C74-02 Quantitative Profiling of Plasma Apelin Isoforms in Critical Illness Using UHPLC-MS/MS

Rationale The apelin and its receptor APJ signaling pathway plays an important role in regulating cardiovascular, pulmonary, and metabolic homeostasis. Apelin is processed into active fragments, including Apelin-13, which can be cyclized to form [Pyr¹]Apelin-13, the predominant isoform in human plasma. This modification enhances peptide stability and prolongs half-life by preventing aminopeptidase degradation. Despite its physiological importance, the role of apelin/APJ signaling in critical illness remains understudied, partly due to challenges in accurately quantifying [Pyr¹]Apelin-13. Methods We quantified Apelin-13 using a commercial ELISA and developed a robust analytical method utilizing parallel-reaction monitoring (PRM) on an Orbitrap mass spectrometer coupled with nano-scale ultra-high performance liquid chromatography (UHPLC) to quantify [Pyr¹]Apelin-13 in EDTA-treated human plasma samples. Results Plasma Apelin-13 levels were significantly lower in critically ill patients with acute respiratory failure (n = 137; mean 170.6 ± 44.7 pg/mL) compared to outpatient controls (n = 25; mean 201.4 ± 14.9 pg/mL; p = 0.0008) using ELISA. Our UHPLC-MS/MS platform, calibrated with authentic [Pyr¹]Apelin-13 standards, demonstrated linear peak responses at femtomolar concentrations, confirming the sensitivity and specificity. In plasma samples from two hospitalized COVID-19 patients, both unoxidized and oxidized [Pyr¹]Apelin-13 isoforms were detectable, validating the feasibility of this approach for clinical samples. Conclusion Total Apelin-13 is reduced in critical illness. Our PRM-based UHPLC-MS/MS method enables precise quantification of [Pyr¹]Apelin-13 isoforms in human plasma, providing a powerful tool for future investigations into apelin signaling in disease states and its potential as a biomarker or therapeutic target.