The Effects of Aspirin Metabolites on Nadh-Methaemoglobin Reductase (Diaphorase) Activity in Human Erythrocytes

The present study was undertaken to know whether aspirin metabolites (sodium salicylate, Na-SA; sodium gentisate, Na-GA; sodium salicylate, Na-SA; sodium gentisate, Na-GA; sodium salicylurate, Na-SU) exert their reported toxic effects by inhibiting the enzyme 'NADH-Methaemoglobin (MHb) reductase (Diaphorase)' in normal adult human erythrocytes. The inhibition of the enzyme activity in normal haemolysates was observed to be highest with Na-SU, followed by Na-GA and Na-SA respectively (92%, 90% and 75% inhibition with 0.13 mM Na-SU, 50 mM Na-GA and 50 mM Na-SA respectively). The Linewearer-Burk plots from the kinetic studies (Vmax) with varying concentrations of the substrate 'NADH' indicated that the inhibition of the enzyme activity by all the three aspirin metabolites were of non-competitive type [Vmax(U)NADH alone: 47.64 ± 4.56, NADH + 10 mM Na-SA: 24.39 ± 2.34, P<0.02; Vmax (U)NADHNADH alone: 16.67 ± 1.12,
NADH + 0.002 mM Na-SU: 7.09 ± 1.05, P<0.01]. The replacement of 'MHb' by the dye '2,6-dichlorobenzeneindophenol' suggested that the inhibitory effects were due to direct interaction of the aspirin metabolites with the enzyme molecules. No evidence was also found that aspirin metabolites interact directly with NADH. It was, therefore, concluded that the aspirin metabolites (Na-SA, Na-GA, Na-SU) exert their reported toxic effects through inhibition of NADH- MHb reductase activity in a non-competitive manner probably by interacting at a region near the 'active site' causing allosteric (conformational) changes in the enzyme molecules. These conformational changes, in turn, inhibit the catalytic activity of the enzyme 'Diaphorase' in human erythrocytes leading to toxic manifestations.