Radiolabels common

The introduction of tritium into molecules is most commonly achieved by reductive methods, including catalytic reduction by tritium gas, PH2], of olefins, catalytic reductive replacement of halogen (Cl, Br, or I) by H2, and metal pH] hydride reduction of carbonyl compounds, eg, ketones (qv) and some esters, to tritium-labeled alcohols (5). The use of tritium-labeled building blocks, eg, pH] methyl iodide and pH]-acetic anhydride, is an alternative route to the preparation of high specific activity, tritium-labeled compounds. The use of these techniques for the synthesis of radiolabeled receptor ligands, ie, dmgs and dmg analogues, has been described ia detail ia the Hterature (6,7). 

The nonquantitative detection of radioactive emission often is required for special experimental conditions. Autoradiography, which is the exposure of photographic film to radioactive emissions, is a commonly used technique for locating radiotracers on thin-layer chromatographs, electrophoresis gels, tissue mounted on sHdes, whole-body animal sHces, and specialized membranes (13). After exposure to the radiolabeled emitters, dark or black spots or bands appear as the film develops. This technique is especially useful for tritium detection but is also widely used for P, P, and 1. 

A wooden or metal containment box surrounding the treated area is commonly used when a small quantity of test material is to be applied. The box, typically rectangular in shape and partially buried beneath the soil surface, serves to isolate the treated area from surrounding soil and protect against wind and water erosion. A one- to two-nozzle application boom that moves along guy wires or tracks is often used to ensure even application. Radiolabeled materials having two or more label positions often serve as replicates in these studies. 

The stoichiometry of the enzyme-inactivator complex has historically been most commonly determined using radiolabeled versions of the inactivator. Alternative methods include incorporation of a fluorescent or chromophoric group into the inactivator, or the use of quantitative LC/MS methods. 

An approach that can be used in determining ADME/PK parameters that is simple to execute and gives confidence that the whole dose is accounted for, is to use a radiolabel. This has been the standard approach for development ADME studies for many years. The common isotopes used are 14C or 3H (tritium). 

Another refinement, that avoids the necessity of developing suitable fecal extraction and chromatographic methods, is to dose the radiolabeled compound by both the i.v. and p.o. routes in two separate studies. Knowing that, by definition, the whole of the i.v. dose must have been bioavailable, a comparison of the proportion of the dose in the urine after the two different routes allows estimation of the percent absorbed. An analogous approach can be used without the use of a radiolabel, when the urine from the two studies is analyzed either for the parent compound or, more usually, for a major common metabolite. Assuming quantitatively identical clearance after both the i.v. and p.o. doses, the ratio of the amounts of analyte in the two experiments gives the absorption. 

The RPA is a sensitive method for quantifying specific RNAs from a mixture of RNAs. This is achieved using a small-volume hybridization of an RNA probe to the RNA under study. Unhybridized probe and sample is then digested with RNAses and the protected probe fragment is visualized after denaturing gel electrophoresis. Commonly, the probe is radiolabeled for maximum sensitivity. Following is a method for RPA detection of R-luc-4 sites and F-luc mRNA. 

Enzymes useful for detection purposes in ELISA techniques (Chapter 26) also can be employed in the creation of highly sensitive DNA probes for hybridization assays. The attached enzyme molecule provides detectability for the oligonucleotide through turnover of substrates that can produce chromogenic or fluorescent products. Enzyme-based hybridization assays are perhaps the most common method of nonradioactive detection used in nucleic acid chemistry today. The sensitivity of enzyme-labeled probes can approach or equal that of radiolabeled nucleic acids, thus eliminating the need for radioactivity in most assay systems. 

Scintigraphic imaging is a noninvasive imaging technique commonly applied in nuclear medicine. Radiolabeled compounds (called radiopharmaceuticals or radiotracers) are administered intravenously to patients for diagnostic or, in certain cases, therapeutic purposes. The in vivo distribution can provide important physiological information about tissue function. 

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. 

To make these substrates suitable for biological assays, the introduction of functional groups that can be traced with the proper analytical techniques is essential. The use of radio-, fluorescent-, and biotin-labeled lipidated peptides has been reported. The synthesis of fluorescent substrates is chemically straightforward and allows for production of larger quantities than the enzymatic synthesis used for radiolabeled peptides and is thus preferred over the use of radioactive compounds. [1 21] Common fluorescent probes can be introduced by conjugation to a free functional group present in the peptide. The fluorescent moiety is... 

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