Federico Fantia, Francesco Della Vallea, Fabiola Eugelioa, Giulia Caionia, Carmine Merolaa, Michele Amorenaa, Manuel Sergia, Dario Compagnonea

aDepartment of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100 Teramo, Italy
bDepartment of Chemistry, Sapienza University of Rome, 00185 Roma, Italy

The investigation on oxysterols profile in a suitable in vivo system could provide useful information on the amount, roles and biological functions of these molecules that are enzymatically generated by cytochrome P450 (CYP450) family, or via autooxidation, or even via both pathways in synergy. Oxysterols, which are oxidized derivatives of cholesterol, are reliable indicators of oxidative stress and lipid oxidation. Zebrafish (Danio rerio), due to their genetic similarity to humans and transparent embryos, serve as an excellent model for such studies. Zebrafish is used in toxicology to estimate the effects of xenobiotics and their teratogenic consequences; this animal model presents several advantageous features as high fecundity, rapid embryonic development (24 h) and external fertilization [1]. The knowledge of the oxysterols profile in zebrafish, during early embryonic stages, provides important information on the role and biological function of these molecules [2]. This research focuses on the determination of oxidative stress in zebrafish embryos by assessing oxysterols as biomarkers at different developmental stages: 3, 24, 48, 72, and 96 hours post fertilization (hpf). The study employs liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the precise quantification and analysis of oxysterols in zebrafish embryos. The methodology involves the rapid extraction of lipids using a modified solid-phase extraction (SPE) technique based on the method described by Fanti et al. [3] in their study on oxysterol profiling in zebrafish embryos exploiting the significant profile mutation during development stages using multivariate analysis. Following extraction, separation and detection are conducted using LC-MS/MS, which offers high sensitivity and specificity. The method was validated according to European Medicines Agency (EMA) guidelines, ensuring robustness and reliability. Results demonstrate that specific oxysterols are significantly elevated in embryos, proposing their use as biomarkers.

Keywords: LC-MS/MS, Oxysterols, Zebrafish, Multivariate Analysisa

References

1. Teame T., Zhang Z. et al., Animal Frontiers, 9 (2019), pag. 68–77. DOI: 10.1093/af/vfz020 

2. Jamadagni P., Patten S. A., et al., NeuroToxicology, 75 (2019), pag. 14-23. DOI: 10.1016/j.neuro.2019.08.007

3. Fanti F., Merola C., et al., Talanta, 220 (2020), pag. 121393 DOI: 10.1016/j.talanta.2020.121393