Radioactive derivative analysis

Another technique which has much potential in analysis, but which to date has had only limited use, is the formation of radioactive derivatives of non-radioactive compounds for quantitation by radiocounting. A radiolabeled reagent is used to form the derivative. This approach has been of use in combination with chromatography. The advantage of this technique is that it avoids problems of sample background which are often associated with spectrophotometric methods. The 14C-methylation of carboxylic acids and the 14 C-acetylation of hydroxyl groups have been studied [39,40]. These methods are quantitative and the sensitivity is dependent on the activity of the radioactive group added to the molecules. The radioactive derivatization of lipids has been reviewed [41]. 

In clinical chemistry the determination of stable elements by radiochemical methods offers no outstanding advantages over alternative methods, but the use of radioisotopes for determining organic compounds is developing rapidly. In isotope dilution methods (G6), a pure but radioactive form of the compound to be measured is mixed with the sample, a fraction is isolated, and its activity is determined. In radio-metric or derivative analysis (W14), a radioactive reagent is allowed to react with the analyte the labeled compound is separated and its activity is measured. The isotopes commonly used are C,... 

In the estimation of unlabelled metabolites extracted from biological materials, enzymes have been used to convert non-radioactive substrates into labelled derivatives. A labelled co-factor is used in a manner similar to the use of a labelled reagent in isotope derivative analysis. A good example of such methods is the double isotope enzymatic assay for histamine [325]. Samples containing unknown amounts of histamine, tracer amounts of H-histamine and " C-S-adenosylmethionine are incubated with a partially purified preparation of histamine methyl transferase from guinea-pig brain. This enzyme is specific for the methylation of histamine [326]. The product of the reaction, 1,4-methylhistamine, is extracted into chloroform and the ratio of determined by liquid scintillation counting, is directly pro-... 

The capture process of the electron capture detector can be very temperature-sensitive. The sensitivity may either increase or decrease with an increase in temperature, depending on the compound involved, as illustrated in Figure 6.24 for three benzene derivatives. Since detector temperature may affect sensitivity it is sometimes possible to improve the analysis by operating at a different temperature. The radioactive source determines the maximum temperature limit for the detector which is listed in Table 6.6. Exact values vary with manufacturer. 

Isotope dilution analysis is applied to the following analysis. Calculate the amount of the compound Y present in the sample and express your answer as percent by weight. A 1-g sample is analyzed for compound Y, molecular weight of 150. A derivative is formed of compound Y and the added radioactive Y (1.5 mCi at a specific activity of 3 mCi/mmol). The derivative, molecular weight of 150 (1 mol of compound Y per mole of derivative), is recrystallized until pure. It has a specific activity of 4.44 x 103 dpm/mg. 

Radiochemical detection is another approach to analysis. The selectivity of such a system makes it very useful for radioactive compounds or suitable derivatives. 

Synaptic neurotransmission in brain occurs mostly by exocytic release of vesicles filled with chemical substances (neurotransmitters) at presynaptic terminals. Thus, neurotransmitter release can be detected and studied by measuring efflux of neurotransmitters from synapses by biochemical methods. Various methods have been successfully employed to achieve that, including direct measurements of glutamate release by high-performance liquid chromatography of fluorescent derivatives or by enzyme-based continuous fluorescence assay, measurements of radioactive efflux from nerve terminals preloaded with radioactive neurotransmitters, or detection of neuropeptides by RIA or ELISA. Biochemical detection, however, lacks the sensitivity and temporal resolution afforded by electrophysiological and electrochemical approaches. As a result, it is not possible to measure individual synaptic events and apply quantal analysis to verify the vesicular nature of neurotransmitter release. 

Figure 6.14 Examples of the application of normal-phase, radio-HPLC to the analysis of de novo biosynthetic pathways in bark beetles (Scolytidae). Derivatization of 14C-labeled ipsenol from Ips paraconfusus (A, B) and 14C-labeled ipsdienol from Ips pini (C, D) leads to expected retention time shifts of radioactivity for each compound and derivative. Derivatization of /. paraconfusus ipsenol with (2/= )-(+)-a-methoxy-a-(trifluoromethyl) phenylacetic acid (MTPA) leads to the formation of only one diastereomer [(4S)-(-)-ipsenoyl-(2 P )-2 -methoxy-2 -phenyl-2 - (trifluoromethyl) phenylacetate] indicating the de novo biosynthesis of highly pure (4S)-(-)-ipsenol (B) Derivatization of I. pini ipsdienol with (1 S)-(-)-camphanic acid leads to the formation of both diastereomers [(4R)- -)- and (4S)-(+)-ipsdienyl-(TS)-camphanates] indicating the de novo biosynthesis of approximately 90 percent-(4/ )-(-)-ipsdienol (D). Figures adapted from Seybold et al. (1995b).

As part of their program of study of neuroamine metabolism in mammals, Davis and co-workers investigated the biotransformations of nor-laudanosoline (87) by rat liver and by brain preparations. They were successful in isolating a catechol O-methyltransferase enzyme system from rat liver which performed methylations of 87 to give two unidentified products (94) and later they obtained soluble enzyme preparations from rat brain and liver which, in the presence of [14C]methyl-5 -adenosylmethio-nine, gave three radioactive metabolites identified by mass spectral analysis as 90, 93, and a ring A monomethyl derivative of 93 (95, 96). 

Since the Schiff base formation is reversible, it should be reduced by sodium borohydride for the fixation of the label. The rate of the reduction of the Schiff base becomes slow as the number of the phosphate groups of the label increases. However, except for adenylate kinase, the NP -PL bound to the proteins were easily fixed by borohydride reduction. After reductive fixation, labeled proteins are cleaved by appropriate methods. The labeled lysine is cleaved by neither trypsin nor lysyl endopeptidase. There are at least three ways to detect the labeled peptide during isolation 1) use of radioactive reagent, 2) use of radioactive sodium borohydride for reduction of the Schiff base, and 3) use of fluorescence derived from the pyridoxyl moiety of the reagent (excitation at 295 nm and emission at 390 nm at acidic pH). The labeled lysyl residue is not positively identified in the amino acid sequence analysis. However, the presence of the label in the peptide isolated can be confirmed by the presence of pyridoxyl lysine in the amino acid analysis. 

As chemists, we know that plant-derived compounds and the synthesized compounds are identical. Assuming they are pure, the only way to tell them apart is through 14C dating Compounds synthesized from petrochemicals have a lower content of radioactive 14C and appear old because their 14C has decayed over time. Plant-derived compounds are recently synthesized from CO2 in the air. They have a higher content of radioactive 14C. Some large chemical suppliers provide isotope-ratio analyses to show that their naturals have high 14C content and are plant-derived. Such a sophisticated analysis lends a high-tech flavor to this twenty-first-century form of Vitalism. 

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