Assessment of the deamination of aminoacetone, an endogenous substrate forsemicarbazide-sensitive amine oxidase

Methylglyoxal, a toxic aldehyde, has been reported to be increased in diabe tes and has been claimed to be related to diabetic complications. Aminoacet one, an intermediate in the metabolism of threonine and glycine, has been p roposed to be an endogenous substrate for semicarbazide-sensitive amine oxi dase (SSAO). Methylglyoxal is the product. An HPLC procedure for the determ ination of SSAO activity toward aminoacetone in vitro is described. It was observed in previous assays that methylglyoxal formed via deamination of am inoacetone was quite unstable and led to erroneous results. o-Phenylenediam ine (o-PD) was therefore employed for derivatization of methylglyoxal, o-PD does not affect SSAO activity and can be included in the enzyme reaction m ixture for continuous trapping of methylglyoxal. This can avoid the loss of methylglyoxal during incubation. Deamination of aminoacetone by human umbi lical artery SSAO was confirmed with this improved assay. The values of K-m and V-max, are 125.9 +/- 20.5 mu M and 332.2 +/- 11.7 nmol/h/mg protein, r espectively. Deamination of aminoacetone was nearly completely inhibited by 1 mM semicarbazide and 1 mu M MDL-72974A, a potent selective SSAO inhibito r, whereas MAO inhibitors clorgyline (1 mM) and deprenyl (1 mM) had no inhi bitory effect. (C) 1999 Academic Press.