Metabolite concentrations, determination lines

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. 

Experimental errors tend to be quite large in biological systems, e.g. 30% in protein concentration measurements. Cell number measurements are generally no better than 5%. At lower viabilities (< 70% viable), accurate determination of viability and cell number is difficult, and the error in each determination may be greater than 10%. Errors in cell and metabolite concentration measurements lead to uncertainties in calculated parameters, such as specific growth, production and consumption rates, therefore a complex profile for these calculated parameters should not be assumed when a straight-line or simple function will suffice. 

Having determined the target tissue, the parent drug and/or one or more of the metabolites in the target tissue are chosen to be the marker residue. The proportion of the marker residue to total residues is obtained at the point on the total residue depletion curve where this line crosses its permitted safe concentration. The level of the marker residue at that point represents the tolerance since it is specified in the Code of Federal Regulations, Title 21, Part 556. 

Another MEKC separation (75 mM SDS, phosphate-borate buffer, pH 9.1) was reported for die determination of ll-nor-A-9-tetrahydrocannabinol-9-carboxylic acid, the major metabolite of A-9-tetrahydrocannabinol present in urine (Wemly and Thormann, 1992a). Sample treatment included basic hydrolysis of urine (5 mL), solid phase extraction, and concentration. The resulting sensitivity was 10 to 30 ng/mL (i.e., comparable to the cutoffs of immunoassays). Again, detection was by on-line recording of peak spectra, by means erf fast-scanning UV detector. 

In addition to enzyme activity, the concentration of a non-electroactive substrate can be determined elec-trochemically by this technique. By keeping the substrate (analyte) as the limiting reagent, the amount of product produced is directly related to the initial concentration of substrate. Either kinetic or equilibrium measurements can be used. Typically, an enzyme that produces NADH is used because NADH is readily detected electrochemically. Glucuronide and sulfate metabolites of pharmaceuticals and lecithin in crude drug formulations have been determined by LCEC through off-line enzymatic reaction. 

An automated in-tube solid-phase microextraction HPLC method for NNK and several metabolites have been developed by Mullett et al. ° In-tube SPME is an on-line extraction technique where analytes are extracted and concentrated from the sample directly into a coated capillary by repeated draw-eject steps. A tailor-made polypyrrole-coated capillary was used to evaluate their extraction efficiencies for NNK and several metabolites in cell cultures. This automated extraction and analysis method simplified the determination of the tobacco-specific A-nitrosamines, requiring a total sample analysis time of only 30 min. 

For the quantification of relatively large amounts of vitamin D, e.g. in pharmaceutical preparations, off-line spectrometric procedures can be considered. Spots eluted from the plates can be quantitated by spectrometric or gas-chromatographic procedures (28). Photodensitometry (reflectance spectrometry at 265 nm) and fluorodensitometry (after spraying the plate) are usually sufficiently sensitive to permit the determination of vitamin D in foods (34-36,38). The low concentrations of vitamin D metabolites in human plasma/serum can only be measured off-line by GC (25(OH)D) (40) or radioligand assays (25(OH)D and l,25(OH)2D) (41-43). 

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