bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of fluvoxamine concentration in plasma by reversed‐phase liquid chromatography
Authors: Wong Steven H. Y. , Kranzler Henry R. , Dellafera Sandra , Fernandes Rosinda
Published: May 2005

Fluvoxamine, a serotonin re‐uptake inhibitor, was quantified in plasma by modifying a previously published procedure for monitoring plasma concentrations of tricyclic antidepressants. Alkalinized plasma samples were extracted with n‐hexane/isoamyl alcohol, followed by back‐extraction with diluted phosphoric acid, The extracts were analysed by reversed‐phase liquid chromatography using a C‐18 column, with phosphate/acetonitrile as the mobile phase. The assay was linear from 10 to 800 μg/L. Precision studies showed within‐run and day‐to‐day coefficients of variation to be 4.5 and 6.8%, respectively. Desipramine interfered with the detection of fluvoxamine. The assay was used to measure a total of 8 plasma samples from 4 alcohol‐dependent patients medicated with fluvoxamine as an adjunct to relapse prevention psychotherapy. In these patients, the plasma concentrations ranged from 54 to 241 μg/L. Dosage of fluvoxamine, duration of treatment, interval between last dosage and blood collection were associated with effects on plasma concentrations that were consistent with the pharmacokinetic profile of fluvoxamine.
Mannitol prevents methionine sulphoxidation mediated electrophoretic heterogeneity of apolipoprotein A‐I
Authors: Nofer Jerzy‐Roch , von Eckardstein Arnold , Assmann Gerd
Published: May 2005

Hybrid isoelectic focusing of apolipoprotein A‐I in polyacrylanide gels with immobilized pH‐gradients under non‐denaturing conditions resulted in the occurrence of additional bands which could prevent the specific and sensitive detection of genetic variants. Hybrid isoelectric focusing of tow chromatographically distinguishable apolipoprotien A‐I isoforms that differ by sulphoxidaton of methionine residues, apo A‐I(Met) and apo A‐I(MetSO), revealed that the additional bands were caused by this post‐translational modification. Several antioxidative additives and conditions were compared for their ability to prevent methionine sulphoxidation in apoliporotein A‐I In the presence of 200 g/L mannitol in the gel, apolipoprotein A/I focused as a single band. Since methionine sulphoxidation in proteins is a general phenomenon either taking place in vivo or in vitro by isoelectric focusing, we conclude that isoelectric focusing in the presence of mannitol will improve the quality of resolution of many proteins in gels with immobilized pH‐gradients.
Determination of oxeladin in human plasma by gas chromatography—mass spectrometry
Authors: Lartigue‐Mattei C. , Galmier M. J. , Chabard J. L. , Beyssac E. , Aiache J. M.
Published: May 2005

A capillary gas chromatographic method with mass‐selective detection was developed for the determination of oxeladin in human plasma. Plasma samples (1 mL) were alkalinized and extracted using 5 mL of hexane: isoamyl alcohol (99:1). The method was demonstrated to be sensitive (limit of quantitation at 1 ng/mL), linear between 1 and 150 mg/mL, accurate and precise enough (mean error and mean coefficient of variation at the limit of quantitation were 2.3 and 13.3%, respectively) to support pharmacokinetic evaluation of the drug at doses down to 30 mg.
Purification of murine immunoglobulin M from spent tissue culture supernatant by one‐step gel filtration chromatography procedure
Authors: Novales‐Li Philipp
Published: May 2005

Optimal purification of immunoglobulin M (IgM) grown as spent tissue culture supernatant can be achieved by a straightforward size‐exclusion chromatography procedure. Preparative isolation of IgM was achieved by precipitation with saturated ammonium sulphate to achieve a 45% solution. IgM was then purified by gel filtration chromatography with a solution of 100 mM Tris+150 mM NaCl at pH 8. Denaturing electrophoresis and Western immunoblotting revealed two prominent bands at 80 and 25 kDa, indicative of the heavy and light chains of IgM respectively. The specific immunoreactivity of this IgM was assessed by a modified enzyme antigen capture assay. In contrast to affinity and ion‐exchange chromatographies, gel filtration allows retention of IgM immunoreactivity.
Sensitive determination of salmon calcitonin, by means of pre‐column derivatization, HPLC and fluorometric determination
Authors: Fukuda Terumi , Ishikawa Kazuyuki , Imai Kazuhiro
Published: May 2005

A high sensitive analytical method for salmon calcitonin (sCT) was established by means of derivatization with a fluorescence labelling agent, DBD‐F, followed by HPLC separation and fluorescence detection at 558 nm with excitation at 430 nm.

Under optimized conditions (DBD‐F, 0.1 M borate buffer (pH 8.5): acetonitrile (70:30, v/v)), three molecules of DBD‐F reacted with one sCT molecule in the presence of 1 mM sodium dodecyl sulphate (SDS) at 50 oC for 3 h. The detection limit for the sCT derivative was 20 fmol. A linear relationship was obtained between the amount of sCT on column and the peak heights in the range of 35–8000 fmol.
High‐performance liquid chromatography and fluorometric detection of arachidonylethanolamide (anandamide) and its analogues, derivatized with 4‐(N‐chloroformylmethyl‐N‐methyl) amino‐7‐N, N‐dimethylaminosulphonyl‐2,1,3‐benzoxadiazole (DBD‐COCl)
Authors: Koga Daisuke , Santa Tomofumi , Hagiwara Kenichi , Imai Kazuhiro , Takizawa Hiromasa , Nagano Tetsuo , Hirobe Masaaki , Ogawa Masako , Sato Taku , Inoue Keizo , Kudo Ichiro
Published: May 2005

The endogeneous ligand for the cannabinoid receptor, arachidonylethanolamide (anandamide) and its analogues, oleinylethanolamide, palmitylethanolamide and eicosapentaenoylethanolamide, were derivatized with a fluorogenic reagent, 4‐(N‐chloroformylmethyl‐N‐methyl)amino‐7‐N, N‐dimethylaminosulphony 1‐2,1,3‐benzoxadiazole (DBDCOCl). They were separated on a reversed phse HPLC with a mobile phase of acetonitrile: water. The fluorometric detection of the derivatives was made at 560 nm with excitation at 450 nm and the detection limits for anandamide was 20 fmol on column. The structures of DBDCO‐ethanolamides were confirmed by liquid chromatography‐atmospheric pressure chemical ionization—mass spectrometry (LCAPCIMS).
Determination of glutathione and related aminothiols by gas chromatography with flame photometric detection
Authors: Kataoka Hiroyuki , Takagi Kiyomi , Makita Masami
Published: May 2005

A selective and sensitive method for the determination of glutathione (GSH) and related aminothiols such as cysteine (Cys), cysteinylglycine (CysGly) and γ‐glutamylcysteine (γ‐GluCys) by gas chromatography (GC) has been developed. GSH and related aminothiols were converted into their N, S‐isopropoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection using a short capillary column (5 m × 0.53 mm i.d.) of cross‐linked DB‐1. The calibration curves were linear in the range 1–25 nmol for GSH and in the range 0.2–5 nmol for other aminothiols, and the detection limits of GSH, Cys, CysGly and γ‐GluCys were approximately 5, 0.2, 0.3 and 0.5 pmol per injection respectively. This method was successfully applied to blood samples without prior clean‐up, and GSH and related aminothiols in these samples could be analysed without any influence from coexisting substances. Overall recoveries of GSH and other aminothiols added to blood samples were 88–107%. The analytical results of free and total blood GSH and related aminothiols in normal subjects are presented.
High‐performance liquid chromatographic separation of penicillamine enantiomers labelled with N‐[4‐(6‐dimethylamino‐2‐benzofuranyl)phenyl]maleimide on a chiral stationary phase
Authors: Nakashima Kenichiro , Ishimaru Tomoko , Kuroda Naotaka , Akiyama Shuzo
Published: May 2005

Penicillamine enatiomers derivatized with N‐[4‐(6‐dimethylamino‐2‐benzofuranyl)phenyl]maleimide (DBPM) were separated and determined by high‐performance liquid chromatography. A fluorogenic reagent, DBPM easily reacted with D‐ or L‐penicillamine to give each two kinds of strong fluorescent derivatives (D1−, D2−, L1− and L2 DBPM), which could be separated on a Pirkle‐type chiral stationary phase using an eluent of 75% aqueous methanol solution containing 0.15 M CH3COONH4 and 0.05 M tetra‐n‐butylammonium bromide. Two of the peaks (D1− or L1‐DBPM), having a shorter retention time than the others, had almost the same retention times (25 min for D1‐DBPM and 25.7 min for L1‐DBPM). The retention times of the peaks eluted later were 28 min and 31.6 min for D2‐ and L2‐DBPM respectively. Linear calibration curves over the range of 2–50 pmol per injection were obtained for D‐and L‐penicillamines with a detection limit of 290 and 350 fmol at respectively at a signal‐to‐noise ratio of 3. Using the proposed method, the absence of contamination of L‐penicillamine in a commercially available D‐penicillamine preparation (capsule) was confirmed.
Reversed‐phase high‐performance liquid chromatography of fullerenes with tetrahydrofuran‐water as a mobile phase and sensitive ultraviolet or electrochemical detection
Authors: Santa Tomofumi , Koga Daisuke , Imai Kazuhiro
Published: May 2005

Fullerenes (C60 and C70) were separated on a reversed‐phase high‐performance liquid chromatography column with a mobile phase of tetrahydrofuran‐water (70:30, v/v) and detected with ultraviolet or electrochemical detection. A baseline resolution of C60 and C70 was achieved with a capacity factor values of 7.6 and 10.8 respectively and a resolution of 3.7. The detection limit for C60 was 0.1 pmol on column and was nearly the same for both detection methods.
The application of affinity chromatography for quantitative determination of metallothionein in physiological fluids by an indirect method based on analysis of metal contentse
Authors: Kabziński Andrzej K. M. , Takagi Toshio
Published: May 2005

An indirect method, based on the analysis of metal contents, for the determination of metallothionein (MT) in body fluids is described. Affinity chromatography gel was used as a solid phase extraction (SPE) support for preconcentration of MT Proteins and metals bound to MT from spiked water and human urine samples. The results showed a good correlation between the concentration of MT proteins added to water or physiological human urine and the concentration of protein indirectly determined by atomic absorption spectrometry (AAS) recovered by the SPE‐affinity chromatography technique and calculated from the metal concentration. The present method can be used in toxicological analysis for determination of MT concentration in physiological fluids in subjects exposed to heavy metals.
Simultaneous determination of nadolol enantiomers in human plasma by high‐performance liquid chromatography using fluorescence‐detection
Authors: Belas Frank J. , Phillips Mark A. , Srinivas Nuggehally R. , Barbhaiya Rashmi H. , Blair Ian A.
Published: May 2005

A high‐performance liquid chromatographic assay is described for the separation and quantification of nadolol isomers in human plasma. The isomers were quantified using reverse‐phase HPLC and fluorometric detection after derivatization with the chiral reagent R(‐)‐1‐(naphthyl)ethylisocyanate [R(‐)‐NEI]. The N‐isopropyl analogue (one isomer) of nadolol was used as the internal standard. The method was reproducible based on precision studies where the percent relative standard deviation was less than 15%. The lower limit of quantitation for each isomer was 2.5 ng/mL. This method was used to evaluate the pharmacokinetic profile of nadolol isomers in human subjects following both single and multiple oral dosing.
The development of a sensitive myo‐inositol analyser using a liquid chromatograph with a post‐label fluorescence detector
Authors: Inamoto Yukiko , Hiraga Yayoi , Hanai Toshihiko , Kinosita Toshio
Published: May 2005

A sensitive liquid chromatographic analysis for myo‐inositol was developed using glycocyamine as the postlabelling reagent. The sensitivty was 500 pmol/injection. The system was applied to analyse myo‐inositol in sera from eight patients with chronic renal failure. The average concentration of serum myo‐inositol was 498.6 ± 257.0 μmol/L before haemodialysis, and 244.0 ± 131.1 μmol/L after haemodialysis. These results indicated that the kidney is the main site of myo‐inositol metabolism.
Relationships between binding of benzodiazepines to alpha‐1‐acid glycoprotein and human serum albumin and their retention on the protein and octadecylsilane columns
Authors: Sadlej‐Sosnowska Nina
Published: May 2005

Correlation between binding parameters of 6 benzodiazepines to free human serum albumin (HSA) and chromatogrphic retention parameters on the HSA stationary phase (HSA‐SP) was better than the similar correlation with alpha‐1‐acid glycoprotein (AGP). Still better correlation was obtained between binding to both proteins and retention on an octadecylsilane (ODS) column. For 12 benzodiazepines the correlations of the retention parameters on both protein columns with the retention on the ODS column were additionally investigated. In the case of HSA this correlation was good.
A novel method for the amino acid sequence/configuration determination of peptides containing D/L‐amino acids utilizing a fluorogenic edman reagent, 7‐N,N‐dimethylaminosulphonyl‐4‐(2,1,3‐benzoxadiazolyl)isothiocyanate (DBD‐NCS)
Authors: Imai Kazuhiro , Matsunaga Hirokazu , Fukushima Takeshi , Santa Tomofumi , Homma Hiroshi , Nakashima Kenichiro , Akiyama Shuzo
Published: May 2005

A novel method for the amino acid sequence and configuration determination of peptides containing D‐ or L‐amino acids is presented. An enkephalin analogue, [D‐Ala2, D‐Leu5]enkephalin (Tyr‐Ala‐Gly‐Phe‐Leu) was derivatized with a fluorogenic Edman reagent, 7‐N,N‐ dimethylaminosulphonyl‐4‐(2,1,3‐benzoxadiazolyl) isothiocyanate (DBD‐NCS), cleaved and cyclized with trifluoroacetic acid at 50 oC for 1 min and the resultant thiazolinaone derivative (DBD‐thiazolinyl‐L‐isomer) on a phenylcarbamylated cyclodextrin column: The separation factor (α) for D‐ and L‐isomers was 1.09. The column eluate was monitored fluorometrically at 524 nm with excitation at 387 nm. The detection limit was about pmol range. The same treatment adoted for the residual peptide, Ala‐Gly‐Phe‐Leu, gave DBD‐thiazolinyl‐D‐Ala (α = 1.09 for D,L‐Phe) with the lesser amount of L‐analogue (about 20%). In the same manner, Gly and L‐Phe (α = 1.09) were detected. The method might be useful for the study of aging of proteins such as eye lens and the amyloid proteins derived from Alzheimer's disease.
Determination of argininosuccinate lyase in human serum by ion‐pair reversed phase liquid chromatography
Authors: Canepari S. , Carunchio V. , Girelli A. M. , Messina A.
Published: May 2005

A new assay for argininosuccinate lyase based on the separation of the enzymatic reaction mixture components by an ion‐pair reversed phase mechanism is reported. The determination of enzyme activity is performed after direct injection by UV detection of the fumarate formed. The chromatographic analysis time is 8 min and a detection limit of 0.4 U/L is achieved. The HPLC method is highly accurate, sensitive and precise. The simple procedure makes this method suitable for the routine determination of ASAL activity in human serum samples.
The quantitative analysis of inhalational anaesthetics in forensic samples by gas chromatography/mass spectrometry/selected ion monitoring
Authors: Maruyama Kyoko , Takatsu Akihiro , Obata Toru
Published: May 2005

The quantitative analysis of volatile anaesthetics for biomedical applications by means of gas chromatography/mass spectrometry/selected ion monitoring (GC/MS/SIM) was studied. Xenon gas was selected as an internal standard for the assay by adding to a closed system, because of its stability and inactivity. In the assay of inhalational anaesthetics, isoflurane and nitros oxide (laughing gas), in forensic samples (serum and cerebrospinal fluid), the calibration of the anaesthetic was linear from 0.12 to 12 nmol/mL in isoflurane and from 30 to 300 nmol/mL in nitrous oxide. Our results suggest that this new method is suitable for the quantitative analysis of inhalational anaesthetics in the biomedical field.
Capillary gas chromatographic analysis of protein amino acids as their N(O,S)‐isobutoxycarbonyl methyl ester derivatives
Authors: Matsumura Sayuri , Kataoka Hiroyuki , Makita Masami
Published: May 2005

A simple and rapid method for the determination of the 22 protein amino acids by capillary gas chromatography is described. The amino acids were converted into their N(O,S)‐isobutoxycarbonyl (isoBOC) methyl ester derivatives and measured by GC with flame ionization detection using a DB‐17 capillary column. Arginine was converted into ornithine by arginase treatment prior to the isoBOC reaction. The N(O,S)‐isobutoxycarbonylation of amino acids with isobutyl chloroformate was completed within 15 s by sonication in aqueous alkaline media. All of the derivatives were quantitatively resolved as single symmetrical peaks within 9 min by GC on a single column. The detection limits of amino acids were 0.2–4.0 ng per injection, and the calibration curves were linear in the range 0.2–50 μg for each amino acid. This method was successfully applied to protein hydrolysate samples. The analytical results of amino acid compositions of several proteins are presented.
Stereoselective analysis of nadolol in human plasma
Authors: Srinivas N. R. , Shyu W. C. , Shah V. R. , Campbell D. A. , Barbhaiya R. H.
Published: May 2005

A steroeselective method, involving a single liquid‐liquid extraction step, was developed and validated for the analysis of nadolol in human plasma. The assay involved extraction of nadolol and desmethyl‐nadolol as internal standard (IS) from alkalinized plasma into dichloromethane. The organic solvent was separated and evaporated under nitrogen at 40°C. A chiral derivatization scheme with (R)‐(−)‐napthylethylisocyanate (50 μL of 0.1% solution in dichloromethane for 60 min) was employed to convert the enantiomers of nadolol into the corresponding diastereomeric derivatives. The residue was reconstitutd in the mobile phase and injected onto a C‐18 column. The mobile phase was a mixture of methanol: tetrahdrofuran: water (52:7:41 by vol) containing about 0.001% v/v of both phosphoric acid and tetramethylethylenediamine. Fluorimetric detection was performed at excitation 230 and emission 330 nm. The assay was specific for the enantiomers of nadolol and the lower limit of quantitation was 2 ng/mL for of the enantiomers. Analysis of quality control samples resulted in precision estimates of 7% RSD for inter‐assay and 10.1% RSD for intra‐assay and the predicted concentrations deviated less than 9.4% of the nominal values for the four enantiomers. The extraction recoveries of the individual enantiomer was about 70%. Stability of nadolol enantiomers were established for four freeze/thaw cycle periods and in the autosampler at 5°C for at least 116 h.
Selective and sensitive determination of pamidronate in human plasma and urine by gas chromatography with flame photometric detection
Authors: Sakiyama Norihisa , Kataoka Hiroyuki , Makita Masami
Published: May 2005

A rapid, selective and sensitive method for the determination of pamidronate in human plasma and urine samples by gas chromatography (GC) has been developed. After deproteinization of the sample with trichloroacetic acid, pamidronate was converted into its N‐isobutoxycarbonyl methyl ester derivative and measured by GC with flame photometric detection (FPD), using a HP‐1 capillary column. The derivative preparation and GC analysis were accomplished within 30 min. The derivative was sufficiently volatile and stable, giving a single and symmetrical peak, and provided an excellent FPD response. The detection limit of pamidronate, at a signal‐to‐noise ratio of 3, was ca. 100 pg injected, and the calibration curve for this compound in the range 20–1000 ng was linear and sufficiently reproducible for quantitative determination. This method could be successfully applied to plasma and urine samples without a preliminary clean‐up except for deproteinization with trichloroacetic acid, and pamidronate could be measured without any influence from coexisting substances. Overall recoveries of pamidronate added to plasma and urine samples were 93–97%. The coefficients of variation for intra‐assay and inter‐assay of pamidronate in these samples were 1.0–7.9% (n = 3) and 4.1–8.3% (n = 6), respectively.
Rapid determination of O‐ and P‐cresol isomers in urine from workers exposed to toluene by high‐performance liquid chromatography using a graphitized carbon column
Authors: Schlatter J. , Astier A.
Published: May 2005

A simple and rapid procedure is described that permits the simultaneous determination of o‐ and p‐ cresol in urine of workers or other individuals exposed to toluene. The urine samples are enzymatically hydrolysed and analysed by high‐performance liquid‐chromatography with a detetion limit of 0.2 mg/L, one fifth of the biological threshold limit value for o‐cresol (1 mg/g ), a specific marker of toluene exposure. A graphitized carbon column Hybercarb‐S, that exhibits an excellent selectivity for aromatic positional isomers, was used with 1% phosphoric acid‐acetonitrile (70:30, v/v) as mobile phase and a detection wavelength of 271 nm. The overall accuracy for o‐cresol determination was 4.5% at 5 mg/L and 8% at 0.5 mg/L. The cresol isomers were sufficiently resolved from endogenous materials to avoid the need for any extraction step, and the method appears suitable for monitoring workers accurately under the permissible level of exposure for occupational medicine purpose.
Metabolic control and compartmentation in single living cells
Authors: Kohen Elli , Kohen Cahide , Hirschberg Joseph G. , Wouters Alain W. , Thorell Bo , Westerhoff Hans V. , Charyulu Komanduri K. N.
Published: May 2005

Microspectrofluorometry of cell coenzymes (NAD(P)H, flavins) in conjunction with sequential microinjections into the same cell of metabolites and modifiers, reveals aspects of the regulatory mechanisms of transient redox changes of mitochondrial and extramitochondrial nicotinamide adenine dinucleotides. The injection of ADP in the course of an NAD(P)H transient produced by glycolytic (e.g. glucose 6‐phosphate, G6P) or mitochondrial (e.g. malate) substrate leads to sharp reoxidation (state III, Chance and Williams, 1955), followed by a spontaneous state III to IV transition, and an ultimate return to original redox steady state. The response to ADP alone is biphasic, i.e. a small oxidation‐reduction transient followed by a larger reverse transient. Similarities between responses to injected ATP and ADP suggest possible intracellular inter‐conversions. Sequential injections of glycolytic and Krebs cycle substrates into the same cell, produce a two‐step NAD(P) response, possibly revealing the intracellular compartmentation of this coenzyme. A two‐step NAD(P)H response to sequentially injected fructose 1,6‐diphosphate and G6P indicates the dynamic or even structural compartmentation of glycolytic phosphate esters in separate intracellular pools. The intracellular regulation and compartmentation of bioenergetic pathways and cell‐to‐cell metabolic inhomogeneities provide the basis on which the quantitative biochemistry of the intact living cell may be reconciled with these in situ findings.
Fibronectin is not a serum spreading factor for HeLa cells
Authors: Michl J. , Baudyšová M. , Sochorová L. , Deyl Z.
Published: May 2005

The spreading of HeLa cells on plastic substratum is mediated by fibronectin‐depleted foetal calf serum but not by fibronectin isolated by gelatin‐Sepharose affinity chromatography. The same is true for freshly explanted chick embryonic chondrocytes. In contrast, BHK cell spreading exceeds 67% after 120 min at 37°C in fibronectin‐supplemented (10 μg ml−1) serum‐free medium. Long‐term cultivation of HeLa cells in Eagle's MEM supplemented with fibronectin‐free serum is associated with the accumulation of cells in mitosis or before cytokinesis; many cells die and the remaining living cells, characterized by marked changes in morphology, multiply very slowly. It can be concluded therefore that fibronectin does not produce spreading in HeLa cells but forces them into mitosis.
Quantitative cytochemistry of blood neutrophils in myelodysplastic syndromes and chronic granulocytic leukaemia
Authors: Schofield K. P. , Stone P. C. W. , Kelsey P. , Leyland M. J. , Stuart J.
Published: May 2005

Quantitative cytochemistry of components of blood neutrophil azurophilic granules (myeloperoxidase, chloroacetate esterase, β‐glucuronidase, and acid phosphatase) and specific granules (lactoferrin) has been performed by scanning and integrating micro‐densitometry in 13 patients with a myelodysplastic syndrome and 11 patients with chronic granulocytic leukaemia. Both patient groups showed a reduction of enzyme activity in azurophilic granules, and also of lactoferrin, consistent with abnormal development of neutrophil granules. These cytochemical changes in blood neutrophils are similar to those found in acute myeloid leukaemia, are consistent with a leukaemic maturation defect, and may be of diagnostic value.
Tumour localization of uroporphyrin isomers I and III and their correlation to albumin and serum protein binding
Authors: El‐Far Mohamed A. , Pimstone Neville R.
Published: May 2005

We were the first to report the superiority of uroporphyrin I (UROP I) as a tumour localizer when compared to haematoporphyrin derivative (HPD). In this study, we compared both isomers of UROP, i.e. I and III, in a KHJJ mammary carcinoma mouse model. Six and 18 h after UROP administration, the tumour, skin and gut porphyrin (P) content was quantitated. Tumour UROP I levels were always at least 50% higher than UROP III in tumour, whereas both isomers were barely detectable in the skin and gastrointestinal tract. We then explored the possibility that tumour P uptake might relate in part to the affinity of circulating P to mouse serum proteins (MSP), in particular, the major binding protein constituent, albumin. Copro‐P III, deutero‐P 2,4 disulphonic acid (DP), proto‐P IX (PP) and heptacarboxylic P I (Hepta I) which in our mouse tumour model do not localize in malignant tissue, were compared to UROP I and III. The P was mixed with 0.775 μM human serum albumin (HSA) at different molar ratios (HSA:P range 2–8) and the unbound P concentration quantitated using an Amicon CF‐25 membrane cone with centrifugation. The percentage free P was significantly higher for UROP I (92–98%) than III (82–95%) and significantly more than that observed with non‐tumour localizing P studied. Similar data were obtained with MSP. This is consistent with the notion that enhanced uptake and retention (particularly UROP I) by malignant neoplastic tissue might reflect a higher affinity for UROP by tumour constituents than by circulating proteins. We conclude, (i) that serum proteins, particularly albumin, may play a significant role in governing the localization of P in tissues and, (ii) UROP clinically is ideally suited as a diagnostic marker of pre‐ or early mucosal malignancy as minimal retention by skin and gut would increase fluorescence specificity and diminish photocutaneous side effects.
Comparison of two cell isolation procedures to study in vitro intestinal wall biotransformation in control and 3‐methyl‐cholanthrene pretreated rats
Authors: Borm Paul J. A. , Koster Andries Sj. , Frankhuijzen‐Sierevogel Ank , Noordhoek Jan
Published: May 2005

Two cell isolation procedures, i.e. a scraping/collagenase‐treatment and a new vibration procedure in EDTA containing medium, were used to isolate intestinal epithelial cells. In both cell populations the metabolism of 7‐ethoxycoumarin and 7‐hydroxycoumarin was studied. Moreover, the time course and extent of induction of both steps in the biotransformation were investigated after oral 3‐methylcholanthrene pretreatment of the rats. Twenty four hours after 3‐methylcholanthrene pretreatment (20 mg kg−1) monooxygenase activity was induced about 6‐fold and 2.5‐fold when studied with cells of the vibratory and enzymic procedures, respectively. Control 7‐ethoxycoumarin deethylase activity and 7‐hydroxycoumarin glucuronidation were about the same when comparing both methods for cell‐isolation. The formation of glucuronides in cells (both methods) is significantly lowered by 3‐MC pretreatment, while sulphation remains unaffected. Results indicate that using enzymic treatment of mucosal scrapings, cell‐populations are obtained containing relatively more differentiated (tip) cells. A number of advantages of the new (vibration) method are: better recovery, viability and reproducibility.
Possible mechanism for target cell lysis by cytotoxic T‐cells
Authors: Friedberg R. C. , Bitensky L. , Chayen J. , Muir I. H. , Askonas B. A. , Taylor P. M.
Published: May 2005

The mechanism of the lysis of target cells by cytotoxic T‐cells (Tc) is still obscure; there is no evidence for transfer of material from the Tc and prior to lysis, despite intimate contact, the plasma membranes of both types of cell appear to remain intact. The effects on the target cell lysosomes of brief contact between anti‐viral Tc and targets bearing both the appropriate histocompatibility and viral antigens, have been examined cytochemically. Both the distribution of acid phosphatase activity and the percentage bound lysosomal naphthylamidase activity indicated that, in virus‐infected target cells exposed to Tc, the lysosomal membranes became totally labilized. Thus the contact between Tc and targets appears to cause sufficient perturbation of the target plasma membrane as to cause the intracellular release of some agent that activates ‘suicide capsule’ lysosomes.
Localization of biotransformational enzymes along the crypt—villus axis of the rat intestine. Evaluation of two cell isolation procedures
Authors: Koster Andries Sj. , Borm Paul J. A. , Noordhoek Jan , Dohmen M. René
Published: May 2005

Rat intestinal epithelial cells were isolated by EDTA‐chelation, combined with gentle shaking (modified Weiser procedure) or with strong longitudinal vibration (Harrison/Webster procedure). Both methods yield large numbers of viable cells and are relatively easy to use. Electronmicroscopical and biochemical data indicate that cell fractions from different levels of the villous region can be obtained only by the modified Weiser procedure. When strong mechanical forces are involved (Harrison‐Webster procedure) the villus epithelium is released according to an all‐or‐nothing process. The biotransformational capacity of cell fractions, obtained from different levels of the villi by the modified Weiser procedure, was investigated. It was shown that the rate of metabolism of 7‐ethoxycoumarin and 1‐naphthol was substantially higher in lower villous cells than in cells isolated from the upper villous region. O‐Deethylation of 7‐ethoxycoumarin decreases from 145 ± 13 pmole/min mg cell protein (72 ± 4% conjugated) in lower villous cells to 62 ± 12 pmole/min mg cell protein (37 ± 6% conjugated) in tip cells. Glucuronidation of 1‐naphthol decreased from 495 ± 23 pmole/min mg cell protein (lower villous cells) to 137 ± 13 pmole/min mg cell protein (tip cells).
The effect of various aldehydes on the respiration of rat liver and hepatoma AH‐130 cells
Authors: Canuto R. A. , Biocca M. E. , Muzio G. , Garcea R. , Dianzani M. U.
Published: May 2005

Some aldehydes, produced during lipid peroxidation of liver lipids, are able to inhibit the respiration of mitochondria and of intact cells both in normal hepatocytes and in Yoshida hepatoma. In mitochondria, the respiratory stimulation produced by addition of ADP and dinitrophenol is decreased more in hepatoma than in normal liver. Two‐ to four‐fold higher concentrations of aldehydes are needed to obtain the same degree of inhibition in normal liver mitochondria as in tumorous organs. The effect of aldehydes on intact cell respiration is absent or very low in hepatocytes, but it is consistently observed in hepatoma cells.
Lysosomal enzymes in human platelets
Authors: Leoncini Giuliana , Balestrero Flavio , Maresca Mitzi
Published: May 2005

In human freshly prepared platelets the following lysosomal enzymes were studied: α‐mannosidase, α‐fucosidase, β‐galactosidase, β‐glucosidase, β‐glucuronidase, β‐N‐acetylglucosaminidase and acid phosphatase. For each of the examined enzymes the conditions providing maximal activity (pH, buffer), kinetic parameters (saturating substrate concentration and Km) as well as heat stability were established. On the basis of these parameters it is suggested that many of the serum glycohydrolases may be platelet derived.
Formation of activated oxygen in the hypoxic rat liver
Authors: Räder Lutz , Siems Werner , Müller Marianne , Gerber Gerhard
Published: May 2005

The biliary GSSG efflux rate of normoxic perfused rat liver was 1·5 ± 0·2 nmol/min/g liver wet weight. The GSSG efflux rate as indicator for the flux through the glutathine peroxidase and, therefore, for an oxidative loading incvreased with the extent of hypoxia. 2·6 ± 0·5 nmol/min/g were released from the severely hypoxic liver. The hydroxyl radical scavenger formate as well as the xanthine oxidase inhibitor allopurinol reduced the efflux rate of GSSG.

GSH was released from the perfused liver at a rate of 15·5 nmol/min/g which was nearly unchanged in severe hypoxia.

The high rate of glucose liberation from the hypoxic liver declined to almost that of the normoxic organ in the presence of formate.

There is an ‘oxidative stress’ during hypoxic liver perfusion which probably originates from increased generation of activated oxygen species in the degradation of purine nucleotides.
Investigation of the role of ubiquinone in rat liver subcellular compartments
Authors: Casu A. , Cottalasso D. , Pronzato M. A. , Rolla C. , Marinari U. M. , Nanni G.
Published: May 2005

The role of ubiquinone in the Golgi apparatus is still unknown, even if it might be considered as a lipid marker of the Golgi compartment because of its high content in these subcellular fractions. In vivo modulation of ubiquinone with ethanol and in vitro pentane extraction show that ubiquinone is not required either for NADH‐ferricyanide reductase, acetaldehyde dehydrogenase activity, or Ca2+ and Mg2+ stimulated ATPases.

Since ubiquinone does not seem to be involved in these enzymic activities in Golgi compartments, other possible functions are discussed, related to a role in membrane fluidity or as a barrier to the propagation of free radicals.
Expression of a particular β‐N‐acetylgucosaminidase isoenzyme in human haematopoietic leukemic cell‐lines
Authors: Orlacchio A. , Emiliani C. , Tabilio A. , Pioda G. B.
Published: May 2005

N‐acetyl‐β‐D‐glucosaminidase (NAG) activity and isoenzyme profiles were studied in myeloid, histiocytic, B‐lymphoid, T‐lymphoid and lymphoblastoid continuous cell lines in order to determine if N‐acetyl‐β‐D‐glucosaminidase isoenzyme expression may help to distinguish among various types of leukemic proliferation.

Total NAG activity in myeloid, histiocytic, erythroleukemic cell lines were higher than Burkitt's lymphoma derived cell lines (B‐lymphoid), T‐ or lymphoblastoid cell lines.

On chromatofocusing by PBE 94 coupled with an automated enzyme assay an intermediate (I) β‐N‐acetyl‐glucosaminidase form, eluting between forms B and A, was found in all leukemic and in Epstein‐Barr virus infected lymphoblastoid cell lines analysed.

The different profiles recorded, the expression of the I form and the different I/B ratios may be useful as markers of tumour proliferation.
Cellular distribution of lysosomal hydrolase activities in the regenerating rat liver
Authors: Baccino Francesco M. , Barrera Giuseppina , Bonelli Gabriella , Messina Maria , Musi Marco , Tessitore Luciana
Published: May 2005

Cathepsins B and D, β‐galactosidase, and acid phosphatase activities were found to be decreased in the regenerating rat liver, the reduction being maximal around the peak of hepatocyte mitoses (30 h). To investigate whether these changes could be heterogeneously distributed among hepatic cells, total cell populations from control or two‐thirds hepatectomized rat livers were dissociated by the collagenase perfusion technique and analysed by different procedures. Isopycnic centrifugation in a Metrizamide gradient satisfactorily resolved hepatocytes and non‐parenchymal cells from control animals but was not adequate when applied to 30‐h regenerating liver cells. Colchicine treatment of the hepatectomized animals, resulted in substantial accumulation of phase M‐hepatocytes. Subpopulations considerably enriched in fast‐sedimenting phase M‐cells were obtained by sedimentation at 1 g of the total liver cell population, and subsequently analysed by isopycnic equilibration. Phase M‐hepatocytes were shown to have markedly reduced levels of β‐galactosidase, acid phosphatase, and cathepsin B activities in comparison, not only with control hepatocytes, but also with those parenchymal cells which were not metaphase‐arrested in the same regenerating livers. Therefore, in partially‐hepatectomized rats, hepatocytes progressing up to metaphase in the first mitotic cycle exhibited a selective depletion of lysosomal enzyme activities. The mechanism(s) underlying this change remain(s) presently unknown.
Cortisol succinate is slowly hydrolysed in organ cultures of porcine articular tissues and is not equivalent to cortisol
Authors: Lawrence Christopher E. , Wright Judith M. , Knight C. Graham
Published: May 2005

The pro‐drug cortisol succinate is frequently used as a substitute for cortisol in organ cultures. We found, however, that in Dulbecco's modified Eagle's medium the time taken for the ester to undergo 50 per cent hydrolysis (t½) to cortisol was 75 h. When 15 per cent heat‐inactivated normal rabbit serum was present t½ decreased to 47 h, but the rate of hydrolysis was not further increased in the presence of porcine articular cartilage or minced synovial tissue. When frozen and thawed synovium was present t½ decreased to 33 h, presumably due to the release of carboxyl‐esterases from the disrupted cells. The level of tetrahydrocortisol was low in all of the cultures. The slow hydrolysis of cortisol succinate resulted in the exposure of the tissues to undersirable fluctuations in the concentration of active hormone, which decreased to low levels at each medium change. Thus, in co‐cultures of porcine synovium and articular cartilage, cortisol had a greater inhibitory effect than cortisol succinate on the breakdown of cartilage matrix caused by synovial tissue.
Characterization and use of polyclonal antibody to Na+, K+‐ATPase: Immunocytochemical localization in salt glands of the duck
Authors: Russo James J. , Merchant Juanita L. , Eager Patricia R. , Barrnett Russell J.
Published: May 2005

The amount of Na+, K+‐ATPase of the avian salt gland increased concomitantly with plasma membrane surface area during salt feeding of ducklings (adaptation), and both enzyme content and membrane surface area decreased upon return to fresh water (deadaptation). In a further study of the enzyme, a marker for plasma membrane biogenesis, polyvalent antibodies were raised to the denatured α‐subunit of the purified ATPase. Antisera did not inhibit enzymatic activity but immunoprecipitated the phosphorylated intermediate of the α‐subunit. Furthermore, the α‐subunit, which was not glycosylated, was immunoprecipitated from homogenates of tissue slices metabolically labelled with [35S]‐methionine, using antisera raised against either duck salt gland or dog kidney α‐subunit. The former antisera also recognized the α‐subunit in the brain, heart, kidney, liver, intestine and skeletal muscle of the duck.

Immunocytochemistry with the antisera raised to the duck salt gland α‐subunit revealed reaction at basolateral as well as apical plasma membrane in the duck salt gland principal cells, with essentially no deposits no deposits on peripheral cells, fibroblasts, erythrocytes, endothelial cells and neural elements. Within the principal cells, immunolabelling was also detected on small vesicles, multivesicular bodies and lysosomes; deposits on extracellular debris and vesicles in the lateral and lumenal spaces were also apparent. The labelling patterns were qualitatively but not quantitatively similar in salt glands of control, adapted and deadapted ducklings, and are discussed in the context of a model for plasma membrane biogenesis and turnover in which degradative events may play a major role.
Growth hormone binding and stimulation of amino acid uptake in epithelial cells of rat ventral prostate
Authors: Prieto J. C. , Carmena M. J.
Published: May 2005

The binding of [125I]‐human growth hormone (hGH) was studied in epithelial cells isolated from rat ventral prostate. Binding and degradation were dependent on time and temperature. The effect of a lysosomotropic agent suggested internalization and lysosomal degradation of the hormone. Dissociation and stoichiometric studies indicated the existence of a single class of GH receptors with a Kd of 0·7 nM and a binding capacity of 46 fmol hGH bound mg−1 cell protein. The receptor appeared to possess a somatotrophic nature since lactogenic hormones such as human placental lactogen and rat prolactin exhibited a very low degree of competition (whereas a variety of unrelated hormones and neuropeptides showed no effect). GH‐stimulated leucine uptake by the cells in a time‐ and dose‐dependent manner, half maximal effect being observed at 0·32 nM GH thus suggesting a direct relationship with the binding step.
Biochemistry of Entamoeba: A review
Authors: Avron Boaz , Chayen Ann
Published: May 2005

Entamoeba histolytica, the protozoan parasite causing amoebiasis, is carried by approximately 10% of the world's population although only a minority of carriers of the organism present active clinical symptoms.

Although Entamoeba are classified as eukaryotes, the biochemistry of these organisms has a number of unusual facets which are reminiscent of prokaryotes. It has indeed been suggested that these cells represent early evolutionary forms that have been successful in surviving unchanged in the protected environment in which they reproduce (the intestine).

Glycolysis in Entamoeba lacks several of the typical eukaryotic glycolytic enzymes. The pentose phosphate shunt enzymes are missing completely. Another unusual feature of the glycolytic path is the utilization of PPi instead of ATP in a number of enzyme reactions e.g. phosphofructokinase. Entamoeba is the only eukaryote in which one of these PPi utilizing enzymes, phosphoenolpyruvate carboxytransferase, has been found.

The respiratory pathway is also an unusual one. Entamoeba are classified as anaerobes but the cells do have an affinity for oxygen. The oxygen is reduced to water at the end of a respiratory chain which is not well understood but which operates withough cytochromes or mitochondria.

The nucleic acid, protein and lipid metabolic pathways have not been well studied and interest has mainly focused on the proteolytic processes of the amoeba which have been implicated in the pathogenic, histolytic behaviour of the parasite. Despite these efforts the mechanism of attack of the parasite and the stimuli that cause it to invade the host are not yet clear. This understandably is the goal of much of the present studies concerning E. histolytica but the organism also deserves study in its own right as an example of an organism that has an unusual biochemistry and may represent an early stage in the evolution of eukaryotic cells.
Adenylate cyclase in membrane fractions of RIN‐A2‐cells: Studies with forskolin, NaF, GppNHp and NEM
Authors: Trier J. K. , Kühn M. , Ammon H. P. T.
Published: May 2005

In crude membrane fractions of rat pancreatic islets and of RIN‐A2‐cells, forskolin and NaF stimulated adenylate cyclase activity. Basal and stimulated enzyme activity was approximately 3 to 6 fold higher in membranes of RIN‐A2‐cells than in membranes of islet cells. In RIN‐A2‐cells GppNHp and NEM inhibited forskolin‐stimulated cyclase system of RIN‐A2‐cells contains inhibitory and stimulatory N‐proteins and that there are critical thiols related to Ni, Ns and/or the catalytic unit. Thus, membrane fractions of RIN‐A2‐cells may be an appropriate model for studies on the adenylate cyclase system of insulin‐producing cells.
Methotrexate: Studies on cellular metabolism. II—Effects on mitochondrial oxidative metabolism and ion transport
Authors: Yamamoto Nair , Lopes Luiz Carlos Vieira , Campello Annibal Paiva , Klüppel Maria Lucia Wambier
Published: May 2005

Effects of methotrexate (MTX) on mitochondrial oxidative metabolism and ion transport were studied. MTX decreases the membrane potential (Δψ) upon energization of the mitochondrial membrane by NAD+‐linked substrates and decreases the amplitude and velocity of swelling induced by glutamate and α‐ketoglutarate. MTX also has an inhibitory effect on the activities of the oxidation enzymes of NAD+‐linked substrates without interfering with the oxidation systems of FAD‐linked substrates. The effects of MTX could be interpreted as a consequence of a decrease in the ionic conductivity of the mitochondrial inner membrane.
Methotrexate: Studies on cellular metabolism. IV. Effect on the mitochondrial oxidation of cytosolic‐reducing equivalents in HeLa cells
Authors: Bastos Ma. Teresa C. C. , Oliveira Ma. Benigna M. , Campello Annibal P. , Klüppel Ma. Lúcia W.
Published: May 2005

The effect of methotrexate (MTX) on the mitochondrial oxidation of cytosolic‐reducing equivalents in HeLa cells was studied. MTX inhibited (100 per cent) malate dehydrogenase activity, but no effect was observed on that of GOT. MTX (0.5 mM) inhibited (100 per cent) the activity of reconstituted enzymatic system MDH–GOT, probably as a consequence of inhibition of malate dehydrogenase activity. MTX decreased pyruvate production (54 per cent), demonstrating its inhibitory action on the malate‐aspartate shuttle. Blockage of the malate–aspartate shuttle by MTX accounts for the decrease in cellular energetic gain. The results obtained are consistent with the view that in HeLa cells, as well as in other tumour cells, the trasport of reducing equivalents from cytoplasmic NADH into the respiratory chain of mitochondria is via the malate‐aspartate shuttle.
Interaction of captan with mammalian microtubules
Authors: Stournaras Christos , Saridakis Ioannis , Fostinis Yannis , Georgoulias Vassilis
Published: May 2005

Using turbidometry, electron microscopy and immunofluorescent microscopy experiments we studied the effect of captan, a widely used pesticide on mammalian microtubules and microfilaments. Turbidometry at 350 nm showed a dose‐dependent inhibition of tubulin assembly incubated with captan. The pesticide, given at equimolar concentration with tubulin (30 μM), caused the total inhibition of microtubule formation, while at lower concentrations (5–20 μM) the inhibition of tubulin polymerization was less extensive. At the same concentration range (5–30 μM), captan also promoted the disassembly of performed microtubules. The results of the in vitro effects of captan with microtubules were confirmed in parallel by electron microscopic studies. In vivo, captan caused also depolymerization of microtubules in cultured mouse fibroblasts as shown by indirect immunofluorescent staining of tubulin. The extent of microtubules disassembly was concentration‐ and time‐dependent. While incubation of the cells with 10 μM captan for 3 h disturbs totally the microtubular structures, incubation with 5 μM captan needs 12 h for the same effect. Recovery of microtubules was observed, when preincubated cells were extensively washed. No interaction of this drug with equimolar concentration of G‐ or F‐actin could be observed in vitro, as shown by polymerization experiments. In line with this, the fluorescent actin pattern in mouse fibroblasts incubated with 10 mM captan for up to 12 h did not seem to be altered. From these results it is concluded that captan interacts in equimolar concentrations with tubulin affecting the assembly and disassembly of microtubules in vitro and in cultures of mammalian cells.