MSACL 2019 EU Abstract

Analysis of Human Monocyte Volatiles by Solid-Phase Microextraction Combined with Two-Dimensional Gas Chromatography-Mass Spectrometry for Biomarker Discovery

Lucie Vaníčková (1,2), Kristyna Pavelicová (2), Kristyna Zemanková (2), Antonio Pompeiano (1,3), Ludmila Mravcová (4), Kamila Bendíčková (3), Marcela Hortová Kohoutková (3), Jan Frič (3)
(1) Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic (2) Mendel University in Brno, Czech Republic (3) International Clinical Research Centre of St. Anne

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Antonio Pompeiano (Presenter) Central European Institute of Technology

Relevant Financial Disclosures (within past 24 months) Grant/Research Support Ministry of Health of the Czech Republic, NV18-06-00529 Salary entral European Institute of Technology, Brno University of Technology Conflict mitigation NOT REQUIRED.

Introduction: Sepsis remains the leading cause of mortality in the intensive care units. Peripheral blood monocytes have central role in sepsis and are source of numerous effector molecules as well as diagnostic or prognostic markers. Volatile substances (VOCs) from serum samples might be used as reliable markers for detection of important metabolism changes related to innate cells exuberant activation. Therefore development of analytical methods for monocyte VOCs detection and identification is crucial for further characterization of monocyte metabolism alterations.
Objectives: The primary objective of this study was to develop and optimize fast and reliable analytical method for monocyte volatiles detection and quantification.

Methods: Monocytes were obtained from healthy individuals blood samples. Subsequently monocytes were treated by LPS or by ZYM in order to simulate bacterial or yeast infection, respectively. Cells were separated form their medium and bought fractions were analyzed by SPME-GC×GC-TOF MS method for the identification and quantification of monocyte VOCs profiles. For the SPME technique optimization, different fiber coatings, incubation conditions and adsorption times were tested. Brombutane was used for internal quantification, while 2-ethyl hexane-1-ol, nonanal and octanal were used as standards for external quantification. The GC×GC-TOF MS method has been developed and optimized for the monocyte VOCs identification. Multivariance statistical analyses were used for the data evaluation.

Results: Among the tested SPME conditions, the DVB/PDMS fiber, 30 min of incubation and 30 min of adsorption were find to be optimal. Different chemical profiles of monocyte VOCs were revealed depending on the treatment. In total, 189 volatiles were identified by the SPME-GC×GC-TOF MS method. The main differences were identified between the VOC profiles of monocytes treated with ZYM and LPS when compared to those produced by untreated monocytes. Some of the compounds, which relative abundance varied in relation to the treatment were identified as 2-ethyl hexane-1-ol, octanal, nonanal, and decanal. The identification and assessment of the peak areas by selective mass traces revealed higher sensitivity levels (0.1-10 ng) for most of the detected compounds. Linear ranges (R2>0.99) were determined with at least five calibration levels in concentration ranges of 0.0001 - 33.3333 mM. The reproducibility was lower than 10 % RSD. Triplicate analyses of the same sample (technical replicates) provided typical coefficients of variations (CVS) of <20% for the majority of metabolite peaks, which were detected in 50% of all samples. Statistical analyses based on MFA and heat map of detected VOCs highlighted possible sepsis biomarkers.

Conclusion: This work represents a first step for development of robust analytical method aiming at the fast and reliable characterization of sepsis VOC markers.