13C-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 13C-NMR resonance of [Fe(13CN)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 1H, 13C, and 31P NMR spectroscopy. Incubating the cells with 13C-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.