Radioactive labelled compounds

Radioactively labelled compounds have been employed in biology for the clarification of metabolic processes since the mid-1940s It has, thus, been necessary to prepare such substances and to check on their punty... 

The presence of a transporter can be assessed by comparing basolateral-to-apical with apical-to-basolateral transport of substrates in polarized cell monolayers. If P-gp is present, then basolateral-to-apical transport is enhanced and apical-to baso-lateral transport is reduced. Transport experiments are in general performed with radioactively labeled compounds. Several studies have been performed with Caco-2 cell lines (e.g. Ref. [85]). Since Caco-2 cells express a number of different transporters, the effects measured are most probably specific for the ensemble of transporters rather than for P-gp alone. P-gp-specific transport has been assayed across confluent cell layers formed by polarized kidney epithelial cells transfected with the MDR1 gene [86], Figure 20.11 shows experimental data obtained with these cell lines. A rank order for transport called substrate quality was determined for a number of compounds [86]. The substrate quality is a qualitative estimate, but nevertheless allows an investigation of the role of the air/water (or lipid/water) partition coefficient, log Kaw, for transport as seen in Fig. 20.11(A). For most of the compounds, a linear correlation is observed between substrate quality and log Kaw- However, four compounds are not transported at all despite their distinct lipophilicity. A plot of the substrate quality as a function of the potential of a... 

Radioactivity Specific, radioactive-labeled compounds Low levels... 

Compound A produces a predominant fragment at miz 264, which corresponds to a neutral loss of 175, which in this case corresponds to the loss of 4-trifluo-romethylbenzylamine [37]. This neutral loss can be used to monitor the presence of other species sharing this common feature. Radioactively labelled compound A was incubated in rat liver microsomes, analysed by LC-MS-MS and the TIC for the neutral loss of a mass of 175 (Figure 6.20a) was obtained. When compared with the radioactivity profile in Figure 6.20b, two extra components were detected in the TIC. This was due to the loss of the radiolabel during metabolism. LC-MS-MS is very useful as a complementary detection method where the radiolabel is lost during metabolism or in situations where a radiolabel is not available. 

Spin at 8000 x g for 20 min and wash the pellet twice with PBS. If unlabeled components were used, dissolve the pellet with 0.1 ml Soln. B and determine the protein content. If a radioactive labeled compound was involved, count for radioactivity. 

At the Mellon Institute he applied l4C tracers to examine the behavior of intermediates in Fischer-Tropsch synthesis over iron catalysts. By adding small amounts of radioactively labeled compounds to the CO/H2 synthesis gas mixtures, he was able to prove that some of these compounds (e.g., small alcohols) are involved in the initiation step of the chain growth process that leads to larger hydrocarbon products. It was during this era that his associates first placed a catalytic reactor into the carrier gas stream of a gas chromatograph and developed the microcatalytic pulse reactor, which is now a standard piece of equipment for mechanistic studies with labeled molecules. While at Mellon Institute Emmett began editing his comprehensive set of seven volumes called Catalysis, which he continued at Hopkins. 

By the use of this type of photoaffinity-labeled retinals at various positions on the ring and side chain, and the use of radioactively labeled compounds, and by carrying out hydrolysis of the retinal from the protein into amino acid(s) or short peptides, it will be possible to determine which amino acid(s) or peptide sequences are close to the chromophore in the binding site. 

Generally, the results of TLC separations are evaluated qualitatively or semi-quan-titatively. Thin layer scanners are available for quantitative work that measure the intensity of reflected light. Alternatively, spots containing a substance can be scratched off the plate, extracted into solution and quantitated. Radioactively-labelled compounds can be detected, and determined quantitatively, using autoradiography. 

Each new experiment should be planned carefully and experimental protocols laid down in advance so you work as safely as possible and do not waste expensive radioactively labelled compounds. In conjunction with your supervisor, decide whether your method of application will introduce enough radioactivity into the system, how you wiU account for any loss of radioactivity during recovery of the isotope and whether there will be enough activity to count at the end. You should be able to predict approximately the... 

There is a large variety of analytical problems that require the separation of radioactive species by column liquid chromatography. This need arises, for instance, in activation analyses, the separation of fission products or the separation of radioactively labelled compounds. Examples of special importance come from the biosciences, where labelled molecules are used in research on metabolism. Whenever a sample contains radioactive species, it is possible to follow their elution from a separation column specifically by radiometric detection. This review outlines briefly the principles of radiometric detection and radiometric detectors, concentrating on problems that arise from the combination of separations by high-performance column liquid chromatography with radiometric detection. 

Biodistribution studies for peptides are frequently performed with radioactively labeled compound. The radioactivity can either be introduced by external labeling with I or by internal labeling of already present atoms, for example, via addition of H, or radioactively labeled amino acids to cell cultures producing... 

Radioactively labeled compounds can be detected by autoradiography. Traditionally, the developed plate is... 

The development and utilization of radioactively labeled compounds was a major step in the study of the biosynthesis of terpenoids. 

For physiological studies, it has been demonstrated that the use of radioactively labeled compounds can be used to follow the fate of a neurotransmitter and its metabolites. Dopamine metabolism can be elucidated through the use of a tritiated derivative, which was followed in vivo in rats. The samples are extracted, from biological tissue by microdialysis and subsequently separated by standard HPLC methods (generally reversed phase on a Cig-bonded stationary phase). Detection of analytes via liquid scintillation measurement offers high sensitivity that, in most instances, is equal to or greater than that obtained from fluorescence spectroscopy or mass spectroscopy. 

Within the constraints referred to above, the synthesis of compounds labelled with stable isotopes follows the standard practices of synthetic chemistry. Unlike radioactive labelled compounds, the syntheses present no radiation hazard nor any special problems of stability. Subsequent sections of this chapter will illustrate the wide range of labelled compounds that have been prepared in order to solve particular problems. 

In 1961/62, the considerable impurity of many of the radioactively labelled compounds commercially available at that time, was demonstrated with the help of TLC [424,... 

Imformation about manufacture and sources of radioactively labelled compounds is to be found on p. 167 and in the list of firms at the end of this book. 

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