Liquid scintillator beta counting of iron-59 in clear and colored systems.
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Iron↗
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Liquid scintillation alpha counting and spectrometry and its application to bone and tissue samples.
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Alpha Particles↗
A comparison of quantitative NMR and radiolabelling studies of the metabolism and excretion of Statil (3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-ylacetic acid) in the rat.
The identification and quantitation of the metabolites of Statil in rat bile and urine were investigated by 1H- and 19F-NMR spectroscopy and liquid scintillation counting. Male Wistar rats received a single oral dose of 100 mg/kg of radiolabelled Statil. Statil is known to produce glucuronide conjugates which are predominantly excreted into the bile in male rats. The complex multiphasic matrix of bile has been shown to make identification of the resonances by 1H-NMR spectroscopy very difficult as Statil appeared to be micellar bound giving rise to very broad signals. This not only impaired unambiguous signal characterisation but also quantification. The partial separation by SPEC-(1)H-NMR spectroscopy enabled the disruption of the micellar matrices and hence enabled the identification of Statil predominantly as aglycone, and to a lesser extent as glucuronide conjugate. In addition, minor acyl migration products of Statil glucuronide could also be detected as they were separated during the SPEC-process. 19F-NMR spectroscopic measurements on whole bile confirmed their presence as a number of overlapped signals could be observed. The selectivity, simplicity and signal dispersion characteristic of 19F-NMR spectroscopy also enabled the calculation of dose related recoveries of Statil related material in the bile and urine samples without the need for a radiolabel. The aim of this work was to investigate the usefulness and limitations of NMR spectroscopy of intact bile and urine as a means of quantifying levels of drug metabolites. The results obtained from NMR spectroscopy are compared with those obtained using scintillation techniques. Scintillation counting yields unequivocal quantification results, provided the label is preserved in metabolites as has been the case here. In general, quantification by 19F-NMR results similar to those obtained by scintillation counting (in agreement within about 20%). However, discrepancies have been observed with very small and broad 19F-NMR signals in bile. Nevertheless, 19F-NMR spectroscopy of bile is a rapid and facile method for assessing metabolite levels of fluorinated drugs.
Animals↗
Punch sampling technique for quantitative identification of tritiated D-amphetamine.
A punch sampling, tissue solubilization, and scintillation counting technique has been devised for the quantitative identification of tritiated d-amphetamine in monkey brain tissue. The method is superior to autoradiography in identifying this water soluble radiochemical, and is easier and more anatomically precise than radiochemical assay of brain homogenates. This rapid and simple technique allows one to survey the whole brain by sampling unit volumes of individual structures verified by light microscopy. The procedure consists of six steps which may be spaced as the laboratory schedule permits: obtaining tissue blocks, preparation for sectioning, punch sampling and sectioning, preparing samples for scintillation counting, histological verification of sample sites, and analysis of scintillation count data. The technique has been successfully used to demonstrate a gradient of radioactivity following experimental intraventricular injections of 3H amphetamine.
Animals↗
A new approach to NEMA scintillation camera count rate curve determination.
The method of determining count rate curves suggested by the National Electrical Manufacturers Association (NEMA) is based upon a single reference count rate. Inaccuracies in the reference value are propagated to the rest of the curve values. NEMA also requires the measurement of individual attenuation factors for every absorber. Errors can easily occur during the measurement and during the subsequent calculations which utilize these factors. An alternative approach is suggested: count rate curves are based upon at least five low count rate values and the determination of individual attenuation factors is eliminated. Count rate curves are generated from graphs of the 1n of the observed count rate against the number of equally thick copper absorber plates, by back-extrapolation of a line which is fitted to the linear low count rate data. The method was successfully implemented in practice. Agreement was found between the results of the NEMA method and the alternative method suggested.
Scintillation Counting↗
Incorporation and retention of radiolabeled S-(+)-and R-(-)-methamphetamine and S(+)- and R(-)-N-(n-butyl)-amphetamine in mouse hair after systemic administration.
We examined the incorporation of unlabeled and tritiated enantiomers of methamphetamine (MA) and a more lipophilic analog N-(n-butyl)-amphetamine (BA) into the hair of pigmented (C57) and nonpigmented (Balb/C) mice after systemic administration. We also compared the ability of extraction methods to remove unlabeled and tritiated MA and BA enantiomers from the hair. R(-)-MA, S(+)-MA, [(3)H]R(-)-MA, [(3)H]S(+)-MA, R(-)-BA, S(+)-BA, [(3)H]R-(-)-BA, and [(3)H]S-(+)-BA were each administered to C57 and Balb/C mice (23 days of age) by i.p. injection at 8.8 mg/kg daily for 3 days. At 44 days of age, hair samples from the animals were treated with a brief methanol wash, a 24-h extraction with pH 6 phosphate buffer, and a final digestion in 1 N NaOH to free residual drugs from the hair. Labeled drugs in the extracts were quantitated by liquid scintillation counting. Unlabeled drugs were quantitated by gas chromatography/mass spectrometry (GC/MS). GC/MS analysis demonstrated MA and BA to be the major (>90%) species present in the blood during the 24 h after administration. Less than 10% of the MA was N-demethylated. No p-hydroxylated metabolites were found. Blood concentrations of tritiated MA and BA enantiomers measured by liquid scintillation counting agreed well with blood concentrations of unlabeled enantiomers measured by GC/MS. Hair concentrations of S(+)-MA were greater than those of R(-)-MA in both mouse strains, paralleling blood concentrations. There were no enantiomeric differences seen with BA hair accumulation in either strain of mouse. Significantly more MA and BA enantiomers were deposited in pigmented than in nonpigmented hair. With labeled and unlabeled compounds, approximately 30% of S(+)-MA and 60% of R(-)-MA in pigmented hair could be removed by a phosphate extraction. A significant amount of drug could not be removed from the hair by extraction. Greater amounts of drug could be extracted from nonpigmented hair than pigmented. Extracted and residual MA and BA concentrations in pigmented hair were significantly greater when labeled compounds were quantitated by liquid scintillation counting than when unlabeled compounds were quantitated by GC/MS. However, radiotracer and unlabeled drug concentrations were the same in nonpigmented hair. The results demonstrate that hair pigmentation is an important determinant in MA and BA deposition, and that MA and BA deposition is not enantioselective. The data demonstrate a significant amount of MA and BA accumulated is not easily amenable to exhaustive aqueous extraction from the hair. The use of tritiated MA and BA enantiomers demonstrates that a significant amount of MA and BA stored in pigmented hair is structurally different from parent MA and BA, perhaps associated with melanin components of hair.
Amphetamine↗
Measurement of cell proliferation by enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to bromodeoxyuridine.
An ELISA was developed and optimized to measure cell proliferation using a monoclonal antibody to bromodeoxyuridine (BrdUrd). Incorporation of BrdUrd into myoblast monolayers, measured as the optical density at 492 nm, increased in response to fetal calf serum, IGF-I and EGF, the ELISA data correlated closely with data obtained by BrdUrd immunocytochemistry (r = 0.984), cell counting (r = 0.972) and tritiated thymidine uptake by liquid scintillation counting (r = 0.990). The BrdUrd ELISA is a useful alternative to measurement of tritiated thymidine uptake by scintillation counting, and has the added advantages of dispensing with the use of radioactivity and of being less labour intensive.
Animals↗
Simple radioactive assay for the estimation of DNA breaks.
The intactness of DNA is an important part of the normal cellular structure. Any change to the DNA in the form of breaks leads to a change in the integrity, which in turn leads to abnormality in the cellular activity. Many discrepancies have been reported among the various methods of detecting DNA damage. Here, a simple, sensitive and reproducible method has been developed for the detection of DNA breaks by radioactive labelling of 5' broken ends. The method was evaluated by studying chemically induced DNA damage by using both organochloride (2,4-dichlorophenoxyacetic acid and lindane) and organophosphorus (sevin and phosphamidon) compounds at different concentrations. Phosphamidon, one of the organophosphorus compounds studied, showed complete degradation of the DNA after treatment. Radioactive analysis of phosphamidon showed higher counts at the lowest concentration (20 microg) of the chemical when compared with the control (2752 scintillation counts per minute, scm). Studies on the chemically induced DNA breaks by radiolabelling revealed that the cumulative effect of the organophosphorus and organochloride compounds showed maximum counts in all the samples (the highest being 2904 scm) when compared with the organophosphorus and organochloride compounds studied separately (the highest being 1881 and 2260 scm, respectively). Radiolabelling studies on the blood samples of 23 pesticide workers by the newly developed assay showed a significant positive correlation (0.893) between the number of years of exposure and the scintillation counts. A maximum of 11,702 scm (for 18 years of exposure) and a minimum of 1682 scm (for 4 years of exposure) were recorded compared with 1253 scm for the negative control. This method can be used effectively for estimation of the DNA breaks, irrespective of its nature.
Agricultural Workers' Diseases↗
Radioassays of yttrium-90 used in nuclear medicine.
Yttrium-90 radioassays are required in nuclear medicine at the gigabecquerel activity level (GBq) for measuring injected activity, and at the becquerel level for measuring individual tissue samples in biodistribution studies. A method of standardizing 90Y for activity using high-efficiency liquid-scintillation counting is described. Solution standards were used to establish the calibration factors for commercial radionuclide calibrators. Detection efficiencies are also presented for liquid-scintillation counting, NaI(T1) bremsstrahlung counting and Cerenkov counting.
Nuclear Medicine↗
Changes in leucine uptake in the retina of the hamster after traumatic detachment.
Protein metabolism was investigated in detached hamster retinas. By sucking off 0.2 ml of aqueous humor from the anterior chamber through limbic insertion of a 27-gauge needle, a tractional force pulled off the neural retina from the retinal pigment epithelium and created a simple detachment without retinal breaks in the right eyes of the hamsters. The left eyes were left untouched as normal controls and sham controls were induced by simple limbic insertion without suction. The animals were sacrificed at selected intervals of 1, 3, 6, 9, 16, 24, 32 days after the operation. Subsequently, scintillation counting and autoradiography were employed to study retinal protein metabolism using leucine uptake as an index. After tritiated leucine uptake, scintillation counting of radioactive substance indicated that detached retinas had taken in less tritiated leucine than normal controls from day 1 to 6 after the operation, but this change had normalized by day 9. For autoradiography, the change in leucine uptake rate was shown to be different in different layers. All the retinal cells seemed to show a decreased leucine uptake with the exception of the outer nuclear layer, in which leucine appeared to be significantly upregulated. This paper illustrates the patterns of protein metabolism and their change after traumatic detachment as well as their possible recovery.
Animals↗
A quantitative immunostaining method for the measurement of UDP-galactose:lactosylceramide galactosyltransferase for the synthesis of globotriaosylceramide in rabbit small intestine and HeLa cells.
Galactosyltransferase is required for the addition of galactose to lactosylceramide (galactose beta 1-4 glucose beta 1-1 ceramide), resulting in the synthesis of globotriaosylceramide (Gb3). We describe a quantitative more sensitive and specific method for the measurement of UDP-galactose:lactosylceramide galactosyltransferase activities in rabbit small intestine and HeLa cell which utilizes the specific binding of Shiga toxin to the product, Gb3. Intestinal microsomal or HeLa cell sonicate preparations were incubated in the presence of lactosylceramide and [14C]UDP-galactose. The lipid reaction products were extracted on C18 Bond-Elut columns, separated by high-performance thin-layer chromatography and exposed to Shiga toxin followed by polyclonal rabbit anti-Shiga toxin antibody and goat anti-rabbit IgG alkaline phosphatase conjugate. Gb3 was visualized with NBT and BCIP and quantitated by densitometry. These data were compared with a standard assay in which, following incubation and lipid extraction, radioactivity was measured by scintillation counting of the isolated lipids. There was a 22-fold increase in enzyme activity by the immunostaining method compared to the usual scintillation counting technique. This is attributable to the exclusion of radioactive lipids other than Gb3 in calculating enzyme activity and the correction for endogenous UDP-galactose. Thus, the immunostaining method provides increased accuracy, sensitivity, and specificity in the assay of galactosyltransferase activity.
Animals↗
A microplate solid scintillation counter as a radioactivity detector for high performance liquid chromatography in drug metabolism: validation and applications.
Sensitive radioactivity detection following high performance liquid chromatography (HPLC) separation remains a challenge in many drug metabolism studies with radiolabeled compounds. In this work, solid scintillation counting (SSC) after fraction collection into 96-well plates was evaluated as an off-line radioactivity detection method, in comparison with conventional liquid scintillation counting (LSC). The impact of counting time and biological matrix on the quantification of radiolabeled metabolites and parent drug in samples from animal and human absorption, distribution, metabolism and excretion (ADME) studies was investigated. Three different approaches were used to test whether reliable quantification by off-line SSC detection, which requires an approximately constant counting yield during the entire chromatographic run, can be realized: (i) the measurement of radioactivity-spiked biological blank samples without HPLC separation as an extreme case of biological background, (ii) the measurement of radioactivity-spiked HPLC fractions of biological blank samples and (iii) the comparison of radiochromatograms obtained by off-line SSC and LSC of real samples from ADME studies with radiolabeled compounds. Situations in which variations in SSC yield during an HPLC run are likely to lead to significant errors in quantitation were identified and are discussed. However, examples from a number of animal or human ADME studies showed that in the majority of cases off-line SSC provides very similar quantitative data, compared with the reference method of off-line LSC radioactivity detection. Approaches for validation of the off-line SSC approach in critical cases are discussed. The main advantages of off-line SSC, compared with off-line LSC, are lower detection limits and a substantially higher throughput. Several applications of off-line SSC detection in ADME studies are shown.
Animals↗
Comparison of three in vitro assays for carcinogen-induced DNA damage.
DNA damage induced by five ultimate carcinogens and five procarcinogens was evaluated by three methods: (1) DNA strand breaks in Crandall feline kidney (CRFK) cells measured by alkaline sucrose gradient centrifugation; (2) unscheduled DNA synthesis (UDS) in CRFK, Syrian hamster embryo (SHE), and rat hepatocyte cultures measured by liquid scintillation counting; and (3) UDS in CRFK, SHE, and rat hepatocyte cultures measured by autoradiography. DNA strand breaks were observed with three procarcinogens and four ultimate carcinogens. Only two procarcinogens and two ultimate carcinogens could be identified by liquid scintillation counting. A positive autoradiographic response for UDS was observed in CRFK cells with four of five ultimate carcinogens and one procarcinogen. SHE cells showed a positive autoradiographic response for UDS with all ultimate carcinogens and three procarcinogens. The autoradiographic system with primary rat hepatocyte cultures was the only one in which a positive response for DNA damage was elicited for all 10 carcinogens.
Animals↗
An assay for estradiol preceptors using a staphylococcal protein-A--estradiol antibody adsorbent.
Antiestradiol antisera were raised in rabbits by immunization with a steroid-protein conjugate Whole antisera and purified antiestradiol antibodies were reacted with heat-killed and formalin-fixed bacteria from the protein-A-bearing strain of Staphilococcus aureus (Cowan I, NCTC 8530). The bacterial immunoadsorbent was used to estimate the estrogen-receptor proteins contained in several specimens from human breast carcinoma. Since the affinity constant for estradiol of antiestradiol antibodies is about 10(8) M-1 while those for breast estrogen-receptor complexes are about 10(9) to 10(10) M-1, it is possible to remove the free steroid from a reaction mixture containing tissue cytosol, radio-labelled estradiol, and the antiestradiol bacterial adsorbent by pelleting the bacteria at low-speed centrifugation. The radiolabelled estradiol molecules bound to their receptors remain in the supernatant and can be easily counted by liquid scintillation counting. The results obtained by this technique on specimens of human breast carcinomas compared more than satisfactorily with those obtained, on separated aliquots from the same specmens, by the dextran-coated charcoal method, according to the EORTC group (1973). The versatility, specificity and stability of the antiestradiol bacterial immunoadsorbent recommend its use, rather than that of charcoal-coated dextran and other non-specific steroid adsorbents, in the mass screening of patients with breast carcinoma which could be amenable to hormonal therapy.
Animals↗
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↗
The uptake of tritiated delta 1-tetrahydrocannabinol by the isolated vas deferens of the rat.
1 Weighed stripped vasa deferentia were incubated in Holman's solution containing (a) [14C]-sorbitol 0.014 mm, (b) [3H]-noradrenaline ([3H]-NA) 12.31 nM, (c) [3H]-tetrahydrocannabinol ([3H]-delta1-THC) 1 mug/ml for 5, 10, 20 and 30 minutes. 2 Tissues were washed, dissolved in Protosol, counted by standard scintillation counting technique and 'drug space' expressed as ct min-1 mg-1 tissue/ct min-l mul-1 bathing fluid. 3 Vasa incubated for 30 min with [14C]-sorbitol were washed for varying lengths of time; 82% clearance had taken place after 2 washes of 5 minutes. 4 The uptake of [3H]-NA was inhibited by the presence of desmethylimipramine (DMI) 10 nM in the bath or by pretreatment of rats with 6-hydroxydopamine (6-OHDA). 5 The uptake of [3H]-delta 1-THC was not inhibited by the presence of DMI. It was reduced but not abolished by 6-OHDA pretreatment.
Animals↗
Measurement of plasma 1,25 dihydroxyvitamin D using a novel immunoextraction technique and immunoassay with iodine labelled vitamin D tracer.
We evaluated a novel assay for the measurement of 1,25 dihydroxyvitamin D (1,25 (OH)2D). Immunoextraction of 1,25(OH)2D is performed using a mini column containing a solid-phase monoclonal antibody followed by radioimmunoassay (RIA) using an 125I-labelled 1,25(OH)2D derivative tracer and Sac-cell separation. The mean recovery of 1,25(OH)2D3 was 101%, linearity was excellent, inter- and intra-assay coefficients of variation were 9, 8 and 13% and 11, 10 and 14% at low, medium and high concentrations of 1,25 (OH)2D3, respectively. The cross-reactivity of vitamin D metabolites was < 0.0015% for 25-hydroxyvitamin D3, 24, 25 dihydroxyvitamin D3 and dihydrotachysterol and 0.54% for 1 alpha calcidol. 1,25 dihydroxyvitamin D2 cross-reactivity was 79%. The detection limit of the assay was 5 pmol/L. Comparison with a commercial radio receptor assay (RRA) and an in-house RIA gave regression equations of y = 0.94x + 11.8 (r = 0.98) and y = 0.91x-1.7 (r = 0.95), respectively, with no major discrepancies between the methods in all patient groups studied. Plasma concentrations of 1,25(OH)2D obtained with the assay were as follows: normal, unsupplemented subjects: mean 88, range 48-155 pmol/L, n = 68, patients with chronic renal failure: mean 11, range 3-36 pmol/L, n = 27, primary hyperparathyroidism: mean 198, range 130-299 pmol/L, n = 23, Paget's disease: mean 92, range 42-149 pmol/L, n = 24, osteomalacia: mean 43, range 27-61 pmol/L, n = 9. A minimum sample volume of 300 microL is required, the hands-on time is significantly less than other commercial assays and the measuring procedure is gamma counting rather than scintillation counting. The assay offers several advantages over previous methods and should allow more laboratories to offer measurement of 1,25(OH)2D as part of their repertoire.
Adult↗
Quantitative receptor autoradiography with tritium-labeled ligands: comparison of biochemical and densitometric measurements.
Tritium-labeled receptor ligands are commonly used for quantitative receptor autoradiography with LKB Ultrofilm. In this study, we compared estimates of tritium concentrations by optical density measurements with direct measurements of tritium concentrations by scintillation counting. We found that predominantly grey matter regions of rat brain showed similar tritium concentrations when measured by either method, but that optical density reading of white matter regions indicated a lower tritium concentration than determined by direct measurement with scintillation counting. We also compared measurements of receptor density by quantitative autoradiography with radioligand-binding measurements on microdissected brain regions. Higher per milligram protein levels of muscarinic receptors were obtained by quantitative autoradiography, comparable to the specific activity of receptors found in purified membranes.
Animals↗