MSACL 2019 EU Abstract

Determination of Membrane Lipid Gangliosides in Human Serum and Cerebral Organoids

Gabriela Dovrtělová (1), Tereza Váňová (2,3), Hana Hříbková (2), Dáša Bohačiaková (2), Zdeněk Spáčil (1)
(1) RECETOX Centre, Masaryk University, Brno, Czech Republic (2) Department of Histology and Embryology, Masaryk University, Brno, Czech Republic (3) International Clinical Research Center (ICRC) St. Anne’s University Hospital, Brno, Czech Republic

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Gabriela Dovrtělová (Presenter) Recetox centre, Masaryk University >> POSTER (PDF)

Presenter Bio: I completed my PhD studies at the Masaryk University in the program of Medical Pharmacology in 2018. As a member of a pharmacokinetic group, I focused on changes and regulation of metabolic activity of liver cytochrome P450. In 2017/2018, I attended a 6 months internship in the group of Dr. Massimo Zuccheti (Istituto Di Ricerche Farmacologiche Mario Negri, Milan, IT) to improve my MS and pharmacokinetic skills at clinical levels. In September 2018, I joined the doctor Spacil’s group (in the RECETOX centre) engaged in metabolomics and targeted proteomics. My current work is focused on the analysis of membrane lipids (gangliosides) using UHPLC/MS system, which play an important role in the pathogenesis of Alzheimer's disease.

Relevant Financial Disclosures (within past 24 months) Grant/Research Support Grant Agency of Masaryk University, Czech Ministry of Education, Youth and Sports, Czech Health Conflict mitigation NOT REQUIRED.

Introduction: Gangliosides (GSs), sialic acid-containing glycosphingolipids, are essential components of cellular membranes, especially in neuronal cells. Besides affecting the function of the membrane in which they are located, these amphiphilic lipids modulate a variety of biological functions through transmembrane signalling.1 GSs content and composition change during aging, which is the most important risk factor for several neurodegenerative diseases.2 Cerebral organoids are a useful model to study composition of membrane GSs in brain tissue or specific cell populations. The brain tissue abundance of GSs expectedly correlates with circulating levels in peripheral blood and perhaps the determination of GSs in human serum can serve as clinically significant marker.
Objectives: The primary aim of our study was to develop an analytical protocol for extraction and detection of selected GSs (aGM1, GM1, GM2, GM3, GD1a, GD1b, GT1b, GQ1b, GD2, and GD3) in cerebral organoids and human serum.
Methods: Several extraction solvent systems were tested (i.e. Folch method,3 water-saturated ethyl acetate, and 80 % of 2-propanol/methanol) to achieve optimal extraction efficiencies. A commercially available pooled human serum and cerebral organoids (~45 days aged) were analysed. SRM-MS assays on a triple-quadrupole mass spectrometer (6495, Agilent Technologies) were used to detect GSs. Stable isotopically labelled internal standards were used for absolute quantification in biological samples.
Results: All targeted GSs were profiled quantitatively in cerebral organoids. The most abundant species in human serum were GM3, GM2, GD1a/b, GD3 and GT1b.
Conclusion: The study presents an analytical protocol to quantify GSs and its application to a biological material (i.e. human serum and cerebral organoids). It i a useful tool for investigation of aging induced alterations in membrane GS composition, essential to elucidate the pathophysiology of neurodegenerative diseases.

(1) Ledeen, R.; Wu, G. Gangliosides of the Nervous System. Methods Mol. Biol. 2018, 1804, 19–55.
(2) Kolter, T. Ganglioside Biochemistry. ISRN Biochem 2012, 2012, 506160.
(3) Folch, J.; Lees, M.; Sloane Stanley, G. H. A Simple Method for the Isolation and Purification of Total Lipides from Animal Tissues. J. Biol. Chem. 1957, 226 (1), 497–509.

This work was supported by the Grant Agency of Masaryk University (GAMU project No. MUNI/G/1131/2017), The Czech Health Research Council, AZV project No. NV19-08-00472 and the Czech Ministry of Education, Youth and Sports (CETOCOEN PLUS CZ.02.1.01/0.0/0.0/15_003/0000469 and LM2015051).