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Hemoprotein quantitation in isolated hepatocytes

D P Jones, S Orrenius, H S Mason

    Biochimica Et Biophysica Acta
    |January 25, 1979
    PubMed
    Summary
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    New methods quantify hemoproteins in isolated hepatocytes. Pretreatment altered cytochrome P-450 and b5 levels, while incubation decreased concentrations, influenced by prior treatments.

    Area of Science:

    • Biochemistry
    • Cell Biology
    • Pharmacology

    Background:

    • Hemoproteins, including cytochromes, are crucial for cellular metabolism and detoxification.
    • Quantifying these proteins in isolated hepatocytes is essential for understanding drug metabolism and toxicity.
    • Previous methods were limited to subcellular fractions, necessitating techniques for whole cells.

    Purpose of the Study:

    • To develop and validate methods for quantifying catalase and various hemoproteins in isolated rat hepatocytes.
    • To assess the impact of in vivo pretreatments (phenobarbital, 3-methylcholanthrene, ethanol) on hepatocyte hemoprotein concentrations.
    • To investigate changes in hemoprotein levels during hepatocyte incubation and the influence of prior treatments on these changes.

    Main Methods:

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  • Established methods for subcellular hemoprotein quantitation were adapted for isolated hepatocytes.
  • Hepatocytes were pretreated in vivo with phenobarbital, 3-methylcholanthrene, or ethanol.
  • Changes in hemoprotein concentrations were measured after varying incubation times and conditions.

    • Main Results:

    • Phenobarbital and 3-methylcholanthrene pretreatments increased cellular concentrations of cytochromes P-450 and b5.
    • Chronic ethanol pretreatment elevated cytochrome P-450 and decreased cytochromes a + a3.
    • Hepatocyte hemoprotein concentrations declined during 4-10 hour incubations, with rates dependent on incubation conditions and prior treatments.

    Conclusions:

    • The developed methods allow for accurate quantitation of key hemoproteins in isolated hepatocytes.
    • In vivo pretreatments significantly alter specific hemoprotein profiles in hepatocytes.
    • Hepatocyte incubation conditions and prior drug exposure influence hemoprotein stability and degradation.