Measurement of LDH activity

The measurement of lactate dehydrogenate (LDH EC. 1.1.1.27) in culture supernatants gives a quantitative value for the loss of cell viability  

The activity of LDH can be measured as the reduction of pyruvate to lactate (Vassault, 1983). The reduction is coupled to the oxidation of NADH to NAD+, which is followed spectrophotometrically at 340 nm. The equilibrium is far on the side of NAD+ and lactate. Because NADH has a high absorbance at 340 nm compared with NAD+, the reaction is measured as the rate of decrease in absorbence at 340 nm. 

Although colorimetric assays based on the reduction of tetrazolium salts have been used, they have been largely superseded because of non-specific side reactions in such assay systems. 

Note NADH must be free of inhibitors. Discard if the powder has become yellow. Stability of solutions 

Buffer is stable at 0-4°C provided that bacterial contamination does not occur. The NADH solution is kept at 0-4°C and must be prepared fresh daily. The pyruvate solution should be dispensed into 1.5-ml aliquots and stored at -20°C. After thawing, each aliquot should be discarded. The pyruvate solution is stable for 2 months. 

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The above commentary and methodology may have given the impression that the only use in cell culture for measurements of LDH activity is in the monitoring of changes in culture viabihty. An alternative application is in the comparison of cell viability between different regions within entrapped cell culture systems, where it may not be possible to determine viable cell numbers directly. 

Scheller et al. (1986a) combined polyurethane-immobilized LOD with an Au/Pd-sputtered carbon electrode. The electrode modification permits H2O2 to be electrochemically oxidized at a potential as low as +450 mV, where interferences by other anodically oxidizable compounds such as NADH and ascorbic acid are largely reduced (Fig. 57). This increased selectivity also enables the measurement of LDH activity. The sensor has been introduced in an FIA manifold. A sample frequency of 200Ai (Fig. 58) and a CV below 1% were obtained with this setup. A platinum electrode with LOD covalently bound to a nylon membrane has been employed for continuous blood lactate determination in an artificial pancreas by Mascini et al. (1985b, 1987). 

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