A rapid method of preparing hepatic parenchymal cells for studying drug metabolism.
Parenchymal cells were prepared from the livers of male Sprague-Dawley rats by collagenase perfusion and purified by a self-generating Percoll gradient. The method consisted of mixing 31% Percoll and 5 x 10(6) cells/ml, followed by centrifugation at 10,000 x g for 10 min. A self-generated gradient provided a rapid and efficient recovery of highly viable parenchymal cells. The parenchymal cells were determined to be very stable during incubation at 37 degrees C for at least 2 h. Cell integrity was evaluated by trypan blue dye exclusion, lactate dehydrogenase leakage, and membrane peroxidation. In addition, drug metabolism and conjugation were evaluated as markers of intracellular integrity. With increasing p-nitroanisole (pNA) concentration, the formation of p-nitrophenol (pNP) increased. The rate of sulfation was maximal at a pNA concentration of 0.25 mM and decreased greatly above 1.0 mM. Glucuronidation increased from 0.25 mM to a maximum rate of 2.0 mM pNA. Above 1.0 mM pNA, nonconjugated pNP increased proportionately to the decrease in sulfation. These results indicate that the cell integrity was maintained, and that these cells can be used as a model for studying drug metabolism.
Brown
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RC
Department of Pharmacology and Nutrition, University of Southern California School of Medicine, Los Angeles 90033.
Bidlack
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WR
D000818:Animals; D000840:Anisoles / Q000378:metabolism; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008054:Lipid Peroxides / Q000032:analysis; D008099:Liver / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains
D000840:Anisoles; D008054:Lipid Peroxides; D007770:L-Lactate Dehydrogenase; C005306:4-nitroanisole