Sphingomyelin and Phosphatidylserine Derived Compounds Found into RBCs of Two Unrelated Individuals with Idiopathic Erythrocytosis with Altered Hemoglobin (Hb) Function Properties and No Hemoglobinopathies - Preliminary Results

Abstract

Sphingomyelin and phosphatidylserine derived compounds were found into Red Blood Cells (RBCs) of two unrelated individuals with idiopathic erythrocytosis, whom were referred to the Laboratory of Hemoglobinopathies - State University of Campinas (State of São Paulo, Brazil) to investigate the presence of hemoglobin (Hb) variants with high oxygen affinity. Both unrelated individuals were 5-year-old age Caucasian Brazilian boys with the following counts: RBC (x 10/6mm3) 6.15 and 7.69, Hb (g/dL) 20.1 and 20, Htc (%) 54 and 58.7, respectively. Most common causes of erythrocytosis, including polycythemia vera (PV) and other secondary erythrocytosis; JAK2, VHL and EPOR mutational screen were previously excluded in the Centro Infantil Boldrini (Campinas, State of São Paulo, Brazil), where the children are followed. No hemoglobin or globin variants were detected by standard methods: alkaline electrophoresis in cellulose acetate (pH 8.9), acidic electrophoresis in agar gel (pH 5.8), cation-exchange HPLC (Variant II, Bio-Rad) and RP-HPLC (Alliance, Waters). Direct genome sequencing of HBA and HBB performed in automated sequencer (ABI PRISM™ 377 DNA Automated Sequencer, Applied BioSystems) after DNA amplification obtained from peripheral leukocytes (QIAamp DNA Blood, Qiagen) did not reveal any mutation. Familial study also did not show any alteration in blood count, hemoglobin or even globin gene mutation in their relatives. Deletional and non-deletional alpha thalassemia was also excluded by Multiplex-gap-PCR and restriction enzyme reaction (Hph and NcoI). Despite of normal results for Hb characterization, functional tests performed with Hb purified by exclusion (Sephadex G-25, Sigma-Aldrich) and ion exchange chromatography (Amberlite MB-3, Sigma-Aldrich) in Hepes buffer indicated some alteration. O2 affinity was evaluated by determining P50 with stripped lysates ([Hb]=70 μM/Heme) in pHs of 6.5, 7.0, 7.5, 8.0 and 8.5, for Bohr effect calculations. Heme-heme cooperativity was determined by Hill coefficient (n) and the activity of allosteric effector in the Hb-O2 ligation was also tested, using inositol hexaphosphate ([IHP]=1mM), a mimetic compound of the intraerythrocytic 2,3-BPG. P50 was slightly decreased (both patients, stripped Hb samples) at extreme acidic and alkaline pH (pH 6.5 and 8.5) leading to altered Bohr effect, in comparison to healthy and erythrocytosis control (caused by cardiopathy, with no Hb variant). P50 was also found decreased at pH 8.5 in the presence of IHP. Heme-heme cooperativity was very decreased at pHs 6.5, 7.0, 8.0 and 8.5, in stripped state as well as in the presence of IHP. In order to investigate the unexpected results mass spectrometry of the purified intraerythrocytic content was performed. RBC lysate was separated from stroma by ultracentrifugation (14.000rpm) and purified by molecular exclusion filtration (Amicon filter - Millipore, 10KDa cutoff) and ion exchange chromatography (Amberlite MB-3, Sigma-Aldrich) for desalinization. Quadrupole Mass spectrometry (MS) with 0.8 ppm MS and MS/MS accuracy (Impact High DefinitionTM, Bruker), preceded by UPLC (Ultra Pressure Liquid Chromatography) system (Shimadzu Nexera) with C18 Polymer RP-column (Supelco apHera C18 Polymer 150 x 2 mm, 5um) was used to separate and identify and compare the compounds to control sample (secondary erythrocytosis with no Hb function alteration). No alteration in peptide, hemoglobin or heme were found. Four small compounds, two derived of sphingomyelin and two derived of phosphatidylserine were identified in both patient samples (C39H79N2O6P, C42H80NO8P, C42H82NO8P, C44H84NO8P) - Figure 1. Sphingomyelin and phosphatidylserine are RBC membrane compounds rich in PO4-, which theoretically could bind b1b2 allosteric 2,3-BPG site in Hb. There is no evidence that these molecules were effectively bound to Hb except that were found in purified Hb samples with some function alteration, particularly with decreased heme-heme cooperativity. Some evidence suggests that analog compounds could be found in higher concentrations in PV or secondary erythrocytosis. To our knowledge it is the first-time that alteration in Hb function was observed in samples with these molecules. These preliminary results should be further validated and at least 10 other similar cases should be better characterized. (Financial support: Fapesp, Faepex-Unicamp, Educorp-Unicamp). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal