LC-MS method for determining the activity of semicarbazide-sensitive amine oxidase in rodents

Abstract

Semicarbazide-sensitive amine oxidase (SSAO) is present in various mammalian tissues and in blood plasma. Elevation of SSAO activity is linked to vascular disorders associated with pathological conditions such as diabetic complications, heart failure and vascular dementia. In the present paper, a high performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS) method is developed to determine the SSAO activity. Methylamine is used as physiological substrate for the enzyme activity assay of SSAO. Formaldehyde, the enzymatic reaction product, was derivatized by dopamine, and separated by a silica-based pentafluorophenyl column. The calibration curve was linear over the range of 0.03–4.00 μM of formaldehyde concentration, with 0.03 μM the lower limit of quantification (LOQ). The inter-day and intra-day precisions ranged from 2.2% to 7.9% and 4.4% to 9.2% respectively for each quality control sample of formaldehyde at 0.06, 0.50, and 2.00 μM. The accuracy ranged from 96.0% to 111.5%. The limit of detection (LOD) for serum SSAO activity was 1 nmol h⁻¹ mg⁻¹ protein. The method was successfully applied for the determination of SSAO activity in both mouse serum and rat tissues. SSAO activity of the serum in diabetes mice was significantly increased compared with the control and was inhibited by 2-bromoethylamine in vivo. The levels of SSAO activity in rat lung, spleen, aorta, kidney and brain tissues were significantly decreased by semicarbazide in vitro.