Metabolism studies with radioactive

While parenteral exposure is not a route posing a significant environmental threat to human health from the isotopes of radium, data acquired in studies using this route are presented here because thousands of persons did acquire radium via this route, and most of the toxicity and metabolic studies with experimental animals have used this route. It is again important to note that effects observed after parenteral administration of radium may be attributed not only to radium itself, but to the presence of any or all of its daughter products and their radioactive emissions in vivo. 

Studies with radioactive copper have helped to delineate more lucidly the disturbance of copper metabolism in the patient with Wilson s disease. Numerous investigators have performed such studies (B8, B9, B32, E2, Jl, M15, M16, M17, 01, 04, S8, S9, SIO, S44). This type of investigation consisted mainly of the oral or intravenous administration of a single dose of radioactive copper, followed by a study of the fate of such a dose in the patients compared to controls. 

The intact animal can be improved for experimental purposes if it is rendered abnormal in some way, by genetic malfunction, by illness, or by operation. Genetic defects, or mutations, are used widely in the study of bacterial metabolism, where they can be read ily induced, for example through irradiation by X-rays or from a radioactive source. Genetic defects frequently reveal themselves in the form of the absence of one specific enzyme, and metabolic studies with such enzymically defective preparations are of the same type as those made possible by the use of a specific enzymic inhibitor which we discussed above. Genetic defects in animals are rarer, but classic cases of the absence of specific enzymes and hence the accumulation of abnormal metabolites are provided in humans by the genetically carried diseases of phenylketonuria and alkaptonuria. In both, unusual substances are excreted in the urine, and the analysis of the reasons for their appearance has led to valuable information about the mechanism of amino acid metabolism in the body. 

Tilmicosin, (20-deoxo-20-(3,5-dimethylpiperidin-l-yl)-desmycosin) is a new macrolide antibiotic which is being developed for treatment of bovine respiratory disease by single subcutaneous injection into cattle. Excretion, tissue residue, and metabolism studies were conducted with " C-tilmicosin-treated cattle and rats. Radioactivity was excreted primarily in the feces after parenteral dosing of cattle and oral dosing of rats. Among edible tissues from treated cattle, liver and kidney contained the highest concentrations of radioactivity. The most abundant metabolite was N-desmethyl tilmicosin. Comparative metabolism studies with cattle and rats indicated that the pattern of metabolism was the same for both species. 

Hydroxypyrimidines do not accumulate in large concentrations in the plant tissues, but are immediately metabolised after absorption through the roots. Therefore, to ensure long-term protection the active substances must be continuously absorbed from the soil. The metabolism of the active substances in the plant has been studied with radioactive dimethirimol-2- K in cuciunbers and in barley treated with radioactive ethirimol-2- HZ (Slade et al., 1972 Calderbank,... 

Experts in drug metabolism carry out time-course studies with radioactive samples. They give a variety of doses and, at intervals, measure plasma radioactivity rather than therapeutic effect. Sensitive instruments aUow for quantitative measurements for example, of the time and concentration at which radioactivity reaches a maximum. To lay a basis for seiecting the animal species used for long-term toxicology studies, these pilot probes of duration may employ several mammalian species. Different species metabolize a single drug dissimilarly, so a suitable animal species handles it as the human one does. 

In the course of our metabolic studies with C -labeled polyenoic fatty acids, we also measured the expired radioactive CO2. Mead and his colleagues (6, 13) and Bernhard (14) have shown that hnoleic and linolenic acids are easily oxidized. In these authors experiments, acids labeled in the carboxy group or uniformly labeled compounds were used. With use of our unsaturated fatty acid labeled close to the methyl end, the appearance of radioactive COa is a valid test for the complete oxida tive degradation of the acid. In any case, the active CO2 can appear only after the oxidation has passed the whole double bond system. 

Recent studies with radioactive iodine have added much to our knowledge of iodine metabolism. Iodine is rapidly and probably completely absorbed from the small intestine at an exponential rate which varies with the level of thyroid activity (Chapter 22). 

MUDGE, G. H. Electrolyte metabolism of rabbit kidney slices studies with radioactive potassium and sodium. American Journal of Physiology, 1953, 17Z, 511-522. 

Metabolic studies with progesterone-2l-C in rodents showed that approximately 10% of the radioactivity was expired as CO2. About 80% of the radioactivity was in the combined excreta and the majority of this in the feces. In bile fistula animals the radioactivity was in the bile this suggests that most of the fecal activity was introduced into the intestines of the normal animals by way of the bile. However, one must again be reminded of species variation, and it is apparent that much work remains to be done in this important area. 

The use of metabolic labelling with radioactive ManNAc has corroborated many of the structural studies and provided an in vivo method of identifying the biosynthetic pathways and subcellular localization of the gangliosides e.g. Caputto et al. 1974, Dreyfus et al. 1975, 1976, ROsner 1975, Landa et al. 1979). 

Often solvents do not extract 100% of the total radioactive residue. In this case, knowledge about the concentration of the target analyte(s) in the extract and the filter cake is necessary. Even if large amounts of radioactivity remain in the solid residual materials, the extraction efficiency may be sufficient if this unextracted radioactivity is permanently bound to the matrix or if it is associated with compounds which are not included in the residue definition. Finally, in all cases a well performed metabolism study can provide the answers needed, even where residues in the edible parts of treated crops or animals do not occur. If incurred residues do not occur, clearly the determination of extraction efficiency is not required. 

Metabolism was studied with halcinonide labelled with carbon-14 in the 2-position of the acetonide group. It was administered intravenously to dogs at a dose of 5 mg/kg. The major portion of the radioactivity was excreted in bile. Radio-autography of bile showed at least 10 distinct metabolites to be present. Four of the metabolites were identified. The two most abundant metabolites, that were identified, accounted for 43% (Figure 8, Ml) and 30%(M2) of the radioactivity. The two minor metabolites (M3 and M4) accounted for 2% each. In dog urine, these four metabolites (Ml-4) accounted for 10, 15, 5 and 18% of the radioactivity, respectively. In dog blood, unchanged halcinonide and metabolites M3 and M4 each accounted for about 15% of the radioactivity. Ml and M2 were not detected. 

Isotope Methods. The isotopes of calcium have relatively short half-lives and are readily counted using liquid scintillation or gamma counters as appropriate to the nuclide. Calcium isotopes may be quantitated in the excreta, blood, tissues or in the whole body. This has made them useful for many nutritional metabolic studies. However, because of safety concerns, radioactive isotopes are cumbersome to work with and many researchers are unwilling to administer them to human beings. This has limited the use of isotopes to those studies in which alternate methods are not available or are imprecise. Methodologies for stable isotopes of calcium, which may be safely used in human being, are becoming available for use in metabolism studies. These will be practical alternatives to radioactive isotopes in the future. 

It is difficult to reconcile the unique chemical structure of tetrodotoxin with that of an animal product. Its structure is not related to that of other animal products by any readily recognized biosynthetic scheme. It is not a terpenoid, not obviously formed from amino acid or carbohydrate units, and apparently not constructed from acetate or propionate units. Nor does it resemble any of the various plant alkaloid patterns. It thus appears to be a very unlikely animal product to result from known biogenetic pathways. In this connection the metabolic incorporation of radioactive precursors using torosa and ]C. granulosa salamanders was studied by Shimizu et al. (47). They observed significant isotopic incorporation into amino acids and steroid metabolites, but they found no such incorporation associated with tetrodotoxin. 

Salicylic acid and Its metabolite were separated by two methods. The first was thin layer chromatography on cellulose with BAW solvent as for the In vivo metabolism studies. A quicker separation was achieved with a polyamide column. The entire 400 pL from an individual assay was placed on top of a 0.8 x 2.0 cm column packed with Polyamide-6 (Accurate Chemical and Scientific Corp.). The salicylic acid metabolite was eluted with 6 mL water but salicylic acid was retained. 3a70B scintillation fluid (Research Product International Corp.) was used to determine the radioactive content of the entire 6 mL of eluant. Separation of salicylic acid and its metabolite by polyamide column chromatography was verified by thin layer chromatography. 

First, the effects of aerobic and anaerobic culture conditions on toxaphene degradation were studied with washed P. putida cells grown on camphor and incubated with no readily usable carbon source. The radioactivities remaining in water after extraction with n-hexane were used as an indicator of metabolic activity. This was further extracted with ethyl acetate after acidification to divide this "total polar metabolites" fraction into aqueous buffer phase and ethyl acetate phase, i.e., the total polar metabolites reported refer to summation of the aqueous buffer and ethyl acetate soluble phases (Table 4). All radioactivities have been corrected by zero time controls and autoclaved 8 hr controls are included in each experiment. 

Metabolism studies in sheep with radiolabeled closantel showed that the parent drug accounted for nearly all the radioactivity in muscle, fat, and kidney. In contrast to tissues in which no metabolism occurred, liver contained two closantel metabolites, 3-monoiodoclosantel and 5-monoiodoclosantel, besides the parent drug. The same metabolites were also identified in feces, although 80-90% of the total radioactivity was due to the parent drug. While amide hydrolysis would also appear to be an alternative metabolism pathway, metabolites that would result from this pathway, such as 3,5-diiodosalicylic acid, have not yet been identified. It might well be that steric hindrance around the amide bonds prevents their hydrolysis (42). 

Kiffe, M., Jehle, A., and Ruembeli, R. (2003). Combination of high-performance liquid chromatography and microplate scintillation counting for crop and animal metabolism studies A comparison with classical on-line and thin-layer chromatography radioactivity detection. Anal. Chem. 75 723-730. 

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