Plasma eicosanoid profiles determined by high-performance liquid chromatography coupled with tandem mass spectrometry in stimulated peripheral blood from healthy individuals and sickle cell anemia patients in treatment

Abstract

Eicosanoids play an important role in homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca(2+) ionophores and Ca(2+)-ATPase inhibitors, as well as natural agonists such as formylmethionine-leucyl-phenylalanine (fMLP), can stimulate eicosanoid biosynthesis. The aims of this work were to develop a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was partially validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient ≥0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of ≤15%, except for the lower limit of quantification, where these values were ≤20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were tested. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli for the production or liberation of eicosanoids. We next compared the eicosanoid profiles of stimulated whole blood samples of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients and those of healthy subjects, mainly for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method can detect significant changes in eicosanoid profiles in stimulated whole blood, which will contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.

Dormant 5-lipoxygenase in inflammatory macrophages is triggered by exogenous arachidonic acid

“…These assays were performed using a Shimadzu HPLC system interfaced with a Hybrid Quadrupole-TOF LC/MS/MS Mass Spectrometer - TripleTOF 5600 + (SCIEX Instruments). For the chromatographic separation of eicosanoids, an aliquot of each previously extracted sample (10 μL) was injected into an HPLC Ascentis Express C18 column (100 × 30 mm, 2.7 μm particle size, Supelco) and the HPLC methodology was performed as previously described 65 . The ESI source operated in negative ion mode for TOF-MS and MRM HR ; scanning was set to an interface heater temperature of 550 °C; ion spray voltage of −4000 eV; curtain gas of 25 psi; ion source gas 1 of 50 psi; and ion source gas 2 of 50 psi.…”

High‐resolution multiple reaction monitoring method for quantification of steroidal hormones in plasma

“…These data associated to specificity, intraday and interday precision, and accuracy give the MRM HR method good sensitivity and selectivity, which is useful to use in routine analysis. The extraction methodology, described by Galvão et al to eicosanoids, also enabled significant recovery of steroids and their metabolites from the serum. As this methodology was described first for eicosanoid extraction, it constitutes an appropriated method to simultaneously analyze eicosanoids and steroids in the same sample and consequently to perform broader lipidomics studies.…”

“…These data associated to specificity, intradayand interday precision, and accuracy give the MRM HR method good sensitivity and selectivity, which is useful to use in routine analysis. The extraction methodology, described by Galvão et al16…”

Evaluation of cardiac function tests in Sudanese adult patients with sickle cell trait

“…Rahimi et al 14 re ported that the LDL-C and total cholesterol levels in SCD subjects were significantly reduced. Also, Galvão et al 15 affirmed the differences between patients and healthy persons but without specifying the type of lipid and the type of SCA.…”