Radio-isotope Labelling

Radioactive isotopes were much used in biosynthetic studies, but stable isotopes are more used now because they can avoid the difficult degradation steps required with radio-labels, as will be illustrated below. Although some common compounds, e.g. C-labelled glucose can be purchased, frequently isotopically enriched compounds have to be synthesized from low molecular mass compounds such as CO2, H20, K CN, or CHj SH and the synthesis of the desired labelled compounds alone may be a major research task. 

The amount of radio-labelled compound obtained at the end of the experiment may be so little that more cold or unlabelled final compound may have to be added to have enough to manipulate. In the studies of the biosynthesis of cholesterol (Chapter 7) unlabelled cholesterol for dilution is available in abundance. That is rarely so with insect substances, and a sample of the cold target compound may have to be synthesized too. 

Sometimes it is useful to watch for the appearance of radioactivity in a particular gland or in a compound to obtain information about the sequence of events in biosynthesis. A hormone or pheromone may only be formed at a certain stage of development, or a gland may be activated. A technique not much used but potentially valuable is to take sections through an insect after it has imbibed a radioactive compound and expose the sections on a photographic plate to see in which organs the activity accumulates (whole body autoradiography). 

Today acetic acid enriched in C would probably be used, which would not require this lengthy degradation. One would look for stronger signals in the NMR spectrum of the product compound for the marked carbon atoms and normal intensity for the unmarked carbon atoms, and also coupling (see later). 

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Radio-isotope labeling or western blotting may also be used to detect synthesized proteins. 

Amongst the earliest applications of labelling was the use of doubly labelled [1, 2- C2] acetate to trace the mode of incorporation of intact acetate units into a wide range of metabolites. This has been one of the major recent developments in biosynthetic methodology and permits information to be obtained which would have been impossible or at best extremely difficult to obtain by classical radio-isotope labelling techniques. The basic concept can be illustrated by a model polyketide system (Fig. 1). 

Although use of radio and stable isotope labels involving the trio of covalently-bonded nitrogenous functions in 3 and in 78, provided evidence that isocyano is the precursor of the isothiocyano and formamido groups [30, 81], it remains to be shown that a biosynthetic equivalent of the in vitro chemically-proven fusion process between isocyano and free sulfur (e.g., cf. Introduction) exists in the cells of sponges. In marine biota, various ionic forms of sulfur in a number of oxidation states, as well as organo-polysulfides are known. However, any association with the isonitrile group and a sulfated species has yet to be established. 

The biosynthesis of the cucujolides was examined in the merchant grain beetle, O. mercator Fauvel, and the rusty grain beetle, C. ferrugineus (Stephens), using radio- and stable isotope-labeled precursors (Vanderwel et al., 1990,1992b). 

The field of immunoassays is large in terms of variety of compounds to be analyzed and the concentrations involved such as millimoles to subpicomoles. One of the optimum requirements is high sensitivity. Immunoassays involving labeling with radio isotopes have been popular in clinical analyses [181]. Some of the advantages and disadvantages of radioimmunoassays are listed in Table 12.32. 

AMS is a special type of MS that has been used to determine various parameters of carotenoid absorption, distribution, and metabolism, and the basics of this technique have been reviewed by Buchholz et al. (2000). A radio-labeled carotenoid (generally labeled with " C) is fed to a subject, and biological sample or expired air are collected. Samples may be analyzed directly, or first extracted and analyzed by HPLC, where fractions containing the putative isotopically labeled parent carotenoid(s) and/or carotenoid metabolites are collected. 

Two-site immunometric or sandwich assays that made use of two or more antibodies directed at different parts of the PRL molecule were next to be developed. As with other two-site IRMA assays, the capture antibody is attached to a solid phase separation system and the second or signal antibody is labeled with a detection molecule (e.g., radio-isotope, enzyme,fluorophor, or chemiluminescence tag ). In some assays, the capture antibody is attached to the wall of test tubes, plastic beads, microtiter plates, ferromagnetic particles, or glass-fiber paper. Other assays have used the strep-avidin approach that couples biotin to the signal antibody with avidin linked to a solid phase. Most of the current immunometric assays for PRL have been adapted to fully automated immunoassay systems. Compared with the older traditional RIA methods, these automated immunometric assays for PRL generally achieve lower detection limits (0.2 to 1.0 ig/L) and improved precision (interlaboratory coefficients of variation of <8% at all concentrations), and have superior specificity (<0.05% crossreactivity with GH). 

Triketides are relatively rare. Triacetic acid lactone (4.2) has been detected in Penicillium patulum. It is also produced by fatty acid synthase in the absence of the reductant NADPH. Radicinin (4.3) is a major phytotoxin isolated from Ahernaria radicina (Stemphyllium radicinum) which causes a black rot of carrots. It is also formed by other Ahernaria species. Its pyrano[4,3- ]pyran structure, the identification of which had eluded purely chemical degradative studies, was established in one of the earlier applications of NMR spectroscopy to natural product structure elucidation. The biosynthesis of radicinin from acetate units was studied in 1970 by both radio-isotope methods using carbon-14 and by carbon-13 enrichment studies with NMR methods of detection. This was one of the first applications of this NMR technique to biosynthetic problems. These results established the labelling pattern for radicinin shown in 4.3. 

Detoxification. A ten minute incubation of both trladlmenol resistant and sensitive U. avenae strains In C1 acetate, labelled 4,4 dimethyl sterols equally In both strains, when trladlmenol was present. After further incubation in the absence of radio-isotope, methyl sterols were no longer labelled In the resistant strain, suggesting that rapid turnover... 

The isotope used to prepare labelled compounds is obtained by irradiation in a nuclear reactor, of solid targets containing atoms of nitrogen (aluminium or beryllium nitride), by neutrons of low energy, known as thermal neutrons, themselves the product of the controlled atomic fission of The radiocarbon formed is next isolated from the target sample by oxidation to Ba " 003, the variety in which it is delivered to chemists. From C02, it is possible to use a plethora of organic chemical reactions to synthesize different compounds in which the radio-isotope can be introduced to a specific position. 

De Graaf, W., Wellsbury, P., Parkes, R.J., and Cappen-berg, T.E., 1996. Comparison of acetate turnover in methanogenic and sulfate-reducing sediments by radio- and stable-isotope labeling and specific inhibitors evidence for isotopic exchange. Appl. Env. Microbiol., 62 772-777. 

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