Endogenous reporter neuropeptides for the measurement of protease dysregulation in Complex Regional Pain Syndrome (CPRS).

M. Bayer, T. Schlereth T, F. Birklein, S. König

Abstract

CRPS is a severe and often disabling syndrome, which develops after trauma in ̃2-3% of all cases; most often after distal radius fractures. Neuropeptides such as bradykinin (BK) and substance P (SP) as well as metalloproteases like angiotensin-converting enzyme (ACE) participate in inflammatory processes and contribute to the pathophysiological mechanisms in CRPS.[1] Modulation of BK signaling is largely determined by ACE which metabolizes the BK nonapeptide into BK1-7 and BK1-5, but degradation by aminopeptidase P (APP, cleaves position 1-2) and carboxypeptidase N (CPN, cleaves position 8-9) is also known.Using TLC-based separation of BK products following serum incubation we have demonstrated that ACE activity is reduced in CRPS patient sera compared to healthy controls. [2] Dabsylationof reporter peptides enables differentiation from peptides naturally occuring in serum as well asadding a chromophore label to eliminate the need for a staining procedure after a TLC-run. Isolated ACE cleaves dabsyl-BK more rapidly than BK and cleavage was only slowed down after adding ACE-inhibitor teprotide whereas BK degradation was completely inhibited as shown bymass spectrometry (Q-TOF Premier, Waters).Following incubation of DBK in serum enzymatic cleavage products of CPN and ACE could be shown by mass spectrometry as well as TLC.For further elucidation of the pathophysiology of CRPS we labeled SP (sequence RPKPQQF-FGLM). After serum incubation for 120 min, fragment KPQQFFGLM is the most abundant. Monitoring of dabsylated neuropeptides holds high promises not only for the investigation ofCRPS.