3.1 Target mass spectrometry to quantify the BK-(1-9) fragments in plasma from rats
BK-(1-9) (RPPGFSPFR) is metabolized in the plasma producing a variety of fragments, which potentially include BK-(1-7) (RPPGFSP) and BK-(1-5) (RPPGF). However, one should note that the detection and quantitation of a peptide in the plasma with the same molecular weight (572.66 Da) and amino acid sequence (RPPGF) of BK-(1-5), for example, does not imply that it is indeed the BK-(1-5) as such fragment could be produced from the hydrolysis of other plasma protein / peptide than BK-(1-9). Therefore, we decided to evaluate whether an acute infusion of BK-(1-9) would alter the concentrations for the potential BK-(1-9) fragments in vivo. We did this to ensure that the putative BK-(1-9) fragments were produced from BK-(1-9) metabolization. As shown in Figure 1A, the plasma levels of BK-(1-7) and BK-(1-5) increased after 15 seconds of BK-(1-9) infusion. The highest plasma concentration for BK-(1-7) was observed after 5 minutes of BK-(1-9) infusion while for BK-(1-5), the peak was at 15 seconds of the administration. This result suggests that indeed BK-(1-7) and BK-(1-5) are peptides endogenously derived from BK-(1-9). To confirm this observation, we gave an i.v. infusion of the heavy-isotope labelled [Pro3(13C5;15N)]-BK-(1-9) in anesthetized male Wistar rats, using the same experimental conditions as above. The results (Figure 1B) showed a similar response profile, in which a rapid increase of the heavy-isotope labelled [Pro3(13C5;15N)]-BK-(1-5) was detected after 15 seconds of [Pro3(13C5;15N)]-BK-(1-9) administration while the highest concentration of the heavy-isotope labelled [Pro3(13C5;15N)]-BK-(1-7) was observed following 5 min of [Pro3(13C5;15N)]-BK-(1-9) administration (Figure 1B).