Effects of Increased Fructose Consumption and Inadequate Copper Intake on the Pathogenesis of Nonalcoholic Fatty Liver Disease (NAFLD): A Feces, Plasma and Liver Metabolomics Study

Importance of the topic

Nonalcoholic fatty liver disease is rising worldwide as metabolic syndrome escalates. Excessive fructose intake and inadequate copper nutrition have been linked to insulin resistance, dyslipidemia, oxidative stress and liver inflammation, driving NAFLD progression. Understanding how dietary factors and micronutrient status modulate liver metabolism is crucial for developing diagnostic biomarkers and nutritional interventions.

Objectives and study overview

This study explored the combined impact of copper deficiency and high‐fructose feeding on metabolic profiles in rats. A total of 84 samples from six experimental groups with varied copper levels (adequate, low, supplemented) and diets (with or without high fructose) were analyzed using comprehensive metabolomics in feces, plasma and liver tissue to identify key metabolic perturbations associated with NAFLD.

Methodology and instrumentation

The workflow included methanol‐water extraction followed by two‐step derivatization: methoximation and MTBSTFA silylation. Analyses were performed on a LECO Pegasus GC‐HRT system and a Pegasus GCxGC‐HRT 4D platform equipped with an Agilent 7890 GC, dual‐oven thermal modulation, Rxi‐5ms and Rxi‐17silms columns, and a time‐of‐flight mass spectrometer operating at 25,000 resolving power and <1 ppm accuracy. Data acquisition rates reached 10 spectra/s for GC‐HRT and 200 spectra/s for GCxGC‐HRT. High‐resolution deconvolution and compound identification used ChromaTOF‐HRT software, while MetPP managed alignment and quantification.

Main results and discussion

• Thirty‐eight metabolites showed significant abundance changes across study groups, including organic acids, amino acids, diacids and fatty acids.
• High fructose combined with copper deficiency disrupted TCA cycle intermediates such as malic, fumaric and succinic acid, indicating mitochondrial stress.
• Amino acids involved in nitrogen metabolism (proline, ornithine) were elevated, whereas glutamate and aspartate decreased under stress conditions, reflecting compromised transamination.
• Fatty acid analysis revealed increased levels of palmitic, stearic and myristic acids in the high‐fructose/low‐copper group, consistent with lipid accumulation in NAFLD.
• GCxGC‐HRT improved chromatographic separation of coeluting metabolites (eg ornithine vs palmitic acid), and high‐resolution accurate mass spectra yielded confident library matches with similarity scores above 900.

Benefits and practical applications of the method

The combined GC‐HRT and GCxGC‐HRT 4D approach delivers superior peak capacity and mass spectral quality, enabling rapid, automated detection of metabolites in complex biological matrices. This methodology supports research on metabolic diseases, nutritional effects, quality control in food safety and biomarker discovery in clinical metabolomics.

Future trends and potential applications

• Integration with LC‐MS/MS and targeted assays to confirm and quantify candidate biomarkers.
• Implementation of machine learning and AI for automated peak deconvolution and pattern recognition in large data sets.
• Expansion to human cohort studies and additional biofluids such as urine and saliva for noninvasive diagnostics.
• Personalized nutrition and precision medicine approaches guided by multi‐omics profiling.

Conclusion

This metabolomics investigation demonstrates that inadequate copper intake exacerbates fructose‐induced metabolic dysregulation in NAFLD. Advanced GC‐MS platforms with high chromatographic resolution and accurate mass measurement provide powerful tools for unraveling diet‐driven metabolic alterations and identifying potential intervention targets.

References

• Y‐S Lai W‐C Chen T‐C Kuo C‐T Ho C‐H Kuo FJ Tseng K‐H Lu S‐H Lin S Panyod L‐Y Sheen J Agric Food Chem 2015 63 7873‐7884
• X Shi X Wei X Yin Y Wang M Zhang C Zhao H Zhao CJ McClain W Feng X Zhang J Proteome Res 2015 14 1174‐1182
• X Wei M Song X Yin DA Schuschke I Koo CJ McClain X Zhang J Proteome Res 2015 14 4050‐4058
• MF Abdelmalek A Suzuki C Guy A Unalp‐Arida R Colvin RJ Johnson AM Diehl Hepatology 2010 51 1961‐1971