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