In metabolism studies

Properties of Radioactive and Stable as Tracers in Metabolic Studies Heavy Isotopes Used... 

Although the radioactive isotope H has been extensively used for studies on the uptake of xenobiotics into whole cells, the intrusion of exchange reactions and the large isotope effect renders this isotope rather less straightforward for metabolic studies. Both deuterium H-labeled substrates, and oxygen and OH2 have, however, been extensively used in metabolic studies, since essentially pure labeled compounds are readily available and mass spectrometer facilities have become an essential part of structural determination. 

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. 

The identification and quantitation of the individual amino acids in a mixture is often required in metabolic studies and investigations of protein structure. The use of thin-layer chromatography or electrophoresis may be adequate to indicate the relative amounts and number of different amino acids in a sample but the use of gas-liquid chromatography or an amino acid analyser is essential for quantitative analysis. 

In metabolism studies, Lech and Costrini (15) observed in vitro acetylation of TFM in liver and kidney extracts of rainbow trout. The nitro group of TFM is apparently first reduced to an amine (reduced TFM), which is then acetylated (Figure 1). In vivo studies of rainbow trout and other species have failed to reveal any significant production of reduced TFM. 

In metabolic studies of /1-lactam antibiotics, there is a major difficulty in distinguishing between enzymatic and chemical reactions. A review of relevant studies shows the importance of the analytical methodology to differentiate the products of metabolic and chemical hydrolysis. 

Nuclear magnetic resonance (NMR) spectroscopy of untreated biological fluids has been used successfully in metabolic studies of penicillins. Connor et al. [153] used this method to investigate the metabolism and urinary excretion of ampicillin and amoxycillin in humans and rats. In addition to the metabolites 5.49 and 5.50, they detected a dimer of amoxycillin (5.51) in rat urine. 

Lactone ring opening can be both enzymatic and nonenzymatic, but it appears that the two mechanistic routes are seldom distinguished in metabolic studies [2], Thus, a number of the reports published on metabolism of lactones assume enzymatic hydration and do not examine what the relative contribution of nonenzymatic processes may be. Like for lactonization reactions, only very few in vitro biochemical studies address the question of relative enzymatic contribution and mechanism. 

Deuterated steroids are also valuable for use in metabolic studies. For example, the isolation of di-deuterotestosterone in urine after oral administration in men of [16,16- H2]DHEA demonstrated that DHEA had potential as an anabolic steroid (Dehennin et al., 1998). Similarly, after treatment of rats with [1- H2]ethanol, mono-deuterotestosterone was isolated from plasma (Alomary et al., 2003). 

In metabolism studies of azo dyes and pigments in the hamster, in vivo cleavage of the benzidine-based dye, Direet Black 38, to benzidine was shown by analysis of the urine. However, studies of the 3,3 -diehlorobenzidine-based pigment. Pigment Yellow 12, showed no evidenee for in vivo cleavage to release 3,3 -diehlorobenzidine (Nony et al. 1980). 

The first studies with isolated human hepatocytes concentrated on the characterization of these hepatocytes, as well as on the improvement of the isolation procedure, and the possibilities of culturing these cells [16,19-24]. Thereafter studies were performed to investigate the metabolism of drugs [25-28], in which emphasis was often put on the activity and concentration of cytochrome P450 isoforms. Nowadays, hepatocytes are more generally used in metabolic studies of specific compounds, in order to unravel potential species differences and... 

The idea of back transformation of a three-dimensional NMR experiment involving heteronuclear 3H/X/Y out-and-back coherence transfer can in principle be carried to the extreme by fixing the mixing time in both indirect domains. Even if one-dimensional experiments of this kind fall short of providing any information on heteronuclear chemical shifts, they may still serve to obtain isotope-filtered 3H NMR spectra. A potential application of this technique is the detection of appropriately labelled metabolites in metabolism studies, and a one dimensional variant of the double INEPT 111/X/Y sequence has in fact been applied to pharmacokinetics studies of doubly 13C, 15N labelled metabolites.46 Even if the pulse scheme relied exclusively on phase-cycling for coherence selection, a suppression of matrix signals by a factor of 104 proved feasible, and it is easily conceivable that the performance can still be improved by the application of pulsed field gradients. 

In metabolic studies with animals it is often difficult to distinguish between processes carried out by the animal and those performed by resident microorganisms, such as the gut microflora. In the following, the transformations refer to those taking place within the marine animal, whether microbially mediated or otherwise. Metabolic studies with marine animals are faced with further complications because water can be an important uptake route. A chemical, in this instance arsenic in its various forms, may undergo microbial conversions in the water, and the resultant metabolites may be accumulated by the marine animal. Thus, careful experimentation may be required to determine what is occurring inside rather than outside the animal. 

Deuteriochloroform was the solvent of choice. The ft,ft-2H2 and , ,/ ,/ -2H4 phenylethylamine, m- and p-tyramine and tryptamine obtained3,4 have been used as tracers in metabolic studies and as internal standards in quantitative mass spectrometry3-5. 

In environmental studies, the analyst is concerned with stable compounds or stable products in metabolic studies, the question of reactive (therefore unstable) products and intermediates is of critical concern. Thus the reaction must be stopped, and the sample must be processed using techniques that minimize degradation. This is facilitated by the fact that the substrate is known, and the range of possible products can be determined by a variety of methods. 

A relatively complex area to address is procedural SOPs for chemical analyses in metabolism studies. One approach is to address the major operations that are common to the studies, for example characterization of metabolites in soil. The SOP can describe the general process, options available in the process and requirements for acceptance or rejection of data. Study-specific procedures that complement the SOPs can be outlined in detail and retained as part of the study records. These study-specific procedures can be prepared in the form of a work sheet and used for entering original documentation, such as the person who performed the procedure and the date it was performed. 

Some of the substitution reactions, mainly at the indole-nitrogen as well as halogenation at C-2 have already been discussed in Volume VIII. Oxidation of 2,3-dihydrolysergic acid derivatives with Fremy s salt leads to derivatives of 12-hydroxylysergic acid (41) which have recently been shown to be significant in metabolic studies (41a). 

The role, if any, of L-glucuronic acid in the metabolism of myo-inositol in vivo is unknown, since this acid has not been found in Nature nor been used in metabolic studies. Apparently, however, the d and l enantiomorphs of glucuronic acid are formed from myoinositol by separate enzymes.131... 

Adlercreutz, H. (1977). Quantitative mass spectrometry of endogenous and exogenous steroids in metabolic studies in man. In "Quantitative Mass Spectrometry in Life Sciences (A. P. De Leenheer and R. R. Roncucci, eds.), Vol. 1, pp. 15-28. Elsevier, Amsterdam. 

One aspect of the metabolism of sulfur mustard that appears to be unresolved is the relative importance of TDG and TDGO as excretion products in man. In metabolism studies in rats, TDGO was present in much higher concentrations than TDG. Furthermore, when 35S 13C4-TDG was administered (i.p.) to rats, approximately 90% of the... 

R. L. Sims, L. M. Mullen, D. B. Milne, Application of inductively coupled plasma emission spectroscopy to multielement analysis of foodstuffs used in metabolic studies, J. Food. Comp. Anal., 3 (1990), 27-37. 

C-Labeled saccharin (la) has been employed in metabolic studies.41... 

Let us now consider some of the types of information one can obtain using stable isotopes in metabolic studies. In essence, one can obtain the same information as with radioisotopes, but with less convenient measurement. Stable Isotopes also have the advantage of an "infinite" half-life, permitting long term studies. For an element like selenium with six stable Isotopes, more than one can be used in the same experiment at the same time. 

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