Enzymatic analysis metabolite concentrations

Enzymes play an important role in biochemical analysis. In biological material—e. g in body fluids—even tiny quantities of an enzyme can be detected by measuring its catalytic activity. However, enzymes are also used as reagents to determine the concentrations of metabolites—e.g., the blood glucose level (C). Most enzymatic analysis procedures use the method of spectrophotometry (A). 

P NMR spectra provide quantitative information on the relative concentrations of phosphorylated metabolites. For spectra acquired under fully relaxed conditions, the area of the P NMR resonance is proportional to the concentration of the metabolite. Absolute quantifications are more difficult to perform but can also be obtained if the concentration of one of the phosphorylated metabolites in the sample (normally ATP) is measured by an independent method, such as enzymatic analysis. Unfortunately, the resonances from the a and p phosphates of ADP overlap under in situ conditions with those from the a and Y phosphates of ATP, thus precluding direct quantification of ADP in situ. The concentration of ADP in situ can be estimated indirectly from the difference in area between the a ATP or y ATP peaks and the area of the P ATP peak, which is almost exclusively derived from ATP. 

As a rule, the combined immobilization of oxidase and peroxidase on the electrode allows the determination of metabolite (S) concentration, even if negligibly small. This type of biosensor is the main one used for detection of any particular compound in blood or other multicomponent system glucose, ethanol, cholesterol, proteins, amino acids, etc. For example, for patients with diabetes a rapid analysis of their blood for glucose concentration is a vitally important procedure. We will not attempt to discuss in full all the existing types of biosensor we will just note that enzymatic and cell biosensors are the most widespread types of these appliances. Enzymatic biosensors are more appropriate to the subject of the current monograph and, therefore, they will be discussed below. 

Fig. 2 The red blood cell has played a special role in the development of mathematical models of metabolism given its relative simplicity and the detailed knowledge about its molecular components. The model comprises 44 enzymatic reactions and membrane transport systems and 34 metabolites and ions. The model includes glycolysis, the Rapaport-Leubering shunt, the pentose phosphate pathway, nucleotide metabolism reactions, the sodium/potassium pump, and other membrane transport processes. Analysis of the dynamic model using phase planes, temporal decomposition, and statistical analysis shows that hRBC metabolism is characterized by the formation of pseudoequilibrium concentration states pools or aggregates of concentration variables. (From Ref...

As a result of these observations, recommendations have been made to include an incubation step in methods of analysis for lincosamides in liver. " If the aim is to detect lincosamide use, then inclusion of an incubation step is necessary to maximize the possibility of detection. However, as indicated earlier, MRLs, where set, are based on the parent compound only with no reference to the metabolites, either by summation of separate measurements or by conversion to a suitable marker. Thus, inclusion of an incubation step could lead to falsely high residue concentrations and the possibility of samples that fall within the legal limits being reported as non-compliant. As a result, analytically, steps should be taken to minimize enzymatic activity to prevent this reverse metabolism. A long-term solution to this issue might be to include the sulfoxides in the legislation as additional markers in some form. 

HPLC/DAD is very important in benzodiazepine analysis. After enzymatic cleavage of the conjugate, the individual metabolites of the benzodiazepines can be clearly distinguished from each other and identified in the chromatogram, so that, for example, the co-consumption of bromazepam when diazepam therapy is in progress can be detected. In sample material of low concentration, for example, the determination of several metabolites of the same starting substance considerably improves the quality of the analytical results. The limit of detection of HPLC is more than adequate in the region of 10-20 ng/ml for routine and confirmatory analysis. 

From metabolic studies, an isotopic caffeine breath test has been developed that detects impaired liver function using the quantitative formation of labeled carbon dioxide as an index. From the urinary excretion of an acetylated uracil metabolite, human acet-ylator phenotype can be easily identified and the analysis of the ratio of the urinary concentrations of other metabolites represents a sensitive test to determine the hepatic enzymatic activities of xanthine oxidase and microsomal 3-methyl demethylation, 7-methyl demethylation, and 8-hydroxylation. Quantitative analyses of paraxanthine urinary metabolites may be used as a biomarker of caffeine intake. Fecal excretion is a minor elimination route, with recovery of only 2-5% of the ingested dose. 

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