Systems based LC-MS metabolite profiling of mice treated with ethanol enriched liquid diets

Significance of the Topic

Understanding how chronic alcohol intake alters endogenous metabolite profiles is critical for elucidating mechanisms of liver injury, developing diagnostic biomarkers and optimizing therapeutic interventions. A non-invasive liquid-diet model in mice offers a practical platform to monitor systemic metabolic responses over time.

Objectives and Study Overview

This study aimed to evaluate a four-week ethanol-enriched liquid diet in mice as an alternative to intragastric cannula feeding, and to apply systems-based LC-MS metabolite profiling to urine, plasma and liver tissue extracts. Key goals included:

• Characterizing ethanol metabolites and lipid alterations at weeks 2 and 4.
• Comparing endogenous metabolic changes against control diet animals.
• Demonstrating the feasibility of combined targeted and untargeted LC-MS approaches.

Methodology and Instrumentation

Animal diets were administered with ethanol-derived calories escalating from 10% in week 1 to 35% in weeks 3–4 (~6.5% v/v alcohol). Sample processing and analysis included:

• Urine: 50 µL diluted in water and analyzed by UHPLC (Phenomenex Kinetex XB-C18, 2.1×100 mm, 1.7 µm) with a 12 min gradient.
• Plasma: 50 µL protein-precipitated with MeOH, analyzed under the same UHPLC conditions.
• Liver tissue: Aqueous extract (MeOH/ACN/H₂O 40:40:20) and organic extract (CHCl₃/MeOH 3:1) were separated over 32.5 min binary gradients to target polar and nonpolar metabolites.
• Detection: LCMS-IT-TOF (Shimadzu) operated in positive/negative switching (m/z 70–1250) with high-accuracy MSn acquisition.
• Data processing: Profiling Solution for untargeted binning and PCA; MetID Solution for targeted ethanol metabolite identification; database searches via LipidMaps, Metlin, HMDB and KEGG.

Main Results and Discussion

Targeted analysis revealed ethanol metabolites (ethyl glucuronide, ethyl sulfate) elevated in week 4 urine only. Liquid-diet mice displayed ethylated lipids (ethyl arachidonate, ethyl linoleate, ethyl DHA) in liver at week 4, aligning with intragastric models but appearing later in this less invasive protocol. Phosphatidylcholines (m/z 496.3398 & 524.3711) and 7-keto-cholesterol were significantly increased in liver and urine samples of week 4 ethanol-treated mice. Untargeted profiling identified a decrease in the osmoprotectant proline betaine and increased fatty acid conjugates (vinylacetylglycine, isobutyrylglycine) in urine, indicating disrupted amino acid and lipid metabolism.

Benefits and Practical Applications

This liquid-diet model simplifies chronic ethanol exposure without surgery, enabling longitudinal sampling of multiple biofluids. Combined targeted and untargeted LC-MS workflows facilitate early biomarker discovery and comprehensive metabolic monitoring relevant to alcohol-related liver disease research and toxicology studies.

Future Trends and Opportunities

Advances in high-resolution LC-MS, microflow chromatography and data-independent acquisition will enhance sensitivity for low-abundance metabolites. Integration with imaging MS and multi-omics platforms (proteomics, transcriptomics) can provide deeper insights into alcohol-induced pathophysiology. Standardized liquid-diet protocols could support translational biomarker validation in clinical cohorts.

Conclusion

The four-week ethanol liquid-diet mouse model combined with systems-based LC-MS profiling successfully identified time-dependent ethanol metabolites and endogenous lipid disturbances. Although metabolic changes were less pronounced than intragastric feeding, the approach offers a practical, non-invasive framework for studying alcohol-induced metabolic stress.

References

• Journal of Proteome Research (2011) 10, 705–713