Assay of brain aldehyde dehydrogenase activity using high-performance liquid chromatography with electrochemical detection.
A method has been developed for assay of aldehyde dehydrogenase (ALDH) in brain tissue or in other tissues containing low ALDH-activity. The aldehyde of dopamine was used as the substrate, and the 3,4-dihydroxyphenylacetic acid formed was measured using high-performance liquid chromatography (HPLC) with electrochemical detection. The aldehyde was prepared enzymatically by incubating dopamine with a monoamine-oxidase preparation from rat liver mitochondria in the presence of Na+-bisulfite in 10 mM K+-phosphate buffer (pH 7.5). Rat brain homogenates were incubated in 50 mM Na+-pyrophosphate buffer (pH 8.8) containing 0.5 mM NAD+ and 5 microM aldehyde. The reaction was terminated with perchloric acid containing Na+-bisulfite to trap excess of the aldehyde. The acid supernatants were injected on a reverse-phase HPLC column and elution was performed with citrate buffer, pH 2.50. The method permits assay with 1-10 mg of brain tissue with an overall precision of 3%. The assay rate was 5-6 samples per hour.
D015102:3,4-Dihydroxyphenylacetic Acid / Q000031:analogs & derivatives / Q000032:analysis; D000444:Aldehyde Dehydrogenase / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry; D011786:Quality Control; D051381:Rats
D015102:3,4-Dihydroxyphenylacetic Acid; C007430:3,4-dihydroxyphenylacetaldehyde; D000444:Aldehyde Dehydrogenase