C-13-NMR studies of transmembrane electron transfer to extracellular ferricyanide in human erythrocytes

Human erythrocytes are known to reduce ferricyanide (hexacyanoferrate) [Fe(CN)(6)](3-) to ferrocyanide [Fe(CN)(6)](2-) in an extracellular reaction that involves the transmembrane transfer of reducing equivalents: potentially these could be either electrons from NADH, formed in glycolysis inside the cells or transmembrane exchange of reduced solutes. The C-13-NMR resonance of [Fe((CN)-C-13)(6)](3-) (which was synthesised in our laboratory) was seen to be very broad while that of ferrocyanide was narrow. This phenomenon formed the basis of a simple non-invasive. procedure to study ferricyanide reduction in high-haematocrit suspensions of erythrocytes. The method should be directly applicable to other cell types In a series of experiments, erythrocyte metabolism was studied in the presence of ferricyanide, using H-1, C-13, and P-31 NMR spectroscopy. Incubating the cells with C-13-labelled glucose enabled the rate of ferricyanide reduction, glucose utilisation, and lactate and bicarbonate production to be measured simultaneously. Various metabolic states were imposed as follows: glycolysis was inhibited with F- and iodoacetate; glucose transport was inhibited with phloretin and cytochalasin B; and anion transport was inhibited with dinitrostilbene 2,2'-disulfonate and p-chloromercuriphenyl sulfonate. Earlier work was confirmed, showing that ascorbate is intimately involved in the reduction reaction; but its main action appears not to be mediated by membrane transport but in a membrane-associated redox-protein complex that is functionally linked to glycolysis. Also, large differences (factors of three) in the rate of the reduction reaction were recorded in erythrocytes from different, apparently healthy, donors.