 0.3 ppb, indicating that the method could determine aluminum with high sensitivity. It was demonstrated that CNDA is a useful metallofluorescent reagent for aluminum. This method has been successfully applied to the determination of aluminum in some tea drinks.

Aluminum↗
PubMed1999

Determination of 4-hydroxy-2-nonenal in primary rat hepatocyte cultures by liquid chromatography with laser induced fluorescence detection.

An HPLC (high performance liquid chromatography) method with laser induced fluorescence (LIF) detection is described for the determination of 4-hydroxy-2-nonenal (HNE) formed from lipid peroxidation in rat hepatocytes. Carbonyl compounds were fluorescently labelled by incubating the hepatocyte samples with a tagging reagent, 4-(2-carbazoylpyrrolidin-1-yl)-7-nitro-2,1,3-benzoxadiazole (NBD-ProCZ), at 60 degrees C for 10 min. The hydrazone derivatives were extracted with a C18 solid phase extraction (SPE) cartridge and separated on a reversed-phase HPLC column. The detection limit was 2.5 fmol or 0.5 nM (5 microL injection) of HNE in the cell homogenate. Method precision (C.V.) was 5% at the 5 nM level. The method has been used to determine free HNE in rat hepatocyte samples treated with several pro-oxidant toxins. A significant HNE increase (from 4 to 27.6 pmol/10(6) cells) was observed with the samples treated by allyl alcohol. The results were in accordance with those for malondialdehyde formation as measured by a thiobarbituric acid (TBA) assay.

Aldehydes↗
PubMed1998

High-performance liquid chromatographic determination of erdosteine and its optical active metabolite utilizing a fluorescent chiral tagging reagent, R-(-)-4-(N,N-dimethylamiosulfonyl)-7-(3-aminopyrrolidin-1-yl)-2 ,1,3-benzoxadiazole.

Chiral separation of racemic M1 metabolized from erdosteine was investigated by reversed-phase chromatography. The sensitive determination of M1 and erdosteine with UV detection was difficult because of their low absorptivity in the effective wavelength region. To improve the sensitivity and separatability, one thiol and two carboxyl groups in the M1 structure were labelled with DBD-F and R-(-)-DBD-APy, respectively. Non-fluorescent DBD-F quantitatively reacted with thiol in M1 at room temperature for 30 min in borate buffer (pH 9.3) to produce the fluorescent derivative. On the other hand, the labelling of two carboxyls was carried out with a chiral fluorescent reagent, R-(-)-DBD-APy, in acetonitrile containing DPPA. The derivatives corresponding to a pair of the enantiomers were completely separated with water-acetonitrile containing 0.1% TFA as the mobile phase by an ODS column. Erdosteine with a carboxyl group was also labelled with R-(-)-DBD-APy and separated together with M1 derivatives. The detection limits (S/N=3) of erdosteine and M1 were 0.37 and 0.22 pmol, respectively. The proposed derivatization and separation methods were applied to simultaneous determination of racemic M1 and erdosteine in rat plasma after administration of erdosteine. The amounts of both enantiomers of M1 were essentially the same in oral and intravenous administrations. In contrast, total amounts (reduced-form and oxidized-form) of S-(-)-M1 in rat plasma were higher than those of R(+)-M1 in both administrations.

Animals↗
PubMed1998

Total resolution of 17 DL-amino acids labelled with a fluorescent chiral reagent, R(-)-4-(3-isothiocyanatopyrrolidin-1-y1)-7-(N,N-dimethylaminosulfonyl)- 2,1,3-benzoxadiazole, by high-performance liquid chromatography.

Total resolution of 17 DL-amino acids after derivatization with a fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfony l)-2,1,3- benzoxadiazole [R(-)-DBD-PyNCS], was studied by reversed-phase liquid chromatography. The reaction of the reagent with amino acids proceeds effectively at 55 degrees C for 20 min in the presence of 1% TEA to produce the corresponding fluorescent diastereomers (excitation at 460 nm, emission at 550 nm). Each pair of the resulting derivatives was efficiently separated with water-acetonitrile containing 1% acetic acid as the mobile phase. Peak resolution was in the range of 0.92 (DL-Arg)-9.8 (DL-Cys). Although mutual separation of some DL-amino acids was possible using the elution solvent, simultaneous resolution of 17 DL-amino acids was difficult with a single chromatographic run, even if some gradient elutions were adopted. Therefore, both gradient and isocratic elution systems were used for total resolution of the DL-amino acids. Thus, 17 DL-amino acids were well resolved by a gradient and an isocratic elution systems. The proposed derivatization and elution methods were applied to the determination of DL-amino acids in yogurt. The results showed that some of the L-amino acids, i.e., Glu, Asp, Ser, Gly, Ala, Thr, Pro, Lys, Phe and Met, were found in the methanol extracts of yogurt. On the other hand, the D-amino acids that were identified in the extracts were D-Glu, D-Asp and D-Ala, and the mean % to each L-amino acid were 11.9% (D-Glu), 27.6% (D-Asp) and 56.7% (D-Ala), respectively.

Amino Acids↗
PubMed1998

Studies of aloe. VI. Cathartic effect of isobarbaloin.

The cathartic effect of isobarbaloin, a stereoisomer of barbaloin (compound principally responsible for the cathartic activity of Aloe), was examined in male rats by oral administration. Individual differences in sensitivity in the laxative activity of isobarbaloin and barbaloin was not found. The cathartic activity (ED50) of isobarbaloin in barbaloin positive rats was 19.2 mg/kg, nearly equal to that of barbaloin (19.5 mg/kg). Also, isobarbaloin administered orally was demonstrated to decompose to aloe-emodin-9-anthrone (active metabolite of barbaloin) as well as to barbaloin. Therefore, it is considered that the mechanism underlying the cathartic effect of isobarbaloin is the same as that of barbaloin.

Administration, Oral↗
PubMed1997

Hepatotoxicity of diisopropyl ester of malonic acid and chloromalonic acids, disinfection by-products of the fungicide isoprothiolane.

The fungicide isoprothiolane (diisopropyl 1,3-dithiolane-2-ylidenemalonate) decomposes to the diisopropyl esters of malonic acid (DM), chloromalonic acid (DCM) and dichloromalonic acid (DDCM) upon aqueous chlorination. In this study, the cytotoxicity of these compounds was examined using rat hepatocytes cultured on Matrigel. DCM and DDCM caused hepatocellular death at concentrations > 0.5 mM, while DM had no effect on the cell viability even at the maximum concentration examined (4 mM). Significant lipid peroxidation, measured as 2-thiobarbituric acid reactive substances, was observed in both DCM- and DDCM-treated hepatocyte cultures, and was significantly enhanced by pretreatment with 0.1 mM bis(p-nitrophenyl)phosphate (BNPP), a carboxylesterase inhibitor. When both BNPP and SKF-525A, a cytochrome P450 inhibitor, were present in the medium, DCM-induced cytotoxicity and lipid peroxidation were significantly suppressed compared to cultures with BNPP-treatment alone. By contrast, the DDCM-induced cytotoxicity was not affected by the combined pretreatment of SKF-525A and BNPP. These results indicate that DCM is metabolically activated by cytochrome P450 in an ester form, while DDCM is activated by a mechanism other than one involving cytochrome P450. To further elucidate the cytochrome P450 isozyme involved in the metabolic activation of DCM, microsomal lipid peroxidation was studied in vitro using microsomes from rats treated with beta-naphthoflavone, musk xylene, phenobarbital, pyrazole, or dexamethasone. Among these preparations, the microsomes from dexamethasone-treated rats showed the most extensive lipid peroxidation in the presence of DCM, and the lipid peroxidation was enhanced by BNPP as observed in hepatocyte cultures. These findings suggest the possible involvement of cytochrome P450 3A in the metabolic activation of DCM.

Animals↗
PubMed1997

Enantioseparation of beta-blockers labelled with a chiral fluorescent reagent, R (-)-DBD-PyNCS, by reversed-phase liquid chromatography.

A fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfony l)-2,1, 3-benzoxadiazole [R(-)-DBD-PyNCS], has been used for the liquid chromatographic resolution of racemic pairs of beta-blockers. The reagents reacts with beta-blockers at 65 degrees C for 90 min in aqueous acetonitrile containing 0.05% triethylamine to produce the corresponding pair of diastereomers. No racemization occurs during the tagging reaction under these conditions. From results of the time-course study of oxprenolol the reactivities of the enantiomers of beta-blockers with R(-)-DBD-PyNCS are comparable. The optimum excitation and emission wavelengths of the resulting derivatives were ca. 460 and 550 nm, respectively. The derivatives of beta-blockers were efficiently resolved by a reversed-phase column with water-acetonitrile containing 0.1% trifluoroacetic acid as the eluent. The resolution (Rs) values of the diastereomers derived from 10 beta-blockers were in the range of 1.54-4.80. The Rs value for timolol was 0.643. The detection limits (signal-to-noise ratio of 2) were one or two orders of magnitude lower with beta-blockers having the iso-propylamino structure (15-300 fmol) than with those having the tert-butylamino structure (1.25-8.00 pmol). The proposed procedure was applied to the determination of R(+)- and S(-)-propranolol in rat plasma and saliva after oral administration of R(+)-propranolol hydrochloride or S(-)-propranolol hydrochloride.

Administration, Oral↗
PubMed1995

Use of derivatization to improve the chromatographic properties and detection selectivity of physiologically important carboxylic acids.

In this review, tagging techniques with reagents used for ultraviolet-visible (UV-Vis), fluorescent (FL), chemiluminescent (CL) and electrochemical detection (ED) for higher carboxylic acids in HPLC are evaluated in terms of the tagging reactions, handling, flexibility, stability of the reagents and the corresponding derivatives, sensitivity and selectivity. Emphasis is given to the applications of these tagging techniques to biologically important carboxylic acids of relatively high molecular mass including free fatty acids, prostaglandins, leukotrienes and thromboxanes etc. Some typical examples are described. Although RIA and GC-MS are powerful techniques for the highly sensitive determination of carboxylic acids, tagging for these techniques is not included in this review because recent progress in tagging methods has been mainly concerned with HPLC detection.

Animals↗
PubMed1995

Induction of rat liver drug-metabolizing enzymes by tetrachloroethylene.

The effect of tetrachloroethylene on Phase I and II drug-metabolizing enzymes in rat liver was examined. Rats were treated orally with tetrachloroethylene daily for five days, at doses of 125, 250, 500, 1,000 and 2,000 mg/kg. The higher doses (> 500 mg/kg) of tetrachloroethylene induced the hepatic microsomal 7-pentoxyresorufin O-depentylase and 7-benzyloxyresorufin O-debenzylase activities associated with the CYP2B subfamily. 7-ethoxyresorufin O-deethylase activity was also induced about 2-fold compared with that of control rats at 500, 1,000, and 2,000 mg/kg dose levels of tetrachloroethylene. However, 7-ethoxycoumarin O-deethylase and 7-methoxyresorufin O-demethylase activities were increased significantly at only the 1,000 mg/kg dose level of tetrachloroethylene (1.4- and 1.5-fold). Although other cytochrome P450-mediated monooxygenase activities such as nitrosodimethylamine N-demethylase, aminopyrine N-demethylase and erythromycin N-demethylase were also induced by tetrachloroethylene, the relative induction to control activity was lower than those of 7-pentoxyresorufin O-depentylase and 7-benzyloxyresorufin O-debenzylase. Western immunoblotting showed that the levels of CYP2B1 and CYP2B2 proteins in liver microsomes were increased at doses of 1,000 and 2,000 mg/kg of tetrachloroethylene. In addition to cytochrome P450-mediated monooxygenases, there was significant induction of the Phase II drug-metabolizing enzymes, DT-diaphorase, glutathione S-transferase activities towards 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene, and UDP-glucuronyltransferase activities towards 4-nitrophenol and 7-hydroxycoumarin. The results indicate that tetrachloroethylene induces both Phase I (CYP2B-mediated monooxygenase) and Phase II drug-metabolizing enzymes (DT-diaphorase, glutathione S-transferase and UDP-glucuronyltransferase) in the rat liver.

Administration, Oral↗
PubMed1994

Sensitive determination of N-terminal prolyl peptides by high-performance liquid chromatography with laser-induced fluorescence detection.

Short-chain peptides with an N-terminal proline (Pro-Gly, Pro-Ile, Pro-Gly-Gly, Pro-Leu-Gly-NH2, and Pro-Thr-Pro-Ser-NH2, etc.) were determined by HPLC with laser-induced fluorescence (LIF) detection. The peptides were quantitatively labelled with 4-(N,N-dimethylaminosulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) at 50 degrees C after 1 h in a 0.1 M borax (pH 9.3)-acetonitrile mixture. The rate of reaction decreases inversely with the molecular weight of the peptides. The mean value of fluorescent emission of the resulting DBD-peptides and DBD-peptide amides was 573 nm (excitation, 453 nm). The proline peptides, including bioactive peptides such as Pro-Leu-Gly-NH2 (release inhibitor of melanocyte-stimulating hormone), Pro-Thr-Pro-Ser-NH2 (IgA1 proteinase inhibitor) and Pro-Asp-Val-Asp-His-Val- Phe-Leu-Arg-Phe-NH2 [FMRF amide-like (Phe-Met-Arg-Phe-NH2) neuropeptide], were well separated by reversed-phase HPLC with water-acetonitrile containing 0.1% trifluoroacetic acid (TFA). The acetonitrile concentration in the mobile phase had a profound effect upon the retention times, and the capacity factors (k') were dependent on the hydrophobicity of the peptides. The structure of DBD-Pro-Leu-Gly-NH2 was identified by LC-atmospheric pressure chemical ionization MS. The chromatographic detection limits (S/N = 2) of the peptides with a 15-mW argon-ion laser at 488 nm were in the 6-28 fmol range. The detection limits were improved to 2-5 fmol with a microbore column. The detectability was two orders of magnitude higher than with a conventional fluorescence detector using xenon arc lamp.

Amino Acid Sequence↗
PubMed1994

The effect of 1,2,3,4-tetrachlorodibenzo-p-dioxin on drug-metabolizing enzymes in the rat liver.

The effects of 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD) on drug-metabolizing enzymes were studied in male and female rats. 1,2,3,4-TCDD (25, 50, 100 and 200 mumol/kg) was administered by i.p. injection once. Among the cytochrome P-450 (P450)-mediated monooxygenase activities tested, 7-ethoxyresorufin O-deethylase (EROD) activities in both male and female rats, which are associated with CYP1A1, were remarkably induced by all doses of 1,2,3,4-TCDD. The relative induction to each control activity were from 3.0- to 24.5-fold and from 2.2- to 16.5-fold, respectively. Also, 1,2,3,4-TCDD increased other CYP1A-mediated monooxygenase activities such as 7-ethoxycoumarin O-deethylase (ECOD) and 7-methoxyresorufin O-demethylase (MROD) in male and female rats dose-dependently (1.4- to 4.3-fold). Western immunoblotting showed that the levels of CYP1A1 and CYP1A2 proteins in liver microsomes were increased by 1,2,3,4-TCDD. Although the activities of other P450-mediated monooxygenases, namely 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD), aminopyrine N-demethylase (APND) and nitrosodimethylamine N-demethylase (NDAND) in both male and female rats were induced at high doses (> or = 50 mumol/kg) of 1,2,3,4-TCDD, the relative level was low compared with those of the CYP1A-mediated monooxygenase such as EROD, ECOD or MROD. In addition to P450-mediated monooxygenase, there was significant induction in the activities of the Phase II drug-metabolizing enzymes, UDP-glucuronyltransferase (UGT) activities towards 4-nitrophenol (4-NP) and 7-hydroxycoumarin (7-HC) and glutathione S-transferase (GST) towards 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB) and DT-diaphorase. These results indicate that 1,2,3,4-TCDD induces both Phase I (CYP1A-mediated monooxygenase) and Phase II drug-metabolizing enzymes (UGT, GST, DT-diaphorase) in the male and female rat liver, and that the alterations of drug-metabolizing enzyme are characteristic of PCDD toxicity.

Animals↗