DEVELOPMENT OF A NOVEL KININ BIOMARKER ASSAY FOR CHARACTERISATION OF BRADYKININ-MEDIATED DISORDERS

Abstract

Introduction

Bradykinin (BK) is involved in various physiological and pathological processes, including angioedema (AE). AE is a predominant manifestation in multiple medical conditions and is generally mediated by BK and/or by histamine. Differentiating BK-mediated from histamine-mediated AE and assessing the role of bradykinin in the pathogenesis of other conditions by measuring kinins remains a challenge. Establishment of a method to accurately measure kinins could aid in identifying, studying, and managing BK-mediated disorders.

Methods

To inhibit ex vivo activation of kallikrein-kinin system (KKS) proteases and degradation of kinins, a protease inhibitor cocktail was developed. An ultra-high performance liquid chromatography-mass spectrometry (UPLC)-MS/MS protocol was optimized to measure BK1-9, BK1-8, BK1-7, BK1-5, and kallidin. Qualification of the UPLC-MS/MS was performed using plasma from healthy volunteers (HV) collected using PI or ethylenediaminetetraacetic acid (EDTA).

Results

The UPLC-MS/MS assay underwent qualification to assess the efficacy of the protease inhibitor cocktail. The range of quantification was 5 to 10,240 pg/mL for BK1-9, BK1-8, BK1-7 and BK1-5 and 20 to 10,240 pg/mL for kallidin. Analysis of the results revealed that kallidin levels were below limit of quantification in HV plasma. BK peptide levels were significantly lower in HV plasma collected using protease inhibitor cocktail vs EDTA. Protease inhibitor cocktail cocktail efficiently inhibited KKS activation and stabilized kinin peptides following 2 freeze and thaw cycles.

Conclusions

The developed BK assay can be used to reliably measure kinin peptides and could become a key tool for identifying, studying, and managing BK-mediated pathologies, including AE.