Plasma bradykinin concentrations during septic shock determined by a novel LC-MS/MS assay

Abstract

Background: Bradykinin is an important mediator of inflammation and vascular permeability and could have an important role in the development of septic shock. Measurement of bradykinin by immunological methods may suffer from interference and lack of specificity. We developed and validated a liquid chromatography mass spectrometry assay (LC-MS/MS) for plasma bradykinin.Methods: We used plasma samples from healthy volunteers (n=19) and patients with septic shock (n=47).Stable isotope bradykinin internal standard was added to samples before solid-phase extraction and quantification by LC-MS/MS. Stability of bradykinin was studied for 12 months.Results: Our assay has good sensitivity (0.1 nmol/l) and a wide linear range (0.1-1000 nmol/l). Bradykinin added to plasma was stable for 12 months at -20 °C when a mixture of protease inhibitors was added at sampling but degraded during repeated freezing and thawing. Bradykinin concentration in plasma from septic shock patients (<0.1-0.6 nmol/l) did not change significantly during shock and recovery but differed slightly from that in healthy individuals (0.5-1.1 nmol/l).Conclusions: Our bradykinin assay was successfully used to determine bradykinin concentrations in plasma samples. Intensive care unit patients with septic shock had low concentrations of plasma bradykinin during both shock and recovery phases.

Sensitive mass spectrometric determination of kinin-kallikrein system peptides in light of COVID-19

“…20,21 However, this sensitivity was not su cient to determine endogenous levels of bradykinin throughout all their plasma samples. 19 In the current study, for the rst time in NLF, concentrations of endogenous levels of speci c kinin peptides were detectable in saline matrix and allowed for their comprehensive determination. Proud et al 1983, measured kinin peptides in eight controls by immunometric detection, seven had levels below the LLOQ (< 20 pg/mL) and one had a level of 100 pg/mL.…”

Targeted LC-MS/MS platform for the comprehensive determination of peptides in the kallikrein-kinin system

“…This leads to the requirement for extensive sample clean-up, including chromatographic separation and multiple SPEs, prior to quantification by immunoassay [2,28]. However, to date, reported quantification limits for bradykinin by LC-MS/MS are quite high-namely, between 94 pg/mL [19] up to 10 ng/mL in plasma or serum [20,21,29]. The presented assay shows a far more sensitive quantification limit of 2 pg/mL for bradykinin.…”

“…For example, quantification limits of 2 ng/mL were achieved for des-Arg(9)-bradykinin by van den Broek et al in 2010 [21]; thus, the presented assay marks an improvement by a factor of 1000. Furthermore, current LC-MS/MS assays or immunoassays require at least 500 μL of plasma/1 mL of blood to establish these (low) quantification limits, whereas the presented assay requires only 150 μL of plasma [2,19,21,28,29]. This reduction enables repeated blood sampling in severely ill patients without increasing the risk of anaemia.…”

“…An ideal surrogate matrix should closely resemble the study samples and be analyte-free [32]. Previous LC-MS/ MS assays for bradykinin used water [19] or bovine plasma as a surrogate matrix due to the endogenous presence of kinins in human matrix [21]. However, different compositions of a surrogate matrix in comparison to study samples might lead to matrix effects affecting the accuracy of the results [32].…”

“…Approaches towards quantification of several kinins simultaneously by nano LC-MS/MS indicated the high potential of this technique [18]. Nevertheless, reported levels of bradykinin applying available LC-MS/MS assays still vary from 200 pg/mL [19,20] up to 160 ng/mL [21] in healthy volunteers. Consequently, other studies have focused on the determination of only the more stable metabolite bradykinin 1-5, whose levels are unaffected by the artificial generation of bradykinin if the degradation of bradykinin is sufficiently inhibited [22,23].…”