Determination of monoamine oxidase activity by HPLC with fluorimetric detection

The aldehyde produced from the oxidative deamination of primary and secondary amines by the monoamine oxidases (EC 1.4.3.4; MAO) or the semicarbazide-sensitive amine oxidases (EC 1.4.3.6; SSAO) may be determined followed reaction at elevated temperatures with 2-diphenylacetyl-1,3-indandione-1-hydrazone (DIH), separation by high-performance chromatography liquid (C-8 column, eluting isocratically with acetonitrile, ammonium acetate, water) and fluorimetric detection (excitation and emission wavelengths 430 and 525 nm). The detection limits for benzaldehyde, p-hydroxybenzaldehyde and 2-phenylacetaldehyde were 125 nM, 150 and 62.3 nM, respectively. Thus the assay is appropriate for determination of amine oxidase activities towards benzylamine, 2-phenylethylamine and tyramine. The fluorescence of the DIH adduct with indole-3-aldehyde was strongly quenched, giving a relatively high detection limit (17.5 microM). The detection limit was lower (3.8 microM) when the absorbance at 430 nm was monitored. Enzyme activities determined by this procedure were shown to be linear with enzyme-protein concentration (rat liver mitochondria). The presence of 1-2 mM semicarbazide, necessary for determining MAO activities in samples also containing SSAO, did not adversely affect the derivatization reaction. The DIH-aldehyde adducts were sufficiently stable to permit their storage at low temperatures prior to assay. The product produced by reaction of 5-hydroxyindole acetaldehyde with DIH had no significant fluorescence and too low an absorbance at 430 nm to allow its determination for assay of activities towards 5-HT. This procedure can also measure succinic semialdehyde (detection limit 240 nM) and thus would be applicable to the determination of GABA transaminase activity.