Abstract
The effect of the redox dye methylene blue on the stability of NADH and on the activity of the enzyme aldehyde dehydrogenase (ALDH; EC 1.2.1.3) was examined. NADH was measured by HPLC with fluorometric or spectrophotometric detection. The ALDH activity assays were carried out by following the formation of 3,4-dihydroxyphenylacetic acid (DOPAC) from 3,4-dihydroxyphenylacetaldehyde (DOPAL) using HPLC and electrochemical detection. Incubation of NADH solutions in the presence of methylene blue resulted in a time-dependent direct oxidation of NADH. Methylene blue inhibited the human erythrocyte and leukocyte ALDHs and the rat liver mitochondrial low- K m ALDH in a concentration-dependent manner. The inactivation was reversible by dilution, and kinetic analysis indicated that methylene blue inhibits the rat liver mitochondrial low- K m and human erythrocyte ALDHs competitively with respect to DOPAL, while no effect of the NAD + concentration was apparent. For the rat liver low- K m ALDH, a K i of 8.4 ± 2.8 μM (mean ± SD; N = 5) was calculated. The inhibition of ALDH and the resulting decrease in the redox effect on the NAD system bound to alcohol dehydrogenase (EC 1.1.1.1) could explain the protective effect of methylene blue against metabolic redox effects of ethanol.
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