Artifacts in the determination of microsomal xenobiotic N-demethylation in the presence of ascorbic acid Tris buffer and Nash reagent

Abstract

1. During investigation of microsomal xenobiotic N-demethylation in the presence of ascorbic acid, large increases in apparent enzymic activity were observed.

2. Examination of incubation components indicated that a non-enzymic interaction between ascorbic acid and Tris buffer, in the presence of acetylacetone and ammonium acetate (Nash reagent), was occurring.

3. The resulting chromophore had an absorption maximum at 412 nm that coincided with the absorption for the chromophore resulting from the interaction of the Nash reagent with the product (formaldehyde) of the enzymic reaction.

4. Strict controls of ascorbic acid potential chemical interaction with incubation components are required in enzymic studies.