Czech Society for Mass Spectrometry

(Czech Mass Spectrometry Conference 2024 - WeP-01)
Targeted profiling of serum oxysterols by UHPLC–MS/MS

Michal Kaleta ^1,2 *, Jana Oklešťková ¹, Miroslav Kvasnica ¹, Ivan Petřík ¹, Ondřej Novák ¹

1. Laboratory of Growth Regulators, Palacký University and IEB CAS, Olomouc
2. Department of Neurology, Faculty of Medicine and Dentistry, Palacký University, Olomouc

Abstract

Oxysterols are a significant group of oxygenated cholesterol derivatives believed to naturally regulate cellular signaling, exhibit immunomodulatory properties, and play physiological roles in processes such as steroid hormone biosynthesis, fatty acid metabolism, and cholesterol homeostasis. They are also likely involved in the pathogenesis of various cardiovascular (e.g., atherosclerosis), neurodegenerative (e.g., Alzheimer's disease), oncological (e.g., breast, lung, stomach cancers), and other diseases. Oxysterol production in the human body primarily occurs through intracellular enzymatic oxidation of cholesterol but can also result from non-enzymatic auto-oxidation. However, comprehensive studies on oxysterol metabolism require reliable analytical approaches. This work presents a high-performance method based on time-efficient sample preparation and sensitive detection using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC–MS/MS), enabling the metabolic profiling of several target oxysterols in human serum. The key sample preparation step involves the precipitation of serum proteins, followed by filtration to remove them. High-efficiency chromatographic separation on a reverse phase allows for simultaneous profiling of ten metabolically related oxysterol compounds in a single experiment, even from serum samples as small as a few microliters. The developed and validated method holds potential for use in targeted metabolomic studies aimed at evaluating changes in oxysterol metabolic pathways under various physiological or pathological conditions. The insights gained could contribute to the discovery of biomarkers for disease prevention, diagnosis, or monitoring progression, as well as improving therapeutic strategies.

* Corresponding author: michal.kaleta@upol.cz

References

1. Borah K. et al.: Redox Biol. 36, 101595 (2020).
2. Chen L. et al.: Chromatographia 82, 553–564 (2019).
3. de Freitas F. A. et al: Cells 11(8), 1251 (2022).

Acknowledgement:

This work was financially supported by the Czech Science Foundation (project GA23-05389S).