Enzymology and subcellular localization of aldehyde oxidation in rat liver: Oxidation of 3,4-dihydroxyphenylacetaldehyde derived from dopamine to 3,4-dihydroxyphenylacetic acid

Abstract

In the presence of ethanol, the metabolism of dopamine in rat liver slices is altered such that the major product is 3,4-dihydroxyphenylethanol, not 3,4-dihydroxyphenylacetic acid (DOPAC). It has been proposed that this metabolic alteration is due to the inhibition of the oxidation of 3,4-dihydroxyphenylacetaldehyde (DOPAL) by acetaldehyde, the first metabolite of ethanol. The oxidation of DOPAL in rat liver slices, however, is not inhibited dramatically by relatively high concentrations of acetaldehyde [A. W. Tank and H. Weiner, Biochem. Pharmac. 28, 3139 (1979)]. Thus, it is possible that acetaldehyde and DOPAL are oxidized by different isozymes of aldehyde dehydrogenase (ALDH) present in different subcellular compartments. Acetaldehyde is oxidized by isozymes of ALDH that are found in the matrix space of the mitochondria of rat liver. The subcellular site of the oxidation of most other aldehydes is not known. Mitochondrial, microsomal and cytosol fractions of the rat liver were isolated by differential centrifugation, and the isozymes of ALDH present in the cytosol and mitochondrial fractions were separated by column isoelectric focusing. Five isozymes of ALDH were isolated from the cytosol, and three isozymes were isolated from the mitochondria. The K m values for acetaldehyde, p-nitrobenzaldehyde and DOPAL for each of the isolated isozymes were determined and were found to range from approximately 1 μM to 1 mM. Each subcellular fraction was incubated with [ethylamine-2- 14C]dopamine to determine its ability to oxidize DOPAL. Partially purified monoamine oxidase was used to generate DOPAL for those incubations which did not contain mitochondria. Intact mitochondria were capable of oxidizing virtually all the DOPAL to DOPAC in the presence or absence of added pyridine nucleotide coenzymes. Cytosol and microsomal fractions were capable of oxidizing the aldehyde, but not to the same extent as the intact mitochondria. ALDH activity present in the mitochondrial matrix space was inhibited by the addition of rotenone. This treatment inhibited formation of DOPAC by 80 per cent in isolated intact mitochondria in the absence of added pyridine nucleotides. Inclusion of rotenone caused the inhibition of DOPAC formation by ca. 50 per cent when intact mitochondria, microsomes and cytosol were incubated together with dopamine. These results suggest that an isozyme of ALDH present in the mitochondrial matrix space is primarily responsible for the oxidation of DOPAL in rat liver, though nonmitochondrial enzymes can contribute to the oxidation.