Metabolite isotope labeling

The application of substrates isotopically labeled in specific positions makes it possible to follow the fate of individual atoms during the microbial degradation of xenobiotics. Under optimal conditions, both the kinetics of the degradation, and the formation of metabolites may be followed— ideally when samples of the labeled metabolites are available. Many of the classical studies on the microbial metabolism of carbohydrates, carboxylic acids, and amino acids used radioactive... 

To understand the evaluation of a CLE, we need to introduce some terms The word isotopomer is a combination of the terms isotope, and isomer. An isotopomer is one of the different labeling states in which a particular metabolite can be encountered [248] that is, a molecule with n carbon atoms has 2" isotopomers. These are usually either depicted using outlined and filled circles for unlabeled and labeled atoms, respectively (see Fig. 14), or are described in text format for example, C 010 would be the isotopomer of a three-carbon molecule labeled at the second position. An isotopomer fraction is the percentage of molecules in this specific labeling state. The positional enrichment is the sum of all isotopomer fractions in which a specific carbon atom in a specific metabolite is labeled [248]. Consequently, the usage of isotopomers enables to account for more information While a molecule with n carbon atoms will yield n positional enrichments, there are 2 — 1 isotopomer fractions (the 2"th measurement is redundant as, by definition, isotopomer fractions must sum up to unity) [260],... 

Although alternative expression systems have been successfully adapted for the production of isotope-labeled proteins (see Sect. 1.5), heterologous expression in E. coli often remains the method of choice for NMR sample preparation. There is a fundamental difference, however, with respect to the kind of medium in which the cells are cultivated. In a so-called chemically defined or minimal medium only one or a very limited number of carbon sources is provided, e. g. glucose or glycerol. All bacterial metabolites have to be biosynthesized by the cells through the various, sometimes lengthy and energy-de-... 

Tab. 1.1 Chemical structures of metabolites involved in selective isotope labeling strategies (from Ref. [14] with kind permission)...

The required data generally are obtained by administering a measured dose of the candidate compound -- often isotopically labelled -- to the rat or mouse either by injection or per os. The animal is housed in a glass metabolism "cage" where it receives food, water, and clean air, and its urine, feces, and respired gases are collected and examined for the parent chemical and its metabolites. Eventual postmortem tissue analysis and calculation of material balance complete the measurements necessary to satisfy the above purposes of metabolism and pharmacokinetic experiments. While in vitro biochemical studies are important adjuncts, it is also apparent that only experiments with intact, healthy, living animals will suffice to meet EPA criteria. 

Additional or different labeled and unlabeled standards can be used. However, note that the incubation as described here includes isotopically labeled L-valine and L-isoleucine and that the metabolites of these branched chain amino acids can interfere with isotopically labeled internal standards for butyrylcarnitine or propi-onylcarnitine. 

The metabolites of interest in urine are separated using reverse-phase HPLC combined with electrospray ionization (ESI)-MS/MS, and detection is performed using multiple-reaction monitoring. Stable-isotope-labeled reference compounds are used as internal standards. 

In some applications like newborn screening and filter paper blood spots, the internal standard that is labeled cannot be mixed with blood. It can only be present in the extraction solvents. Therefore, only the extracted metabolites can be quantitatively measured. I have denoted a term called pseudo-isotope dilution to account for the differences between traditional isotope dilution and the technique commonly used in newborn screening by MS/MS. A special analysis is capable using this technique, however, in terms of an extraction efficiency experiment. With isotope-labeled standards you can perform an experiment whereby a traditional isotope-dilution technique (internal standard added to liquid blood and spotted) is compared to pseudo-isotope dilution techniques (internal standard is added to the extraction matrix). The ratio of the results of these two analysis (pseudo/traditional) is the extraction efficiency. 

In the determination of the sequence of intermediates of a specific pathway, one single probe, the use of isotopically labelled precursors, has proven of pivotal importance. With the appropriate safeguards, which we shall consider below, the administration of a compound isotopically labelled in a recognizable pattern and the subsequent isolation of a labelled metabolite in which the distribution of label can be recognized as occurring without randomization constitutes de facto evidence of a precursorial relationship. 

Amphetamine is metabolized by deamination, oxidation, and hydroxylation. Figure 4.1 illustrates the metabolic scheme for amphetamine. Deamination produces an inactive metabolite, phe-nylacetone, which is further oxidized to benzoic acid and then excreted in urine as hippuric acid and glucuronide conjugates. In addition, amphetamine is also converted to norephedrine by oxidation and then this metabolite and the parent compound are / -hydroxylated. Several metabolites, including norephedrine, its hydroxy metabolite, and hydroxy amphetamine, are pharmacologically active. The excretion of amphetamine depends on urinary pH. In healthy men who were administered 5 mg of isotopically labeled d,l-amphetamine, approximately 90% of the dose was excreted... 

Mutlib, A., Lam, W., Atherton, J., Chen, H., Galatsis, P., and Stolle, W. (2005). Application of stable isotope labeled glutathione and rapid scanning mass spectrometers in detecting and characterizing reactive metabolites. Rapid Commun. Mass Spectrom. 19 3482-3492. 

It has been shown by using NMR spectroscopy in conjunction with isotopelabelling studies that there is a significant degree of deacetylation followed by reacetylation (futile deacetylation) of paracetamol metabolites in vivo in the rat [58]. If this also occurs in humans, then it may help to explain the observed incidence of nephrotoxicity of paracetamol in that the process would result in levels of the potent nephrotoxin 4-aminophenol in vivo. Confirmation of the levels of futile deacetylation in individual metabolites of isotopically labelled paracetamol in man has been achieved by using directly coupled HPLC-NMR... 

Another application of LC-NMR in natural products chemistry concerns biosynthetic studies employing feeding experiments with stable isotope-labelled compounds. H NMR spectra allow the determination of the amount of isotopic label incorporated into metabolites, e.g. by observing signals that arise from /-couplings of protons to 13C-labelled nuclei [41,42],... 

Martens-Lobenhoffer et al. [119] used chiral HPLC-atmospheric pressure photoionization tandem mass-spectrometric method for the enantio-selective quantification of omeprazole and its main metabolites in human serum. The method features solid-phase separation, normal phase chiral HPLC separation, and atmospheric pressure photoionization tandem mass spectrometry. The internal standards serve stable isotope labeled omeprazole and 5-hydroxy omeprazole. The HPLC part consists of Agilent 1100 system comprising a binary pump, an autosampler, a thermo-stated column component, and a diode array UV-VIS detector. The enantioselective chromatographic separation took place on a ReproSil Chiral-CA 5 ym 25 cm x 2 mm column, protected by a security guard system, equipped with a 4 mm x 2-mm silica filter insert. The analytes were detected by a Thermo Scientific TSQ Discovery Max triple quadrupole mass spectrometer, equipped with an APPI ion source with a... 

Isotope-labeled glucose (e.g., I3C, 14C) Metabolism changes Measure glucose converted to labeled lactate and other metabolites. 70-72... 

Stable-isotope-labeled compounds are the most important standard reagents used in most MS-based assays that require accurate quantification. Measurement of metabolites using stable isotopes is commonly referred to as isotope... 

The quantification of metabolites in dried blood spots primarily ensures that the quality of the isotopes standards is excellent in terms of chemical and isotopic purity. When using MS/MS, it is essential that the fragments produced by the collision cell and the product ions detected ensure that both labeled and unlabeled metabolites are identical. Most importantly, the choice of the isotope label and the structural positions must be such that they are stable and do not exchange with other isotopes during sample preparation. Finally, it is imperative that the mass shift is sufficiently high (at least 3 Da) for small molecules less than 1000 Da and that the label occurs at a mass free from other compound interference. Figure 4 illustrates the concepts of quantification using stable isotope with Phe measurement in a dried blood spot as an example. 

Denaro CP, Wilson M, Jacob P El, et al. Validation of urine caffeine metabolite ratios with use of stable isotope-labeled caffeine clearance. Clin Pharmacol Ther... 

VAn Langenhove, A., Costello, C. E., Biller, J. E., Biemann, K., and-Browne, T. R. (1982). A gas chromatographic/mass spectrometric method for the simultaneous quantitation of 5,5-diphenylhydantoin, its p-hydroxylated metabolite and their stable isotope labeled analogs. Clin. Chim. Acta 115, 263-275. 

An important difference between isotope balancing and metabolite balancing is the fact that the isotope label, e.g., due to reversible reactions, may be transported in the opposite direction to the net flux. Thus, reversible reactions, or... 

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