, Volume 17, Issue 1, pp 45–52 | Cite as

A specific microassay for evaluating hepatic LDH activity in co-cultures of hepatocytes with other cells

  • M. Teresa Donato
  • José V. Castell
  • M. José Gómez-Lechón


This study describes the development of a simple, rapid and reproducible microassay for determining the intracellular LDH activity of rat hepatocytes present in a co-culture system with other cells. The procedure involves treatment of cellular homogenates with an anti-LDH antiserum that specifically inhibits the LDH activity of rat hepatocytes. The assay is performed in 96-well plates and LDH activity can be measured directly in the same wells using a colorimetric method. The difference in LDH activity values measured before and after antiserum incubation reflects the LDH content of the hepatocytes in the sample. The advantages of this method are the small number of cells required, a reduction in sample handling and the possibility of differentiating LDH activity in hepatic and non-hepatic cells. The possible applications of this technique as a parameter for biochemical data and as a test for cytotoxicity studies in co-cultures are also discussed.

Key words

Co-cultures cytotoxicity test hepatocytes LDH activity microassay 


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  1. Akrawi M, Rogiers V, Vandenberghe Y, Palmer CNA, Vercruysse A, Shephard EA and Phillips IR (1993) Maintenance and induction in co-cultured rat hepatocytes of components of the cytochrome P450-mediated mono-oxygenase. Biochem. Pharmacol. 45:1583–1591.Google Scholar
  2. Begue J, Guguen-Guillouzo C, Pasdeloup N and Guillouzo A (1984) Prolonged maintenance of active cytochrome P-450 in adult rat hepatocytes co-cultured with another liver cell type. Hepatology 4:839–842.Google Scholar
  3. Bergmeyer HV and Bernt E (1974) Lactate dehidrogenase: UV-assay with piruvate and MADH. In: Bergmeyer HV (ed.) Methods in Enzymatic Analysis. Vol 2 (pp. 574–579) Academic Press, New York.Google Scholar
  4. Conner J, Vallet-Collom I, Daveau M, Delers F, Hiron M, Lebreton JP and Guillouzo A (1990) Acute-phase-response induction in rat hepatocytes co-cultured with rat liver epithelial cells. Biochem. J. 266:683–688.Google Scholar
  5. Dnato MT, Castell JV and Gómez-Lechón MJ (1991a) Co-cultures of hepatocytes with epithelial-like cell lines: expression of drugbiotransformation activities by hepatocytes. Cell Biol. Toxicol. 7:1–14.Google Scholar
  6. Donato MT, Gómez-Lechón MJ and Castell JV (1990) Drug metabolizing enzymes in rat hepatocytes co-cultured with cell vitro Dev. Biol. 26:1057–1062.Google Scholar
  7. Donato MT, Gómez-Lechón MJ and Castell JV (1991b) Rat hepatocytes cultured on a monkey kidney cell line: expression of biotransformation and hepatic metabolic activities. vitro 5:435–438.Google Scholar
  8. Gómez-Lechón MJ, López P and Castell JV (1984) Biochemical functionality and recovery of hepatocytes after deep-freezin vitro 20:826–832.Google Scholar
  9. Goulet F, Normand C and Morin O (1988) Cellular interactions promote tissue-specific function, biomatrix deposition and junctional communication of primary cultured hepatocytes. Hepatology 8:1010–1018.Google Scholar
  10. Guguen-Guillouzo C, Clement B, Baffet G, Beaumont C, Morel-Chany E, Glaise D and Guillouzo A (1983). Maintenance and reversibility of active albumin synthesis by adult rat hepatocytes co-cultures with another epithelial cell type. Exptl. Cell. Res. 143:47–54.Google Scholar
  11. Kuri-Harcuch W and Mendoza-Figueroa T (1989) Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions. Differentiation 41:148–157.Google Scholar
  12. Lowry OH, Rosenbrough NJ, Farr AL and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.Google Scholar
  13. Maier P (1988) Development ofin vitro toxicity test with cultures of freshly isolated rat hepatocytes. Experientia 44:807–817.Google Scholar
  14. Montoya A and Castell JV (1987) Long-term storage of peroxidase-labelled immunoglobulins for use in enzyme immunoassay. J. Immunol. Methods 99:13–20.Google Scholar
  15. Morin O and Normand C (1986) Long-term maintenance of hepatocyte functional activity in co-culture: requirements for sinusoidal endothelial cells and dexamethasone. J. Cell. Physiol. 129:103–110.Google Scholar
  16. Omura T and Sato R (1964) The carbon monoxide-binding pigment of liver microsonmes. J. Biol. Chem. 239:2370–2378.Google Scholar
  17. Perrot N, Chesné C, De Waziers I, Conner J, Beaune PH and Guillouzo A (1991) Effects of ethanol and clofibrate on expression of cytochrome P-450 enzymes and epoxide hydrolase in cultures and cocultures of rat hepatocytes. Eur. J. Biochem. 200:255–261.Google Scholar
  18. Ponsoda X, Jover R, Castell JV and Gómez-Lechón MJ (1991) Measurement of intracellular LDH activity in 96-well cultures: a rapid and automated assay for cytotoxicity studies. J. Tiss. Cult. Meth. 13:21–24.Google Scholar
  19. Rogiers V, Vandenberghe Y, Callaerts A, Verleye G, Cornet M, Mertens K, Sonck W and Vercruysse A (1990) Phase I and phase II xenobiotic biotransformation in cultures and co-cultures of adult rat hepatocytes. Biochem. Pharmacol. 40:1701–1706.Google Scholar
  20. Utesch D and Oesch F (1992) Dependency of thein vitro stabilization of differentiated functions in liver paranchymal cells on the type of cell line used for vitro Cell. Dev. Bio. 28A:193–198.Google Scholar
  21. Voss JU and Seibert H (1992) Toxicity of glycols and allyl alcohol evaluated by means of co-cultures of microcarrier-attached rat hepatocytes and Balb/c 3T3 mouse fibroblasts. ATLA 20:266–270.Google Scholar

Copyright information

© Kluwer Academic Publishers 1995

Authors and Affiliations

  • M. Teresa Donato
    • 1
  • José V. Castell
    • 1
  • M. José Gómez-Lechón
    • 1
  1. 1.Unidad de Hepatología Experimental, Centro de InvestigaciónHospital Universitario La FeValenciaSpain

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