N-hydroxy-N-arylacetamides. III: Mechanism of haemoglobin oxidation by N-hydroxy-4-chloroacetanilide in erythrocytes in vitro.

N-Hydroxy-4-chloroacetanilide(N-hydroxy-4C1AA) was the most active, and N-hydroxy-2-acetylaminofluorene(N-hydroxy-2AAF) the least active compound among six N-hydroxy-N-arylacetamides, in forming ferrihaemoglobin(HbFe3+) in bovine erythrocytes in the presence of 11 mM glucose. N-Hydroxy-4C1AA oxidized 25 equiv. of HbFe2+, both in the presence and absence of glucose or lactate. Therefore, its catalytic properties did not depend on metabolic regeneration by the NADPH- or NADH-dependent erythrocyte reductases. In contrast, N-hydroxy-4-chloroaniline(N-hydroxy-4C1A) oxidized 760 equiv. of HbFe2+ in the presence of glucose, but only 81 equiv. of HbFe2+ in the presence of lactate. These results indicate that the catalytic activity depended on the metabolic regeneration from 4-chloronitrosobenzene(4-C1NOB) by NADPH-dependent erythrocyte reductases. A relationship was established between HbFe3+ concn. and the concn. of N-hydroxy-4C1A and 4-C1NOB(determined together), 4-chloroacetanilide(4-C1AA) and 4-chloroaniline(4-C1A), indicating co-oxidation of N-hydroxy-4C1AA and oxyhaemoglobin in erythrocytes and partial reduction of the newly formed 4-C1NOB to 4-C1A. In rat blood in vitro incubated with N-hydroxy-4C1AA, 4-C1NOB concn. increased with increasing HbFe3+ concn., indicating that 4-C1NOB was formed by co-oxidation of oxyhaemoglobin and N-hydroxy-4C1AA, and not by enzymic N-deacetylation.