Measurement of the extracellular release of lactate dehydrogenase in cultured primary rat hepatocytes
Commonly used acronym: LDH assay
Scope of the method
- Human health
- Basic Research
- In vitro - Ex vivo
- Animal derived cells / tissues / organs
Description
- Hepatotoxicity
- Hepatocytes
- cytotoxicity
- LDH
- Toxicology
- Hepatotoxicity
- Primary hepatocytes
- cytotoxicity
This method assesses general cytotoxicity. Upon disruption of the cell membrane, lactate dehydrogenase (LDH) is released. LDH catalyzes the interconversion of pyruvate and lactate with concomitant interconversion of reduced (NADH) and oxidized (NAD+) nicotinamide adenine dinucleotide. The principle of the assay described in the current standard operating procedure is based on this reaction. In particular, the consumption of NADH is spectrophotometrically assessed and serves as a measure that is proportional to the LDH activity.
Spectrophotometer
- History of use
Pros, cons & Future potential
Easy-to-apply method
Cell membrane damage is a rather late and rough marker of cytotoxicity that mainly indicates necrosis and that may yield false negative results
References, associated documents and other information
Bergmeyer H.U. (1974) Lactate dehydrogenase. In: Methods of enzymatic analysis, (ed. H.U. Bergmeyer), pp. 574-579. New York, USA: Academic Press
Elaut G., Henkens T., Papeleu P., Snykers S., Vinken M., Vanhaecke T. and Rogiers V. (2006) Molecular mechanisms underlying the dedifferentiation process of isolated hepatocytes and their cultures. Current Drug Metabolism 7: 629-660
Gómez-Lechón M.J., Lahoz A., Gombau L., Castell J.V. and Donato M.T. (2010) In vitro evaluation of potential hepatotoxicity induced by drugs. Current Pharmaceutical Design 16: 1963-1977
Jaeschke H., Gores G.J., Cederbaum A.I., Hinson J.A. Pessayre D. and Lemasters J.J. (2002) Mechanisms of hepatotoxicity. Toxicological Sciences 65: 166-176
McKim J.M. (2010) Building a tiered approach to in vitro predictive toxicity screening: a focus on assays with in vivo relevance. Combinatorial Chemistry and High Throughput Screening 13: 188-206
Papeleu P., Vanhaecke T., Henkens T., Elaut G., Vinken M., Snykers S. and Rogiers V. (2006) Isolation of rat hepatocytes. Methods in Molecular Biology 320: 229-237
Seglen P.O. (1976) Preparation of isolated rat liver cells. Methods in Cell Biology 13: 29-83
Van Calsteren V. (2010) Analyse van de herhaalde toedieningstoxiciteitstesten met cosmetische ingrediënten bestudeerd op Europees niveau. Dissertation Master in Pharmaceutical Sciences - Vrije Universiteit Brussel, Rogiers V. (promotor) and Pauwels M. (co-promotor)
Vinken M., Decrock E., De Vuyst E., Leybaert L., Vanhaecke T. and Rogiers V. (2009) Biochemical characterisation of an in vitro model of hepatocellular apoptotic cell death. Alternatives to Laboratory Animals 37: 209-218
Contact person
Kaat LeroyOrganisations
Vrije Universiteit Brussel (VUB)Pharmaceutical and Pharmacological Sciences
In Vitro Toxicology and Dermato-Cosmetology
Belgium
Brussels Region