Study of bradykinin metabolism in human and rat plasma by liquid chromatography with inductively coupled plasma mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry.
Bradykinin is a small peptide that acts mainly as a hormone by activating specific receptors that confer protection against the development of hypertension. The efficacy of bradykinin is influenced by the activities of various kininases present in plasma and blood. In this study, both human and rat plasma were incubated with a labelled form of bradykinin (at 4 and 12.5 microM), that will be referred to as bromobradykinin. The metabolic fate of bromobradykinin was monitored by liquid chromatography coupled to an orthogonal acceleration time-of-flight mass spectrometer (oaTOF). Quantification measurements of the bromine-containing metabolites were performed on-line, via flow splitting, by inductively coupled plasma mass spectrometry (ICPMS). The data obtained highlighted that the mechanism(s) of bradykinin metabolism in human and rat plasma are different, with the metabolism of bradykinin in rat plasma being much more aggressive than that observed in human plasma. In addition to the known bradykinin metabolites, e.g. [1,5], [1,7] from ACE, [1,8] from carboxypeptidase and [2,9] from aminopeptidase activity, we have identified the presence of new bradykinin metabolites in both human and rat plasma. These have been identified as fragment [5], the amino acid phenylalanine, which was present in both the human and rat plasma and the fragments [2,8] and [4,8] in rat plasma. To our knowledge it is the first time that these fragments have been recorded in human and rat plasma. The occurrence of these new fragments provides evidence for the presence of potentially new enzymes and mechanisms of bradykinin metabolism. The method described here provides a powerful technique for monitoring the activity of the many kininases involved in bradykinin metabolism such as ACE (angiotensin I converting enzyme), carboxypeptidase N and aminopeptidase P. In addition, this procedure could be used as a screening assay for selecting and monitoring the actions of inhibitors of the enzymes implicated in bradykinin metabolism directly in plasma or serum.
Marshall
Peter
P
GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage SG1 2NY, UK. psm0667@gsk.com
Heudi
Olivier
O
McKeown
Stephen
S
Amour
Augustin
A
Abou-Shakra
Fadi
F
D000818:Animals; D001120:Arginine / Q000737:chemistry; D001711:Biotransformation; D001920:Bradykinin / Q000031:analogs & derivatives / Q000097:blood / Q000138:chemical synthesis / Q000493:pharmacokinetics*; D002138:Calibration; D002853:Chromatography, Liquid; D006801:Humans; D007202:Indicators and Reagents; D008297:Male; D013058:Mass Spectrometry; D009590:Nitrogen Radioisotopes / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D013056:Spectrophotometry, Ultraviolet
D007202:Indicators and Reagents; D009590:Nitrogen Radioisotopes; C450919:bromobradykinin; D001120:Arginine; D001920:Bradykinin