ZEBRAFISH LIPIDOMICS VIA LC-MS AND CHEMOMETRICS: DIET AND EXERCISE-ASSOCIATED LIPID ALTERATIONS

This study presents an evaluation of the effects of diet and exercise on the lipidome of zebrafish (Danio rerio) biological models. To this end, the animals were subjected to different dietary conditions and daily exercise routines and were divided into four groups (normal-fat diet - NFD, normal-fat diet submitted to exercise - NFD-E, high-fat diet- HFD, and high-fat diet submitted to exercise - HFD-E). Following the experimental period, the animals were euthanized and subjected to lipidomic analysis using liquid chromatography coupled with mass spectrometry (LC-MS). For total lipid extraction, the whole zebrafish body was homogenized in methanol (100 mg.mL-1) to obtain a homogenate, which was then processed using the Bligh and Dyer extraction protocol, involving methanol, water, and chloroform. In this protocol, lipids are extracted into the chloroform phase, which is subsequently collected, dried, and reconstituted in methanol for LC-MS analysis. LC-MS analyses were performed using an LC-20AD liquid chromatography system (Shimadzu, Kyoto, Japan) coupled to a micrOTOF-Q III mass spectrometer (Bruker, Bremen, Germany). Chromatographic separation was achieved on a C18 column (2.1 × 50 mm, 2.0 µm) maintained at 60 °C. A 5 µL aliquot of each sample was injected, and separation was carried out under a gradient elution mode at a flow rate of 0.300 mL min⁻¹. Mass spectrometric analysis was conducted in both positive and negative ionization modes using an electrospray ion source. Sample injection order was randomized within analytical batches to minimize bias, and blank injections were included after every three sample analyses to monitor background signals and potential carryover. Spectral data were processed using DataAnalysis software version 5.0 (Bruker Daltoniks, Bremen, Germany).