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Sample treatment optimization for fish stool metabolomics.

Takeshi Hano1, Mana Ito1, Katsutoshi Ito1

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PubMed
Summary

Optimizing fecal metabolite analysis in fish is crucial for understanding gut health. This study developed a novel method using gas chromatography-mass spectrometry (GC-MS) with NaCl rinsing for improved fish metabolomics.

Keywords:
FecesGas chromatography-mass spectrometryMetabolomicsMethod optimization

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Area of Science:

  • Marine biology
  • Analytical chemistry
  • Fish health

Background:

  • Gut microbiota are vital for organism health, and fecal metabolites reflect these changes.
  • Fecal metabolomics in fish is challenging due to metabolite instability in aquatic environments.
  • Understanding fish gut microbiota dynamics is essential for aquaculture and environmental monitoring.

Purpose of the Study:

  • To develop and optimize a novel sample preparation method for fish fecal metabolomics using gas chromatography-mass spectrometry (GC-MS).
  • To determine the optimal rinsing solvent and frequency for preserving fecal metabolites in red sea bream (Pagrus major).
  • To enhance the reliability of fecal metabolite profiling for assessing fish health and environmental impacts.

Main Methods:

  • Developed and optimized a gas chromatography-mass spectrometry (GC-MS) method for fish fecal sample preparation.
  • Investigated the efficacy of different rinsing solvents (NaCl, artificial seawater) and rinsing frequencies (0, 1, 3, 5 times).
  • Analyzed metabolite recovery, focusing on amino acids, organic acids, fatty acids, sugars, and sterols.

Main Results:

  • Sterilized 2.5% NaCl solution was superior to 3.0% artificial seawater (ASW) for rinsing, yielding higher recovery of 57 out of 86 identified metabolites.
  • Three or more NaCl rinses were optimal for robust and stable detection of encapsulated metabolites.
  • Rinsing was found to be unnecessary for analyzing sugars, amino acids, and sterols, indicating solvent and frequency optimization is metabolite-dependent.

Conclusions:

  • The optimized GC-MS method with NaCl rinsing improves fecal metabolite analysis in fish.
  • Appropriate selection of rinsing solvent and frequency is critical for accurate fish metabolomics.
  • This methodology supports the assessment of fish health, gut microbiota dynamics, and environmental influences in aquaculture.