An integrated LC-MS approach to the metabolome profile in the whole-body tissue of adult zebrafish

Abstract

Zebrafish (Danio rerio) has served as a crucial experimental model in biomedical and environmental research. Compared to the more commonly used embryonic and juvenile stages, adult zebrafish possesses a complete inter-organ metabolic network, which is advantageous in the development of a chronic disease model. However, there are very few comprehensive studies on metabolomics methods for adult zebrafish. Herein, we have implemented an integrated approach that combines hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) to maximize metabolite detection and optimized key parameters in sample pretreatment, including tissue homogenization, extraction solvents, the extraction solvent addition method, and re-dissolution solvents. As such, we have established an untargeted metabolomics analysis method, utilizing ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) technology, which is suitable for whole-tissue analysis. This method has demonstrated satisfactory precision, accuracy, linearity, and sample stability in methodology validation tests. Through metabolite database retrieval using custom scripts, a total of 620 metabolites were annotated, highlighting the complementarity of RPLC and HILIC, as well as the method's extensive coverage. Its applicability was verified through a 20 days observation period experiment, screening out 110 differential variables associated with zebrafish growth. Of these, 23 are known compounds that are primarily related to glycerophospholipid metabolism, phenylalanine, tyrosine and tryptophan biosyntheses, and phenylalanine metabolism. This study has clarified the experimental conditions for metabolomics studies based on adult zebrafish, providing novel methodological approaches for related disease or environmental exposure research.