Reliable measurement of plasma kinin peptides: Importance of preanalytical variables

Abstract

Background: The kallikrein-kinin system is involved in many (patho)physiological processes and kinin peptides are considered potential clinical biomarkers. Variance in blood specimen collection and processing, artificial ex vivo bradykinin formation, and rapid degradation of kinins have contributed to divergence in published plasma levels, therefore limiting their significance. Thus, reliable preanalytical settings are highly required.

Objectives: This study aimed to develop and evaluate a standardized preanalytical procedure for reliable kinin quantification. The procedure was based on identification of the most impactful variables on ex vivo plasma level alterations.

Methods: Suitable protease inhibitors and blood specimen collection and handling conditions were systematically investigated. Their influence on plasma levels of seven kinins was monitored using an established in-house liquid chromatography-tandem mass spectrometry platform.

Results: In nonstandardized settings, ex vivo rise of bradykinin was found to already occur 30 seconds after blood sampling with high interindividual variation. The screening of 17 protease inhibitors resulted in a customized seven-component protease inhibitor, which efficiently stabilized ex vivo kinin levels. The reliability of kinin levels was substantially jeopardized by prolonged rest time until centrifugation, phlebotomy methodology (eg, straight needles, catheters), vacuum sampling technique, or any time delays during venipuncture. The subsequently developed standardized procedure was applied to healthy volunteers and proved it significantly limited interday and interindividual kinin level variability.

Conclusion: The developed procedure for blood specimen collection and handling is feasible in clinical settings and allows for determination of reliable kinin levels. It may contribute to further elucidating the role of the kallikrein-kinin system in diseases like angioedema, sepsis, or coronavirus disease 2019.

Keywords: blood specimen collection; bradykinin; factor XII; kallikrein‐kinin system; phlebotomy.

FIGURE 1

Overview of the conducted investigation regarding the preanalytical variables. The preanalytical conditions were optimized step by step based on the results of the previous step (top‐down). The general workflow was consistent for all experiments, including fresh blood samples and direct analysis without long‐term storage. LC‐MS/MS, liquid chromatography–tandem mass spectrometry

FIGURE 2

Addressed targets by the customized protease inhibitor within the kallikrein‐kinin system (A). Targets were assigned based on known characteristics of inhibitors. The kininases involved in kinin metabolism are shown with their catalytic type and cleavage site in (B). BK, bradykinin; FA, formic acid; FXIIa, activated factor XII; HDMB, hexadimethrine bromide; HK, high‐molecular‐weight kininogen; KBK, kallidin; LK, low‐molecular‐weight kininogen; PKa, plasma kallikrein, TK, tissue kallikrein

FIGURE 3

Self‐formation of bradykinin and formation of its metabolites after blood sampling. Spontaneous contact activation without in vitro activation in the absence of inhibitors in healthy volunteer in EDTA S‐Monovettes (A) was monitored. The ratios of metabolite to bradykinin formation from 5 to 30 minutes are further displayed (B)

FIGURE 4

Effects of distinct inhibitors on the formation of bradykinin in citrate plasma. The relative abundance of bradykinin 0.5 and 4.5 hours after blood sampling is shown. (A) Results of the screening of multiples protease inhibitors is shown. (B) The evaluation of distinct concentrations of the best‐performing inhibitors HDMB and nafamostat and their combination are depicted. Mean values with standard deviation and individual data points (n = 3)

FIGURE 5

Development of the customized protease inhibitor. The percentage change of spiked kinin levels (1 ng/mL) in plasma after 3 h of benchtop storage using different protease inhibitor approaches is shown. Mean values (n = 3) with their coefficient of variations (CV) are presented. The dashed line represents the maximum deviation of ±15% according to the US Food and Drug Administration guideline. HDMB, hexadimethrine bromide

FIGURE 6

Impact of blood sampling conditions on kinin levels. Box‐whisker‐plots show the impact of the tube size (A), time delays (B), collection devices (C) and the collection technique (D). Experiments were carried out at least in triplicate (A: n = 3; B: n = 3 or n = 6, respectively; C: n = 3, n = 6 or n = 9; D: n = 6). An interexperimental control using Safety Multifly needles 21G with 200 mm tubing into 1.2‐mL S‐Monovettes under aspiration was run for each experiment

FIGURE 7

Box‐whisker‐plots of the kinin levels for interday (A) and interindividual (B) variability using the developed standardized procedure. Only kinin levels above the lower limit of quantification were shown. N = 3