Metabolite concentrations, determination

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. 

Each reversible reaction is associatedwith a reversibility parameter y = —, entirely determined by the equilibrium value and the metabolite concentrations. 

Figure 12, Chapter 3. Nonaqueous fractionation (NAF), enables the determination of metabolite concentrations and enzyme activities at the subcellular level. The figure is adapted from [203].

After oral administration, drug and metabolite concentrations in blood, urine, and feces can easily be monitored. In contrast, topical application to the skin usually aims at a local treatment. Therefore, the main interest lies in determining the drug level within the skin, in order to evaluate the dermal bioavailability of compounds or assess the bioequivalence between different formulations. In the following sections, appropriate analytical techniques will be presented. 

The EDI of phthalates in China, Germany, Taiwan, and US populations are shown in Table 7. The calculation was based on phthalate metabolite (primary and secondary) concentrations, the model of David [137] and the excretion fractions according to various authors [23,28,143,144]. DEHP median values are very close or clearly exceed the TDIs and RfD values (Table 4). The median values for the rest of PAEs are below levels determined to be safe for daily exposures estimated by the US (RfD), the EU and Japan (TDI) (Table 4). However, the upper percentiles of DBP and DEHP urinary metabolite concentrations suggested that for some people, these daily phthalate intakes might be substantially higher than previously assumed and exceed the RfD and TDIs. 

Benomyl was extracted and isolated by the procedure of Austin and Briggs (7). After isolation and concentration, benomyl and its metabolites were determined colorimeCrically. 

LSD metabolism was investigated using MS-MS. Metabolites were determined using MS-MS. The main metabolite was 2-oxo-3-hydroxy-LSD (O-H-LSD) present in urine at concentrations of 2.5 and 6.6 pg/L, respectively, for case 1 and 2, but it was not detected at all in plasma. Nor-LSD was also found in urine at 0.15 and 0.01 pg/L levels. Nor-iso-LSD, lysergic acid ethylamide (LAE),... 

Tyrbing et al. [166] studied the stereoselective disposition of omeprazole and its formed 5-hydroxy metabolite in five poor metabolizers, and five extensive metabolizers of 5-mephenytoin. After a single oral dose of omeprazole (20 mg), the plasma concentrations of the separated enantiomers of the parent drug and the 5-hydroxy metabolite were determined for 10 h after drug intake. In poor metabolizers, the area under the plasma concentration versus time curve [AUC(0-8)] of (+) omeprazole was larger... 

Most subjects provided insufficient data to estimate the half-life of the carboxy-late metabolite. Nevertheless, sufficient data were available from 14 patients, from which an estimate of ILX651-C-carboxylate half-life could be determined. Neither day of administration, BSA, nor dose had any influence on ILX651-C-carboxylate half-life. The least-squares mean ILX651-C-carboxylate half-life was 8 h, which was considerably longer than the half-life of tasidotin. Between-subject variability in metabolite half-life was 17%. Given a parent half-life of less than 1 h, metabolite concentrations were not formation rate-limited. Given the half-life of the metabolite, little accumulation of the metabolite would be expected. 

Data should be available about the uniformity of the fermentation conditions and cell propagation, and about the maintenance of the product yield (cell concentration and viability, nutrient and metabolite concentrations, product concentration, etc.). The criteria as to when to discard a culture should be established (when they are not included in the uniformity specifications mentioned). The characteristics of the host cell and vector at the end of production cycles should be observed. If pertinent, the nucleotide sequence of the insert coding the cloned DNA-derived product should be determined at least once after the culture is carried out on a large scale. 

Because modulation of enzyme activities depends on metabolite concentrations, which in turn are determined by the entire metabolic network, the overall response time for these controls can be on the order of seconds. This is the same as the time scale for changes in environmental conditions (e.g., pH, dissolved oxygen concentration) encountered by cells as they circulate through the nonuniform contents of a large-scale bioreactor. Therefore, beyond the complexities of enzyme activity control in the steady state, dynamic properties of this control system are important. The circulation pattern in a bioreactor has major effects on product formation [28]. Lack of understanding of transient responses of cell metabolism is one central obstacle to systematic scale-up of laboratory results (obtained in idealized,... 

Concentration of the drug and related metabolites are determined using appropriate analytical methods such as HPLC, LC-MS or LC-MS/MS techniques. The... 

Whereas the radioactivity measurement alone does not allow distinguishing between drug and metabo-lite(s), samples obtained from the described studies should be also used for standard determination of the drug and its known metabolites, receiving information about the drag and the known metabolite kinetics directly. The gap between radioactivity concentrations and the concentrations determined by direct bioana-lytical methods defines the contribution of unknown metabolites. 

Always keep in mind that the radioactivity represents the sum of the original compound and/or radiolabeled metabolites and not the drug itself. Therefore, it is worthwhile to determine also the drug and known metabolites directly by bioanalytical methods with samples withdrawn from the same study. The comparison of both, the radioactivity concentrations and the sum of concentrations determined by specific methods, allows estimation of the gap (of unknown metabolites) which often develops time-dependently for instance at the beginning of the study radioactivity concentrations in plasma should fit to the drug concentration. Usually, the gap (as a percentage of the total radioactivity at a certain time point) between both concentrations increases with time. 

Quantitative values on the unbound drug or metabolite concentration-time profile in different tissues or sub-regions of tissues are determined. 

Nonequilibrium reactions can be detected by determining metabolite concentrations in the tissue of interest. Conventionally, a tissue sample is rapidly frozen by compression between metal plates that have been cooled to 77 K by immersion in liquid nitrogen (freeze-clamping). This procedure rapidly halts any enzymatic processes that might alter the metabolite concentrations the concentrations can then be determined by enzymatic or chemical assays. Recently, 31P-NMR spectroscopy has shown considerable value in measuring the concentrations of such metabolites as ATP, ADP, AMP, phosphate, and phosphocreatine in living cells or tissues. 

This principle relates to experiments in which a metabolic pathway is perturbed, for example, by adding an inhibitor or activator of one of the enzymes in the pathway. Following such a perturbation, the metabolite concentrations before and after a control enzyme in the pathway will change in opposite directions. This arises because such an enzyme catalyzes a slow, often rate-determining, step in the pathway. For example, if the perturbation decreases the activity of the enzyme, there will be an increase in the concentration of its substrate(s) and a decrease in the concentration of its product(s). This theorem is strictly valid only for nonbranching linear segments of metabolic pathways. 

The total radioactivity minus the parent compound concentration (determined by the bioanalytical method) in a specimen estimates the amount of metabolites present. If the difference is minimal and does not change over time, the extent of metabolism is low. For plasma or serum specimens, a small difference indicates that metabolites are not present in systemic circulation. For bile or urine specimens, high levels of radioactivity suggest a primary route of elimination for the parent and metabolites. For a drug candidate cleared primarily by metabolism, a preliminary metabolite profile in urine and bile can determine the number of potential metabolites. When the level of a metabolite in a matrix is high, attempts to isolate and identify the metabolite can be undertaken. If sufficient quantities are obtained, the metabolite s pharmacologic and toxicologic... 

Becker JB, Adams F, Robinson TE (1988) Intraventricular microdialysis A new method for determining monoamine metabolite concentrations in the cerebrospinal fluid of freely moving rats. J Neurosci Methods 24 259-269. 

As a result of GC-MS analyses, 103 metabolites were determined, of which 66 were successfully identified and 18 were used to create a diagnostic model. Of these 18 metabolites, 5 (suberic acid, glycine, L-tyrosine, L-threonine, and succinic acid) had significantly higher levels in patients with HCC than in healthy volunteers (p < 0.05). Other metabolites (oxalic acid, xylitol, urea, phosphates, propanoic acid, threonine, pimelic acid, butyric acid, trihydroxypentanoic acid, hypoxanthine, arabinofuranose, dipeptide of hydroxyproline, and tetrahydroxypentanoic acid) showed higher levels in healthy volunteers (p < 0.05). In addition, Wu et al. determined the levels of AFP using an ELISA test in serum from the same patients and healthy volunteers as in the metabolomic study of urine samples. An AFP concentration above 20 ng/mL suggests a positive result and the presence of... 

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