Tritium assay

Apparent tritium exchange as calculated from the tritium assay. e Correction as calculated from the C14 assay. 

Assay of the tritiated species is conveniently carried out by a combination of gas-liquid chromatography and gas-phase effluent counting (Cacace, 1961). The coimters described by Wolfgang and MacKay (1958) are to be recommended. The more recent use of ionization chambers and other developments is reviewed by Cacace (1961). Some of the more detailed aspects of tritium assay are discussed by Lee et al. (1962). 

There should be specific, saturable binding to the receptor, accompanied by pharmacological characteristics appropriate to the functional effects, demonstrable using a radioactive, eg, tritium or iodine-125, ligand to label the receptor. Radioligand binding assays (1,6) have become a significant means by which to identify and characterize receptors and enzymes (see Immunoassays Radioactive tracers). Isolation of the receptor or expression of the receptor in another cell, eg, an oocyte can be used to confirm the existence of a discrete entity. 

Binding assays for the saxitoxins were conducted with homogenized rabbit brain and saxitoxin exchange-labelled with tritium at C-11 (92, 93). If the various saxitoxins were available with suitably intense radiolabels, then the equilibrium dissociation constant, K, could be measured directly for each. Since only saxitoxin is currently available with the necessary label, the binding experiments instead measure the ability of a compound to compete with radiolabelled saxitoxin for the binding site. The value obtained, Kj, corresponds to the uilibrium dissociation constant, K, that would be observed for the compound if it were measured directly. Affinity is defined for this assay as the reciprocal of Kj. The affinities of several of the saxitoxins (94) are summarized in Figure 11, expressed relative to saxitoxin and plotted on a logarithmic scale. 

An azetidine motif was also present in two series of CBi antagonist compounds disclosed by Vernalis Research [335, 336]. In the former, compound (560) was claimed to have an affinity of 285 nM in transfected HEK293 cells using tritium-labelled (382). Among the preferred indications were psychosis, schizophrenia, smoking cessation and eating disorders associated with excessive food intake. Compound (561) was claimed to have an affinity of 0.8 nM in the same binding assay [336]. 

Figure 7.3 Dose-response effects of MDMA on the release of preloaded [3H]5-HT (left panel) and [3H]DA (right panel) from synaptosomes in vitro. [3H]Transmitter release is expressed as percent of tritium retained in tissue. Various concentrations of MDMA were incubated with or without the 5-HT uptake blocker fluoxetine (10 n/W) in [3H]5-HT assays, whereas various concentrations of MDMA were incubated with or without the DA uptake blocker GBR12909 (10 n/W) in [3H]DA assays. Data are mean SD for three separate experiments, each performed in triplicate. See Baumann et al.39 for methods.

One of the first pieces of evidence for the mechanism of this reaction involved an attempt to develop a new assay for the activity of tyrosine synthase, which converts phenylalanine to tyrosine. A tritium was placed in the para position of phenylalanine, and it was assumed that oxidation of this position would lead to the loss of tritium and the rate of this loss would be a measure of the activity of the enzyme (Fig. 4.76). 

However, when the results of this assay were compared to other assays, it was found to underestimate the activity of the enzyme. Further analysis revealed that some of the tyrosine contained tritium in the meta position this was referred to as the NIH shift because the early mechanistic studies were performed at the National Institutes of Health (143). The... 

The radiochemical assays were done as follows At the end of a polymerisation experiment, when the conductivity had become constant, a ten-fold excess of tritiated water was added from a burette (see Figure 1), the cell was warmed rapidly to room temperature, and any polymer which had been precipitated during the polymerisation was allowed to re-dissolve. It was always noted that no hydrolysis occurred until the solutions reached 0 °C. This could be seen from a rapid drop of conductivity to a very low value. The solvent and most of the tritiated water were then distilled out, within about 15 minutes. The polymer was then dissolved in toluene, also run from a burette into the reaction vessel, which was then cut from the vacuum line. The polymer was precipitated in methanol and prepared for the determinations of radioactivity and DP. For the radiochemical assay the polymers were dissolved in toluene, re-precipitated in methanol, dried, weighed, re-dissolved in toluene, and the activity determined. The processes of precipitation and dissolution were repeated until the activity of the polymer became constant, (up to 7 repetitions). It was assumed that when the activity had become constant, all the excess of tritium had been removed. 

A representative selection of the polymerisations described above were terminated with tritiated water, and one of them with 14C-labelled 2-propanol, and the polymers were worked up and assayed as described in the Experimental Part the number of tritium atoms per polymer molecule is shown in Table 1. 

The conductivity changes accompanying and following the polymerisation of five portions of norbornadiene added to an aluminium bromide solution at -63 °C are shown in Figure 6 (Experiment No. R4). The polymer, which was precipitated during the reaction, was subsequently found to be insoluble and therefore presumably crosslinked. Since this polymer was, therefore, unsuitable for radiochemical assay, another experiment (RIO) was done with norbornadiene in a mixture of methyl and ethyl bromide at -125 °C to prevent cross-linking. The polymer was soluble and the number of tritium atoms per molecule of polymer was much greater than for polyisobutylene. 

This assay is normally carried out only if positive effects have been obtained in earlier in vitro tests. The UDS test measures the DNA repair synthesis which occurs after excision and removal of a stretch of DNA containing the region of damage, induced in hepatocytes of animals treated with the test chemicals. UDS is measured by the uptake of radioactively labelled nucleotide, usually tritium-labelled th)unidine, into the DNA of the damaged hepatocytes. Animals, usually male rats, are treated with the test chemical, and... 

Alamar blue assay, Tetrazole reduction (MTT, WST assay) Tritium-thymidine (3H-TdR) incorporation, bromodeoxyuridine (BrdU) incorporation... 

The existence of photoreversible, but not of heat-reversible, absorbance change in irradiated poly dI dC was taken to prove that the photoproducts are entirely dimers (in contrast to those in poly C irradiations where the product is almost entirely the hydrate82a). It was possible to detect dimers of uracil as well as those of cytosine, by means of the much slower photoreversal of uracil dimers. In the acid hydrolysates of irradiated dl-dC, both uracil dimers and uracil could be identified. Enzymatic hydrolysis (snake venom phosphodiesterase) does not split pyrimidine dimers, and the products of such hydrolysis of irradiated tritium-labeled poly dl dC contained trinucleotides shown by radioactivity to contain cytosine dimers. Thymine dimers were formed in the photolysis of the poly dA dT, and were detected and assayed by the same methods. The yield of thymine dimers in irradiated poly... 

Samples of Sedan ejecta were collected around the crater lip and along several transects of the ejecta field. A 10-inch diameter hole is dug with a conventional posthole auger at each sampling station. Discrete samples are taken at depths of 6 inches, 1 foot and at 1-foot intervals below that to a depth of 5 or 6 feet. The sample is passed through a 2-mm. sieve and collected into 1-quart wide-mouthed Mason jars. Samples are shipped to the Lawrence Radiation Laboratory (Livermore, Calif.), where aliquots are taken from the jars and lyophilized on a large vacuum manifold. Individual glass traps are utilized on the manifold and extracted water from each ejecta sample is collected separately. The extracted water is assayed for tritium with a model 3375 Packard liquid... 

Laboratory studies of tritium leaching from Sedan ejecta have shown that a front of almost undiluted soil water is pushed from a soil column when the column is leached with successive aliquots of water. A sample of ejecta was obtained from the maximum tritium zone at 4 feet on Sedan crater lip. This material was placed in four long glass columns, 72 mm. in diameter and 30 cm. long. Separate aliquots of water (equal to 1 inch of rainfall) were poured on the top of the soil columns each day. The column was allowed to drain, and each aliquot of leachate was collected daily and assayed for tritium. Figure 5 shows the elution of tritium from one of the large columns of Sedan ejecta. Each data point in Figure 5 represents the concentration of tritium in the daily collection of leachate. 

For general purpose tracer work, however, and particularly in polymer chemistry, the liquid scintillation counter surpasses all other instruments in its sensitivity and adaptability. There is no question on the author s mind that at the present time such an instrument would be the first choice, particularly where tritium, carbon-14 or sulphur-35 were involved. Samples for assay are dissolved in a phosphor whose major solvent usually consists of toluene, toluene-alcohol, or dioxan. Many polymers and low molecular weight compounds are readily soluble in these solvents. Prospective users should not be deterred by alleged complications due to "variable quench effects" as these effects are readily corrected for via internal or external standards or the channels ratio method (7, 46, 91). Dilution quench corrections, though valid, are tedious and unnecessary. Where samples are insoluble in phosphor they may be suspended (e.g. as gels or as paper cut from chromatograms, etc.) or they can be burnt and the combustion products absorbed in a suitable phosphor solution. A modification of the Schoniger flask combustion technique is particularly suitable for this purpose (43—45). 

Rates of Enolization Reactions. For a better understanding of the transformation and oxidation reactions of reducing sugars, methods have been developed to measure the primary rates of enolization (18). One of these methods depends on the rate at which tritium ions are released from aldoses-2- to the solvent. This is measured by separation of the water-, sublimation, and radiochemical assay of the water as the reaction proceeds. The rate constant is calculated from the first-order equation ... 

A large scale preparation of E. coli 045 was subjected to enzyme purification using the assay for 3,5-epimerase. Protamin sulfate precipitation, ammonium sulfate fractionation was followed by DEAE-chroma-tography. The fraction containing enzymatic activity, as measured by tritium exchange, was eluted from the DEAE column early. This fraction was incapable of producing any net synthesis of TDP-6-deoxy-L-... 

The counting of tritium in water is a special problem about which much has been written. Current methods for assay of tritium in water have a range of 0.1 -5000 TU, where a tritium unit (TU) has the value of 7.2 dpm/L. The most desirable feature of a tritium measuring system is that it be capable of measuring a large number of samples rapidly, simply, and cheaply as possible with an uncertainty of +10% or better. It is generally more important to assay 100 samples with an uncertainty of +10% than to assay 10 samples with an uncertainty of +3%. 

Isotopic dilution techniques were used to determine residual, tritium-labeled gibberellic acid in potatoes, grapes, and various products derived from barley The seeds, the young plants, or the fruit were treated with labeled gibberellic acid and analyzed at the end of the growth period by extraction of the labeled residue in the presence of carrier gibberellic acid, isolation of a pure crystalline specimen, and subsequent assay by liquid scintillation counting ... 

Fig. 9 Schematic representation of the scintillation proximity assay of (S)-propanolol using imprinted microspheres. The light green area represents the aromatic antenna element, (a) The bound, tritium-labeled (S)-propranolol triggers the scintillator to generate a fluorescent light, (b) When the tritium-labeled (S)-propranolol is displaced by the unlabeled (S)-propranolol, it is too far away from the scintillator antenna to transfer efficiently the radiation energy therefore, no fluorescence can be generated (as described in [62])...

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