bluuebunny/crossref_metadata_2025 huggingface dataset:
A simple method for the quantification of famotidine in human plasma and urine by paired‐ion high performance liquid chromatography
Authors: Imai Yasuhiko , Kobayashi Shin‐Ichiro
Published: May 2005

A new method for the quantification of famotidine consists of a simple extraction procedure and paired‐ion high performance liquid chromatography with ultraviolet detection. The method has good accuracy and precision and should be suitable for the routine measurement of plasma and urine samples for clinical studies.
Analysis of chlortetracycline by high performance liquid chromatography with postcolumn alkaline‐induced fluorescence detection
Authors: Bryan Peter D. , Hawkins Kristina R. , Stewart James T. , Capomacchia Anthony C.
Published: May 2005

A high performance liquid chromatographic method for the analysis of chlortetracycline (CTC) using postcolumn fluorescence detection has been developed. After chromatographic separation of CTC on a polystyrenedivinylbenzene copolymer column, a highly fluorescent derivative isochlortetracycline (iso‐CTC) was formed postcolumn in an on‐line reaction coil with the addition of 25% NaOH (w/v). Chromatographic separation was achieved on a PRP‐1 column, 15 cm × 4.6 mm, with 27:73 acetonitrile:0.2% perchloric acid (v/v), at 1.0 mL/min. Fluorescence derivatization was achieved by the on‐line addition of 25% NaOH (w/v), at a flow rate of 0.2 mL/min, into the column eluant in a post‐column reaction coil. The reaction coil was 9 m of teflon (1/16 in o.d., 0.3 mm i.d.) knitted into a six‐sided coil. The fluorescent derivative was detected at λex 355 nm and λem >389 nm. Using this method after a simple sample cleanup, CTC can be detected in milk at 0.04 μg/mL, which is comparable to that obtained by microbiological assays. The detection method was linear between 0.02 μg/mL and 4 μg/mL. Because of the chromatographic separation, the method is more selective than microbiological assays and more sensitive than ultraviolet detection. With the chromatographic system described, the keto tautomeric forms of CTC and 4‐epi‐CTC are separated in a system which minimizes their formation on‐column. In acidic aqueous organic solutions, the keto tautomer of CTC is the only product formed to any significant amount.
Drug monitoring of clomipramine and desmethylclomipramine in depressed patients using a new liquid chromatographic assay
Authors: Diquet Bertrand , Thomaré Patrick , Bocquentin Muriel , Diviné Catherine
Published: May 2005

A method has been developed for the quantitative analysis of clomipramine and its major metabolite desmethylclomipramine in plasma, using normal phase chromatography with UV detection at 254 nm. This rapid (6 min) and highly sensitive methodology (detection limits 0.5 ng/mL and 2 ng/mL for clomipramine and desmethylclomipramine, respectively; S/N = 3, 0.001 aufs) allows pharmacokinetic studies and drug monitoring of the two compounds. Using the described methodology we report an application which involved 10 depressed inpatients.
A high performance liquid chromatographic method for the determination of febantel and its major metabolites in lamb plasma
Authors: Landuyt J. , Debackere M. , Delbeke F. , McKellar Q.
Published: May 2005

A high performance liquid chromatographic (HPLC) method for the determination of the anthelminthic pro‐benzimidazole febantel and its major metabolites in lamb plasma has been developed. Samples were extracted after addition of albendazole as internal standard, NH4OH and distilled diethyl ether. The extracted phase was dried under a stream of nitrogen redissolved in methanol and chromatographed by HPLC. Detection was by UV absorbance at 292 nm. Recovery from the plasma was 97.2, 97.1, 54.5 and 88.0% for febantel, fenbendazole, oxfendazole and oxfendazole sulphone respectively, and within‐day and between‐day coefficients of variation were 4.03, 4.69, 3.57 and 5.06% and 4.25, 3.73, 5.12 and 4.12%, respectively, for febantel, fenbendazole, oxfendazole and oxfendazole sulphone. The specificity and sensitivity of this method (limit of detection in plasma 0.025 μg/mL and ⩽0.0125 μg/mL for febantel and its metabolites, respectively) were sufficiently high to enable us to characterize the time course of the drug in the plasma after oral administration of therapeutic doses to sheep.
Rapid liquid chromatographic assay of glutathione in cultured cells
Authors: Leroy Pierre , Nicolas Alain , Thioudellet Christine , Oster Thierry , Wellman Maria , Siest Gérard
Published: May 2005

A rapid, sensitive and selective method for the assay of glutathione in cultured cells has been developed using ion‐pair reversed phase rapid high performance liquid chromatography. The use of a 4 μm particle, 5 cm long column (Superspher 100 RP 18 end‐capped) allowed complete analysis of glutathione within 3 min. A postcolumn derivatization reaction with o‐phthaladehyde and fluorometric detection made the assay fully selective with regard to other endogenous thiols and sensitive (the detection limit was 0.5 ng of glutathione injected). The linearity range was between 0.1 and 2.0 μg/mL with good repeatability (relative standard deviation less than 5% for the lowest concentration quantitated). Recoveries of GSH from cultured cell samples were above 98%. The rapid analysis enabled the processing of a large number of samples in a short time (up to 20 per hour). The method was applied to the measurement of the intracellular glutathione amount in V79 fibroblasts along cell growth in culture.
Purification and characterization of hedgehog liver metallothioneins
Authors: Pan Aihua , Tie Feng , Duan Zhenwen , Ma Hongbao , Li Liangyuan , Ru Binggen
Published: May 2005

Two forms of liver metallothioneins (MTs) were purified from hedgehog exposed to zinc, using gel filtration on Sephacryl S‐100 and DEAE Sepharose Fast Flow chromatography. The peptide chain weight of both MT‐1 and MT‐2 was found to be about 10,000, as determined by high performance liquid chromatography. This value was higher than that calculated from amino acid analysis. The amino acid composition of hedgehog liver MT‐1 and MT‐2 resembles that of liver to MTs from rabbit and other species. Their distinctive features include an extremely high cysteine content, about 33% of all the amino acid residues, and an absence of aromatic amino acids and histidine. In addition, a rapid method for the determination of MTs during animal tissue purification has been established. The samples were directly added in an ammoniacal solution of a Co(II) salt for recording linear sweep polarograms. By comparison with the commonly used metal determination method, our method is direct, rapid, credible and suitable for all the MTs or MT‐like samples.
Micropreparative ion exchange high performance liquid chromatography: Applications to microsequence analysis
Authors: Nice E. C. , Fabri L. , Hammacher A. , Andersson K. , Hellman U.
Published: May 2005

Data on the characterization of anion and cation micropreparative (50X1.6 mm i.d.) HPLC columns is presented. It is shown how subnanomole quantities of protein can be efficiently recovered from such columns, rendering them compatible for use in multidimensional chromatographic strategies for the purification of trace biological samples. By selection of appropriate solvent systems (e.g., buffer‐free sodium chloride solutions), the small eluant peak volumes can be loaded directly onto the gas phase sequencer, and N‐terminal sequence data obtained. The potential of the technique is illustrated for the purification of a GTPase activating protein (GAP‐3).
High performance liquid chromatographic analyses of compounds related to nucleic acids by 3‐(1‐naphthoylamino)‐ and 3‐(1‐anthroylamino)‐propyl‐modified silica gel packing materials
Authors: Akiyama Shuzo , Nakashima Kenichiro , Yamada Kyoko , Kuroda Naotaka
Published: May 2005

The development of column packing materials was tried as a contribution to studies related to the further modification of 3‐aminopropyl‐modified silica gel. Consequently, we prepared two types of gel, 3‐(1‐naphthoylamino)‐ and 3‐(1‐anthroylamino)‐propyl‐modified silica gels (NAPS and AAPS). Since NAPS and AAPS, each having an aromatic ring, were expected to behave in a separation mode by a π‐π or hydrophobic interaction, their application to the analyses of the compounds related to nucleic acids was carried out. Adenosine nucleotides were consequently separated by the gels in a single analysis by a single buffer elution with UV detection. NAPS could be also successfully used for the measurement of ATPase activity in fish meat (Sillago japonica). The mobile phase consistently used in the analysis was an aqueous phosphate buffer.
High performance liquid chromatography of penicillins with penicillin‐enhanced luminol chemiluminescence detection
Authors: Nakashima Kenichiro , Kawaguchi Shinki , Akiyama Shuzo , Schulman Stephen G.
Published: May 2005

A high performance liquid chromatographic method with chemiluminescence detection for the determination of penicillin G and ampicillin is reported. The method is based on the enhancement of the luminol chemiluminescence with β‐lactam antibiotics. The linear relationship was obtained between the peak height and the concentration of penicillin G or ampicillin up to 15 nmol per 20 μL injection. Detection limits were 1 nmol for penicillin G and 0.5 nmol for ampicillin with a signal‐to‐noise ratio of 2. Relative standard deviations for five replicate measurements of 4 nmol/injection each of penicillin G and ampicillin were 2.1 and 2.3%, respectively.
A rapid and sensitive determination of salmon calcitonin in solutions containing bovine serum albumin or gelatin
Authors: Fukuda Terumi , Yun Arifuku , Imai Kazuhiro
Published: May 2005

A sensitive and selective reversed phase high performance liquid chromatographic method was introduced for the determination of salmon calcitonin (sCT) in solutions containing bovine serum albumin or gelatin. The method was based on the sensitive fluorescence detection of sCT by postcolumn derivatization with o‐phthal(di)aldehyde. The sample was loaded onto a reversed phase column, purified by column switching and separated with linear gradient elution using the ion pair technique. A linear relationship was obtained between the peak height and the amount injected in the concentration range 1–40 ng. The detection limit was 10 ng/mL (S/N = 3) at a sample injection volume of 100 μL.
Resolution of enantiomers of amino acids by HPLC
Authors: Bhushan R. , Joshi Shalini
Published: May 2005

Application of HPLC as a prime tool in the area of enantiomeric resolution has opened doors of success and varied interest. Use of chiral reagents either indirectly (as derivatization reagent) or directly (added to stationary or mobile phase) has led to achieve resolution of a wide range of compounds. Amino acids, being important molecules with simple structure and easy availability, have been extensively studied. A bibliographic survey on HPLC resolution of amino acids and derivatives along with a brief discussion on general methods of enantiomeric separation has been presented.
Quantitation and identification of two cholecystokinin peptides, CCK‐4 and CCK‐8s, in rat brain by HPLC and fast atom bombardment mass spectrometry
Authors: Qureshi G. Ali , Bednar Ivan , Min Qian , Södersten Per , Silberring Jerzy , Nyberg Fred , Thörnwall Madeleine
Published: May 2005

A sensitive HPLC method has been described for quantitation of two cholecystokinin (CCK) peptides in discrete rat brain regions. Separation and quantitation was performed by the reversed‐phase HPLC combined with electrochemical detection. Analytical recoveries of the tetrapeptide (CCK‐4) and octapeptide‐sulphate (CCK‐8s) were 96% and 94%, respectively. The between assay coefficient of variation (CV) was less than 3% for both peptides. The within‐assay CV was 4% and 6% for CCK‐4 and CCK‐8s and the detection limit was 2 and 10 pmol/mL, respectively.

For identification of structures, the peptides were fractionated by semi‐preparative HPLC using a novel SMART system for micropurification. The fractions were analysed by fast atom bombardment mass spectrometry (FAB MS) which confirmed the presence of both CCK‐4 and CCK‐8s in the rat brain tissue.
Isolation and purification of amyloglucosidase from Halobacterium sodomense
Authors: Chaga Grigoriy , Porath Jerker , Illéni Tibor
Published: May 2005

Amyloglucosidase from Halobacterium sodomense was purified by a combination of hydrophobic interaction chromatography and immobilized metal ion affinity chromatography at analytical and preparative scale with 75% recovery. The enzyme was found to be a dimer of two different subunits with molecular weights of 72,000 and 82,000 D, respectively, combining in a 175,000 D native protein. The specific activity, KM, and amino acid composition of the enzyme was determined.
A simple method for plasma cannabinoid separation and quantification
Authors: Alemany G. , Gamundí A. , Nicolau M. C. , Saro D.
Published: May 2005

A sensitive (up to nanogram level) method for resolving a cannabinoid mixture in plasma is described. Cannabinoids were extracted with a C‐18 Sep Pak cartridge and derivatized with dansylchloride. Then the derivatives were resolved on thin layer HPTLC silica plates which were developed and quantified by fluorescence densitometry at 340 nm.
A fully automated HPLC method for the determination of catecholamines in biological samples utilizing ethylenediamine condensation and peroxyoxalate chemiluminescence detection
Authors: Prados Pablo , Higashidate Sakae , Imai Kazuhiro
Published: May 2005

A fully automated in‐line extraction reversed‐phase high‐performance liquid chromatography (HPLC) method with chemiluminescence detection was developed for the analysis of human and rat plasma catecholamines (CAs), norepinephrine (NE), epinephrine (E) and dopamine (DA). N‐Methyldopamine (N‐MeDA) was used as an internal standard. The method involves collection of plasma samples, which are first diluted with a sample dilution buffer containing N‐MeDA, and in‐line extraction of CAs using a carboxylic acid small resin precolumn (SERUMOUT‐CEX). This pre‐extraction process was coupled with an HPLC system including reversed‐phase mode separation on an analytical column (TSK gel ODS‐80Ts), fluorogenic derivatization with ethylenediamine (ED) and finally postcolumn peroxyoxalate chemiluminescence reaction detection using bis [4‐nitro‐2‐(3,6,9‐trioxadecyloxycarbonyl)phenyl]oxalate (TDPO) and hydrogen peroxide. The optimized mobile phase compositions, flow rates, operation timing for the adsorption and desorption of CAs in the precolumn, the separation in the analytical column and the optimum fluorogenic and chemiluminogenic reaction conditions were investigated.

The detection limit for all the CAs was about 1 fmol (signal‐to‐noise ratio is 2). Excellent linearity of the calibration curves for CAs was observed in the range from 5 to 500 fmol for each CA using the internal standard. The relative standard deviations of the method for determining NE (183 fmol), E (23.6 fmol) and DA (6.1 fmol) in 50 μL of human plasma (n = 3) were 2.8, 2.7 and 3.1%, respectively, for the within‐day assay and 5.0, 3.8 and 4.0%, respectively, for the between‐day assay. The method was applicable to the determination of CAs in 25–50 μL of human or rat plasma.
Experimental models for optimization of insulin separation on reversed phase columns
Authors: Dimov N. , Simeonov S.
Published: May 2005

Experimentally defined relationships have been found to describe adequately the retention and separation of bovine and porcine insulins as well as their desamido products on reversed phases C4 30 nm and C18 10 nm pore diameter. The equations are valid for a region of initial acetonitrile concentrations from 16 to 31% and gradient rates from 0.04 to 0.60%/min. The peak heights showed an exact non‐linear relationship with the time interval between the first and last peak of interest, independent of their retention time.

The number of experiments required for obtaining the parametric estimates of the models depends on the particular task, but in all cases is less than eight.

The relationships found permit the correct choice to be made in advance both for analysis conditions for a particular column and for different requirements of insulin analysis.
Direct determination of mitoxantrone in plasma by high performance liquid chromatography using an automatic precolumn‐switching system as sample clean‐up procedure
Authors: Catalin Jacques , Peloux Anne F. , Coloma Fabrice , Payet Brigitte , Lacarelle Bruno , Cano Jean P. , Just‐Landi Sylvaine
Published: May 2005

A high‐performance liquid chromatography method which uses direct injection and a column‐switching valve for determination of mitoxantrone in plasma is described. After addition of internal standard, plasma was deproteinized by adding 5‐sulphosalicylic acid reagent. The supernatant was injected onto an enrichment precolumn flushed with washing solvent (methanol and water 5:95).

Absorbed mitoxantrone was backflushed from the precolumn into an analytical column C18 Nucleosil 250 × 4 mm with a gradient elution (solvent A, ammonium formate buffer 1.6 M, pH 4.3; solvent B, acetonitrile and water 40:60; linear gradient from 45 to 55% of B for 30 min was programmed) at a flow rate of 1.3 mL/min. Detection was carried out at 665 nm.

This method showed obvious advantages over conventional extraction procedures in terms of speed and facility of sample handling.
Quantitative analysis of neuroactive amino acids in brain tissue by liquid chromatography using fluorescent pre‐column labelling with o‐phthalaldehyde and N‐acetyl‐L‐cysteine
Authors: Soto‐Otero R. , Méndez‐Alvarez E. , Galán‐Valiente J. , Aguilar‐Veiga E. , Sierra‐Marcuño G.
Published: May 2005

A high‐performance liquid chromatographic method for the determination of aspartic acid, glutamic acid, glutamine, glycine, taurine, and γ‐aminobutyric acid in brain tissue is described. Amino acids were derivatized with o‐phthalaldehyde/N‐acetyl‐L‐cysteine, a fluorescent labelling mixture, in the presence of 0.1 M borate buffer pH 9.5. The derivatization reaction was sensitive to the pH and concentration of borate buffer. A drift in the fluorescent response less than 4% was obtained with the reported conditions after 4 h of reaction. The resolution of the amino acid derivatives was accomplished in a reversed‐phase column with a methanol gradient in 50 mM acetate buffer pH 5.5. These conditions also allowed the separation of the major tissue free physiological amino acids. L‐Norvaline was used as an internal standard for both peak identification and quantification. Within‐day and between‐day precision were less than 6.2%, and the accuracy ranged from 99.1 to 104%. The applicability of the method was demonstrated in a study in rats in which the levels of the assayed amino acids in discrete areas of brain were examined.
Glutathione and cysteine measurement in biological samples by HPLC with a glassy carbon working detector
Authors: Carvalho F. D. , Remião F. , Valet P. , Timbrell J. A. , Bastos M. L. , Ferreira M. A.
Published: May 2005

An HPLC method using a glassy carbon working detector for measuring cysteine and reduced glutathione (GSH) in biological samples was developed. Oxidized glutathione (GSSG) was also measured after reduction with glutathione reductase. This study was conducted in human plasma. Cysteine and GSH standard curves were linear in the physiological range, presenting detection limits of 22.0 pmol and 6.0 pmol respectively. Plasmatic results found were 43.92 ± 4.15, 4.50 ± 0.65, and 0.19 ± 0.12 mM (means ± SE), for cysteine, GSH and GSSG, respectively. Peak specificity for cysteine and reduced glutathione was certified by their disappearance after treatment with N‐ethylmaleimide.
Simultaneous determination of antidepressant drugs and metabolites by HPLC. Design and validation of a simple and reliable analytical procedure
Authors: Joron S. , Robert H.
Published: May 2005

An analytical procedure suitable for routine use has been developed and validated for the simultaneous separation and quantification of most of the antidepressant drugs (bicyclic, tricyclic and tetracyclic) and their metabolites in human serum by reversed‐phase high performance liquid chromatography with ultraviolet detection. The method has good sensitivity and specificity, without the need for long and complex extraction procedures. The results obtained with three different analytical columns were compared and an improvement in specificity by selection of column and detection wavelength used was achieved.
Measurement of theophylline metabolites produced by reaction with hepatic microsome by high performance liquid chromatography following solid phase extraction
Authors: Konishi Hiroki , Yamaji Akira
Published: May 2005

An analytical method has been developed with which to measure the microsomal enzyme activities responsible for oxidative theophylline metabolism. Three metabolites: 3‐Methylxanthine (3‐MX); 1‐methylxanthine (1‐MX); 1,3‐dimethyluric acid (1,3‐DMU), with acetaminophen as an internal standard (IS), were separated by solid phase extraction using a Sep‐Pak C18 cartridge, followed by high performance liquid chromatography on a reversed‐phase column with isocratic elution using 25 mM acetate buffer containing 4% acetonitrile and 2.5 mM tetra‐n‐butylammonium hydrogen sulphate (pH 5.25) as the mobile phase. The analytes were clearly resolved and no interference with foreign peaks was observed. A linear relationship was obtained for the metabolites over the concentration range of 0.5–5.0 μg/mL, and their analytical recovery was almost 100%. This method can be used to assess drug interactions involving alterations in the biotransformation of theophylline.
A comparison of fluorescence versus chemiluminescence detection for analysis of the fluorescamine derivative of histamine by HPLC
Authors: Walters Denise Lowe , James John E. , Vest Floyd B. , Karnes H. Thomas
Published: May 2005

Fluorescence and chemiluminescence detection were compared for HPLC analysis of the fluorescamine derivative of histamine. The kinetic behaviour of the chemiluminescent response for the derivative was characterized in a static system. An HPLC method was optimized for the derivative using fluorescence detection. Fluorescence detection was linear over the range of 166–1666 pg on column for the fluorescamine–histamine derivative with a limit of detection of 13 pg on column. Using a detector designed for optimal use with chemiluminescence, the chemiluminescence response of the fluorescamine derivative was linear over a range of 1.66–16.6 ng on column with a limit of detection of 1.0 ng on column. These results exemplify a case in which superior detectibility is provided by fluorescence over chemiluminescence, and contradicts many reports comparing fluorescence to chemiluminescence. The authors conclude that chemiluminescence should be considered when indicated by conditions established for separation that are favourable for the observation of chemiluminescence. These conditions include sufficiently low excitation energies corresponding to an excitation maximum greater than 400 nm, favourable dipole character of analytes, mobile phases of high organic content, and an appropriate pH of the mobile phase.
Fluidized‐bed receptor‐affinity chromatography
Authors: Spence Cheryl , Schaffer Carol Ann , Kessler Stephen , Bailon Pascal
Published: May 2005

A multipurpose fluidized‐bed receptor‐affinity purification system based upon the biological recognition between an immobilized receptor and its soluble protein ligands is described. The fluidized affinity sorbent consists of a soluble form of interleukin‐2 receptor chemically bonded to an aldehyde derivative of controlled pore glass beads, which have a pore diameter of 1000 Å and a particle density of 1.2–1.3 g/mL. The fluidized‐bed separation device used in this study consists of a specially designed column fitted at the inlet end with a perforated distributor plate covered with a screen and the top outlet with an adjustable piston. The fluidized‐bed consisting of a loose gel matrix permits the unimpeded passage of cell debris and particulate matter, while the target protein is captured by the affinity beads. Purification of the humanized‐anti‐Tac monoclonal antibody is used as a model system to determine the operational parameters. Also, fluidized‐bed receptor‐affinity chromatography has been successfully employed in the purification of recombinant interleukin‐2 and single chain anti‐Tac(Fv)‐Pseudomonas exotoxin immunotoxin from unclarified inclusion body extracts. Overall, fluidized‐bed receptor‐affinity chromatography is found to be a productive affinity method suitable for the purification of recombinant human interleukin‐2 and related molecules.
HPLC separation of pentazocine enantiomers in serum using an ovomucoid chiral stationary phase
Authors: Kelly J. W. , Stewart J. T. , Blanton C. D.
Published: May 2005

A stereospecific HPLC method was developed for the analysis of (−) and (+) pentazocine in human serum. Each enantiomer and the internal standard nalophine were isolated from serum using a liquid–liquid extraction procedure. Recoveries of 99.05 ± 5.37 and 97.42 ± 2.78% were obtained for (−) and (+) pentazocine, respectively. Resolution of the enantiomers was obtained by using an ovomucoid chiral stationary phase with a mobile phase of methanol: acetonitrile: 10 mM phosphate buffer, pH 5.8 (20:5.3:74.7 v/v/v). A resolution (Rs) value of 1.80 was obtained for the pentazocine enantiomers. Linear calibration curves were obtained in the 10–100 ng/mL range for each enantiomer in serum. The detection limit based on a signal‐to‐noise ratio of 3 was 5 ng/mL for each enantiomer in serum using fluorescence detection with excitation at 275 nm and emission set at 335 nm. The lowest quantifiable level was found to be 10 ng for each enantiomer. Precision and accuracy of the method were in the 3.8–4.8% and 1.3–4.2% ranges, respectively.
Comparison of standard chromatographic procedures for the optimal purification of soluble human brain acetylcholinesterase
Authors: Novales‐Li Philipp
Published: May 2005

With the view of purifying soluble human brain acetylcholinesterse (AChE) into its separate isoforms, various preparative chromatographic procedures were compared. Chromatofocusing of cerebrospinal fluid (CSF) AChE revealed two major activity peaks, whilst that of caudate nucleus AChE showed one major peak, Both CSF and caudate nucleus AChE eluted at isoelectric points (pI) of between 5.5 and 5.2 Chromatofocusing failed to separate AChE into its individual isoforms, based on qualitative isoelectric focusing. Preparative purification by affinity chromatography showed a better AChE yield with the use of procainamide as a ligand, vis‐à‐vis acridinium. Maximum recovery for CSF and caudate nucleus AChE was 10 and 43% using acridinium and procainamide, respectively. Qualitative analysis by SDS‐PAGE of affinity‐purified AChE revealed four major bands between 50 and 62kDa, corresponding to the catalytic subunits of AChE as verified by an anti‐AChE polyclonal antibody. A size‐exclusion column also allowed brain AChE purification, with the latter eluting at a putative molecular mass of 310 kDa. Unfortunately, cation‐exchange using the state‐of‐the‐art SMART system failed to separate AChE into its isoforms. AChE aggregation is given as one major obstacle precluding good resolution of isoforms.
Interaction of carboxymethyl‐γ‐cyclodextrin with anticancer drugs studied by charge‐transfer thin‐layer chromatography
Authors: Cserháti Tibor
Published: May 2005

The interaction between 22 anticancer drugs and carboxymethyl‐γ‐cyclodextrin (CM‐γ‐CD) was studied by reversed‐phase charge‐transfer thin‐layer chromatography and the relative strenth of interaction was calculated. CM‐γCD formed inclusion complexes with 11 compounds, the complex always being more hydrophilic than the uncomplexed drug. The inclusion forming capacity of drugs differed considerably according to their chemical structure. Both principal component analysis and cluster analysis found a relationship between the inclusion complex forming capacity and hydrophobicity parameters of anticancer drugs. This result suggests that the preponderant role of hydrophobic interactions is in inclusion complex formation.
High‐resolution separation of PCR product and gene diagnosis by capillary gel electrophoresis
Authors: Baba Yoshinobu , Tomisaki Riyo , Sumita Chinuyo , Tsuhako Mitsutomo , Miki Tetsuro , Ogihara Toshio
Published: May 2005

High‐resolution separation of a PCR product from a mixture of DNA restriction fragments was achieved using capillary gel electrophoresis. The capillary gel electrophoretic separation gives an excellent resolution of two fragments of the 500‐bp PCR product and the 506‐bp DNA fragment, which differs by only 6 bp, and the complete separation of a broader chain lenght range of DNA fragments up to 12kbp within 20min. The plate number of gel‐filled capillary was achieved to be 2 million plates per meter. Capillary gel electrophoresis is applied to the gene diagnosis for heart disease through apolipoprotein E genotyping. The advantages and limitations of capillary gel electrophoresis in the application to PCR analysis and DNA diagnosis are discussed.
Simple determination of formaldehyde in dimedone adduct form in biological samples by high performance liquid chromatography
Authors: Sárdi Éva , Tyihák Ernö
Published: May 2005

A high performance liquid chromatographic method for the determination of endogenous formaldehyde in dimedone adduct form in biological samples is described. The simple procedure involves extraction of the formaldehyde of different binding force in biological samples with methanol containing dimedone as the capture molecule and separation on a C18 reversed phase column with methanol as eluent.
TLC studies on certain aminoglycoside antibiotics
Authors: Bhushan R. , Joshi Shalini
Published: May 2005

Aminoglycoside antibiotics were separated by thin layer chromatography. Separation was achieved on untreated silica gel layers using various combinations of solvent systems, dioxane:ammonia, methanol:ammonia and propanol:ammonia. These antibiotics were visualized with the help of iodine vapours.
Effect of column temperature and nitrogen flow rate on the separation of cis‐permethrinic acid isomers on a (2, 3, 6‐Tri‐O‐methyl)‐β‐cyclodextrin coated capillary column
Authors: Payer Károly , Forgács Esther , Cserhàti Tibor
Published: May 2005

The separation efficiency of a capillary column coated with (2, 3, 6‐tri‐O‐methyl)‐β‐cyclodextrin was determined at the temperature and flow rate ranges of 80–120°C and 0.3–1.5 mL/min respectively using (+) and (−) cis‐permethrinic acid methylesters as test compounds. The dependence of the retention differences, sum of peak widths and separation factor on the temperature and flow rate was calculated with step regression analysis. The column exhibited acceptble isomer separation capacity at each temperature and flow rate proving the excellent separation power of the cyclodextrin coating. The differences between the retention times of isomers depended on the logarithm and on the reciprocal value of temperature, the effect of flow rate was negligible. The sum of their peak widths depended reciprocally on the temperature and logarithmically on the flow rate, the impact of temperature being higher than that of flow rate. The significant impact of the reciprocal value of temperature can be explained by the inclusion complex formation between cyclodextrin and cis‐permethrinic acid methylester isomers. The resolution depended on the square of column temperature and flow rate, indicating local optimums at each temperature and flow rate.
Simultaneous determination of catecholamines, their basic metabolites and serotonin in urine by high‐performance liquid chromatography using A mixed‐mode column and an eight‐channel electrochemical detector
Authors: Mashige Fumiko , Matsushima Yoshikazu , Miyata Chizuko , Yamada Rika , Kanazawa Hideko , Sakuma Ichiro , Takai Nobuharu , Shinozuka Noriko , Ohkubo Akiyuki , Nakahara Kazuhiko
Published: May 2005

A high‐performance liquid chromatographic method for simultaneous determination of free catecholamines, their basic metabolites and serotonin in human urine was developed. The compounds were separated on a precolumn of cation‐exchange resin and a mixed‐mode (C18/cation‐exchange) column and determined by an eight‐channel electrochemical detector. By this method, free norepinephrine (NE), epinephrine (E), dopamine (DA), normetanephrine (NMN), metanephrine (MN), 3‐methoxy‐tyramine (3‐MT) and serotonin (5‐HT) were determined in 40 min with inter‐assay precision of 1.9, 3.2, 1.2, 1.6, 1.6, 5.5 and 3.2%, respectively. Detection limits were between 0.05 (E) and 0.3 (3‐MT) pmol. The mean values of analytical recovery for NE, E, DA, NMN, MN, 3‐MT, 5‐HT were 99, 104, 104, 99, 102, 109 and 99% respectively.
BioCARS—a novel nonlinear optical technique to study vibrational spectra of chiral biological molecules in solution
Authors: Koroteev Nikolai I.
Published: May 2005

A description is given of a novel coherent nonlinear optical technique, acronymed as BioCARS, which can be considered as a nonlinear variant of vibrational optical activity with no background (unlike spontaneous Raman optical activity) and with the capability of studying the whole vibrational spectra of biomolecules in water solutions (unlike vibrational circular dichroism). The BioCARS technique is based on a coherent five‐wave mixing process in macroscopically noncentrosymmetric liquid solutions of chiral biomolecules. It is the generalization for chiral (optically‐active) liquids of the well‐known CARS (Coherent Anti‐Stokes Raman Spectroscopy) technique. The theoretical background of the BioCARS technique is developed, the calculation of the relevant material parameter, which describes the BioCARS process,—a fourth‐order nonlinear susceptibility—is given for a simple model chiral medium, and selection rules for BioCARS spectra are discussed. © 1995 John Wiley & Sons, Inc.
Taking a risk and playing it safe: the use of actors in interagency child protection training
Authors: Lexton Ann , Smith Martin , Olufemi David , Poole Gaye
Published: May 2005

Interagency training in child protection is an important part of engaging professionals in a common approach to their collective responsibility for safeguarding children. Training is a key method for empowering professionals to carry out their complex role in child protection with appropriate knowledge, skills and confidence. In Buckinghamshire, experiential learning involving actors has met with some success as part of the interagency child protection training programme. The following paper proposes the argument for using actors, describes how this is done and evaluates its effectiveness for participants, trainers and actors. It is suggested that this method has ample rewards if properly planned, particularly for practitioners in lessons for practice. A close working relationship must be developed between actors and trainers. Scenarios that seem real can be portrayed and skills can then be practised in the knowledge that no one is really being harmed but valuable lessons for real‐life situations can be learned. A central aspect is learning together in a supportive environment away from a blame culture. Copyright © 2005 John Wiley & Sons, Ltd.
Correlations between common tests for assessment of liver damage: Indices of the hepatoprotective activity of promethazine in carbon tetrachloride hepatotoxicity
Authors: Reddrop Colin J. , Cheeseman Kevin H. , Slater T. F.
Published: May 2005

The effects of promethazine (PM) on different aspects of the hepatotoxic action of CCl4 in the rat were investigated with the objective of finding rapid and reliable indicators of hepatoprotective effects. The study was based on definitive histological assessment of liver damage caused by CCl4 in the presence and absence of PM: PM (78 μmol kg−1, i.p.) protected against CCl4‐induced hepatic necrosis 24 h after a low dose of CCl4 (1.3 mmol kg−1) but not against a higher dose (13.0 mmol kg−1). The large increases in plasma activities of GOT, GPT and LDH produced by dosing with CCl4 were partially inhibited by the administration of PM. PM and CCl4 caused a synergistic and long‐lasting decrease in body temperature (2–3°C for 8–10 h). Modifying the toxicity with PM, together with a low dose of CCl4, helped to minimize secondary effects of CCl4, to clarify the sequence of toxic events, and to assess the sensitivity of some standard tests of hepatotoxicity. Simultaneous measurement of over 20 commonly used biochemical screening tests in individual animals 3 or 6 h after treatment permitted direct correlation of a wide variety of concentrations, activities and effects. For example, liver CHCl3 concentrations (as a measure of CCl4 metabolism) correlate strongly with increases in diene conjugation of microsomal lipids (as a measure of CCl4‐induced lipid peroxidation); malonaldehyde production appears to be less sensitive as a measure of lipid peroxidation in vivo than diene conjugation. The changes induced in each parameter and the correlations between them are discussed with reference to the overall nature of the hepatotoxic reaction and its modification by PM.
Role of transferrin in uptake of non‐physiological metals into cells
Authors: Planas‐Bohene Felicitas , Taylor David M. , Duffield John R. , Darai Gholamreza
Published: May 2005

At physiological concentrations of citrate the uptake of 59Fe, 67Ga, and 239Pu into human type B lymphocytes of splenic origin is the same in viable and in non‐viable cells. Addition of transferrin has no effect on the uptake into non‐viable cells but in viable cells it increases the uptake of Fe and Ga but decreases that of Pu. Uptake decreases as transferrin concentration increases although this is less marked with Ga.
Is there any connection between heat inactivation of spectrin‐dependent ATPase and loss of smooth biconcave shape of red cells?
Authors: Mirčevová Libuše , Kodíček Milan , Mařík Tomáš
Published: May 2005

Spectrin‐dependent ATPase activity was measured in membranes from native human erythrocytes and erythrocytes heated for 20 min at different temperatures. This activity was found to decline when the erythrocytes were heated at 48°C and higher. The break in ATPase activity corresponds to morphological changes in erythrocytes found by Crome and Mollioson [Brit. J. Haematol. (1964) 10, 137]. The role of spectrin‐dependent ATPase in erythrocyte shape maintenance is discussed.
Platelet membrane fatty acids in thrombocytosis due to myeloproliferative disorders
Authors: Leoncini G. , Maresca M. , Balestrero F. , Piana A. , Benatti U. , Armani U.
Published: May 2005

The fatty acid composition of platelet membranes has been analysed in patients with thrombocytosis due to myeloproliferative disorders, who had not taken any drugs. A significant increase in palmitic and oleic acid, together with a decrease in stearic, linoleic and arachidonic acids was observed. The fatty acid pattern of platelet membranes was also analysed in patients during treatment with ASA (acetylsalicylic acid). ASA ingestion completely normalizes the platelet content of palmitic acid and partially that of stearic and arachidonic acid, whereas it has no effect on the level of linoleic acid and raises that of oleic acid. The altered pattern of fatty acids observed in patients may interfere with platelet function by decreasing membrane fluidity. Treatment of patients with ASA seems to act on platelet membranes by partially normalizing the fatty acid composition.
Nude mice injected with DNA released by antigen stimulated human tlymphocytes produce specific antibodies expressing human characteristics
Authors: Anker P. , Jachertz D. , Maurice P. A. , Stroun M.
Published: May 2005

Nude mice were injected with DNA purified from the nucleoprotein complex released by T lymphocytes previously exposed in vitro to inactivated herpes or poliovirus. After five days the serum of these mice was tested for its virus neutralizing activity. Results show that injected nude mice synthesize antiherpetic or antipolio antibodies depending on the antigen used to sensitize the T lymphocytes in vitro. These antibodies were not found in the serum of uninjected control mice or mice injected with inactivated herpes or polio viruses. Mice injected with DNA release by human T cells produced antibodies carrying human allotypes since they could be neutralized by anti‐allotype sera. Moreover their antiviral activity was inhibited by anti‐human IgM or IgG. However, the mice which were injected with DNA released by antigen stimulated murine T lymphocytes produced antiviral antibodies which were not neutralized by anti‐human allotype sera.
Characteristics of taurine transport in rat hepatocytes in primary culture
Authors: Ohkuma Seitaro , Tamura Jun‐ichi , Kuriyama Kinya , Mukainaka Teruo
Published: May 2005

Characteristics of taurine transport in rat hepatocytes maintained in primary culture for 24 h (cultured hepatocytes) have been investigated. The uptake of [3H] taurine by cultured hepatocytes at 2°C was unsaturable, whereas that at 37°C consisted of unsaturable and saturable processes. The saturable transport system was sodium‐dependent and consisted of two processes with low and with high affinities. The latter process (Km, 76.9 μM; Vmax, 0.256 nmole/mg protein/min; activation energy (EA), 37.8 kcal mol−1) was competitively inhibited by 2,4‐dinitrophenol and ouabain, as well as by taurine analogues such as hypotaurine and guanidinoethyl sulphonate. The Vmax and EA values found in cultured hepatocytes at 37°C were 6.0 and 6.8 times higher than those found in freshly isolated hepatocytes. These results indicate that taurine transport in hepatocytes in primary culture consisted of unsaturable, and saturable, sodium and energy‐dependent carrier‐mediated transport processes, respectively. The facilitation of the latter transport system by primary culture of hepatocytes is also suggested.
Functional alterations of the endoplasmic reticulum and the detoxification systems during diethyl‐nitrosamine carcinogenesis in rat liver
Authors: Garcea R. , Canuto R. A. , Biocca M. E. , Muzio G. , Rossi M. A. , Dianzani M. U.
Published: May 2005

The mixed‐function oxidase system shows a number of variations in the liver of diethyl‐nitrosamine (DEN) treated rats. These include a decrease of the cytochrome P450 content and of the aminopyrine demethylase activity both in the hyperplastic nodules and in the hepatoma. Processes of detoxification, such as the glutathione system, show some modifications. These alterations are in accordance with the decrease of glutathione peroxidase and the increase of γ‐glutamyltranspeptidase during diethyl‐nitrosamine carcinogenesis.
Stimulation by cholera toxin of ADP‐ribosylation of membrane proteins, adenylate cyclase and insulilin release in pancreatic islets
Authors: Svoboda Michael , Garcia‐Morales Pilar , Dufrane Simon P. , Sener Abdullah , Valverde Isabel , Christophe Jean , Malaisse Willy J.
Published: May 2005

In rat pancreatic islet membranes exposed to [α–32P]NAD, cholera toxin stimulated the labelling of three peptides with Mr close to 22000, 42000 and 48000, respectively. In the islets, the toxin‐stimulated ADP‐ribosylation of the heavy form of the Ns α‐subunit predominated over that of the light from, in mirror image of the situation found in the exocrine pancreas. When intact islets were preincubated with cholera toxin, the adenylate cyclase activity of a subcellular particulate fraction was increased. The responsiveness of adenylate cyclase to GTP was also augmented, but that to NaF was decreased. In intact islets, the production of cyclic AMP and the glucose‐stimulated release of insulin were also enhanced after pretreatment with cholera toxin. These findings reveal the presence in pancreatic islets of the guanyl nucleotide regulatory protein of adenylate cyclase, with an unusual predominance of the heavy from of the Ns α‐subunit.
Alpha‐Lactalbumin as a lysosomal enzyme‐releasing factor
Authors: Komine Senichi , Nakanishi Keiko , Anzaio Tadashi , Yoshimoto Akio
Published: May 2005

In the early stage of mammary gland involution, biochemically detectable lysosomal damage occurs. The mechanism(s) underlying this damage is not well understood. We found that α‐lactalbumin from mouse milk induced the release of enzymes from the lysosomes of mouse mammary epithelial cells in vitro, and this induction also occurred with bovine α‐lactalbumin. This enzyme release was accelerated by the addition of whey proteins with a molecular weight of 50 000 to 60 000. We also found that the lysosomal membrane of mammary epithelial cells had a strong affinity for α‐lactalbumin.
A catabolin‐like factor from porcine kidney
Authors: Monserrat J. Juan , Dingle John T.
Published: May 2005

Explants of pig kidney cortex and medulla release a catabolin‐like factor (CLF). The CLF of both kidney cortex and medulla can be precipitated with ammonium sulphate, mainly in the 60–95 per cent fraction. By gel chromatography the kidney CLF showed a major active fraction at a molecular weight of around 22 500.

Whole glomeruli and dissociated glomerular cells in culture also released into the medium a CLF that could be bioassayed in live cartilage but displayed no effect on dead cartilage. The possible role of such a local hormone is discussed.
Triiodothyronine receptor sites in serum‐free cultured hepatocytes from adult rat liver
Authors: Goglia F. , Gallo G. , Palmero S. , Voci A. , Fugassa E.
Published: May 2005

Nuclear T3 specific binding sites were characterized by Scatchard analyses of L‐125I‐T3 binding to nuclei extracted from freshly isolated and 1,2 and 6 day‐cultured hepatocytes. The results demonstrate a marked decrease in T3 binding capacity of nuclei extracted from 1 day‐cultured cells followed by an almost complete recovery within 6 days. The affinity constant value of nuclear receptor sites is significantly decreased in 1 day‐cultured cells with a subsequent partial recovery. The affinity and capacity pattern of nuclear T3 binding sites appears to be in line with the delayed responses of hepatocyte primary cultures to T3.
The relationship between tissue preparation and function; methods for the study of control of aldosterone secretion: A review
Authors: Vinson G. P. , Hinson J. P. , Raven P. W.
Published: May 2005

The study of the control of aldosterone synthesis and secretion by the rat adrenal gland has over the past thirty years involved the application of many different in vivo and in vitro techniques. In this review the relationship between the data that each of these methods has produced is compared. There are striking differences in overall steroid production rates, and in the qualitative nature of the steroid profile which the various methods produce. In particular, aldosterone is secreted at higher rates in vivo, and when whole tissue preparations are used in vitro, than in incubations of isolated glomerulosa cells. In addition, while corticosterone is a major product of glomerulosa tissue in vitro, the available evidence suggests that it is not a major glomerulosa product in vivo.