Abstract
The outbreak of COVID-19 has raised interest in the kinin–kallikrein system. Viral blockade of the angiotensin-converting enzyme 2 impedes degradation of the active kinin des-Arg(9)-bradykinin, which thus increasingly activates bradykinin receptors known to promote inflammation, cough, and edema—symptoms that are commonly observed in COVID-19. However, lean and reliable investigation of the postulated alterations is currently hindered by non-specific peptide adsorption, lacking sensitivity, and cross-reactivity of applicable assays. Here, an LC–MS/MS method was established to determine the following kinins in respiratory lavage fluids: kallidin, bradykinin, des-Arg(10)-kallidin, des-Arg(9)-bradykinin, bradykinin 1-7, bradykinin 2-9 and bradykinin 1-5. This method was fully validated according to regulatory bioanalytical guidelines of the European Medicine Agency and the US Food and Drug Administration and has a broad calibration curve range (up to a factor of 103), encompassing low quantification limits of 4.4–22.8 pg/mL (depending on the individual kinin). The application of the developed LC–MS/MS method to nasal lavage fluid allowed for the rapid (~ 2 h), comprehensive and low-volume (100 µL) determination of kinins. Hence, this novel assay may support current efforts to investigate the pathophysiology of COVID-19, but can also be extended to other diseases.
Highlights
The outbreak of COVID-19 has raised interest in the kinin–kallikrein system
This study aimed to develop and validate a novel LC–MS/MS method characterized by a broad calibration curve range to comprehensively and sensitively determine KKS peptides (bradykinin, kallidin, desArg[9]-bradykinin, des-Arg[10]-kallidin, bradykinin 1-7, bradykinin 1-5 and bradykinin 2-9) to enable reliable insights into their alterations in COVID-19—or other disease states—in comparison to controls
Analyzing the contour plots of the D-optimal optimization model for the original injection solvent composition and the contour plots with restricted organic amount, showed clearly that high acidic amounts were necessary for the injection solvent, as well as the highest organic amount that would be compatible with peak shapes of the hydrophilic peptides (Fig. 2)
Summary
The outbreak of COVID-19 has raised interest in the kinin–kallikrein system. Viral blockade of the angiotensin-converting enzyme 2 impedes degradation of the active kinin des-Arg[9]-bradykinin, which increasingly activates bradykinin receptors known to promote inflammation, cough, and edema—symptoms that are commonly observed in COVID-19. The application of the developed LC–MS/MS method to nasal lavage fluid allowed for the rapid (~ 2 h), comprehensive and low-volume (100 μL) determination of kinins This novel assay may support current efforts to investigate the pathophysiology of COVID-19, but can be extended to other diseases. It is assumed that cleavage of active des-Arg[9]-bradykinin into the inactive bradykinin 1-7 is considerably reduced during SARS-CoV-2 infection In this context, viral attenuation of ACE 2 activity contributed to the pathogenesis of lung inflammation that was concomitant with increased bradykinin-1 receptor expression. Saline lavage fluids from the respiratory tract would likely be the most suitable matrix to investigate alterations in the KKS peptide levels, as these fluids originate from the main area of viral infection and clinical symptoms in mild to severe courses (e.g. cough, nasal congestion, pulmonary inflammation and edema). Design of experiments (DoE) has proven its usefulness as a lean tool for method development of multifactordependent settings and contributes to signal increase in LC–MS/MS29,30
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