Pyruvate and acetate oxidation by leukocytes in vitro. Activation of the pyruvate dehydrogenase complex by uncoupling of oxidative phosphorylation

Abstract

Pyruvate oxidation by normal intact leukocytes has been systematically studied to define optimal conditions for detection of enzymatic defects in this process. Leukocytes were isolated by dextran sedimentation and lymphocytes by Ficoll centrifugation. Cells were incubated for 2 h with [1-¹⁴C]-pyruvate, [2-¹⁴C]-pyruvate or [1-¹⁴C]-acetate as substrate. The specific oxidative capacity of lymphocytes was almost three times higher than that of granulocytes from the same blood. Oxidation of both pyruvate and acetate was highly dependent on the substrate concentration in the medium reaching a plateau between 0.5 and 1.0 mmol/1. Addition of succinate (1 mmol/1) stimulated oxidation of [1-¹⁴C]-pyruvate by 30%. Uncoupling of phosphorylation by addition of carbonyl cyanide chlorophenylhydrazone (CCCP) (0.1 μmol/1) increased oxidation of [1-^14C]-pyruvate by 200% and of [1-¹⁴C]-acetate by 70%. Addition of CCCP plus succinate caused further stimulation of pyruvate oxidation (+40%), but not of acetate oxidation. It is therefore concluded that: (1) Lymphocytes are better than mixed leukocytes for oxidative studies. (2) Unlabelled substrate should be added at optimal concentrations. (3) The pyruvate dehydrogenase complex is normally only partially active in lymphocytes. (4) Stimulation of oxidation by CCCP greatly enhances the flux through the PDH step thus facilitating the detection of defects in pyruvate oxidation.

Key Words:

• congenital lactic acidosis
• pyruvate dehydrogenase deficiency