Measurement of Metabolite Concentrations

The experimental deduction of reaction mechanisms relies on the precise and accurate measurement of concentration changes of reacting species. With increasing system complexity, and thus number of analytes, separation methods are preferred to classical enzymatic assays, primarily for their capacity to analyze simultaneously a few to 

The analytical system of choice is expected to provide simultaneous access to the metabolites of interest and globally relevant cofactors. In addition, it is desirable to have a platform that can be flexibly extended for purposes of untargeted metabolome profiling. From this perspective, two conceptually different systems hold prime attention high-pressure liquid chromatography and capillary electrophoresis. 

Even more difficult than the separation is the detection of central carbon metabolites. Direct UV detection is suitable only for nucleotides and some Krebs cycle intermediates 

This method allows the quantification of inorganic anions as well, although particular care is necessary to limit strong background signals originating from salt contaminations. 

A breakthrough was achieved with the advent of electrospray interfaces for the coupling of HPLC to mass spectrometers (MS). We discuss MS in detail in a following section. In conjunction with chromatography, MS provides subpicomole sensitivity and high-resolution m/z-based separation of chromatographically unresolved analytes 

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Measurements of metabolite concentrations in muscle fibers before and after fatiguing stimulation have shown that ATP decreases from 6 to 4.6 mM and PCr decreases from 35 to 2.4 mM with a calculated increase in Pj from 3 to 38 mM (Dawson et al., 1978 Nassar-Gentina et al., 1978). The free ADP concentration was calculated to increase from 30 to 200 pM. At the same time pH decreased from 7.0 to 6.5 (Dawson et al., 1978 Juel, 1988 Westerblad and Lannergren, 1988). The effect of these metabolic changes has been studied in skinned muscle fibers, i.e., fibers in which the cell membrane has been removed. The skinning of the fibers... 

The scaled elasticities of a reversible Michaelis Menten equation with respect to its substrate and product thus consist of two additive contributions The first addend depends only on the kinetic propertiesand is confined to an absolute value smaller than unity. The second addend depends on the displacement from equilibrium only and may take an arbitrary value larger than zero. Consequently, for reactions close to thermodynamic equilibrium F Keq, the scaled elasticities become almost independent of the kinetic propertiesof the enzyme [96], In this case, predictions about network behavior can be entirely based on thermodynamic properties, which are not organism specific and often available, in conjunction with measurements of metabolite concentrations (see Section IV) to determine the displacement from equilibrium. Detailed knowledge of Michaelis Menten constants is not necessary. Along these lines, a more stringent framework to utilize constraints on the scaled elasticities (and variants thereof) as a determinant of network behavior is discussed in Section VIII.E. 

As already discussed in Section VII.B.2, reactions close to equilibrium are dominated by thermodynamics and the kinetic properties have no, or only little, influence on the elements of the Jacobian matrix. Furthermore, thermodynamic properties are, at least in principle, accessible on a large-scale level [329,330]. In some cases, thermodynamic properties, in conjunction with the measurements of metabolite concentrations described in Section IV, are thus already sufficient to specify some elements of the Jacobian in a quantitative way. 

Metabolites, biologically relevant compounds with mass less than 1000 Da, are the downstream products of cellular functions and represent virtually every metabolic function in living systems. Altered metabolic levels often represent defects in the functions of specific genes, transcripts, or enzymes. Hence, measurement of metabolite concentrations using biofluids or tissue specimens offers many avenues, for example, for diagnosing and managing diseases assessing... 

The use of fluorescent proteins as biochemical sensors allowing measurement of metabolite concentrations, enzyme activity or protein-protein interaction is a more active application for these type of proteins [76]. Such indicators can be further divided into molecules with single chromophores versus composites in which the light emission dependents on the energy transfer between two chromophores. 

Metabolomics is the technology behind the measurement of metabolite concentrations, fluxes, and secretions in cells and tissues (metabolome). Metabolomics is at the cross-roads of genotype/phenotype interactions, where the interrelationship of metabolic pathways is considered to be the fundamental component of an organism s phenotype. Metabolomics is distinguished by the fact that metabolites are not directly linked to the genetic code but actually are products of a concerted action of many enzyme networks and other regulatory proteins (243). Metabolites also are more complex than nucleic acids, which are composed of four nucleotides, or proteins, which in turn are composed of 20 different amino acids. As a result, metabolites cannot be... 

Elucidating the dynamics of this uptake system is an essential step to critically assess the reliability of approximations based on the unstructured Monod type of rate expression. To address this issue, we applied the tools of measurements of metabolite concentrations and in vivo diagnosis to E. coli (Noissomit-Rizzi et al., to be published). Both sampling techniques were used during continuous culture of Escherichia coli W3110. In what follows, only those metabolites will be discussed which are related to the uptake system. Also, a first attempt towards kinetic analysis of the PTS system will be presented. 

An update of a previous study (Axelson et al. 1978), Axelson (1986) evaluated an expanded cohort of 1,424 men (levels of trichloroethylene exposure inferred from measured urinary metabolite concentrations) and found a significant increase in incidences of bladder cancer and lymphomas, and a lower than expected incidence of total cancer mortality. A further update of this work (Axelson et al. 1994) expanded the cohort to include 249 women, tracking cancer morbidity over 30 years, and found no correlation between exposure concentration or exposure time and cancer incidence at any site. The highest standardized incidence ratio noted in this study was 1.56 (95% Cl of 0.51-3.64) for 5 cases of non-Hodgkin s lymphoma observed in men. Although four of these cases occurred in persons exposed for at least 2 years, and 3 cases had a latency of 10 years or more, urinary levels of TCA showed that 4 of the 5 cases were exposed to the lowest levels of trichloroethylene (urinary levels of TCA 0-49 mg/L). The study authors mentioned that a urinary TCA level below 50 mg/L corresponds to a trichloroethylene exposure concentration of about 20 ppm. The study authors concluded that "this study provides no evidence that trichloroethylene is a human carcinogen, i.e., when the exposure is as low as for this study population."... 

This can be carried out in vitro (in brain slices, cultured cell preparations) or in vivo and involves penetrating the experimental tissue with a carbon-fibre electrode of 5-30 pm in diameter (Fig. 4.9). This serves as an oxidising electrode and the Faradaic current generated by the oxidation of solutes on the surface of the electrode is proportional to their concentration. Obviously, only neurotransmitters which can be oxidised can be measured in this way so the technique is mainly limited to the study of monoamines and their metabolites. The amplitude of each peak on the ensuing voltammogram is a measure of solute concentration and individual peaks can be identified because different... 

As in the case in the analysis of food samples, the introduction of relatively inexpensive MS detectors for GC has had a substantial impact on the determination of methylxanthines by GC. For example, in 1990, Benchekroun published a paper in which a GC-MS method for the quantitation of tri-, di-, and monmethylxanthines and uric acid from hepatocyte incubation media was described.55 The method described allows for the measurement of the concentration of 14 methylxanthines and methyluric acid metabolites of methylxanthines. In other studies, GC-MS has also been used. Two examples from the recent literature are studies by Simek and Lartigue-Mattei, respectively.58 57 In the first case, GC-MS using an ion trap detector was used to provide confirmatory data to support a microbore HPLC technique. TMS derivatives of the compounds of interest were formed and separated on a 25 m DB-% column directly coupled to the ion trap detector. In the second example, allopurinol, oxypurinol, hypoxanthine, and xanthine were assayed simultaneously using GC-MS. 

Exposure is represented by pharmacokinetic parameters demonstrating the local and systemic burden on the test species with the test compound and/or its metabolites. The area under the matrix level concentration-time curve (AUC) and/or the measurements of matrix concentrations at the expected peak-concentration time Cmax, or at some other selected time C(llme, are the most commonly used parameters. Other parameters might he more appropriate in particular cases. 

FIGURE 23-22 The composition of blood. Whole blood can be separated into blood plasma and cells by centrifugation. About 10% of blood plasma is solutes, of which about 10% consists of inorganic salts, 20% small organic molecules, and 70% plasma proteins. The major dissolved components are listed. Blood contains many other substances, often in trace amounts. These include other metabolites, enzymes, hormones, vitamins, trace elements, and bile pigments. Measurements of the concentrations of components in blood plasma are important in the diagnosis and treatment of many diseases. 

Measurement of the concentration of the active metabolite can be used to assess clopidogrel efficacy for patients who have a pharmacokinetic mechanism for platelet resistance. Mega et al. showed that patients who are intermediate or slow metabolizers... 

Note that amitriptyline, imipramine, and other tertiary amines give rise to the corresponding demethylated metabolites in urine samples. Measurement of plasma concentrations of tiiese (hugs is rarely necessary and should only be cMried out by specific gas chromatographic procedures. 

Immunoaffinity-based assays are routinely developed for new biologicals and products of the biotechnology industry as part of their characterization as new agents. In contrast, assays used for pharmacokinetic studies of new chemically synthesized entities are less likely to be immunoaffinity based because analysts are required to measure accurately the concentration of the administered parent drug. Metabolism of the parent drug can result in metabolites that are structurally very similar and that cross-react with antibodies to the parent drug, but exhibit different pharmacological activity. For this reason, determination of the structures of these metabolites and, commonly, the measurement of their concentrations... 

An HPLC method for the more difficult measurement of plasma free metanephrines was first described in 1993. This method, like those involving measurements of plasma or urinary deconjugated metanephrines, requires a preana-lytical cation-exchange extraction and purification step. The low plasma concentrations of free metanephrines present several technical challenges. In particular, low levels of interfering substances, such as acetaminophen, tend to be more troublesome to measurements of plasma concentrations of the free metabolites than to the higher decon-... 

Cyclosporine is slowly absorbed, and peak concentrations are reached in 4 to 6 hours. Cyclosporine is 90% protein bound and concentrated in erythrocytes. " The degree of concentration in erythrocytes is temperature dependent in vitro for this reason, measurement of plasma concentration is not recommended. " The optimal specimen for analysis is whole blood. The ehmination profile of cyclosporine is biphasic. An early elimination phase with an apparent half-life that typically ranges from 3 to 7 hours is followed by a slower elimination phase with an apparent half-life ranging from 18 to 25 hours. The volume of distribution is 17L/kg. Cyclosporine undergoes extensive metabolism by CyP 3A4. Many of the 31 known metabolites of cyclosporine are inactive. One of the major metabolites, hydroxylated at the... 

Schwertner et al. introduced a salting-out procedure, in combination with single extraction using chloroform-isopropanol (95 5). 100 pi plasma samples could be effectively extracted with 2 ml chloroform-isopropanol. Theophylline was derivatized with pentafluoroben-zyl chloride, and 3-isobutyl-l-methylxanthine was used as an internal standard. This standard is similar to theophylline in extractability, derivatization rates, stability and chromatographic properties. Accurate measurements of plasma concentrations ( + 0.22 ug/ml) could be obtained with little or no interference from theophylline metabolites and other coextract-able material. A packed column with 3 OV-17 on Gas Chrom Q and temperature programming from 150°C to 250°C was used in combination with an electron capture detector. 

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