Determination of platelet monoamine oxidase activity by high-performance liquid chromatography with electrochemical detection.
A procedure for determining human platelet monoamine oxidase (MAO) with dopamine (DA) as substrate is described. High-performance liquid chromatography (HPLC) with electrochemical detection (ED) was used to separate and detect components of the reaction mixture. The method for platelet preparation was also improved and only 2 ml of blood were required. Following a 10-min incubation of the platelet preparation with DA in 0.1 M Tris buffer (pH 9.0), excess DA substrate was removed by adsorption on a cation-exchange resin. The reaction product, 3,4-dihydroxyphenylacetaldehyde, was adsorbed on acid-washed alumina, eluted with 0.1 M perchloric acid and analyzed by HPLC. Simple, clean chromatograms were obtained with good reproducibility using 3,4-dihydroxybenzylamine as an internal standard. The within-sample, between-samples and between-day relative standard deviations were 0.9, 3.7 and 6.1%, respectively. The apparent Michaelis constant and maximum velocity were 0.10 mM and 0.37 nmol/min.mg protein, respectively. This HPLC-ED method offers a good alternative to methods using radioactivity.
Ogata
T
T
Division of Pediatric Pharmacology and Critical Care, Rainbow Babies and Childrens Hospital, Cleveland, OH.
Myers
C
C
Blumer
J L
JL
D001792:Blood Platelets / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004298:Dopamine; D004563:Electrochemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008995:Monoamine Oxidase / Q000032:analysis*
D008995:Monoamine Oxidase; D004298:Dopamine