Development and validation of an LC-MS/MS method for detection and quantification of in vivo derived metabolites of [Pyr1]apelin-13 in humans

Abstract

[Pyr1]apelin-13 is the predominant apelin peptide isoform in the human cardiovascular system and plasma. To date, few studies have investigated [Pyr1]apelin-13 metabolism in vivo in rats with no studies examining its stability in humans. We therefore aimed to develop an LC-MS/MS method for detection and quantification of intact [Pyr1]apelin-13 and have used this method to identify the metabolites generated in vivo in humans. [Pyr1]apelin-13 (135 nmol/min) was infused into six healthy human volunteers for 120 minutes and blood collected at time 0 and 120 minutes after infusion. Plasma was extracted in the presence of guanidine hydrochloride and analysed by LC-MS/MS. Here we report a highly sensitive, robust and reproducible method for quantification of intact [Pyr1]apelin-13 and its metabolites in human plasma. Using this method, we showed that the circulating concentration of intact peptide was 58.3 ± 10.5 ng/ml after 120 minutes infusion. We demonstrated for the first time that in humans, [Pyr1]apelin-13 was cleaved from both termini but the C-terminal was more susceptible to cleavage. Consequently, of the metabolites identified, [Pyr1]apelin-13(1–12), [Pyr1]apelin-13(1–10) and [Pyr1]apelin-13(1–6) were the most abundant. These data suggest that apelin peptides designed for use as cardiovascular therapeutics, should include modifications that minimise C-terminal cleavage.

The emerging role of the apelinergic system in kidney physiology and disease

“…The longest secretable form is apelin-55 [8,9]. Several enzymes that process apelin to generate both active fragments and inactive fragments lacking various N-terminal and Cterminal residues have been identified [8,10], and the list of "orthodox" isoforms is toolimited.…”

Prolyl Carboxypeptidase Mediates the C-Terminal Cleavage of (Pyr)-Apelin-13 in Human Umbilical Vein and Aortic Endothelial Cells

“…We also observed cleavage of (pyr)-apelin-13 between Ser6 and His7 in HAoEC, but not in HUVEC. This (pyr)-apelin-13 (1)(2)(3)(4)(5)(6) fragment was previously identified as a metabolite of (pyr)-apelin-13 in human plasma of healthy volunteers [49]. Murza et al also detected the cleavage site between Ser6 and His7 in rat, mouse and human plasma, but only when (pyr)-apelin-13 was first cleaved between Leu5 and Ser6, so (pyr)-apelin-13 (1)(2)(3)(4)(5)(6) was not formed [27].…”

On Methods for the Measurement of the Apelin Receptor Ligand Apelin

“…Apelin-55 (named proapelin), -36, -17, -13 and its pyroglutamated form (pyr-13), and -12, are considered the main isoforms and can activate the AR 8 – 10 . However, recent evidence reveals that various enzymatic processes are likely to be involved in apelin processing and many fragments, both active and inactive, lacking various numbers of N-terminal and C-terminal residues have been detected 11 , 12 , indicating that the list of “conventional” isoforms above is probably too restrictive. In addition, while pyr-13 apelin and apelin-17 have been determined to be the major isoforms of apelin in the circulation 8 , 9 , 13 , their relative contribution remains uncertain and potentially dependent on the study population.…”

“…Lastly, detection of naturally circulating apelin peptides with mass spectrometry (MS) has been attempted. With this approach, one group was able to detect pyroglutamated apelin at a level of a few pg/mL in samples of healthy subjects 8 , 11 , while others were unable to detect any native apelin 12 , 17 .…”

“…In addition, the very short half-life of the apelinergic peptides, probably a few minutes 12 , 18 , further complicates the measurement. While several methods, including acidification, cooling and protease inhibitors have been used, the most reliable way to preserve apelinergic peptides before measurement has yet to be determined.…”