Single Metabolites

Watson discusses the biological activity of algal odour compounds that are the primary cause of foul source-water odour and have been treated by industry as metabolic waste with focus on their removal (Watson 2003). That these compounds can also have multiple functions in the intact ecosystem is elaborated in this contribution. 

In the field of fresh water plankton chemical ecology microcystins from cyanobacteria have stimulated much research and discussion. A critical reflection on the ecological function of these non-ribosomal peptides has recently been published (Babica et al. 2006). Surveys of methods for the quantification of these peptides (McElhiney and Lawton 2005 Msagati et al. 2006) and on the effects on fish (Malbrouck and Kestemont 2006) can be found as well. 

Even if this selection of review articles is not comprehensive, it well reflects the current activities in the field of plankton ecology. Exciting new progress can be expected during the next few years, especially because of the advanced application of new 

Anderson TR (2005) Plankton functional type modelling running before we can walk J Plankton Res 27 1073-1081 

Caldwell GS, Watson SB, Bentley MG (2004) How to assess toxin ingestion and postingestion partitioning in zooplankton J Plankton Res 26 1369-1377 Cembella AD (2003) Chemical ecology of eukaryotic microalgae in marine ecosystems. Phycologia 42 420 147 

---

For the quantitative description of the metabolic state of a cell, and likewise which is of particular interest within this review as input for metabolic models, experimental information about the level of metabolites is pivotal. Over the last decades, a variety of experimental methods for metabolite quantification have been developed, each with specific scopes and limits. While some methods aim at an exact quantification of single metabolites, other methods aim to capture relative levels of as many metabolites as possible. However, before providing an overview about the different methods for metabolite measurements, it is essential to recall that the time scales of metabolism are very fast Accordingly, for invasive methods samples have to be taken quickly and metabolism has to be stopped, usually by quick-freezing, for example, in liquid nitrogen. Subsequently, all further processing has to be performed in a way that prevents enzymatic reactions to proceed, either by separating enzymes and metabolites or by suspension in a nonpolar solvent. 

Consider the simplest case of a metabolic network, namely, a single metabolite S that is synthesized and consumed by the reactions and vcon, respectively. The time dependence of the concentrations S(t) is then described by the corresponding mass balance equation... 

Concentrations of metabolites outside the reference ranges may constitute a typical pattern indicating the presence of an inborn error of purine or pyrimidine metabolism. However, altered excretions of purine and pyrimidines may also be a secondary phenomenon due to the presence of other metabolic disorders, such as a deficiency of the urea cycle [15]. Increased concentration of a single metabolite or combinations of metabolites may also result from bacterial contamination, sample conditions, medication, or dietary compounds [6]. 

If a metabolite is responsible for the toxic effect of a compound, then induction of the enzyme responsible may increase that toxicity. However, if there is only one route of metabolism and elimination is dependent on this, then only the rate of metabolism to the single metabolite will be increased rather than the total amount. This may not... 

Molecules with structures as diverse as carbamoyl-phosphate, tryptophan, and cytidine triphosphate are feedback inhibitors of the E. coli glutamine synthase. The feedback inhibition is cumulative, with each metabolite exerting a partial inhibition on the enzyme. Why would complete inhibition of the glutamine synthase by a single metabolite be metaboli-cally unsound ... 

Cyclosporin A is slowly but extensively metabolized. The biotransformation pathway and the pattern of the generated metabolites are similar in humans and animals. Approximately 17 single metabolites have been detected so far, all of which are present in considerably lower plasma concentration than cyclosporin A itself [46]. Eleven ether-extractable compounds have been isolated from urine of dog and man and from rat bile and faeces using preparative HPLC and thin-layer chromatography [43]. Structural assignments for these... 

Second, some reactants that make up a free inter-converting pool are combined into a single metabolite [204], For example the two glycolytic intermediates di-hydroxyacetone phosphate and glyceraldehyde 3-phosphate are represented by the lumped species T3P (for triose-3-phosphate). 

Because the half-life of the epoxide intermediate is short, immediate rearrangement or reaction may lead to a single metabolite or a variety of substituted metabolites. The intermediacy of an epoxide intermediate can be inferred by the identification of para-and meta-hydroxylated and dihydrodiol metabolites, although their relative abundances will vary with substitution and steric considerations. Acetanilide, like phenobarbital discussed previously, exemplifies the aromatic compounds that rearrange rapidly following CYP-mediated arene epoxide formation leading to a single metabolite, as shown in Scheme 11.10. 

In any case, both biosensors and biosensing devices have been coupled to microdialysis and are considered among the non-separation-based methods [83]. The drawback of biosensing approaches is that they are usually able to measure just one analyte at a time, in contrast with separation-based methods such as chromatography and electrophoresis, which allow the detection of several analytes. However, if the primary interest is not the identification of unknown compounds, but, for example, the monitoring of variations in a single metabolite or drug, the optimization of therapeutic responses, or the control of a bioprocess via a marker analyte, the use of a specific sensor, which can be employed in a continuous manner, can provide useful information, and can also help to avoid the analysis of hundreds of samples or to reduce the number of animals necessary for a study. 

Captafol is poorly absorbed from the gastrointestinal tract. The liver and the gastrointestinal tract are the primary sites of metabolism of captafol. Captafol is eliminated via urine, feces, and expired air. The major single metabolite, tetrahydrophthalimide (THPI), was detected in blood, urine, and feces, but most of the activity in the blood and urine was in the form of more water-soluble metabolites. Following oral administration in animals, captafol is hydrolyzed to THPI and dichloroacetic acid. THPI is degraded to tetrahydrophthalimidic acid and further down to phthalic acid and ammonia. 

In most tissue samples, three metabolite peaks are apparent, whereas only a single metabolite is produced in peripheral blood cells (see Note 9). For systems using the Berthold LB506C detector (see Note 10), the software allows quantitation of the counts contained in each peak. Alternatively, fraction collection and scintillation counting can be applied. From the counts per peak for metabolites (see Note 11), the activity of an individual sample can be calculated using the equation ... 

The major function of xenobiotic metabolism can be seen as the elimination of physiologically useless compounds, some of which may be harmful as witnessed by the tens of thousands of toxins produced by plants. The function of toxin inactivation justifies the designation of detoxification originally given to reactions of xenobiotic metabolism. However, the possible pharmacological consequences of biotransformation are not restricted to detoxification. In the simple case of a xenobiotic having a single metabolite, four possibilities exist ... 

Metabolism studies are required in the laboratory animal species used to determine the toxicological NOEL, as well as each food-producing animal species. ADIs are based on total residues of drug plus all metabolites, whereas MRLs comprise a single, quantifiable marker residue, most commonly the parent compound but in some instances a single metabolite or a mixture of compounds. To establish the MRL for each tissue, food consumption estimates are made on the basis of an assumed standard meal (the so-called food basket), as discussed further in Section 2.5.2.2. 

Finally, gaps in the analytical coverage still remain, due to the lack of suitable procedures in the open literature for compounds with defined marker residues. These include fiorfenicol, tiamulin, and tulathromycin. Where any method exists, it has tended to focus on the parent drug or a single metabolite, which may not be appropriate for either screening or confirmatory purposes. 

---