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

Abstract

1. N-Hydroxy-4-chloroacetanilide(N-hydroxy-4ClAA) 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.

2. N-Hydroxy-4ClAA 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.

3. In contrast, N-hydroxy-4-chloroaniline(N-hydroxy-4ClA) 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-CINOB) by NADPH-dependent erythrocyte reductases.

4. A relationship was established between HbFe³⁺ concn. and the concn. of N-hydroxy-4ClA and 4-CINOB(determined together), 4-chloroacetanilide(4-ClAA) and 4-chloroaniline(4-CIA), indicating co-oxidation of N-hydroxy-4ClAA and oxyhaemoglobin in erythrocytes and partial reduction of the newly formed 4-CINOB to 4-CIA.

5. In rat blood in vitro incubated with N-hydroxy-4CIAA, 4-CINOB concn. increased with increasing HbFe³⁺ concn., indicating that 4-CINOB was formed by co-oxidation of oxyhaemoglobin and N-hydroxy-4CIAA, and not by enzymic N-deacetylation.