Radiolabelling development

The methods for detection and quantitation of radiolabeled tracers are deterrnined by the type of emission, ie, y-, or x-rays, the tracer affords the energy of the emission and the efficiency of the system by which it is measured. Detection of radioactivity can be achieved in all cases using the Geiger counter. However, in the case of the radionucHdes that emit low energy betas such as H, large amounts of isotopes are required for detection and accurate quantitation of a signal. This is in most cases undesirable and impractical. Thus, more sensitive and reproducible methods of detection and quantitation have been developed. 

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. 

Other methods of sensitive detection of radiotracers have been developed more recently. Eourier transform nmr can be used to detect (nuclear spin 1/2), which has an efficiency of detection - 20% greater than that of H. This technique is useful for ascertaining the position and distribution of tritium in the labeled compound (14). Eield-desorption mass spectrometry (fdms) and other mass spectral techniques can be appHed to detection of nanogram quantities of radiolabeled tracers, and are weU suited for determining the specific activity of these compounds (15). 

In one example (Lawrence and Casida 1984, Abalis et al. 1985) rat brain microsacs were used to test the action of cyclodiene insecticides such as dieldrin and endrin on the GABA receptors contained therein. The influx of radiolabeled CL into the microsacs via the pore channel of the receptor was inhibited by these chemicals. A similar assay was developed using microsacs from cockroach nerve. Assays with this preparation showed again the inhibitory effect of a cyclodiene (this time heptachlor epoxide) on CL influx. Also, that microsacs from cyclodiene resistant cockroaches were insensitive to the inhibitory effect of picrotoxinin, which binds to the same site on the GABA receptor (Kadous et al. 1983). 

Van Emon et al. ° developed an immunoassay for paraquat and applied this assay to beef tissue and milk samples. Milk was diluted with a Tween 20-sodium phosphate buffer (pH 7.4), fortified with paraquat, and analyzed directly. Fortified paraquat was detected in milk at less than 1 pgkg , a concentration which is considerably below the tolerance level of 10 pg kg Ground beef was extracted with 6 N HCl and sonication. Radiolabeled paraquat was extracted from ground beef with recoveries of 60-70% under these conditions. The correlation coefficient of ELISA and LSC results for the ground beef sample was excellent, with = 0.99, although the slope was 0.86, indicating a significant but reproducible difference between the assays. 

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. 

Terminal transferase labeling was originally developed using radiolabeled (typically 32P) nucleoside triphosphates (Roychoudhury et al., 1979 Tu and Cohen, 1980). Later, the technique was extended to the use of nonradioactive nucleotide derivatives (Kumar et al., 1988). 

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