Determination of S-period and cell cycle time of 19S hemolysin producing spleen cells of mice cultured in vitro

Summary The cell cycle time and the doubling time of mouse spleen cells producing 19S hemolytic antibody against sheep red blood cells were determined in vitro.The doubling time of the number of hemolytic plaque forming cells was found to be 4–7 hours. By a combination of the hemolytic plaque assay and the double labeling method with ³H- and ¹⁴C-thymidine the duration of the S-period and the cell cycle time were determined to be 8–9 hours and 13–15 hours, respectively.The disagreement in doubling time and cycle time of PFC is discussed and a possible explanation by cell recruitment is presented.Herrn Prof. Dr. W. Maurer zum 65. Geburtstag gewidmet.     

H2AX phosphorylation as a genotoxicity endpoint

The γH2AX focus assay, based on phosphorylation of the variant histone protein H2AX, was evaluated as a genotoxicity test in immortalised wild-type mouse embryonic fibroblasts (MEFs) treated for 4 h with a panel of reference compounds routinely used in genotoxicity testing. The topoisomerase II poison etoposide (0.006–60 μg/ml), the alkylating agent methyl methanesulfonate (1.3–65 μg/ml) and the direct DNA-damaging agent bleomycin (0.1–10 μg/ml) all produced a positive concentration–response relationship. The non-genotoxic compounds ampicillin (0.035–3500 μg/ml) and sodium chloride (0.058–580 μg/ml) showed no such response with increased concentrations. The H2AX phosphorylation results were compared with the outcome of two standard in vitro genotoxicity tests, namely the micronucleus and comet assays. Compounds that produced measurable DNA damage in the focus assay generated micronuclei at comparable concentrations. In this study, the focus assay identified genotoxic agents with the same specificity as the comet assay.These results were substantiated when H2AX phosphorylation was analysed using flow cytometry in the murine cell line L5178Y, growing in suspension. The data were in concordance with the manual scoring focus assay. To further this investigation, the γH2AX flow cytometry was compared to the in vitro micronucleus flow cytometry and mouse lymphoma assay using the same cell population after MMS treatment. The median γH2AX value increased significantly above the control at all four MMS concentrations tested. The percentage of micronucleus events in the in vitro micronucleus flow test and the mutation frequency in the mouse lymphoma assay were also significantly increased at each MMS concentration. The current data indicate that H2AX phosphorylation could be used as a biomarker of genotoxicity, which could predict the outcome of in vitro mammalian cell genotoxicity assays.     

Competitive Immunochemical Determination of Antigens Using Conjugates Containing Co(II) and Ni(II)

A new variant of competitive heterogeneous immunoassay for certain proteinaceous antigens has been developed. The assay is based on the use of the target protein conjugated with Co(II) or Ni(II) ions and immobilized antibodies. The effect of catalytic hydrogen release allows quantitation of the metal ion labels by voltammetry at the final step of the assay. The conjugates have been characterized by spectrophotometry, voltammetry, atomic adsorption spectrometry, and nuclear magnetic relaxation. Based on the use of the conjugate RNase–diethylenetriaminepentaacetic acid–Co(II) (10 : 4 : 4), a competitive immunoassay for RNase has been developed, detecting the target protein in the range 2 × 10^–2–2 × 10^–4 mg/ml.     

Reversed-phase high-performance liquid chromatographic analysis of atenolol enantiomers in rat hepatic microsome after chiral derivatization with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate

A reversed-phase high-performance liquid chromatographic method for the determination of the enantiomers of atenolol in rat hepatic microsome has been developed. Racemic atenolol was extracted from alkalinized rat hepatic microsome by ethyl acetate. The organic layer was dried with anhydrous sodium sulfate and evaporated using a gentle stream of air. Atenolol racemic compound was derivatized with 2,3,4,6-tetra-O-acetyl-β- -glycopyranosyl isothiocyanate at 35°C for 30 min to form diastereomers. After removal of excess solvent, the diastereomers were dissolved in phosphate buffer (pH 4.6)–acetonitrile (50:30). The diastereomers were separated on a Shimadzu CLC-C18 column (10 μm particle size, 10 cm×0.46 cm I.D.) with a mobile phase of phosphate buffer–methanol–acetonitrile (50:20:30, v/v) at a flow-rate of 0.5 ml/min. A UV–VIS detector was operated at 254 nm. For each enantiomer, the limit of detection was 0.055 μg/ml (signal-to-noise ratio 3) and the limit of quantification (signal-to-noise ratio 10) was 0.145 μg/ml (RSD <10%). In the range 0.145–20 μg/ml, intra-day coefficients of variation were 1.0–7.0% and inter-day coefficients of variation were 0.4–16.5% for each enantiomer. The assay was applied to determine the concentrations of atenolol enantiomers in rat hepatic microsome as a function of time after incubation of racemic atenolol.     

Nodularin in feathers and liver of eiders (Somateria mollissima) caught from the western Gulf of Finland in June–September 2005

Nodularins are cyanobacterial hepatotoxins, which may cause intoxication at very low exposure levels. The nodularin-producing cyanobacterium Nodularia spumigena usually forms massive blooms in much of the Baltic Sea during the summer season. Breast feathers and liver samples from common eider (Somateria mollissima) were analysed for nodularins by liquid chromatography–mass spectrometry (LC–MS) and enzyme-linked immunosorbent assay (ELISA). Fifteen eiders from the western Gulf of Finland were caught by hunters between June and September 2005. Blue mussels (Mytilus edulis), a dietary component of the birds, were also obtained by diving near the same marine area and time as the collection of the ducks. Eider breast feathers contained 6–52 μg nodularin-R (Nod-R)/kg dry weight (dw) by ELISA, and 8–43 μg Nod-R/kg dw by LC–MS. No Nodularia filaments were adhered to feather samples according to light microscopy assessment. Liver samples from the same individuals contained Nod-R between 3 and 48 μg/kg dw by LC–MS. Mussel samples from the area contained Nod-R at concentrations of 12–80 μg/kg dw by LC–MS. Analysis of bird feathers offers a facile and non-invasive means of assessing the exposure of birds to nodularins.     

A nanogram-level colloidal gold single reagent quantitative protein assay

We have developed a nanogram-level quantitative protein assay based on the binding of colloidal gold to proteins adhered to nitrocellulose paper. The protein–gold complex produces a purple color proportional to the amount of protein present, and the intensity of the stain is quantified by densitometry. Typical assays require minimal starting material (10–20 μl) containing 1 to 5 μg protein. A small volume (2 μl) of protein solution is applied to nitrocellulose paper in a grid array and dried. The nitrocellulose is incubated in colloidal gold suspension with gentle agitation (2–16 h), rinsed with water, and scanned. Densitometric analysis of the scanned images allows quantitation of the unknown sample protein concentration by comparison with protein standards placed on the same nitrocellulose grid. The assay requires significantly less sample than do conventional protein assays. In this report, the Golddots assay is calibrated against weighed protein samples and compared with the Pierce Micro BCA Protein Assay Kit. In addition, the Golddots assay is evaluated with several known proteins with different physical properties.     

Determination of acyclovir by ultrafiltration and high-performance liquid chromatography

A simple, sensitive and rapid high-performance liquid chromatographic (HPLC) procedure to determine total serum acyclovir concentrations is described. The assay involves a heat inactivation step at 56°C to prevent risk of infection, ultrafiltration as a pretreatment step prior to ion-pair reversed-phase liquid chromatography using guanosine as internal standard, and ultraviolet detection at 254 nm. This method has excellent recovery (97–100%), linearity (0.5–100 mg/l) and precision (1.2–8.0% coefficient of variation). The detection limit is 50 μg/l. The assay proved to be suitable for therapeutic drug monitoring of acyclovir.     

High-performance size-exclusion chromatographic analysis of dextran–methylprednisolone hemisuccinate in rat plasma

A size exclusion chromatographic method is presented for the measurement of the concentrations of a macromolecular prodrug of methylprednisolone (MP), dextran–methylprednisolone succinate (DEX–MPS), in rat plasma. After precipitation of the plasma (100 μl) proteins with perchloric acid, the samples are injected into a size exclusion column with a mobile phase of water:acetonitrile:glacial acetic acid (75:25:0.2) and a flow-rate of 1 ml/min. The DEX–MPS conjugate, detected at 250 nm, elutes at a retention time of 6.5 min, free of endogenous peaks. Excellent linear relationships (r²=0.997) were found between the detector response and the concentrations of DEX–MPS in the range of 2–100 μg/ml (MP equivalent), with intra- and inter-run C.V.s of <6% and error values of <5%. The application of the assay was also demonstrated by measurement of the plasma concentrations of DEX–MPS after single 5 or 10 mg/kg doses of the conjugate administered intravenously to rats.     

Determination of a novel selective inhibitor of type 1 5α-reductase in human plasma by liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry

A sensitive and specific assay of human plasma for the determination of (5α,7β,16β)-16[(4-chlorophenyl)oxy]-4,7-dimethyl-4-aza-andronstan-3-one (I), a selective inhibitor of human type 1 5α-reductase, has been developed. The method is based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS–MS) detection. The analyte (I) and internal standard, Proscar (II), were isolated from the basified biological matrix using a liquid–liquid extraction with methyl-tert.-butyl ether (MTBE). The organic extract was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The MS–MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring using the precursor→product ion combinations of m/z 430→114 and 373→305 was used to quantify I and internal standard (II), respectively. The assay was validated in the concentration range of 0.5 to 500 ng/ml in human plasma. The precision of the assay, expressed as coefficient of variation (C.V.), was less than 7% over the entire concentration range, with adequate assay specificity and accuracy. The HPLC–MS–MS method provided sufficient sensitivity to completely map the 24 h pharmacokinetic time-course following a single 0.5 mg dose of I.     

Simultaneous determination of theophylline, enoxacin and ciprofloxacin in human plasma and saliva by high-performance liquid chromatography

A simple reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of theophylline, ciprofloxacin and enoxacin in plasma and saliva. The biological fluid samples were extracted with methylene chloride-isopropyl alcohol prior to isocratic chromatography on a Waters C₁₈ μBondapak column. Ultraviolet detection was carried out at 268 nm. The assay in linear for ciprofloxacin and enoxacin (0.05–10 μg/ml), and theophylline (0.1–20 μ/ml). The assay can be used to investigate the interaction of these two fluoroquinolones with theophylline.     

Determination of ritodrine in biological fluids of the pregnant sheep by fused-silica capillary gas chromatography using electron-capture detection

A sensitive and selective gas chromatographic assay method employing splitless injection, fused-silica capillary columns and electron-capture detection is reported for the quantitation of the tocolytic drug, ritodrine, in a variety of biological fluids obtained from the pregnant ewe and fetus. This method has improved sensitivity and selectivity over previously published assay procedures. A 25 m × 0.31 mm I.D., cross-linked 5% phenylmethylsilicone, fused-silica capillary column was employed for all analyses. Linearity of response was observed over the range 2.5–75 ng of ritodrine base per 0.05–0.5 ml of biological fluid, representing ≈ 1–75 pg at the detector. The coefficient of variation was less than 10% over the range 2.5–75 ng of added ritodrine. The minimum quantifiable amounts is ≈ 2.5 ng from a 0.5-ml biological fluid sample. Applicability of this method to biological fluids, obtained from ovine subjects, is demonstrated by the analysis of samples obtained during the course of ritodrine placental transfer studies.     

Solid-phase extraction and liquid chromatographic quantitation of quinfamide in biological samples

This paper describes a high-performance liquid chromatographic method for the assay of quinfamide and its main metabolite, 1-(dichloroacetyl)-1,2,3,4,-tetrahydro-6-quinolinol, in plasma, urine and feces. It requires 1 ml of biological fluid, an extraction using Sep-Pack cartridges and acetonitrile for drug elution. Analysis was performed on a CN column (5 μm) using water–acetonitrile–methanol (40:50:10) as a mobile phase at 269 nm. Results showed that the assay was linear in the range between 0.08 and 2.0 μg/ml. The limit of quantitation was 0.08 μg/ml. Maximum assay coefficient of variation was 14%. Recovery obtained in plasma, urine and feces ranged from 82% to 98%.     

Chemotaxis towards sugars inChlamydomonas reinhardtii

Chemotaxis ofChlamydomonas reinhardtii 137c cells towards maltose and sucrose was observed by capillary assay. The threshold concentration was approximately 10^–5 m for maltose and 10^–3 m for sucrose. The peak accumulation of cells occurred at 10^–3 m for maltose and 10^–2 m for sucrose. A selection procedure for chemotaxis mutants was developed. Mutant strain CHE-1 was not attracted by maltose; mutant strain CHE-2 was not attracted by sucrose. Addition of attractant fully inhibited photoaccumulation of cells. After a period of time the photoresponse of cells recovered. Under the conditions of our experiments the period of adaptation lasted 15–20 min in wild-type cells and 4–5 min in mutant cells on the given sugar. Glucose and acetate did not attract cells ofChlamydomonas. Added to the medium, these compounds had no effect on the photoaccumulation of cells.     

Evaluation of different techniques for pregnancy diagnosis in sheep

Fifty Corriedale ewes were used in this study to evaluate pregnancy diagnosis in sheep. Ewes were bred under a pen mating system and pregnancy diagnosis was initiated from day 15 post-mating, applying the diagnostic techniques of trans-abdominal real-time B-mode ultrasonography, Preg-alert (A-mode ultrasonography), the Doppler ultrasonic fetal pulse detector or the plasma progesterone concentration assay (EIA). These tests were repeated fortnightly on all the ewes until the onset of lambing. The accuracy of trans-abdominal real-time B-mode ultrasonography (68%) at days 15–30 of pregnancy increased to 100% by days 61–75 and remained constant until lambing. The accuracy of the Preg-alert (56%) diagnosis at days 31–45 increased to 94% by days 91–105 of gestation and then decreased to 82% from days 136 of gestation to lambing. The accuracy of both the Doppler ultrasound (56%) at days 31–45 and plasma progesterone assay (98%) at days 15–30 of gestation increased to 100% at days 76–90 and 46–60 of gestation, respectively and remained constant until parturition. The mean plasma progesterone concentration at days 0–6 (1.41 ± 0.21 ng/ml) increased to 4.0 ± 0.87 ng/ml at days 16–30 (days 18.23 ± 0.78) post-mating. Animals returning to estrus recorded less than 1 ng/ml at days 18.23 ± 0.78 post-mating. The accuracy of both the B-mode ultrasonic technique (78%) and plasma progesterone assay (98%) was significantly higher (P < 0.05) than the accuracy obtained with the A-mode and Doppler ultrasound (both 56%) at days 31–45 of gestation. The study concluded that real-time B-mode ultrasonography is the earliest, most accurate, safest, fastest and most economical method of pregnancy diagnosis in sheep at farm level. The A-mode and Doppler methods can also be used under field conditions, where sophisticated laboratory facilities are not available. Plasma progesterone assays (EIA) can be used as a means of early pregnancy diagnosis in organized sheep farms with fair accuracy.     

Quantification of epimeric budesonide and fluticasone propionate in human plasma by liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry

A highly sensitive and selective liquid chromatography–atmospheric pressure chemical ionization tandem mass spectrometry assay was developed and validated for simultaneous determination of epimeric budesonide (BUD) and fluticasone propionate (FP) in plasma. The drugs were isolated from human plasma using C₁₈ solid-phase extraction cartridges, and epimeric BUD was acetylated with a mixture of 12.5% acetic anhydride and 12.5% triethylamine in acetonitrile to form the 21-acetyl derivatives following the solid-phase extraction. Deuterium-labelled BUD acetate with an isotopic purity >99% was synthesized and used as the internal standard. The assay was linear over the ranges 0.05–10.0 ng/ml for epimeric BUD, and 0.02–4.0 ng/ml for FP. The inter- and intra-day relative standard deviations were <14.3% in the assay concentration range.     

Surface-enhanced Raman spectroscopy combined with atomic force microscopy for ultrasensitive detection of thrombin

Indole-3-acetic acid (IAA) amide conjugates play an important role in balancing levels of free IAA in plant cells. The GH3 family of proteins conjugates free IAA with various amino acids. For example, auxin levels modulate expression of the Oryza sativa (rice) GH3-8 protein, which acts to prevent IAA accumulation by coupling the hormone to aspartate. To examine the kinetic properties of the enzyme, we developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay system. Bacterially expressed OsGH3-8 was purified to homogeneity and used to establish the assay system. Monitoring of the reaction confirms the reaction product as IAA–Asp and demonstrates that production of the conjugate increases proportionally with both time and enzyme amount. Steady-state kinetic analysis using the LC–MS/MS-based assay yields the following parameters: V/E_t^IAA = 20.3 min⁻¹, K_m^IAA = 123 μM, V/E_t^ATP = 14.1 min⁻¹, K_m^ATP = 50 μM, V/E_t^Asp = 28.8 min⁻¹, K_m^Asp = 1580 μM. This is the first assignment of kinetic values for any IAA–amido synthetase from plants. Compared with previously described LC- and thin-layer chromatography (TLC)-based assays, this LC–MS/MS method provides a robust and sensitive means for performing direct kinetic studies on a range of IAA-conjugating enzymes.     

Simultaneous on-line extraction and analysis of sirolimus (rapamycin) and ciclosporin in blood by liquid chromatography–electrospray mass spectrometry

We developed a sensitive and specific semi-automated liquid chromatography–electrospray mass spectrometric (HPLC–ESI-MS) assay for the simultaneous quantification of sirolimus and ciclosporin in blood. Following a simple protein precipitation step, the supernatants were injected into the HPLC system and extracted on-line. After column switching, the analytes were backflushed from the extraction column onto the analytical narrow-bore column and eluted into the ESI-MS system. The assay was linear from 0.4 to 100 μg/l sirolimus and from 2 to 1500 μg/l ciclosporin. The mean recoveries of sirolimus and ciclosporin were 98 and 96%, respectively. The mean interday precision/accuracy was 8.6%/−4.8% for sirolimus and 9.3%/−2.9% for ciclosporin.     

Measurement of dexfenfluramine metabolism in rat liver microsomes by gas chromatography–mass spectrometry

A specific and useful method was developed for the determination of dexfenfluramine metabolism by microsomal systems utilising GC–MS. The synthesis of two metabolites 1-(3-trifluoromethylphenyl)propan-2-ol (‘alcohol') and 1-(3-trifluoromethylphenyl)-1,2-propanediol (‘diol') via straightforward routes, were confirmed by MS and NMR spectra. The conditions for extraction from alkalinised microsomal mixtures of the metabolites nordexfenfluramine, 1-(3-trifluoromethylphenyl)propan-2-one (‘ketone'), alcohol and diol, their conversion to trifluoroacetate derivatives and analysis by GC–MS–SIM are described. Calibration curves were constructed between 48 and 9662 nM and fitted to quadratic equations (r²>0.999). The method precision was good over low (121 nM) medium (2415 nM) and above medium (9662 nM) concentrations for all metabolites; the within- and day-to-day coefficients of variation ranged between 2.5–12.4% and 6.7–17.5%, respectively. The accuracy, measured as bias, was very good both within- and day-to-day (range: −0.4–12.6%, 0.8–18.9%). For most metabolites, the C.V. for the assay and bias increased at 121 nM. Dexfenfluramine metabolism by rat liver microsomes was investigated using the assay method and showed a concentration dependent increase in nordexfenfluramine and ketone metabolites over the substrate range of 5–200 μM.     

Determination of ceftazidime in plasma using high-performance liquid chromatography and electrochemical detection: Application for individualizing dosage regimens in elderly patients

This study describes a sensitive HPLC–electrochemical detection method for the analysis of ceftazidime, a third-generation cephalosporin, in human plasma. The extraction procedure involved protein precipitation with 30% trichloroacetic acid. The separation was achieved on a reversed-phase column (250×4.6 mm I.D., 5 μm) packed with C₁₈ Kromasil with isocratic elution and a mobile phase consisting of acetonitrile–25 mM KH₂PO₄–Na₂HPO₄ buffer, pH 7.4 (10:90, v/v). The proposed analytical method is selective, reproducible and reliable. The assay has a precision of 0.2–15.1% (C.V.) in the range of 5–200 μg ml⁻¹. (corresponding to 0.5 to 20 ng of ceftazidime injected onto the column), and is optimised for assaying 50 μl of plasma. The extraction recovery from plasma was approximately 100%. The method was highly specific for ceftazidime and there was no interference from either commonly administered drugs or endogenous compounds. This assay was used to measure ceftazidime in elderly patients for therapeutic drug monitoring.