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

• Published in: Analytical and bioanalytical chemistry Vol. 408; no. 13; pp. 3613 - 3623
• Main Authors: Galvão, Alyne Fávero, Petta, Tânia, Flamand, Nicolas, Bollela, Valdes Roberto, Silva, Célio Lopes, Jarduli, Luciana Ribeiro, Malmegrim, Kelen Cristina Ribeiro, Simões, Belinda Pinto, de Moraes, Luiz Alberto Beraldo, Faccioli, Lúcia Helena
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01-05-2016; Springer; Springer Nature B.V
• Subjects: Analytical Chemistry; Anemia, Sickle Cell - blood; Anemia, Sickle Cell - drug therapy; Anemia, Sickle Cell - therapy; Biochemistry; Biosynthesis; Blood; Blood Transfusion; Case-Control Studies; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatography; Chromatography, High Pressure Liquid - methods; Drug therapy; Eicosanoic acid; Eicosanoids - blood; Food Science; Health aspects; High performance liquid chromatography; Human; Humans; Infectious diseases; Ionization; Laboratory Medicine; Lipids; Liquid chromatography; Mass spectrometry; Methods; Monitoring/Environmental Analysis; Patients; Physiological aspects; Plasma; Reference Values; Research Paper; Samples; Scientific imaging; Sickle cell anemia; Statistical analysis; Statistical methods; Stimulation; Stimuli; Tandem Mass Spectrometry - methods; Brazil; HPLC-MS/MS; Human plasma; Method validation; Lipidome; Eicosanoid; Chronic red blood cell (RBC) transfusion; Sickle cell anemia (SCA); Hydroxyurea (HU); Whole blood stimulation
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-016-9445-8

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, LTB
4
, LTE
4
, TXB
2
, and PGE
2
. 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.
Graphical abstract
Profiles of eicosanoids produced after whole blood stimulation in healthy volunteers and patients with SCA under treatment. Blood cells from patients with SCA under treatment with HU or chronic blood transfusions produce a different profile of eicosanoids after stimulation of whole blood with A23187 or thapsigargin when compared to those of healthy volunteers. The quantification of eicosanoids was performed in a HPLC-MS/MS using MRM mode.