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PubMed1999

Procedures for the determination of 222Rn exhalation and effective 226Ra activity in soil samples.

Two methods for measuring 222Rn exhalation and effective 226Ra in soil samples were studied. In the first determination, the method employed was based on the adsorption of radon onto activated charcoal and subsequent measurement of the activity of its daughters with an HPGe (high-purity germanium) detector. In the second, vials containing an aqueous suspension of the sample, mixed with an insoluble high efficiency mineral oil scintillation cocktail, were measured using a low-level liquid scintillation counter. Studies of optimum sampling time, efficiency in both procedures, variation of 226Ra efficiency with quenching, as well as the effect of sample amount and granulometry upon the quenching parameter, were carried out. The two methods were applied to the determination of 222Rn exhalation and effective 226Ra in environmental samples.

Charcoal↗

PubMed1983

An enzymic radiochemical method for determining phosphatidylglycerol in amniotic fluid.

We describe an enzymic quantification of phosphatidylglycerol in amniotic fluid. Phosphatidylglycerol is hydrolyzed in alkali and the resulting glycerol is then enzymatically phosphorylated with adenosine 5'-[gamma-32P]triphosphate to yield glycero[32P]phosphate. After removal of excess [gamma-32P]ATP by charcoal, the radioactivity of the glycerophosphate is measured in a liquid scintillation counter. Triglyceride in the amniotic fluid is hydrolyzed by lipase before extraction and thus does not interfere with the analysis. This method is specific for phosphatidylglycerol. Preliminary studies suggest that a phosphatidylglycerol value greater than or equal to 10 nmol/mL correlates with fetal lung maturity.

Amniotic Fluid↗

PubMed2000

Detection of p56(lck) kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384- and 1536-well format.

p56(lck) is a lymphocyte-specific tyrosine kinase that plays an important role in both T-cell maturation and activation. We have developed a homogeneous assay in which p56(lck) catalyzes the transfer of the gamma-phosphate group from [gamma-(33)P]ATP to a biotinylated peptide substrate. The labeled peptide is then captured on a streptavidin-coated scintillation proximity assay (SPA) bead or imaging proximity bead. The SPA is counted in a microplate scintillation counter and the imaging proximity assay is counted in a charge-coupled device-based imaging system called LEADseekertrade mark, recently launched as a homogeneous imaging system by Amersham Pharmacia Biotech. We show, via time-dependence assays and inhibitor studies, that this assay can be performed in 1536-well microplate format using imaging proximity as the method of detection. The results compare favorably with the same assay performed in 384-well microplate format using both SPA and imaging proximity as the detection methods. From this study, we conclude that a kinase assay can be performed in 384- and 1536-well format using imaging as the detection method, with significant time savings over standard scintillation counting. In addition, we show cost saving advantages of 1536- over 384-well format in terms of reagent usage, higher throughput, and waste disposal.

Amino Acid Sequence↗

PubMed2002

Uptake rate measurement of some amino acids on normal and treated yeast cells to xenobiotics using 14C labelled amino acid.

Benzo(alpha)pyrene (BP) and 7,12--dimethylbenz(alpha) anthracene (DMBA) are potent carcinogens for mammals, which are able to affect the normal metabolic processes. The influence both of BP and DMBA to the transport rate of individual 14C labeled amino acids (14C-lysine; 14C-valine; 14C-leucine or 14C-tyrosine) in yeast Saccharomyces cerevisiae strain A3 were studied by introducing about one microCi (37 kBq) of individual 14C labeled amino acid into 30 ml liquid ethanol media that contained BP (0.001% v/v) or DMBA (0.001% v/v), then followed by inoculating a known concentration of yeast suspension in such a manner to get the initial optical density (OD) of new cultures were about 0.10. Uptake rates were determined at certain intervals after inoculation, using a liquid scintillation counter. The results show that BP had the tendency to increase the uptake rate while DMBA showed a reversed effect on the use of amino acids. It was also found that tyrosine was absorbed faster than valine as well as leucine and this was different with the result reported by the former investigators.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2002

A radioassay for phosphofructokinase-1 activity in cell extracts and purified enzyme.

Phosphofructokinase-1 plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of the glycolytic flux in a tissue sample. The method most commonly employed to determine phosphofructokinase-1 activity is based on oxidation of NADH by the use of aldolase, triosephosphate isomerase, and alpha-glycerophosphate dehydrogenase. This method suffers from several disadvantages, including interactions of the auxiliary enzymes with phosphofructokinase-1. Other methods that have been used also require auxiliary enzymes or are less sensitive than a coupled assay. Here, we propose a direct method to determine phosphofructokinase-1 activity, without the use of auxiliary enzymes. This method employs fructose-6-phosphate and ATP labeled with 32P in the gamma position ([gamma-32P]ATP), and leads to the formation of ADP and fructose-1,6-bisphosphate labeled with 32P ([1-32P]fructose-1,6-bisphosphate). Activated charcoal is used to adsorb unreacted [gamma-32P]ATP, and the radioactive product in the supernatant, [1-32P]fructose-1,6-bisphosphate, is analyzed on a liquid scintillation counter. The proposed method is precise and relatively inexpensive, and can be applied to determine phosphofructokinase-1 activity in cellular extracts as well as in the purified enzyme.

Adenosine Triphosphate↗

PubMed1987

Observations on tracheal mucociliary clearance in horses.

The rate of movement up the mucociliary escalator of Technetium99m tagged to macroaggregated albumin was measured in 7 normal horses and 8 with respiratory disease. The activity at 2 sites in the neck, separated by 12-15 cm, was measured using a hand held scintillation counter and rate meter. In normal horses the Technetium complex moved as a bolus up the neck with a mean rate of 1.58 cm/min. In the horses with respiratory disease the Technetium complex was less likely to move as a discrete bolus and in three horses it was not possible to measure the transport rate. The remainder had a mean transport rate of 1.19 cm/min.

Animals↗

PubMed1998

Myocardial fatty acid metabolism in diabetic mice with 125I-BMIPP.

In patients with diabetes mellitus, the existence of diabetic cardiomyopathy was substantiated. This study was undertaken to evaluate the myocardiac fatty acid metabolism of diabetic mice (n = 21) and controls (n = 21) in 125I-BMIPP in fasted and unfasted states. 125I-BMIPP of 370 kBq was given and thirty minutes later, animals from both groups were killed. Samples of hearts, liver and other organs were removed, weighed and then counted in a scintillation counter. The percent injected dose/g of hearts of diabetic mice was significantly reduced compared to controls in unfasted (p < 0.05) and fasted (p < 0.01) groups. These findings may reflect impaired fatty acid utilization of the hearts in diabetic mice compared to controls.

Animals↗

PubMed1978

[A method for on-line acquisition and semi-automated reporting of unilateral clearance studies of the kidneys (author's transl)].

A four-channel scintillation counter for clearance studies has been connected on-line to a computer. A program has been written which allows the computer to calculate the total clearance rate and the relative contribution of each kidney. The results, together with a semi-automated report, are issued as a letter by teletype. The accuracy of the program has been checked by manual evaluation of the clearance curves.

Computers↗

PubMed1981

Steroid sulphatase activity in the skin biopsies of various types of ichthyosis.

A simple method was developed for the determination of steroid sulphatase activity in a skin biopsy for routine use. In this method, 6 mg of minced tissue is incubated in 1 ml of Krebs-Ringer bicarbonate buffer with radioactive dehydroepiandrosterone sulphate (20,000 c.p.m., S.A. 1.1 Ci/mmol) for 4 h. After the incubation the liberated unconjugated dehydroepiandrosterone and its possible metabolites are separated from the conjugated compound by extraction with ethyl acetate-ethyl ether 10:90 (v/v). The organic phase is counted in a scintillation counter. The results are expressed as c.p.m. values per 6 mg tissue wet weight. Steroid sulphatase activity was measured in skin biopsies from nine control subjects and from thirteen patients with various types of ichthyosis. In the different groups studied, the ranges of the c.p.m. values were as follows: controls (n = 9) 377-1802; X-linked ichthyosis (n = 5) 140-214; ichthyosis vulgaris (n = 5) 607-1320; ichthyosiform erythroderma (n = 2) 2146-2214; lamellar ichthyosis (n = 1) 2185. The reagent blank varied from 180 to 259 c.p.m. In X-linked ichthyosis the c.p.m. values were always less than in the corresponding reagent blank, indicating that no enzyme activity was present in the tissue. In other types of ichthyosis, steroid sulphatase activity was normal. The method described is easy to accomplish in any clinical laboratory where scintillation counting is possible.

Female↗

PubMed1984

Clearance from cerebrospinal fluid of intrathecally administered beta-endorphin in monkeys.

Five adult male monkeys (Macaca mulatta) weighing 7.1-9.9 kg were given synthetic human beta-endorphin (800 micrograms) and [14C]methoxy-inulin (50 microCi) in 400 microliters of normal saline intrathecally. Serial samples of cerebrospinal fluid were drawn through a previously positioned indwelling spinal catheter and were assayed for concentrations of beta-endorphin (determined by radioimmunoassay) and inulin (determined by liquid scintillation counter). Spinal fluid concentrations of beta-endorphin and inulin peaked and declined in a parallel manner. The clearance ratio (calculated from the reciprocal of the ratio of the areas under the respective curves of elimination of the two species) remained remarkably similar from animal to animal, giving a mean value of 1.060 +/- 0.090 (SEM). This ratio, being near unity, suggests that beta-endorphin is eliminated from spinal fluid in a fashion similar to that of inulin, which is removed exclusively by bulk absorption.

Absorption↗

PubMed2006

Scintillation proximity assay for DNA binding by human p53.

Many DNA binding proteins are known to regulate gene expression. When that binding is altered, a disease state can result. A common method for measuring DNA binding, namely electrophoretic mobility shift assay (EMSA) is often used but it is not amenable to rapid screening of many samples. As an alternative method, we have developed a DNA binding assay for the tumor suppressor protein p53 in a 96-well microtiter plate format using scintillation proximity assay (SPA) beads. We have shown this assay to be sensitive (as little as 0.5 ng p53 can be detected), quick (assay completed in as little as 15 min), and easily quantitated using a microtiter plate scintillation counter We also used the assay to analyze the kinetics of the DNA binding to p53. The specificity of this p53 DNA binding SPA was confirmed using competition by oligonucleotides either from the same gene or from mutated versions of this sequence. Thus, SPA is a good alternative to gel shift assays for DNA binding and may be useful for the analysis of multiple tumor cell samples or for high-throughput screens for compounds affecting DNA binding by proteins of interest.

Animals↗

PubMed1999

[Diagnostic pneumology using model aerosols].

Clearance of Aerosol Particles from the Airways: Measurement of the efficiency and kinetics of particle clearance from the airways is dependent on the site of particle deposition within the lungs. Insoluble particles deposited in the alveolar region are mainly cleared by macrophages over a period of hundreds of days, whereas particles from the tracheobronchial airways are mainly cleared within hours or days by mucociliary clearance. We introduce a method for mucociliary clearance measurements called 'Radio-Bolus-Scintigraphy'. A small volume of radiolabelled aerosol particles sandwiched in clean air (aerosol bolus) is inhaled near the end of a tidal breath which leads to a preferential deposition of the particles in the airways. Particles were Fe3O4 labelled with 99mTc with an aerodynamic particle diameter of 3.5 microns (monodisperse). The retention of the particles within the lungs was detected by Gamma Camera and Human Scintillation Counter. 26 healthy subjects volunteered in this study (14 nonsmokers and 12 smokers). The half width of the mucociliary clearance was found to be 2.4 h in nonsmokers and 3.3 h in smokers (p < 0.05). Intersubject variability was small, 24 hours after inhalation 55% (+/- 6%) of the inhaled particles were still found in the airways of the subjects. No difference was found between nonsmokers and smokers. Good intersubject reproducibility makes this method useful in therapy control as well as in early diagnosis of changes in mucociliary clearance kinetics and efficiency.

Aerosols↗

PubMed1999

Determination of 222Rn in natural water samples from health resorts in the Sudety mountains by the liquid scintillation technique.

A method involving a Wallac 1414 WinSpectral alpha/beta liquid scintillation counter for determining 222Rn in aqueous samples is described. Samples were collected from 23 springs and 4 taps in health resorts of the Klodzka valley in the Sudety mountains in Poland. Half of the investigated spring water samples were radon enriched with an activity concentration higher than 74 Bq/l. In the tap waters the radon concentration level is very low or below the lower limit of detection. Owing to the statistical nature of radioactive decay the uncertainty of the measurement was determined as the standard deviation of 222Rn activity. The method introduced is fast and simple and does not require a chemical sample preparation procedure.

Fresh Water↗

PubMed1980

Attenuation of I- 125 radiation by chloroform and other dense solvents, and its relevance to radioimmunoassays: concise communication.

The counting efficiency of iodine- 125 in well-type scintillation counters is reduced when the labeled compound is dissolved or suspended in chlorinated hydrocarbons or potassium iodide solution. The reduction is probably caused by absorption of the weak gamma and x-rays of I- 125 by the halogen atoms in the solvent or solute molecules. This phenomenon may introduce artifacts into procedures involving radioiodinated compounds and organic solvents and KI, but it could also be useful in differentiating bound from free labeled ligands by their differential solubility (and attenuation) in dense solvents. The reduction in counting efficiency can be overcome by evaporation of the solvent, or by the use of emitters with higher energy radiation.

Cardiolipins↗

PubMed1984

Chemiluminescence induced by phagocytosis of Escherichia coli by polymorphonuclear leucocytes.

Chemiluminescence emitted by phagocytosing human polymorphonuclear leucocytes stimulated by Escherichia coli was measured using a liquid scintillation counter equipped with a multichannel analyser. In the presence of the amplifying agent luminol, light emission can be divided into two channels, one of which ('high energy') appears to correlate directly with phagocytic activity of the PMNL, and the other ('low energy') with the background luminol dioxygenation by the cells. Measuring in the 'high energy' window also eliminates the normal 'out of coincidence' background. The method is applicable to measuring opsonizing capacity of different sera, and responds to PMNL number, age, composition of assay medium and the integrity of the stimulating bacteria. Other bacterial strains produce a similar response, as does the artificial stimulator zymosan. Low temperature and anaerobiosis, which inhibit phagocytic killing, also suppress light emission.

Escherichia coli↗

PubMed1992

[Effects of ouabain on 201Tl and 99mTc MIBI kinetics in rat myocardium--in vivo study using miniature CsI detector].

The aim of this study is to investigate the effect of ouabain (inhibitor of Na-K ATPase) in the myocardial uptake and clearance of 201Tl and 99mTc MIBI using rats. Time activity curves of three groups were measured by CsI miniature detector for 15 min after the administration of each radiopharmaceutical. Groups were divided as follows; 1) control group (CON group), 2) early phase as 15 min after the administration of ouabain (OU15 group), and 3) late phase as 120 min after the administration of ouabain (OU120 group). Main organs of rats including myocardium were resected at 1 min and 15 min after the administration of 201Tl or 99mTc MIBI under the anesthesia of pento-barbital sodium. The uptake (%ID/g) of 201Tl or 99mTc MIBI was measured by well type scintillation counter. The uptake of 201Tl of OU15 group was significantly higher than CON group (OU15: 10.81 +/- 1.90, CON: 1.86 +/- 0.42) and cleared more rapidly. On the contrary, OU120 group showed lower uptake (1.70 +/- 0.21) and slower clearance than OU15 group. Time activity curve of OU15 group indicated the accelerated washout compared with CON group. However, OU120 group showed significantly the reduced washout (CON group: 19%, OU15 group: 22%, OU120 group: 9%). On the other hand, the uptake and time activity curves of 99mTc MIBI were not influenced by ouabain administration. In conclusion, myocardial uptake and clearance of 99mTc MIBI were not related to Na-K ATPase activity, while those of 201Tl were markedly influenced by Na-K ATPase.

Animals↗

PubMedDate not supplied

Radiolocalization of 99mTc-labeled fragments in a melanoma xenografted model.

A xenograft model of human malignant melanoma was used to compare, in terms of tumor localization, the specific antimelanoma monoclonal antibody (MoAb) 225.28S with an irrelevant antibody (4C4). Both specific and non-specific 99mTc-labeled fragments were injected in 12 nude mice bearing subcutaneous tumor. The animals were then sacrificed at 6 and 24 hours post-injection and immediately dissected. Radioactivity of the tumor and normal tissues was measured in a well scintillation counter and autoradiography of tumor, liver and kidneys was also obtained. Tumor localization of 99mTc-labeled MoAb 225.28S fragments was highly specific compared with 99mTc-labeled irrelevant antibody 4C4. With the exception of the kidneys, already at six hours there was a satisfactory tumor-to-normal tissue ratio, which improved at 24 hours. However, the percentage of injected dose per gram of tumor decreased with time, probably due to the weaker bond of radiolabeled Fab' fragments to tumor cells. These results would indicate 99mTc-fragments of the antimelanoma MoAb 225.28S as a suitable radiotracer in clinical nuclear medicine.

Animals↗

PubMed2006

Quantification of [3H]docetaxel in feces and urine: development and validation of a combustion method.

Most radiolabeled biological samples require extensive sample preparation to reduce quenching interference before quantification of radioactivity is possible. Clearly, a more rapid and simple method ensuring a constant count rate and optimal counting efficiency has important advantages. We report on the development and analytical method validation of a rapid and simple combustion method to quantify [3H]docetaxel excreted in human feces and urine. A 3-day validation procedure was performed; quality control (QC) samples, prepared in blank feces and urine, were combusted 5 times and aliquots of the produced tritiated combustion water were counted in a liquid scintillation counter. The validation runs demonstrated adequate precision (below 7.6%) across all QC levels. Sensitivity at the lowest QC level was excellent and recovery of radioactivity constant (ranging from 85 to 91.8%). Clinical applicability of the method was tested in a cancer patient receiving docetaxel and a tracer amount of [3H]docetaxel; during the first 72 h after [3H]docetaxel infusion, 60% of total radioactivity was excreted in the collected feces and urine, which is within the expected range. Combustion of tritiated feces and urine samples is a simple, rapid, sensitive, precise and reproducible method with high recovery. It can be applied to quantify [3H]docetaxel excretion after i.v. administration.

Antineoplastic Agents, Phytogenic↗