Lactate dehydrogenase techniques

In addition to enzyme activity, the concentration of an nonelectroactive substrate can be determined electrochemically by this technique. By keeping the substrate (analyte) 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 which produces NADH is used because NADH is readily detected electrochemically. Lactate has been detected using lactate dehydrogenase, and ethanol and methanol detected using alcohol dehydrogenase... 

Part—I has three chapters that exclusively deal with General Aspects of pharmaceutical analysis. Chapter 1 focuses on the pharmaceutical chemicals and their respective purity and management. Critical information with regard to description of the finished product, sampling procedures, bioavailability, identification tests, physical constants and miscellaneous characteristics, such as ash values, loss on drying, clarity and color of solution, specific tests, limit tests of metallic and non-metallic impurities, limits of moisture content, volatile and non-volatile matter and lastly residue on ignition have also been dealt with. Each section provides adequate procedural details supported by ample typical examples from the Official Compendia. Chapter 2 embraces the theory and technique of quantitative analysis with specific emphasis on volumetric analysis, volumetric apparatus, their specifications, standardization and utility. It also includes biomedical analytical chemistry, colorimetric assays, theory and assay of biochemicals, such as urea, bilirubin, cholesterol and enzymatic assays, such as alkaline phosphatase, lactate dehydrogenase, salient features of radioimmunoassay and automated methods of chemical analysis. Chapter 3 provides special emphasis on errors in pharmaceutical analysis and their statistical validation. The first aspect is related to errors in pharmaceutical analysis and embodies classification of errors, accuracy, precision and makes... 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Assay of lactate dehydrogenase illustrates this technique. This enzyme catalyzes the oxidation of lactate, yielding pyruvate and NADH as shown in Figure 6-17. NADH produced in this reaction can be used along with the intermediate electron carrier, phenazine methosulfate (PMS), to... 

Considerable interest was aroused by the finding of Wieme and Lauryssens (W16) in 1962 that there is a change in the electrophoretic isoenzyme pattern of lactate dehydrogenase in diseased human muscle. The major isoenzyme of lactate dehydrogenase in most normal muscles moves slowest on electrophoresis (LDH 5), but in myopathic muscle the proportion of LDH 5 may be considerably reduced. This finding has been confirmed and extended by numerous workers, utilizing various techniques for isoenzyme differentiation (e.g., BIO, E5). The abnormal pattern is seen in most, but not all, cases of Duchenne dystrophy and in a variety of other muscular disorders. It may be evident in the very early stages of Duchenne dystrophy (P2) and is seen even in some female carriers of the disease (E3). 

Deng, H., Zheng, J., Sloan, D., Buegnee, J., Callender, R. (1989) Classical Raman spectroscopic studies of NADH and NAD+ bound to lactate dehydrogenase by difference techniques, Biochemistry 28, 1525-1533. 

S. Olsen, J. Ruzicka, E.H. Hansen, Gradient techniques in flow injection analysis. Stopped-flow measurement of the activity of lactate dehydrogenase with electronic dilution, Anal. Chim. Acta 136 (1982) 101. 

Roels et al. [38] points out that the analytical techniques identified in Table 1 are not easily available and are not well-suited for routine biomonitoring of occupational or environmental exposures. Instead, indirect biomarkers such as urinary enzymes are often used with success to evaluate mercury exposure and injury. Zalups [35] identifies numerous methods used to detect renal tubular injury induced by mercury. These methods monitor the urinary excretion of enzymes that leak from injured and necrotic proximal tubules, including lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and N-acetyl-P-D-glucosaminidase (NAG). Although advocated by Zalups (35) to detect renal tubular injury, Mason et al. (48) questions the practical utility of such biomarkers in occupational surveillance. According to Mason et al., small increases in NAG, leucine... 

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