title: 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.
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, Ribeirão Preto, São Paulo, 14040-903, Brazil.
authors.forename: Alyne Fávero
authors.initials: AF
authors.lastname: Galvão
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, Ribeirão Preto, São Paulo, 14040-903, Brazil.
authors.forename: Tânia
authors.initials: T
authors.lastname: Petta
authors.affiliation: Département de Médecine, Faculté de Médecine Université Laval, Centre de Recherche de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec, 2725, chemin Sainte-Foy, Québec, G1V 4G5, Canada.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Flamand
authors.affiliation: Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, São Paulo, 14049-900, Brazil.
authors.forename: Valdes Roberto
authors.initials: VR
authors.lastname: Bollela
authors.affiliation: Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, São Paulo, 14049-900, Brazil.
authors.forename: Célio Lopes
authors.initials: CL
authors.lastname: Silva
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, Ribeirão Preto, São Paulo, 14040-903, Brazil.
authors.forename: Luciana Ribeiro
authors.initials: LR
authors.lastname: Jarduli
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, Ribeirão Preto, São Paulo, 14040-903, Brazil.|Centro de Terapia Celular, Centro Regional de Hemoterapia do Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Rua Tenente Catão Roxo 2501, Ribeirão Preto, São Paulo, 14049-900, Brazil.
authors.forename: Kelen Cristina Ribeiro
authors.initials: KC
authors.lastname: Malmegrim
authors.affiliation: Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, São Paulo, 14049-900, Brazil.
authors.forename: Belinda Pinto
authors.initials: BP
authors.lastname: Simões
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes, 3900, Ribeirão Preto, São Paulo, 14049-901, Brazil.
authors.forename: Luiz Alberto Beraldo
authors.initials: LA
authors.lastname: de Moraes
authors.affiliation: Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, Ribeirão Preto, São Paulo, 14040-903, Brazil. faccioli@fcfrp.usp.br.
authors.forename: Lúcia Helena
authors.initials: LH
authors.lastname: Faccioli
mesh_terms: D000755:Anemia, Sickle Cell / Q000097:blood / Q000188:drug therapy* / Q000628:therapy; D001803:Blood Transfusion; D016022:Case-Control Studies; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015777:Eicosanoids / Q000097:blood*; D006801:Humans; D012016:Reference Values; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015777:Eicosanoids
keywords: Chronic red blood cell (RBC) transfusion; Eicosanoid; HPLC-MS/MS; Human plasma; Hydroxyurea (HU); Lipidome; Method validation; Sickle cell anemia (SCA); Whole blood stimulation