Identifying changes in ethanol metabolism and endogenous metabolic profiles following the administration of alcohol as a liquid diet to mice

Importance of the Topic

The use of a liquid diet to administer ethanol offers a minimally invasive model to study how chronic alcohol intake alters systemic metabolism. By tracking changes in endogenous metabolites and specific ethanol markers, researchers can gain insight into metabolic stress responses, identify potential biomarkers of exposure, and advance our understanding of alcohol-related pathologies.

Study Objectives and Overview

This study evaluated a four-week ethanol-enriched liquid diet in mice as an alternative to intragastric cannulas. A systems-based metabolomics approach was applied to urine, plasma, and liver tissue to:

• Identify ethanol metabolites and their kinetics.
• Characterize alterations in endogenous lipid and small-molecule profiles.
• Compare findings with prior intragastric feeding models.

Methodology

Mice received a defined liquid diet with escalating alcohol content: 10% of caloric intake in week 1, 20% in week 2, and 35% in weeks 3–4 (equivalent to ~6.5% v/v ethanol). Biological samples were collected weekly:

• Urine: diluted and injected directly.
• Plasma: protein-precipitated with methanol then centrifuged.
• Liver: dual extraction into methanol/acetonitrile/water for aqueous metabolites, followed by chloroform/methanol for lipids.

Data processing included untargeted profiling, spectral binning, and principal components analysis to detect ions of significance.

Used Instrumentation

• Nexera UHPLC system with a Phenomenex Kinetex C18 column.
• LCMS-IT-TOF mass spectrometer operated in positive/negative switching mode (m/z 70–1250).
• Profiling Solution software for data alignment and statistical analysis.
• Database searches against LipidMaps, Metlin, HMDB, and KEGG for metabolite identification.

Main Results and Discussion

Key findings at week 4 of ethanol feeding included:

• Detection of urinary ethanol markers ethyl sulfate and ethyl glucuronide confirming recent exposure.
• Emergence of ethylated lipids in liver tissue—ethyl arachidonate, ethyl linoleate, and ethyl DHA—absent in controls and early time points.
• Elevated phosphatidylcholine signals (m/z 496.34 and 524.37) in liver and urine of treated mice.
• Reduction of retinol and retinol palmitate in liver, indicating perturbation of vitamin A metabolism.
• PCA differentiated sample types but showed only subtle separation between control and ethanol groups, suggesting modest metabolic shifts under this feeding regimen.

Benefits and Practical Applications

• Non-invasive liquid diet model enables prolonged high-level ethanol exposure without surgical intervention.
• Identification of lipid and small-molecule biomarkers may inform diagnostic assays for alcohol consumption and liver stress.
• Systems-based profiling supports integration of metabolomics into toxicology and nutritional studies.

Future Trends and Potential Applications

Advancements may include targeted quantification of identified biomarkers, extension to human dietary studies, integration with transcriptomics or proteomics, and development of real-time monitoring platforms for ethanol-induced metabolic changes.

Conclusion

This work demonstrates that a four-week ethanol liquid diet induces specific metabolic alterations detectable in urine, plasma, and liver. The approach confirms established ethanol markers and reveals ethylated lipid species as early indicators of chronic alcohol exposure. The liquid diet model offers a practical alternative to surgical feeding methods for metabolic investigations.

References

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