Radioactive carbon, metabolism studies with

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. 

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. 

This paper is the only one in the liquid chromatography portion of this symposium which will attempt to deal with chromatography specifically from the viewpoint of the pesticide metabolism chemist. A residue analyst knows what compound he must analyze for, and develops his method with the properties of that substance in mind. On the other hand, the pesticide metabolism chemist has a different problem. At the conclusion of the treatment, exposure, and harvest phases of a radiolabeled metabolism study, he divides his material into appropriate samples, and extracts each sample with selected solvents to obtain the radioactive materials in soluble form. Typically these extracts consist of low levels (ppm) of carbon-14 labeled metabolites in a complicated mixture of normal natural products from the plant, animal, or soil source. The identity of each metabolite is unknown, and each must be isolated from the natural background and from other labeled metabolites in sufficient quantity and in adequate purity for identification studies, usually by mass spectrometry. The situation is rather like looking for the proverbial "needle in the haystack" when one does not know the size, shape,or composition of the needle, or even how many needles there are in the stack. At this point a separation technique must be selected with certain important requirements in mind. 

Three general questions about pesticides in agricultural products and the environment must be answered where is it and its metabolites, how much of these are there and what are the metabolites. The Metabolism Section of the Biochemistry Department uses a variety of analytical techniques to answer these questions. Metabolism studies are generally done with radioactive carbon which provides a convenient method for... 

The metabolic formation of carbonyl sulfide from carbon disulfide was confirmed in an in vivo study (Dalvi and Neal 1978). After intraperitoneal injection of 14C-carbon disulfide in nonpretreated rats, carbonyl sulfide was excreted by the lung in greater quantities than carbon dioxide. Pretreatment with phenobarbital, however, resulted in a greater amount of excretion of carbon dioxide than carbonyl sulfide. In both experiments, excretion of 14C-carbonyl sulfide and carbon dioxide accounted for 14-43% of the total administered radioactivity, with about twice as much carbon dioxide. These results indicate that phenobarbital treatment caused induction of cytochrome P-450 which catalyzed the conversion of carbon disulfide to carbonyl sulfide faster in pretreated rats than in rats not pretreated with phenobarbital. The role of the cytochrome P-450 monooxygenase system in catalyzing carbonyl sulfide formation was also confirmed by in vitro studies (Dalvi et al. 1974, 1975). The rate of carbonyl sulfide formation was NADPH-dependent and increased with microsomes obtained from phenobarbital-treated rats. 

Why are these isotopes important in biochemistry and medicine The isotopes we have mentioned occur at very low natural abundance , e.g. in the world around us only about 1 carbon atom in 10 (a million million) is C. However, with the advent of nuclear physics and specifically the Manhattan Project, the atomic bomb project in World War 11, radioactive isotopes started to be produced artificially, and this meant that chemical compounds could be radioactively labelled , either uniformly (e.g. in every carbon position) or selectively (i.e. with radioactive enrichment in particular positions). In the case of carbohydrate metabolism, it was possible to study the relative importance of glycolysis and PPP by comparing the release of radioactivity from glucose, specifically labelled either in carbon 1 or in carbon 6. If you look at Topic 28, you will see that in the initial reactions of the PPP the CO2 that is produced comes entirely from the Cl position. Over time, as the later molecular rearrangements come into play, C6 atoms could also eventually be released but not initially. On the other hand, if you revisit Topics 13 and 14, you will see that, because the sugar phosphate is split down the middle into two triose phosphate halves that are then handled identically, CO2 released in the oxidation of pyruvate to acetyl CoA will be derived equally from Cl and C6. This allows biochemists to assess the relative activities of PPP and glycolysis in different tissues or in the same tissue over time. This is how it was possible to estimate (Topic 28) that 30% of glucose breakdown in liver is via PPP. 

Many gold phosphine complexes have been found to be potent cytotoxic agents, to have antitumor activity against P388 leukaemia and to be active against a spectrum of transplantable tumour models . It has been of particular interest to obtain some of them labeUed with radioactive isotopes of gold and carbon for cellular, binding, metabolism and biodistribution studies. 

Radiolabeled isotopes are frequently used in drug metabolism studies by virtue of the fact that radioactive species do not occur naturally, can be readily traced, and provide a convenient method of measuring very low concentrations of unknown metabolites in complex biological matrices, with very high sensitivity and specificity. The most frequently used radioisotopes in drug metabolism studies are S-emitting nuclides such as carbon-14 and... 

The most rewarding procedure has involved the feeding of specifically Relabelled possible precursors to an appropriate culture foUowed by isolation of the metabolite(s). Screening of the product for radioactivity affords a preliminary indication of the precursors. Subsequent repetition with appropriately C-labelled precursors and examination of the product by NMR provides data on the exact location in which the precursors have been incorporated without the necessity for degradative studies. This information is adequate in many cases to deduce the metabolic pathway. In carbon-13 studies, both proton NMR using the satellite method, and C-NMR have been employed. 

The introduction of isotopic carbon into a fatty acid molecule provides the unexcelled method by which a fatty acid may be tagged. Three isotopes have been employed in metabolic studies (1) C", a radioactive isotope ((3+) with a half life of about 20 minutes (2) C, a stable isotope and (3) C, a low-level, radioactive isotope (/3 , 0.154 m.e.v.) with a half... 

The total amount of labeled drug to prepare, in terms of radioactivity, depends on the anticipated studies, the radioisotope, and methods for detecting the radioactivity. In a typical drug-metabolism study Involving 3 animal species and man, using liquid scintillation counting techniques, approximately one mCi of a carbon-14 or sulfur-35 labeled drug will suffice. Similar studies with tritium would require 2 to 4 mCl since the detection efficiency for tritium by the same techniques is only one-half to one-fourth that for carbon-14 and sulfur-35. 

The histidine catabolic pathway is discussed under Folate in Chapter 9. The material reveals that histidine is catabolized to produce glutamate. Glutamate in turn, can be converted to a-ketoglutarate and completely oxidized to CO in the Krebs cycle. In the study depicted in Figure 8,26, the dietary histidine was spiked with I Cjhistidine, The term "spiked" means that only a very small proportion of the histidine contained carbon-14. The metabolic behavior of the radioactive histidine, which can be followed, mirrors the metabolic fate of nonradioactive histidine in the diet. All of the CQz exhaled by the rats can be easily collected, The " COj present in the rat s breath can be measured by use of a liquid scintillation counter. The amount of CO2 produced directly mirrors the proportion of histidine, absorbed from the diet that was degraded the rat s body. 

The course of carbon-14-radioactivity derived from oral (l- C)ascorbic acid in plasma and several tissues was studied in male guinea pigs up to 320 h after intake. The excretion of label was followed in respiratory carbon dioxide, urine, and feces. The evaluation by pharmacokinetic principles yielded an overall half-life of 61 h and a body pool of 21 mg with a total turnover of about 10 mg/d. The total turnover of ascorbate is lower than the daily intake (16 mg/d), indicating incomplete absorption. Ascorbic acid seemed to be bound in several tissues (adrenals, testes) to a higher percentage than in plasma. The maximum rate of excretion as carbon dioxide occurred at 0.5 h, whereas peak concentration of radioactivity in plasma was reached at 1.5 h. Therefore, presystemic metabolism must be considered. 

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