Bioactivity Assay Of Relaxin Indirectly Quantified By LC-MS/MS

Backgroud: The peptide hormone relaxin belongs to the insulin-IGF family,with pleiotropic effects reported in various reproductive tissues during pregnancyincluding growth and softening of the cervix, inhibition of uterine contractility,vasodilation of blood vessels, regulation of cardiovascular function, stimulating cellproliferation, neovascularization, tissue remodeling and metastasis, with potential tobe a new drug of multiple functions. Unlike insulin related receptor belongs to asubgroup of membrane-associated receptor tyrosine kinase, relaxin appears to signalthrough a member of the G-protein-coupled receptor (GPCR) family with seventransmembrane domains. It is important to assess bioactivity of relaxin forexperimental and clinical purposes, but the available method lacks specificity andreproducibility.Objective: This study describes a reliable bioactivity assay of porcine relaxinbased on cyclic adenosine3’,5’-monophosphate accumulation in THP-1cellsquantified by a liquid chromatography-tandem mass spectrometry (LC-MS/MS)method. The precision and sensitivity of the LC-MS/MS method for the EC50determination of pRLX were compared with those of a competitive enzymeimmunoassay assay (ELISA), confirming the potential application of the establishedLC-MS/MS method in the bioactivity assay of pRLX.Method: In this study, a LC-MS/MS method to quantify the cyclic adenosine3’,5’-monophosphate was developed to determine the bioactivity of pRLX. Firstly, arapid, reliable and reproducible LC-MS/MS method for the determination ofintracellular cAMP levels was developed and validated. Then, the LC-MS/MS methodwas employed to analyze the intracellular cAMP levels in THP-1cells stimulated byserial concentrations of pRLX. Thus, the EC50was calculated to assess the bioactivityof pRLX. Finally, the reproducibility, precision and sensitivity of the LC-MS/MSmethod for the EC50determination of pRLX were evaluated in comparison with acommercially available ELISA assay to confirm the potential application of theestablished LC-MS/MS method in the bioactivity assay of pRLX.Results: As a result, the LC-MS/MS was based on a positive multiple reactionmonitor (MRM) of cAMP with a stable internal standard,8-Br-cAMP (150ng·mL-1)and a protein precipitation procedure by HClO4. The standard curve of cAMP was linear from5.0ng·mL-1to992.0ng·mL-1(r2≥0.992), with limits of detection andquantification of0.5ng·mL-1and5.0ng·mL-1. The intra-day accuracy and precision(expressed as relative standard deviation) were94.9~102.2%and1.7~7.2%, whileinter-day accuracy and precision were95.4~106.7%and3.8~9.9%, respectively.When measured by LC-MS/MS, the pRLX sample showed a time-anddose-dependent stimulation of cAMP with an EC50of40.9ng·mL-1. The standardcurve of cAMP measured by ELISA (Neweast Biosciences) was linear from0.2ng·mL-1to137.1ng·mL-1(0.4-250.0pmol·mL-1，r≥0.971), When measured by ELISA,the pRLX sample showed a time-and dose-dependent stimulation of cAMP with anEC50of54.4ng·mL-1.Conclusion：This method provides a determination for bioactivity of pRLX incombination of LC-MS/MS and cell activity test. Compared with the commercialenzyme linked immunosorbent assay (ELISA) kits, the LC-MS/MS indicated higherprecision and selectivity.