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1.
Drug Discov Ther ; 7(5): 172-7, 2013 Oct.
Article in English | MEDLINE | ID: mdl-24270380

ABSTRACT

Glutathione (GSH) is a tri-peptide that plays an important role in protecting cells and tissues against oxidative stress. So far a lot of analytical methods of glutathione have been reported. This brief review presents an overview of the analysis of glutathione in biological samples by high-performance liquid chromatography (HPLC) in recent five years, focusing on the sample pretreatment, derivatization and mass spectrometric detection.


Subject(s)
Chromatography, High Pressure Liquid/methods , Glutathione/analysis , Oxidative Stress , Humans , Mass Spectrometry/methods
2.
Drug Discov Ther ; 7(1): 9-17, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23524938

ABSTRACT

Liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) has been frequently utilized for the sensitive and selective determination of the trace level compounds in biological samples. In LC/ESI-MS/MS, chemical derivatization is sometimes used to enhance the detection sensitivity of the analytes. This review presents an overview of the derivatization reagents in LC/ESI-MS/MS that have been applied to the low molecular weight compounds in recent five years (2008-2012).


Subject(s)
Chromatography, Liquid/methods , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , Hydrophobic and Hydrophilic Interactions , Indicators and Reagents/chemistry , Molecular Weight , Sensitivity and Specificity
3.
Br J Pharmacol ; 164(1): 83-92, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21418186

ABSTRACT

BACKGROUND AND PURPOSE: Lymphangioleiomyomatosis (LAM) is characterized by the abnormal growth of smooth muscle-like cells (LAM cells) and cystic destruction of the lung parenchyma. LAM cell-derived matrix metalloproteinases (MMPs) are thought to play a prominent role in the tissue destruction. The aim of this study was to determine whether doxycycline, a known MMP inhibitor, can inhibit LAM cell proliferation or mitochondrial function and/or modulate MMPs and their tissue inhibitors (TIMPs). EXPERIMENTAL APPROACH: Wild-type and tuberous sclerosis complex-2 (TSC2)-null mouse embryonic fibroblasts (MEFs) were cultured in DMEM containing 10% fetal bovine serum (FBS). Human LAM cells were derived from the lungs of LAM patients and airway smooth muscle cells from control subjects. Cells were stimulated with FBS with or without doxycycline for up to 9 days. Proliferation was assessed by manual cell counts and MTT assay, MMP production by zymography and ELISA, and TIMP production using elisa. KEY RESULTS: Doxycycline did not change FBS-induced proliferation in MEFs or human cells. However, doxycycline did reduce metabolic activity of both wild-type and TSC2-null MEFs and LAM cells, but had no effect on control cells. Furthermore, doxycycline reduced MMP-2 from MEFs and decreased active-MMP-2 from LAM cells but had no effect on TIMP-1 and TIMP-2 from human LAM cells. CONCLUSIONS AND IMPLICATIONS: Doxycycline decreased MMP levels and cell metabolic activity, which raises the possibility of therapeutic efficacy in LAM.


Subject(s)
Doxycycline/pharmacology , Lymphangioleiomyomatosis/drug therapy , Lymphangioleiomyomatosis/metabolism , Matrix Metalloproteinase Inhibitors , Tumor Suppressor Proteins/deficiency , Animals , Cell Proliferation/drug effects , Cells, Cultured , Female , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/enzymology , Fibroblasts/metabolism , Humans , Lymphangioleiomyomatosis/pathology , Matrix Metalloproteinase 2/biosynthesis , Matrix Metalloproteinase 2/metabolism , Mice , Middle Aged , Mitochondria/drug effects , Myocytes, Smooth Muscle/metabolism , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tissue Inhibitor of Metalloproteinase-2/metabolism , Tuberous Sclerosis Complex 2 Protein , Tumor Cells, Cultured , Tumor Suppressor Proteins/metabolism
4.
Am J Med Genet A ; 149A(6): 1219-23, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19449432

ABSTRACT

Peroxisomal biogenesis disorders represent a group of genetically heterogeneous conditions that have in common failure of proper peroxisomal assembly. Clinically, they are characterized by a spectrum of dysmorphia, neurological, liver, and other organ involvement. To date, mutations in 13 PEX genes encoding peroxins have been identified in patients with peroxisomal biogenesis disorders. Mutations in PEX13, which encodes peroxisomal membrane protein PEX13, are among the least common causes of peroxisomal biogenesis disorders with only three mutations reported so far. Here, we report on two infants whose clinical and biochemical profile was consistent with classical Zellweger syndrome and whose complementation analysis assigned them both to group H of peroxisomal biogenesis disorders. We show that they harbor two novel mutations in PEX13. One patient had a genomic rearrangement resulting in a 147 kb deletion that spans the whole of PEX13, while the other had an out-of-frame deletion of 14 bp. This represents the first report of a PEX13 deletion and suggests that further work is needed to examine the frequency of PEX13 mutations among Arab patients with peroxisomal biogenesis disorders.


Subject(s)
Frameshift Mutation , Membrane Proteins/deficiency , Sequence Deletion , Zellweger Syndrome/genetics , Base Sequence , Fibroblasts/metabolism , Fibroblasts/pathology , Gene Rearrangement , Genetic Complementation Test , Humans , Infant , Membrane Proteins/genetics , Molecular Sequence Data , Zellweger Syndrome/metabolism
5.
Drug Discov Ther ; 1(2): 108-18, 2007 Oct.
Article in English | MEDLINE | ID: mdl-22504396

ABSTRACT

Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESIMS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is frequently used to enhance the MS/MS detectability. The derivatization improves the separation and ionization efficiency. Moreover, the generated derivatives give particular product ions by CID (collision induced dissociation), which allow for the sensitive detection. In this review, we present an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS, focusing on the applications involving small molecules in biomatrices.

6.
Biomed Chromatogr ; 15(5): 295-318, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11507712

ABSTRACT

Fluorogenic and fluorescent labeling reagents having a benzofurazan (2,1,3-benzoxadiazole) skeleton such as 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-N,N-dimethylaminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (DBD-F), 4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole (ABD-F), ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H), 4-N,N-dimethylaminosulfonyl-7-hydrazino-2,1,3-benzoxadiazole (DBD-H), 4-nitro-7-N-piperazino-2,1,3-benzoxadiazole (NBD-PZ), 4-N,N-dimethylaminosulfonyl-7-N-piperazino-2,1,3-benzoxadiazole (DBD-PZ), 4-(N-chloroformylmethyl-N-methyl)amino-7-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole (DBD-COCl) and 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isothiocyanate (DBD-NCS) are reviewed in terms of synthetic method, reactivity, fluorescence characteristics, sensitivity and application to analytes.


Subject(s)
Benzoxazoles/chemistry , Fluorescent Dyes/chemistry , Sensitivity and Specificity , Spectrometry, Fluorescence
7.
Biomed Chromatogr ; 15(5): 319-27, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11507713

ABSTRACT

D-amino acid-containing peptides with biological activities have been isolated from invertebrates and amphibians, and partial racemization of amino acid residues in mammalian peptides associated with aging and diseases have been discussed. Here, we review the amino acid configuration determination methods in these peptides and recent progress of simultaneous determination method for sequence and configuration of amino acid residues. The applicability of C-terminus sequence analysis and mass spectrometry to configuration determination of amino acids is also discussed.


Subject(s)
Amino Acids/analysis , Peptides/chemistry , Amino Acid Sequence , Animals , Humans , Protein Conformation
8.
Anal Chem ; 73(10): 2165-70, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11393836

ABSTRACT

During the course of our studies of the development of fluorogenic reagents having a 4,7-disubstituted benzofurazan structure, we previously proposed 7-acetylamino-4-mercapto-2,1,3-benzoxadiazole (AABD-SH) as a fluorogenic reagent for carboxylic acids. Since then, progress has made it possible to estimate the fluorescence quantum yields of the 4,7-disubstituted benzofurazan compounds on the basis of the PM3 calculation of their S1-T2 energies. Subsequently, a new fluorogenic reagent, 4-mercapto-7-methylthio-2,1,3-benzoxadiazole (MTBDSH) was designed and synthesized. In the presence of condensation reagents, triphenylphosphine (TPP) and 2,2'-dipyridyl disulfide (DPDS), MTBD-SH readily reacted with n-caprylic acid within 1 min at room temperature. The derivatives of five carboxylic acids (n-caprylic acid, n-capric acid, lauric acid, myristic acid, and palmitic acid) were well-separated on a reversed-phase column and were fluorimetrically detected at 519 nm with excitation at 391 nm. The detection limits (S/N = 3) were 2.4-5.0 fmol. Thus, MTBD-SH had properties that were considered to be superior. For carboxylic acids, itwas superior not only to AABD-SH, but also to many other conventional reagents. The superiority was examined in terms of its reactivity and sensitivity and the avoidance of interfering peaks that were derived from the reagent itself or degradation products in the chromatogram.


Subject(s)
Carboxylic Acids/analysis , Fluorescent Dyes/chemistry , Oxadiazoles/chemistry , Chromatography, High Pressure Liquid , Fluorescence
9.
Cancer Res ; 61(11): 4365-70, 2001 Jun 01.
Article in English | MEDLINE | ID: mdl-11389061

ABSTRACT

The mechanism of hepatocarcinogenesis in hepatitis C virus (HCV) infection is still undefined. One possibility is the involvement of oxidative stress, which can produce genetic mutations as well as gross chromosomal alterations and contribute to cancer development. We recently showed that after a long period, the core protein of HCV induces hepatocellular carcinoma (HCC) in transgenic mice with marked hepatic steatosis but without inflammation, indicating a direct involvement of HCV in hepatocarcinogenesis. To elucidate the biochemical events before the development of HCC, we examined several parameters of oxidative stress and redox homeostasis in a mouse model of HCV-associated HCC. For young mice ages 3-12 months, there was no significant difference in the levels of hydroperoxides of phosphatidylcholine (PCOOH) and phosphatidylethanolamine in liver tissue homogenates between transgenic and nontransgenic control mice. In contrast, the PCOOH level was increased by 180% in old core gene transgenic mice > 16 months old. Concurrently, there was a significant increase in the catalase activity, and there were decreases in the levels of total and reduced glutathione in the same mice. A direct in situ determination by chemiluminescence revealed an increase in hydroperoxide products by 170% even in young transgenic mice, suggesting that hydroperoxides were overproduced but immediately removed by an activated scavenger system in young mice. Electron microscopy revealed lipofuscin granules, secondary lysosomes carrying various cytoplasmic organelles, and disruption of the double membrane structure of mitochondria, and PCR analysis disclosed a deletion in mitochondrial DNA. Interestingly, alcohol caused a marked increase in the PCOOH level in transgenic mice, suggesting synergism between alcohol and HCV in hepatocarcinogenesis. The HCV core protein thus alters the oxidant/antioxidant state in the liver in the absence of inflammation and may thereby contribute to or facilitate, at least in part, the development of HCC in HCV infection.


Subject(s)
Hepacivirus , Hepatitis C, Chronic/complications , Hepatitis C, Chronic/metabolism , Liver Neoplasms, Experimental/metabolism , Liver Neoplasms, Experimental/virology , Oxidative Stress , Animals , Catalase/metabolism , DNA Damage , DNA, Mitochondrial/metabolism , Glutathione/metabolism , Hepacivirus/genetics , Hepacivirus/metabolism , Hepatitis C, Chronic/virology , Hydrogen Peroxide/metabolism , Inflammation/metabolism , Inflammation/virology , Lipid Peroxidation , Liver/metabolism , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria, Liver/metabolism , Mitochondria, Liver/pathology , Reactive Oxygen Species/metabolism , Viral Core Proteins/biosynthesis , Viral Core Proteins/genetics
10.
J Pharmacol Exp Ther ; 294(2): 510-5, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10900226

ABSTRACT

It has been a desire to develop orally effective therapeutic agents that restore the liver function in chronic injury. Here we demonstrated that trans-4-L-hydroxyprolyl-L-serine (JBP923) and cyclo-trans-4-L-hydroxyprolyl-L-serine (JBP485), which was previously isolated from hydrolysate of human placenta, exhibit potent antihepatitis activity after their oral administration. The increase in bilirubin concentration and activities of liver cytosolic enzymes in serum caused by alpha-naphthylisothiocyanate intoxication in rats were significantly countered both after i.v. and oral administration of these dipeptides, whereas glycyrrhizin, which has been used in the treatment of chronic hepatitis, is active only after its i.v. administration. Antihepatitis activity of dipeptides results, at least partially, from their direct effect on hepatocytes because glutamic-oxaloacetic transaminase and lactate dehydrogenase activities in the medium of hepatotoxin-exposed primary cultured hepatocytes were reduced by these compounds. When comparing the plasma concentration-time profile of JBP923 after its i.v., oral, and portal vein injection, it is suggested that JBP923 is almost completely absorbed from gastrointestinal lumen, and hepatic first-pass removal is minor. JBP923 inhibited the proton-dependent transport of glycylsarcosine in brush-border membrane vesicles, suggesting that peptide transport system(s) may recognize JBP923. Thus, these dipeptides are potent antihepatitis reagents that are still active after oral administration and may be useful for clinical applications.


Subject(s)
Chemical and Drug Induced Liver Injury/drug therapy , Dipeptides/pharmacology , Liver/drug effects , Peptides, Cyclic/pharmacology , 1-Naphthylisothiocyanate , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Carbon Radioisotopes , Cells, Cultured , Chemical and Drug Induced Liver Injury/etiology , Cytosol/enzymology , Dipeptides/blood , Dipeptides/pharmacokinetics , Gastric Mucosa/metabolism , Glycyrrhizic Acid/pharmacology , Intestinal Absorption , Intestinal Mucosa/metabolism , Intestines/drug effects , Liver/enzymology , Male , Microvilli/drug effects , Microvilli/metabolism , Peptides, Cyclic/blood , Peptides, Cyclic/pharmacokinetics , Rabbits , Rats , Rats, Wistar
11.
Jpn J Pharmacol ; 83(1): 39-45, 2000 May.
Article in English | MEDLINE | ID: mdl-10887939

ABSTRACT

We previously reported a highly sensitive chemiluminescence high-performance liquid chromatographic method to determine catecholamines in plasma. In this study, we employed this method to measure the cardiac function and plasma norepinephrine (NE) concentration in conscious rats. Benidipine, 1,4-dihydropyridine calcium antagonist (4 mg/kg), and beta-blocker (propranolol, 30 mg/kg) were administered orally to conscious spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats, and blood pressure, heart rate and plasma NE levels were measured. Plasma NE concentration was used as an index of sympathetic nervous system activity in conscious rats. The basal plasma NE levels were significantly higher in SHRs than in WKY rats (P<0.05), indicating the activity of the basal sympathetic nervous system in SHRs was elevated. The sensitivity of the baroreflex-mediated sympathetic nervous response was reduced in SHRs as compared to that in WKY rats. The concomitant administration of benidipine and a beta-blocker decreased heart rate without affecting the baroreflex-mediated sympathetic nervous response, indicating that propranolol might suppress mainly the cardiac beta-adrenoceptor. The present study suggested the high activity of the basal sympathetic nervous system and the reduced response of the baroreflex-mediated sympathetic nervous system in SHRs compared to WKY rats in the conscious condition.


Subject(s)
Hypertension/physiopathology , Norepinephrine/blood , Sympathetic Nervous System/physiopathology , Animals , Blood Pressure/drug effects , Dihydropyridines/administration & dosage , Dihydropyridines/therapeutic use , Heart Rate/drug effects , Male , Propranolol/administration & dosage , Propranolol/therapeutic use , Rats , Rats, Inbred SHR , Rats, Inbred WKY
12.
Analyst ; 125(2): 293-6, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10820891

ABSTRACT

A fully automated and highly sensitive method with a semi-microcolumn liquid chromatography system for the determination of rat plasma catecholamines (CAs) was developed. Automated on-line extraction of CAs in diluted plasma using a precolumn packed with strong acidic cation exchange resin was coupled with separation of CAs on a semi-microcolumn (250 x 1.5 mm id). fluorogenic derivatization with ethylenediamine and finally postcolumn peroxyoxalate chemiluminescence detection utilizing bis[2-(3,6,9-trioxadecanyloxycarbonyl)-4-nitrophenyl]oxalate (TDPO) and hydrogen peroxide. The detection limits were 0.91, 0.36 and 1.1 fmol for norepinephrine (noradrenaline), epinephrine (adrenaline) and dopamine, respectively, at a signal-to-noise ratio of 3. A good linearity of the calibration curve for each CA was observed in the range of 5.0 to 500 fmol for each CA using N-methyldopamine (N-MeDA) as an internal standard. The RSD for the proposed method (n = 5) were 3.7-9.5% for the intra-day assay and 6.6-10.0% for the inter-day assay. The volume of rat plasma required for the determination of CAs was 10 microliters.


Subject(s)
Catecholamines/blood , Animals , Chromatography, Liquid/methods , Luminescent Measurements , Male , Rats , Rats, Sprague-Dawley
13.
Anal Chem ; 72(4): 732-9, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10701257

ABSTRACT

On the basis of the relationship between the fluorescence characteristics of the benzofurazan compounds and the Hammett constants (sigma p), a new fluorescence Edman reagent, 7-methylthio-4-(2,1,3-benzoxadiazolyl) isothiocyanate (MTBD-NCS) was designed and synthesized. MTBD-thiohydantoin (TH)-amino acid derivatives produced by the Edman sequencing method gave fluorescence, whereas other degradation byproducts such as MTBD-thiocarbamoyl (TC)- or carbamoyl (CA)-amino acids did not fluoresce. MTBD-NCS was applicable as an Edman sequencing reagent to the simultaneous determination of both the sequence and D/L-configuration of amino acids in peptides. Boron trifluoride (BF3) and HC1/methanol were adopted as the cyclization/cleavage and conversion reagents to suppress the amino acid residue racemization. The MTBD-TH-amino acids were separated on a reversed-phase column for amino acid sequencing, and their enantiomers were resolved on two types of polysaccharide-based chiral stationary phases for D/L-configuration determination. The method was successfully applied to the sequence and D/L-configuration determination of D-amino acid-containing peptide [D-Ala2]-deltorphin II.


Subject(s)
Fluorescent Dyes/chemical synthesis , Isothiocyanates/chemical synthesis , Oxadiazoles/chemical synthesis , Peptides/chemistry , Sequence Analysis, Protein/methods , Amino Acids/analysis , Chromatography, High Pressure Liquid/methods , Fluorescent Dyes/chemistry , Indicators and Reagents , Isothiocyanates/chemistry , Oxadiazoles/chemistry , Protein Conformation , Stereoisomerism
14.
Biomed Chromatogr ; 14(2): 133-6, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10694709

ABSTRACT

The fluorescent product obtained by the oxidation of 7-N, N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) (DBD)-thiocarbamoyl (TC)-proline with NaNO(2)/H(+) in the modified Edman sequencing procedure was identified as the corresponding thiazolyl compound, N-[(8-dimethylaminosulfonyl)thiazolo[5,4, e]benzo[2,1,3]oxadiazol-5-yl]-L-proline, formed by the attack of the sulfur atom of the thiocarbamoyl group on the benzofurazan skeleton. The reaction mechanism for the formation of the fluorescent compound from DBD-TC primary and secondary amines is also discussed.


Subject(s)
Amino Acids/chemistry , Fluorescent Dyes/chemistry , Sequence Analysis, Protein/methods , Chromatography, High Pressure Liquid/methods , Oxidation-Reduction , Spectrometry, Fluorescence
15.
Biomed Chromatogr ; 13(5): 335-9, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10425023

ABSTRACT

Enantiomeric separation and detection of D,L-aspartic acid (Asp) derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) by capillary electrophoresis (CE) using modified cyclodextrins as chiral selectors was studied. Heptakis(2,3, 6-tri-O-methyl)-beta-cyclodextrin(TM-beta-CD) was most effective for enantiomeric separation of NBD-D,L-Asp with optimum conditions of 30 mM TM-beta-CD in 50 mM phosphate buffer (pH 4.0) and the limit of detection (LOD) attained was 100 nM for each enantiomer. The method proposed in the present study was convenient for both D- and L-Asp determination since the other amino compounds migrated differently and D-Asp in bio-samples such as rat pineal gland and foods was determined with a simple sample pretreatment and a short analysis run time.


Subject(s)
Aspartic Acid/analysis , Animal Feed/analysis , Animals , Cyclodextrins , Electrophoresis, Capillary , Indicators and Reagents , Liliaceae/chemistry , Male , Pineal Gland/chemistry , Rats , Rats, Sprague-Dawley , Stereoisomerism , Wine/analysis
16.
Yakugaku Zasshi ; 119(6): 436-43, 1999 Jun.
Article in Japanese | MEDLINE | ID: mdl-10376003

ABSTRACT

Effects of mouth wash (mouth rinsing and gargling) on the removal of drug residues in both mouth and pharynx after the use of pressurized aerosol metered-dose inhaler (MDI) were studied. The concentration of beclomethasone dipropionate (BM) in mouth wash after a splay of Becotide inhaler was measured by the method using HPLC. The total amount of the removed BM was measured by a sum of the concentrations of BM in 4 or 5 times of mouth washes in the following 4 kinds of methods. In method 1, mouth wash was done with 5 times of water change after a splay of MDI on wetted mouth. In method 2, mouth wash was done with 5 times of change water on dried mouth. In method 3, mouth wash was done with 4 times of change saliva on wetted mouth. In these methods, the actual inhalation of BM was not done. In method 4, mouth wash was done with 5 times of change water after a splay and a inhalation on wetted mouth. The mouth wash procedures removed totally 47.9%, 51.1%, 31.3%, and 33.3% of a splayed amount of BM in each method, respectively. It was required for the removal of 90% of the totally recovered BM to do one time of mouth wash in method 1, two times in method 2, three times in method 3, and two times in method 4, respectively. These data suggest that the mouth wash procedure is shown to have prophylactic benefit for candidiasis induced by steroid delivered by MDI.


Subject(s)
Beclomethasone/analysis , Mouthwashes , Administration, Inhalation , Adult , Aerosols , Beclomethasone/administration & dosage , Beclomethasone/adverse effects , Candidiasis, Oral/prevention & control , Chromatography, High Pressure Liquid , Female , Humans , Male , Mouthwashes/chemistry
17.
Anal Biochem ; 270(2): 257-67, 1999 Jun 01.
Article in English | MEDLINE | ID: mdl-10334843

ABSTRACT

A method for amino acid sequence and D/L configuration identification of peptides by using fluorogenic Edman reagent 7-[(N, N-dimethylamino)sulfonyl]-2,1,3-benzoxadiazol-4-yl isothiocyanate (DBD-NCS) has been developed. This method was based on the Edman degradation principle with some modifications. A peptide or protein was coupled with DBD-NCS under basic conditions and then cyclized/cleaved to produce DBD-thiazolinone (TZ) derivative by BF3, a Lewis acid, which could significantly suppress the amino acid racemization. The liberated DBD-TZ amino acid was hydrolyzed to DBD-thiocarbamoyl (TC) amino acid under a weakly acidic condition and then oxidized by NaNO2/H+ to DBD-carbamoyl (CA) amino acid which was a stable and had a strong fluorescence intensity. The individual DBD-CA amino acids were separated on a reversed-phase high-performance liquid chromatography (RP-HPLC) for amino acid sequencing and their enantiomers were resolved on a chiral stationary-phase HPLC for identifying their D/L configurations. Combination of the two HPLC systems, the amino acid sequence and D/L configuration of peptides could be determined. This method will be useful for searching D-amino-acid-containing peptides in animals.


Subject(s)
Amino Acids/analysis , Amino Acids/chemistry , Fluorescent Dyes , Indicators and Reagents , Isothiocyanates , Oxadiazoles , Amino Acid Sequence , Animals , Chromatography, High Pressure Liquid/methods , Drug Stability , Hydrogen-Ion Concentration , Hydrolysis , Peptides/chemistry , Sequence Analysis/methods , Solvents , Stereoisomerism
18.
Anal Biochem ; 269(2): 386-92, 1999 May 01.
Article in English | MEDLINE | ID: mdl-10222014

ABSTRACT

A highly specific and sensitive automated high-performance liquid chromatographic method for the simultaneous determination of catecholamines (CAs; norepinephrine, epinephrine, and dopamine) and their 3-O-methyl metabolites (normetanephrine, metanephrine, and 3-methoxytyramine) is described. Automated precolumn ion-exchange extraction of diluted plasma is coupled with HPLC separation of CAs and their 3-O-methyl metabolites on an ODS column, postcolumn coulometric oxidation, fluorescence derivatization with ethylenediamine, and finally peroxyoxalate chemiluminescence reaction detection. The detection limits were about 3 fmol for norepinephrine, epinephrine, and dopamine, 5 fmol for normetanephrine, and 10 fmol for metanephrine and 3-methoxytyramine (signal-to-noise ratio of 3). Fifty microliters of rat plasma was used and 4-methoxytyramine was employed as an internal standard. The relative standard deviations for the method (n = 5) were 2.5-7.6% for the intraday assay and 6.3-9.1% for the interday assay. The method was applicable to the determination of normetanephrine and metanephrine in 50 microl of rat plasma.


Subject(s)
Blood Chemical Analysis/methods , Catecholamines/blood , Chromatography, High Pressure Liquid/methods , Animals , Blood Chemical Analysis/instrumentation , Blood Chemical Analysis/statistics & numerical data , Chromatography, High Pressure Liquid/instrumentation , Chromatography, High Pressure Liquid/statistics & numerical data , Fluorescent Dyes , Luminescent Measurements , Male , Metanephrine/blood , Normetanephrine/blood , Oxalates , Oxidation-Reduction , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and Specificity
19.
Biomed Chromatogr ; 13(1): 27-32, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10191940

ABSTRACT

Nicardipine, a dihydropyridine type calcium channel blocker, was infused at two flow-rates into spontaneously hypertensive (SH) and control normotensive Wistar-Kyoto (WKY) rats (young, 6-week-old and adult, 23-week-old, n = 5) under pentobarbital anesthesia, to cause hypotension. Mean arterial blood pressure and the concentrations of plasma amino acids and norepinephrine (NE) were measured before infusion and at each step of the infusion. The reduction in blood pressure caused by nicardipine induced a decrease in plasma L-arginine concentration in both young and adult SH rats, this effect being larger in adult rats. There was no significant change in plasma levels of L-arginine in age-matched WKY rats. The concentration of other amino acids did not change in both rat strains. On the contrary, there was an increase in plasma NE concentration in both SH and WKY rats after infusion with nicardipine. Plasma L-arginine concentration showed a good inverse correlation with the logarithm of plasma NE concentration in SH and WKY rats and the correlation was expressed as Y = -alpha log(X) + m (Y, plasma L-arginine concentration (nmol/mL); X, plasma NE concentration (pmol/mL); alpha, a slope; and m, an intercept). alpha, 43.0 and 4.35 for 23-week-old SH and WKY rats, respectively, and 17.0 and 4.0 for 6-week-old SH and WKY rats, respectively. The present data together with previous data suggest a direct noradrenergic stimulation of the synthesis of nitric oxide (NO) from L-arginine. The findings also indicate an impairment of the L-arginine metabolism or pools in SH rats compared with WKY rats. The deficiency of L-arginine increases with the age of SH rats and could be related to the development and maintenance of hypertension due to inefficient production of NO.


Subject(s)
Arginine/blood , Hypertension/blood , Hypotension/blood , Animals , Antihypertensive Agents/pharmacology , Calcium Channel Blockers/pharmacology , Hypotension/chemically induced , Male , Nicardipine/pharmacology , Norepinephrine/blood , Rats , Rats, Inbred SHR , Rats, Inbred WKY
20.
Anal Chem ; 71(23): 5367-71, 1999 Dec 01.
Article in English | MEDLINE | ID: mdl-21662733

ABSTRACT

During the course of our studies, we found the relationship between the fluorescence characteristics (the fluorescence intensity and the maximum excitation and emission wavelengths) of benzofurazan compounds and the sum and difference of Hammett substituent constants (σp) at the 4- and 7- positions. This prompted us to design a useful fluorogenic derivatization reagent having the benzofurazan skeleton for alcohols along this line of thought. Accordingly, the fluorogenic derivatization reagents, which have no fluorescence themselves, 7-N,N-dimethylaminosulfonyl-4-(2,1,3-benzoxadiazolyl) isocyanate (DBD-NCO), 7-phenylsulfonyl-4-(2,1,3-benzoxadiazolyl) isocyanate (PSBD-NCO), and 7-methylsulfonyl-4-(2,1,3-benzoxadiazolyl) isocyanate (MSBD-NCO), were synthesized. Among the derivatives derived from the three reagents, that from PSBD-NCO was most strongly fluorescent. PSBD-NCO reacted with 1-octanol within 4 h in acetonitrile solution in the absence of a catalyst at 60 °C. The derivatives with four alcohols (1-octanol, 1-nonanol, 1-decanol, and 1-undecanol) were separated on a reversed-phase column and detected fluorimetrically at 490 nm with the excitation at 368 nm. The detection limits were at the 10-femtomole level. PSBD-NCO was superior to other fluorescent-labeling reagents with regard to the avoidance of the interfering peaks derived from the reagents themselves and degradation products in the chromatogram. The effectiveness of our approach is disccussed in terms of the development of new fluorogenic reagents.

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