Glucose enzymatic analysis

Glucose [50-99-7] urea [57-13-6] (qv), and cholesterol [57-88-5] (see Steroids) are the substrates most frequentiy measured, although there are many more substrates or metaboUtes that are determined in clinical laboratories using enzymes. Co-enzymes such as adenosine triphosphate [56-65-5] (ATP) and nicotinamide adenine dinucleotide [53-84-9] in its oxidized (NAD" ) or reduced (NADH) [58-68-4] form can be considered substrates. Enzymatic analysis is covered in detail elsewhere (9). 

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). 

Bergmeyer, H.U., Brent, E., Schmidt, F. and Stork, H. (1974) Enzymatic analysis of glucose, in Methods of Enzymatic Analysis (ed. H.U. Bergmeyer), Academic Press, London, pp. 1196-201. 

H.-L. Lee and S.-C. Chen, Microchip capillary electrophoresis with electrochemical detector for precolumn enzymatic analysis of glucose, creatinine, uric acid and ascorbic acid in urine and serum, Talanta, 64 (2004) 750-757. 

W20. Williams, D. L., Doig, A. R., and Korosi, A., Electrochemical-enzymatic analysis of blood glucose and lactate. Anal. Chem. 42, 118-121 (1970). 

In many cases, process control comprises only the on-line monitoring of temperature and pH value as well as an accompanying enzymatic analysis of glucose and lactic acid. Since this represents an indirect process control, product yields may only be marginally improved according to these analytical results. In contrast, a number of instrumental methods are now available which allow the analysis of essential components of the fermentation process within a short time, so that a timely intervention in the process is possible and the product yield may be optimized. 

Reproduced with permission from Vormbrock R (1988) UV-method with glucose dehydrogenase. In Bergmeyer HU (ed.) Methods of Enzymatic Analysis, vol. VI, ch. 2.4.2. Weinheim VCH-Verlag VCH-Verlag. 

The quantification of glucose in the blood, using enzymatic techniques (glucose oxidase) or by ultraviolet (UV)-visible absorption spectrophotometry, may provide some clarification as to the cause of suspicious death (for example, hyper- or hypoglycemic comas). However, the results are only valid if the time between the death and the analysis is relatively short. 

Glucose analyzers based on enzyme electrodes have been brought onto the market in the United States, Japan, France, Lithuania, and Germany. As compared with the convential enzymatic analysis, the main advantages of such analyzers are the extremely low enzyme demand (a few milliunits per sample), the simplicity of operation, and the high analytical quality. 

Figure 72. Several measurements with known glucose concentration are plotted on a diagram calculated from the theoreticar AW values for this aliquot. Note the thermopile and the set-up was not optimized further. The only drawback is the fact that both enzymes are lost after each determination as they are part of the receptor buffer solution. This is also the case in traditional enzymatic analysis with photometric evaluation. Here, only a so-called. spectral-line photometer can work without further calibration...

In many cases, process control comprises only the online monitoring of temperature and pH value and an accompanying enzymatic analysis of glucose and lactic acid. Because this represents an indirect process control, product yields... 

Lang G and Michal G (19T ) D-Glucose-6-phosphate and D-fructose-6-phosphate. in Bergmeyer HU, ed. Methods of enzymatic analysis (2nd English edn.), pp. 1238-12 2. Weinheim. Verlag Chemie. 

FIGURE 16.12 Enzymatically synthesized amylose"-type nb/lcb glucans ( ) with a significant amount of the substrate glucose-1-PO4 separated on Sephacryl S-SOO/S-IOOO (60 + 9S x 1.6 cm) 3-ml fractions were collected for further analysis normalized (area = 1.0) eluogram profiles (ev) constructed from an off-line determined mass of carbohydrates for each of the pooled fractions flow rate 0.42 ml/ min V,xd = 126 ml, V , = 273 ml eluent O.OOS M NaOH. 

A second principle used widely for glucose analysis, is that of the oxidation of glucose enzymatically, mediated by the action of glucose oxidase with the formation of gluconic acid and hydrogen peroxide (22). In this procedure it is the hydrogen peroxide which is usually assayed for determination of glucose. This method suffers from the action of inhibitors which occur, particularly with patients in a diabetic coma and these need to be removed. 

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