Determination of pyruvate oxidation rate and citric acid cycle activity in intact human leukocytes and fibroblasts.
We measured pyruvate oxidation in intact leukocytes and fibroblasts by measuring 14C02 production. The optimal pyruvate concentration appeared to be higher than that usually applied. Activities remained constant during the incubation and were proportional to the amount of tissue protein added. Man values (+/-SD) were 2.8 +/- 0.9 nmol/h per 10(6) cells and 37 +/- 14 nmol/h per mg of protein for leukocytes and fibroblasts, respectively, for [1-14C]pyruvate oxidation; and 2.1 +/- 0.8 nmol/h per 10(6) cells and 18 +/- 7 nmol/h per mg of protein, respectively for [2-14C]pyruvate oxidation. We compared oxidation rates of pyruvate and 2-oxoglutarate by intact cells with those of isolated mitochondria. The ratio of 14CO2 production vs. activity of mitochondrial marker enzyme demonstrated that the rate of pyruvate oxidation can adequately be assayed in intact cells, but that the permeability of the cell membrane is rate-limiting in the oxidation of 2-oxoglutarate. No significant oxidation of other intermediates of the citric acid cycle was found, presumably owing to a low rate of transport of these substances across the cell membrane.
D001692:Biological Transport; D002245:Carbon Dioxide / Q000097:blood / Q000378:metabolism; D002463:Cell Membrane Permeability; D002952:Citric Acid Cycle; D005347:Fibroblasts / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007655:Ketoglutarate Dehydrogenase Complex / Q000097:blood / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000097:blood / Q000378:metabolism; D007962:Leukocytes / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D010084:Oxidation-Reduction; D011768:Pyruvate Dehydrogenase Complex / Q000097:blood / Q000378:metabolism; D011773:Pyruvates / Q000097:blood / Q000378:metabolism*
D007656:Ketoglutaric Acids; D011768:Pyruvate Dehydrogenase Complex; D011773:Pyruvates; D002245:Carbon Dioxide; D007655:Ketoglutarate Dehydrogenase Complex