Measurement of the extracellular release of lactate dehydrogenase in cultured primary rat hepatocytes

Commonly used acronym: LDH assay

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Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Basic Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
Rat
Type of cells/tissues/organs
Primary rat hepatocytes

Description

Method keywords
  • Hepatotoxicity
  • Hepatocytes
  • cytotoxicity
  • LDH
Scientific area keywords
  • Toxicology
  • Hepatotoxicity
  • Primary hepatocytes
  • cytotoxicity
Method description

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.

Lab equipment

Spectrophotometer

Method status
  • History of use

Pros, cons & Future potential

Advantages

Easy-to-apply method

Challenges

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

References

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

Associated documents

Contact person

Kaat Leroy

Organisations

Vrije Universiteit Brussel (VUB)
Pharmaceutical and Pharmacological Sciences
In Vitro Toxicology and Dermato-Cosmetology
Belgium