Liquid Scintillation Counting (liquid + scintillation_counting)

Distribution by Scientific Domains

Selected Abstracts

Measurement of specific radioactivity in proteins separated by two-dimensional gel electrophoresis

Shaobo Zhou
Abstract We report a method to quantify the specific radioactivity of proteins that have been separated by 2-DE. Gels are stained with SyproRuby, and protein spots are excised. The SyproRuby dye is extracted from each spot using DMSO, and the fluorescence is quantified automatically using a plate reader. The extracted gel piece is then dissolved in hydrogen peroxide and radioactivity is quantified by liquid scintillation counting. Gentle agitation with DMSO for 24,h was found to extract all the SyproRuby dye from gel fragments. The fluorescence of the extract was linearly related to the amount of BSA loaded onto a series of 1-D gels. When rat muscle samples were run on 2-DE gels, the fluorescence extracted from 54,protein spots showed a good correlation (r = 0.79, p < 0.001) with the corresponding spot intensity measured by conventional scanning and image analysis. DMSO extraction was found not to affect the amount of radioactive protein left in the gel. When a series of BSA solutions of known specific radioactivity were run on 2-DE gels, the specific radioactivity measured by the new method showed a good correlation (r = 0.98, p < 0.01, n = 5) with the specific radioactivity measured directly before loading. Reproducibility of the method was measured in a series of 2-DE gels containing proteins from the livers of rats and mice that had been injected with [35S]methionine. Variability tended to increase when the amount of radioactivity in the protein spot was low, but for samples containing at least 10,dpm above background the CV was around 30%, which is comparable to that obtained when measuring protein expression by conventional image analysis of SyproRuby-stained 2-DE gels. Similar results were obtained whether spots were excised manually or using a spot excision robot. This method offers a high-throughput, cost-effective and reliable method of quantifying the specific radioactivity of proteins from metabolic labelling experiments carried out in,vivo, so long as sufficient quantities of radioactive tracer are used. [source]

A systematic investigation into the recovery of radioactively labeled proteins from sodium dodecyl sulfate-polyacrylamide gels

Shaobo Zhou
Abstract We report the results of a systematic investigation designed to optimize a method for quantifying radioactivity in proteins in sodium dodecyl sulfate-polyacrylamide gels. The method involves dissolving appropriately sized pieces of gel in hydrogen peroxide and heating to 70C overnight followed by liquid scintillation counting. H2O2 had no effect on the count rates of [14C]bovine serum albumin (BSA) when counted in a conventional liquid scintillation system, and the count rates remained stable for several days. Temperatures below 70C resulted in incomplete extraction of radioactivity from gels containing [14C]BSA, but there was also a significant reduction in count rates in samples incubated at 80C. At 70C recovery was not affected by the amount of sample loaded onto the gel or by the staining procedure (Coomassie Brilliant Blue or SYPRO Ruby). Recoveries were in the range of 89,94%, and the coefficient of variation for five replicate samples was 5,10%. This method offers a reliable way of measuring the amount of radioactivity in proteins that have been separated by electrophoresis. It may be useful, for example, in quantitative metabolic labeling experiments when it is necessary to know precisely how much tracer has been incorporated into a particular protein. [source]

Stereoselective renal tubular secretion of levocetirizine and dextrocetirizine, the two enantiomers of the H1 -antihistamine cetirizine

M. Strolin Benedetti
Abstract Competition for uptake and/or efflux transporters can be responsible for drug interactions. Cetirizine is mainly eliminated unchanged in urine through both glomerular filtration and tubular secretion. The aim of this study was to investigate whether the eutomer, levocetirizine, and the distomer, dextrocetirizine, have a similar tubular secretion. The renal clearance associated with tubular secretion was calculated from the renal clearance of levocetirizine and dextrocetirizine obtained in a study in healthy volunteers. The values of the unbound fraction in plasma were obtained in an in vitro study of the binding of 14C-cetirizine and 14C-levocetirizine to human plasma proteins using equilibrium dialysis and chiral high-performance liquid chromatography (HPLC) with on-line liquid scintillation counting. The unbound fraction was 0.074 for levocetirizine and 0.141 for dextrocetirizine. The tubular secretion of dextrocetirizine (44.5 mL/min) is higher than that of levocetirizine (23.1 mL/min), which may have consequences for drug interactions at the renal level. The higher tubular secretion for dextrocetirizine may be due to the higher free fraction available for secretion or to a higher affinity for (a) renal transporter(s) mediating the secretion pathway. [source]

PMN responses in chronic periodontal disease: evaluation by gingival crevicular fluid enzymes and elastase-alpha-1-proteinase inhibitor complex

Rainer Buchmann
Abstract Objectives: In the present trial, the hypothesis was examined that the local PMN responses in untreated and treated chronic periodontitis can be differentiated by gingival crevicular fluid lysosomal enzyme activities and elastase-alpha-1-proteinase inhibitor complex. Methods: In nine subjects (average age 49.2 7.1 years) with chronic periodontitis, clinical parameters and markers of the PMN-derived inflammatory tissue response in gingival crevicular fluid (GCF) were assessed before and 6 months after surgical periodontal therapy. Myeloperoxidase (MPO), beta-N-acetyl-hexosaminidase (beta-NAH) and cathepsin D (CD) were analyzed as indicators of the PMN-associated host tissue destruction, and elastase-alpha-1-proteinase inhibitor complex (alpha-1-EPI) as the major serum protein inactivating PMN elastase. The total activities of the lysosomal enzymes MPO and beta-NAH were evaluated spectrophotometrically, the CD levels by liquid scintillation counting with [14C] hemoglobin as substrate, and the total alpha-1-proteinase inhibitor complex using a sandwich-immunoassay. Results: The clinical parameters revealed a statistical significant decrease at the 6-month reexamination. PD levels dropped from 5.40 to 2.88 mm (change 2.52 1.04 mm), the CAL scores from 6.67 to 4.43 mm (change 2.24 0.77 mm). The 30 s GCF volumes dropped from 129.8 to 68.6, displaying a change of 61.1 18.6, p , 0.05. The decrease in total MPO, beta-NAH and CD levels (medians: 1.7/0.6 U MPO, 0.035/0.020 U beta-NAH, 1.3/0.5 ng CD) following therapy was associated with a significant drop in total GCF amounts of alpha-1-EPI from 76.3 ng at baseline to 52.4 ng after 6 months. Conclusion: The clinical healing in chronic periodontal disease is associated with a downregulation of the local PMN responses following periodontal therapy. The reorganization of periodontal tissues is characterized by a decrease of lysosomal enzyme activities and the alpha-1-proteinase inhibitor complex in gingival crevicular fluid. [source]

Summary findings of the fourth international radiocarbon intercomparison (FIRI)(1998,2001)

Elisabetta Boaretto
Abstract Interlaboratory comparisons have been widely used in applied radiocarbon science. These are an important part of ongoing quality assurance (QA) programmes, which are vital to the appropriate interpretation of the evidence provided by the 14C record in Quaternary applications (including climate change and environmental reconstruction). International comparisons of laboratory performance are an essential component of the quality assurance process in radiocarbon dating. If the user community is to have confidence in radiocarbon results, it needs to be assured that laboratories world wide are producing measurements that are reliable and in accordance with ,good practice'. The findings from the most recent (completed in 2001) and extensive (more than 90 participating laboratories) radiocarbon intercomparison (FIRI) are reported here. This study was designed (i) to assess comparability, or otherwise, of the results from different laboratories and (ii) to quantify the extent and possible causes of any interlaboratory variation. The results demonstrate that there are no significant differences amongst the main measurement techniques (gas proportional counting, liquid scintillation counting and accelerator mass spectrometry (AMS)) but there is evidence of small laboratory offsets relative to known age samples for some laboratories. There is also evidence in some cases of underestimation of measurement precision. Approximately 10% of all results were classified as extreme (outliers) and these results were generated by 14% of the laboratories. Overall, the evidence supports the fact that radiocarbon laboratories are generally accurate and precise but that, notwithstanding internal QA procedures, some problems still occur, which can best be detected by participation in independent intercomparisons such as FIRI, where the results allow individual laboratories to assess their performance and to take remedial measures where necessary. The results from FIRI are significant in that they show a broad measure of agreement between measurements made in different laboratories on a wide range of materials and they also demonstrate no statistically significant difference between measurements made by radiometric or AMS techniques. Copyright 2002 John Wiley & Sons, Ltd. [source]

Uptake of pesticides from water by curly waterweed Lagarosiphon major and lesser duckweed Lemna minor

Renato F de Carvalho
Abstract The uptake of pesticides from water by two aquatic plants, the submersed Lagarosiphon major (Ridley) Moss and the floating duckweed Lemna minor L., was measured over periods of up to 72 h. Twelve non-ionised pesticides and analogues, chosen to span a wide range of physicochemical properties, and one analogue (3,5-D) of the phenoxyacetic acid herbicide 2,4-D were studied. Concentrations of the parent compound were determined in the plants following extraction and separation by chromatography. Quantification was by liquid scintillation counting for the 14C-labelled compounds and by high-performance liquid chromatography for the four non-radiolabelled commercial pesticides. Uptake for all compound and plant combinations had reached equilibrium by 24 h. Accumulation of compound in the plant could be described well for most non-ionised compounds by equilibration into the aqueous phase in the plant cells together with partitioning onto the plant solids, this latter process becoming dominant in Lagarosiphon for compounds with log Kow > 1 and in Lemna for compounds with log Kow > 1.8. Lipophilic compounds with log Kow > 4 were concentrated more than 100-fold on a fresh-weight basis. However, the uptake of isoproturon and chlorotoluron was up to threefold less than expected from their Kow values, and their behaviour was better explained using solvation descriptors. Uptake of the acid 3,5-D was dependent on solution pH, this compound being strongly taken up at lower pH by the process of ion trapping, as previously observed in barley roots. Aquatic vegetation can thus rapidly accumulate pesticides, and could be an important sink especially for lipophilic pesticides reaching well-vegetated waters. Copyright 2007 Society of Chemical Industry [source]