Spectroscopy, lactate dehydrogenase

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. 

Zipp, A., Watson, C. and Greyson, J. (1978). Reflectance spectroscopy as an analytical tool in the clinical laboratory Application to the quantitative determination of lactate dehydrogenase in serum. Clin. Chem. 24, 1009, Abstr. 105. 

Stevens, J.F., Tsang, W. and Newall, R.G. (1983). Measurement of the enzymes lactate dehydrogenase and creatine kinase using reflectance spectroscopy and reagent strips. J. Clin. Pathol. 36, 1371-1376. 

UTP, GTP, CTP, and ITP can act as the phosphate donor. This enzyme catalyses one of the rate-limiting reactions of glycolysis. It may be estimated by u.v. spectroscopy using lactate dehydrogenase in an indicator reaction [591]. 

High-resolution H n.m.r. spectroscopy has been used to probe the conformations of a number of o-ribofuranosylamine derivatives and such rigid molecules as 2,2 -cyclonucleosides and nucleoside 3, 5 -phosphates in aqueous solution. H N.m.r. spectroscopy has also been used to study details of the intramolecular association and conformations of a- and j8-linked pyridine ribo-nucleosides and their 5 -phosphates. The results were analysed in terms of base-D-ribose, o-ribose-side-chain, and base-side-chain interactions and the conformational restraints imposed by the cis HO-2-HO-3 interaction in jS-nucleo-tides and the additional cis HO-2 -base interaction in a-nucleotides. H N.m.r. measurements - including measurements of nuclear Overhauser effects and paramagnetic relaxations effected by Mn + cations - have been used to investigate the preferred conformation about the jV-glycosidic bond of 8-amino-, 8-methyl-amino-, and 8-dimethylamino-adenylic acid, all of which competitively inhibit the coenzyme NADH in the reaction with chicken-muscle lactate dehydrogenase. The primary and secondary amines were shown to prefer anti conformations, whereas the tertiary amine prefers a syn conformation. 

Metabolism Global metabolic profile via metabolomics (e.g. NMR spectroscopy, liquid chromatography-mass spectrometry (LC-MS), Lactate/pyruvate ratio, glucose and amino acid consumption, succinate dehydrogenase levels... 

Figures 4 and 5 illustrates the use of NMR spectroscopy to study the metabolism of (l-i C)glu-cose in primary cultures of neurons and astrocytes. A simplified scheme of the metabolism of (l-i C)glu-cose in neural cells is given in Figure 4. Briefly, (1-i C)glucose is metabolized to (3- C)pyruvate through the Embden Meyerhoff glycolytic pathway. The (3-i C)pyruvate produced can be transaminated to (3- C)alanine, reduced to (3-i C)lactate or enter the tricarboxylic acid (TCA) cycle through the pyruvate dehydrogenase (PDH) or pyruvate carboxylase (PC) activities. A net increase in (3-i C)lactate reveals increased aerobic glycolysis and is normally observed under hypoxic conditions in normal cells. If (3-i C)pyruvate enters the TCA cycle though PDH it produces (2-i C)acetyl-coenzyme A first, and subsequently (4-i C)a-ketoglutarate. In contrast, if (3-i C)pyruvate is carboxylated to (3-i C)oxalacetate by...

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