Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
PLoS One ; 9(1): e85216, 2014.
Article in English | MEDLINE | ID: mdl-24416365

ABSTRACT

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.


Subject(s)
Cysteine/metabolism , Diabetes Mellitus/blood , Liver Cirrhosis/blood , Renal Insufficiency/blood , Serum Albumin/metabolism , Aged , Amino Acids, Branched-Chain/administration & dosage , Bilirubin/chemistry , Biomarkers/blood , Chronic Disease , Cysteine/chemistry , Diabetes Mellitus/diagnosis , Diabetes Mellitus/drug therapy , Diazepam/chemistry , Female , Glycation End Products, Advanced/blood , Humans , Liver Cirrhosis/diagnosis , Liver Cirrhosis/diet therapy , Male , Middle Aged , Models, Molecular , Oxidation-Reduction , Oxidative Stress , Protein Binding , Renal Insufficiency/diagnosis , Renal Insufficiency/diet therapy , Serum Albumin/chemistry , Spectrometry, Mass, Electrospray Ionization , Tryptophan/chemistry , Warfarin/chemistry
2.
Anal Bioanal Chem ; 405(25): 8093-103, 2013 Oct.
Article in English | MEDLINE | ID: mdl-23736350

ABSTRACT

Here, we describe a novel method for L-phenylalanine analysis using a sandwich-type immunometric assay approach for use as a new method for amino acid analysis. To overcome difficulties of the preparation of high-affinity and selectivity monoclonal antibodies against L-phenylalanine and the inability to use sandwich-type immunometric assays due to their small molecular weight, three procedures were examined. First, amino groups of L-phenylalanine were modified by "N-Fmoc-L-cysteine" (FC) residues and the derivative (FC-Phe) was used as a hapten. Immunization of mice with bovine serum albumin/FC-Phe conjugate successfully yielded specific monoclonal anti-FC-Phe antibodies. Second, a new derivatization reagent, "biotin linker conjugate of FC-Phe N-succinimidyl ester" (FC(Biotin)-NHS), was synthesized to convert L-phenylalanine to FC-(Biotin)-Phe as a hapten structure. The biotin moiety linked to the thiol group of cysteine formed a second binding site for streptavidin/horseradish peroxidase (HRP) conjugates for optical detection. Third, a new semi-sandwich-type immunometric assay was established using pre-derivatized L-phenylalanine, the monoclonal anti-FC-Phe antibody, and streptavidin/HRP conjugate (without second antibody). Using the new "semi-sandwich" immunometric assay system, a detection limit of 35 nM (60 amol per analysis) and a detection range of 0.1-20 µM were attained using a standard L-phenylalanine solution. Rat plasma samples were analyzed to test reliability. Intra-day assay precision was within 6% of the coefficient of variation; inter-day variation was 0.1%. The recovery rates were from 92.4 to 123.7%. This is the first report of the quantitative determination of L-phenylalanine using a reliable semi-sandwich immunometric assay approach and will be applicable to the quantitative determination of other amino acids.


Subject(s)
Immunoassay/methods , Phenylalanine/blood , Animals , Antibodies, Monoclonal/immunology , Female , Haptens/immunology , Limit of Detection , Mice , Mice, Inbred BALB C , Phenylalanine/immunology , Rats , Rats, Sprague-Dawley
3.
Int J Biomed Sci ; 5(3): 293-301, 2009 Sep.
Article in English | MEDLINE | ID: mdl-23675150

ABSTRACT

Albumin (Alb) is mixture of reduced and oxidized forms. It is physiologically significant to determine Alb(red)%, which is the proportion of reduced Alb in the sum of Alb. However, reduced Alb in both blood and plasma samples is easily converted to oxidized Alb. Accordingly, the stabilization of Alb in samples is necessary to determine precise Alb(red)% values. Alb stabilization in blood or plasma was achieved by pH control and buffer dilution. At least a 50-fold dilution with 50 mmol/l phosphate buffer (pH 6.0) was required for human plasma. For human blood, a 10-fold dilution with 0.5 mol/l sodium citrate buffer (pH 4.3) was required. To measure Alb(red)%, treated samples were applied to HPLC or LC-ESI-TOFMS. We also developed a "pre-incubation method", to accelerate the oxidative reaction in plasma by heating at 37°C. Alb(red)% values were maintained around the initial value for 48 h after stabilizing human plasma and 72 h after stabilizing human blood. Accelerating the oxidative reaction in plasma produced large differences in the Alb(red)% values between normal and model disease samples. Precise Alb(red)% values were routinely obtained under the stabilization control. Additionally, pre-incubation of the plasma before measurement is useful to enhance the difference between normal and disease samples.

4.
Rinsho Byori ; 56(5): 409-15, 2008 May.
Article in Japanese | MEDLINE | ID: mdl-18546891

ABSTRACT

Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases; however, little is known regarding the pathological and physiological significance of oxidation due to poor characterization of the precise structural and functional properties of oxidized HSA. Here, we characterize both structural and functional differences between reduced and oxidized HSA. Using LC-ESITOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HSA. Based on this structural information, we prepared standard samples of purified HSA, e.g. nonoxidized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA. Using these standards, we demonstrated several differences in functional properties of HSA, including protease susceptibility, ligand-binding affinity and antioxidant activity. From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions. In addition, we determined blood and plasma sampling conditions for accurate measurement of the oxidized albumin ratio in plasma using EST-TOFMS screening.


Subject(s)
Biomarkers/blood , Serum Albumin/physiology , Humans , Oxidation-Reduction
5.
J Biotechnol ; 128(1): 93-111, 2007 Jan 30.
Article in English | MEDLINE | ID: mdl-17055605

ABSTRACT

Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.


Subject(s)
Amino Acids/biosynthesis , Amino Acids/metabolism , Models, Biological , Bioreactors , Biosynthetic Pathways , Carbon Isotopes , Chromatography, Liquid , Escherichia coli/metabolism , Proteins/metabolism , Tandem Mass Spectrometry
6.
FEBS J ; 273(14): 3346-57, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16857017

ABSTRACT

Human serum albumin (HSA) exists in both reduced and oxidized forms, and the percentage of oxidized albumin increases in several diseases. However, little is known regarding the pathophysiological significance of oxidation due to poor characterization of the precise structural and functional properties of oxidized HSA. Here, we characterize both the structural and functional differences between reduced and oxidized HSA. Using LC-ESI-TOFMS and FTMS analysis, we determined that the major structural change in oxidized HSA in healthy human plasma is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HSA. Based on this structural information, we prepared standard samples of purified HSA, e.g. nonoxidized (intact purified HSA which mainly exists in reduced form), mildly oxidized and highly oxidized HSA. Using these standards, we demonstrated several differences in functional properties of HSA including protease susceptibility, ligand-binding affinity and antioxidant activity. From these observations, we conclude that an increased level of oxidized HSA may impair HSA function in a number of pathological conditions.


Subject(s)
Antioxidants/metabolism , Serum Albumin/chemistry , Serum Albumin/metabolism , Amino Acid Sequence , Chromatography, Liquid , Computer Simulation , Cysteine/metabolism , Electron Spin Resonance Spectroscopy , Humans , Hydrolysis , Ligands , Models, Molecular , Molecular Sequence Data , Molecular Weight , Oxidation-Reduction , Peptide Fragments/chemistry , Protein Binding , Protein Conformation , Serum Albumin/analysis , Serum Albumin/isolation & purification , Spectrometry, Mass, Electrospray Ionization , Spectroscopy, Fourier Transform Infrared , Structure-Activity Relationship , Trypsin/pharmacology
7.
Biomed Chromatogr ; 19(10): 788-95, 2005 Dec.
Article in English | MEDLINE | ID: mdl-15971288

ABSTRACT

We report the simultaneous determination of the carboxylic acids related to the tricarboxylic acid (TCA) cycle, which plays an important role in producing adenosine triphosphate (ATP) and generating energy in mitochondria. Seven carboxylic acids from the TCA cycle, and pyruvic acid and 2-methylsuccinic acid, as an internal standard, were derivatized with a fluorescent reagent for carboxyl groups, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ), in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 4-N,N-dimethyaminopyridine as the coupling reagents, at 60 degrees C for 120 min. Subsequently, the excess DBD-PZ was removed efficiently using a cation-exchange cartridge, SDB-RPS (Empore). These fluorescent derivatives were separated well from each other on an octadecyl silica column (TSKgel ODS-80Ts, 250 x 4.6 mm, i.d.) with an eluent of acetonitrile-water containing 1% formic acid at a flow rate of 0.8 mL/min, and were detected fluorometrically at 560 nm, with excitation at 450 nm. The validation data were satisfactory in the range of 2.5-100 microm citric acid, isocitric acid, 2-oxoglutaric acid, succinic acid and fumaric acid. The detection limit (S/N = 3) for citric acid was 2 fmol on the column. The structures of these derivatives were confirmed by high-performance liquid chromatography-mass spectrometry, which proved that their carboxylic groups were completely labeled with DBD-PZ, except for oxaloacetic acid. This HPLC method was successfully applied to the analysis of TCA cycle metabolites in rat urine. The method will also be useful for metabolome research, such as for target analyses of metabolites with carboxyl groups, not only in urine but also in cells and organs.


Subject(s)
Carboxylic Acids/analysis , Chromatography, High Pressure Liquid/methods , Citric Acid Cycle , Animals , Carboxylic Acids/urine , Fluorescent Dyes/chemistry , Male , Oxadiazoles/chemistry , Piperazines/chemistry , Rats , Rats, Sprague-Dawley , Spectrometry, Fluorescence/methods , Spectrometry, Mass, Electrospray Ionization , Sulfonamides/chemistry
8.
Anal Chem ; 76(7): 1896-902, 2004 Apr 01.
Article in English | MEDLINE | ID: mdl-15053649

ABSTRACT

A miniaturized pepsin reactor was prepared inside a fused-silica capillary (i.d. 75 microm) by coating a pepsin-containing gel on a photopolymerized porous silica monolith. The pepsin-encapsulated film was prepared by a sol-gel method. The sol-gel reaction was optimized so that the sol solution containing pepsin forms a thin film on the photopolymerized sol-gel (PSG) monolith that was initially fabricated at the inlet of the capillary. Pepsin was encapsulated into the gel matrix without losing its activity. The large surface area of the PSG monolith enabled the immobilized pepsin to achieve a high catalytic turnover rate, and the porous nature of the PSG promotes penetration of large molecular proteins into the column. The immobilized pepsin-digested peptides and proteins, and the resulting mixture of peptide fragments, could be directly separated in the portion of the capillary where no PSG monolith exists. The durability and repeatability of the fabricated pepsin-coated column was tested and found to be satisfactory. An acidic solution consisting of 0.5 M formic acid was used as the running buffer, because it suppresses the adsorption of proteins or peptides on the inner surface of the capillary as well as enables direct connection of the output of the capillary electrophoresis column to a mass spectrometer. The on-line digestion of insulin chain beta and lysozyme provides identification of the proteolytic peptides. Recovery was achieved for 100% of the insulin chain beta amino acid sequence and 73% of the lysozyme amino acid sequence.


Subject(s)
Chromatography, Gel/methods , Electrophoresis, Capillary/methods , Pepsin A/metabolism , Peptide Mapping/methods , Proteins/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Amino Acid Sequence , Animals , Digestion , Dipeptides/metabolism , Gels , Hydrogen-Ion Concentration , Insulin/metabolism , Molecular Sequence Data , Muramidase/metabolism , Peptides/chemistry , Peptides/isolation & purification , Photochemistry , Proteins/chemistry , Proteins/metabolism , Surface Properties
9.
J Immunol Methods ; 274(1-2): 63-75, 2003 Mar 01.
Article in English | MEDLINE | ID: mdl-12609533

ABSTRACT

Anti-idiotype antibodies recognizing the variable regions of a particular anti-hapten antibody are valuable tools, which can be used in sensitive hapten immunoassays based on a noncompetitive format. Here, we describe the production and characterization of monoclonal anti-idiotype antibodies against idiotopes on the variable regions of an antibody showing high affinity and specificity to 11-deoxycortisol (11-DC). 11-DC is the biosynthetic precursor of cortisol and a diagnostic index for the assessment of pituitary-adrenal function. BALB/c or A/J mice were repeatedly immunized with the anti-11-DC antibody conjugated with keyhole limpet hemocyanin and their spleen cells were then fused with P3/NS1/1-Ag4-1 myeloma cells. Seven kinds of anti-idiotype antibodies were generated, one of which was a beta-type antibody recognizing the paratope and others which were alpha-type antibodies recognizing the framework region. A noncompetitive ELISA based on idiotype-anti-idiotype reactions was established using one of these alpha-type antibodies in combination with the beta-type antibody and with the anti-11-DC antibody. This noncompetitive assay system provided improved sensitivity (detection limit: 1.0 pg=2.9 fmol), which is approximately 10 times higher than the corresponding competitive enzyme immunoassay, and offered a practical specificity for clinical use. Appropriate serum 11-DC levels were obtained for normal subjects [0.16+/-0.09 (S.D.) microg/l (n=6), ranging from 0.086 to 0.316 microg/l] using the present assay system.


Subject(s)
Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/immunology , Cortodoxone/immunology , Enzyme-Linked Immunosorbent Assay , Immunoglobulin Variable Region/immunology , Animals , Antibodies/immunology , Antibodies, Anti-Idiotypic/biosynthesis , Antibodies, Monoclonal/biosynthesis , Antibody Affinity , Antibody Specificity , Cortodoxone/blood , Cortodoxone/chemistry , Female , Haptens/immunology , Humans , Mice , Mice, Inbred BALB C , Sensitivity and Specificity
10.
J Immunol Methods ; 272(1-2): 1-10, 2003 Jan 15.
Article in English | MEDLINE | ID: mdl-12505707

ABSTRACT

We have established a noncompetitive enzyme-linked immunosorbent assay (ELISA) for the group-specific determination of 7-N-acetylglucosaminide of ursodeoxycholic acid (UDCA 7-NAG) and its glycine- and taurine-amidated metabolites (UDCA 7-NAGs) in human urine. These metabolites are expected to be a diagnostic marker for patients with primary biliary cirrhosis (PBC). This assay is based on the idiotype-antiidiotype reaction where the analyte was captured by an excess amount of anti-UDCA 7-NAG antibody, and the unoccupied paratope was blocked with a beta-type antiidiotype antibody. The hapten-occupied antibody was then selectively detected with a biotin-labeled alpha-type antiidiotype antibody. The amount of bound biotin, increasing proportionally to the increase in the analyte, was colorimetrically determined using a peroxidase-labeled streptavidin. This assay provided subfemtomole range sensitivity (detection limit 118 amol) and allowed group-specific measurement of the UDCA 7-NAGs in urine without any pretreatment. The present ELISA revealed that significant amounts of UDCA 7-NAGs are excreted even in healthy subjects. Daily excretion rates for healthy males were determined to be 246+/-184 (S.D.) microg (n=5) as the glycine-amidated UDCA 7-NAG equivalent. Randomly collected urine specimens from patients with PBC (n=7) were also measured, and the assay values (standardized to creatinine excretion) ranged from 1.82 to 13.4 microg/mg Ucre with the average of 5.41+/-4.53 (S.D.) microg/mg Ucre.


Subject(s)
Acetylglucosamine/analogs & derivatives , Acetylglucosamine/immunology , Acetylglucosamine/urine , Enzyme-Linked Immunosorbent Assay/methods , Ursodeoxycholic Acid/analogs & derivatives , Ursodeoxycholic Acid/immunology , Ursodeoxycholic Acid/urine , Acetylglucosamine/chemistry , Antibodies, Anti-Idiotypic , Biomarkers/urine , Case-Control Studies , Enzyme-Linked Immunosorbent Assay/statistics & numerical data , Humans , Immunoglobulin Idiotypes , Liver Cirrhosis, Biliary/diagnosis , Liver Cirrhosis, Biliary/urine , Male , Reference Values , Sensitivity and Specificity , Ursodeoxycholic Acid/chemistry
SELECTION OF CITATIONS
SEARCH DETAIL
...