ISOLATION OF LIVER-CELLS WITH CA-2+ AND K+ CHELATING-AGENTS - BIOCHEMISTRY AND CELL MORPHOLOGY

  • 1 January 1981
    • journal article
    • research article
    • Vol. 31  (4) , 217-228
Abstract
Cell morphology, glutamic pyruvic (GTP) and glutamic oxalacetic transaminases (GOT) concentrations and the ability to produce glucose or urea from different substrates (pyruvate, alanine, fructose, lactate and glutamine) were studied in isolated mouse and rat liver cells in the presence of Ca2+ and K+ chelating agents (0.1 M sodium perchlorate and 0.027 M sodium citrate with 1 mg/ml bovine albumin; ionic strength: 0.198, pH: 7.4). The chelating agent was perfused through the portal vein of an in situ liver, at low pressures (8 ml/min) at 20.degree. C for 15 min. Cell dispersion was obtained by cutting liver lobes and massaging the tissue with a plastic spatula. Wash and cell concentration may be obtained by sedimentation or centrifugation in Krebs III, glucose 150 mg %, improved with 0.16 M pyruvate, 0.1 M fumarate and 0.16 M glutamate. This procedure furnished 53.06 .+-. 3.33 .times. 106 cells, which was highly significant (P < 0.001) with respect to saline controls: 6.11 .+-. 1.91 .times. 106. After staining with Papanicolaou, hematoxylin-eosin and periodic acid Schiff, the cellular material obtained was classified optically into: normal isolated parenchymal liver cells, hepatocyte clumps, burst cells, normal blood or reticuloendothelial cells, cellular debris and noncellular material. Cell morphology showed that a constant perfusion (8 ml/min) with a minimal mechanical treatment, 82.5% of liver cells appears normal. Biochemical study showed that transaminases are indeed lost, but this loss is below the amount capable of effecting metabolic blockade (3/4 of transaminases remain in liver cells; GOT in cells, 692 .+-. 218; GPT in cells, 264 .+-. 94; GOT in supernatant; 152 .+-. 29 and GPT in supernatant, 79 .+-. 12 mUI/106 cells, after recovering 60 min at 37.degree. C). Conversion of substrates (sodium pyruvate 10 mM, 20 mM DL-alanine, 10 mM fructose and 20 mM DL sodium lactate) into glucose was statistically significant with respect to the baseline when the liver cells were isolated and recovered (rat liver cells, basal: 25.37 .+-. 3.73; pyruvate: 54.04 .+-. 7.98; DL-alanine: 62 .+-. 10.07; fructose 264.67 .+-. 20.51; DL-lactate: 78.05 .+-. 17.99 mmol/106 cells). Urea production from 5 mM DL-glutamine was statistically highly significant to the basal with rat liver cell isolated and recovered (basal: 160.60 .+-. 3.76; DL-glutamine: 608.47 .+-. 16.15 mmol/106 cells). Liver cells isolated with Ca2+ and K+ chelating agents used as described above are apparently of value for biochemical studies.