Bradykinin Measurement by LC-MS/MS in Hereditary Angioedema Subjects Enhanced by Cold Activation

Background Bradykinin (BK) is a key mediator responsible for swelling episodes in hereditary angioedema due to C1INH deficiency/dysfunction (HAE-C1INH). Current BK measurement faces many challenges primarily related to very low levels and instability, and is not feasible for application in clinical settings. Objective This study aimed to develop a novel method to overcome the issues in current protease inhibitor-based methods for measuring endogenous BK and its metabolites. Methods The blood from HAE-C1INH and healthy volunteers were collected and subjected to cold activation for contact system. Cold-induced BK and its major metabolites were measured via Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). The protocol was established according to the US FDA bioanalytical validation guidelines as a CLIA laboratory-developed test. The BK measurement was optimized based on blood sample types, collection methods, time windows, and temperature/storage conditions. Results EDTA whole blood samples without protease inhibitors incubated at 4 degree for 1 to 3 days produced over 100-fold differences in total BK levels between HAE-C1INH subjects and healthy volunteers (324.3 +/- 54.7 ng/mL, n=33; vs 2.3 +/- 0.3 ng/mL, n = 43; mean +/- S.E.M., p < 0.001). The sensitivity and specificity were 90.9% and 97.1% respectively. BK levels highly correlated with the plasma kallikrein activity in the same samples. Conclusions Whole blood under cold activation demonstrated remarkable elevation of BK levels in HAE-C1INH subjects, while minimally affecting healthy individuals. The assay has validated accuracy, precision and stability. It may serve as a reliable and robust tool for HAE diagnosis and management. Clinical Implication Cold-induced Bradykinin measurement can be used as a new biomarker for diagnosis, disease monitoring and guiding therapeutic options for HAE-C1INH and other bradykinin-mediated angioedema (AE-BK), with or without identifiable genetic mutations.