Plasma and tissue angiotensin-converting enzyme 2 activity and plasma equilibrium concentrations of angiotensin peptides in dogs with heart disease

Larouche-Lebel, Éva; Loughran, Kerry A.; Oyama, Mark A.; Solter, Phil F.; Laughlin, Danielle S.; Sánchez, Melissa D.; Assenmacher, Charles-Antoine; Fox, Philip R.; Fries, Ryan C.

Briefly, plasma conditioning for equilibrium analysis was performed at 37 °C followed by stabilization through the addition of an enzyme inhibitor cocktail (Attoquant Diagnostics, Vienna, Austria). Equilibrated plasma samples were further spiked with stable isotope-labeled internal standards for each AP at a concentration of 200 pg/ml. The samples then underwent C-18-based solid-phase-extraction and were subjected to LC-MS/MS analysis using a reversedphase analytical column operating in line with a Xevo TQ-S triple quadruple mass spectrometer (Waters Corporation, Milford, MA). Internal standards were used to correct for peptide recovery of the sample preparation procedure for each AP in each individual sample. Analyte concentrations were reported in pg/ml and were calculated considering the corresponding response factors determined in appropriate calibration curves in original sample matrix, on condition that integrated signals exceeded a signal-to-noise ratio of 10. The lower limit of quantification and validation data are shown in Supplemental Tables 2 and 3 Angiotensin 1-9 (Ang1-9) 3.5