A strategy using isotopic fine structure to reveal potential biomarkers showing the effects of traditional Chinese medicines on Alzheimer disease in rats

Importance of the Topic

Alzheimer disease (AD) is a relentless neurodegenerative disorder characterized by progressive memory loss and cognitive decline. Despite extensive research, its pathogenesis remains elusive and no curative therapy is available. Metabolomic profiling offers a powerful avenue for uncovering biochemical biomarkers that reflect disease progression and therapeutic response. Investigating traditional Chinese medicines (TCM) such as Rhodiola crenulata extract (RCE) may reveal metabolic signatures linked to their neuroprotective effects, thus supporting novel treatment strategies.

Aims and Overview of the Study

This study aimed to develop a metabolomic strategy based on accurate mass measurement and isotopic fine structure (IFS) analysis using magnetic resonance mass spectrometry (MRMS) to identify serum biomarkers altered in an AD rat model and to evaluate how RCE modulates these biomarkers. The workflow integrated high-performance liquid chromatography (HPLC) MRMS screening with fraction collection and direct-infusion (DI) MRMS to achieve decisive elemental formula assignment.

Methodology

An experimental AD model was established by bilateral hippocampal injection of amyloid β1–42 peptide in rats. Cognitive performance was assessed via the Morris water maze. Serum samples underwent:

• HPLC-MRMS profiling to detect features with mass accuracy <1 ppm.
• Fractionation of target peaks followed by DI-MRMS to obtain ultra-high resolution isotopic spectra.
• Seven-step isotopic fine structure analysis:
1. Detection of monoisotopic and A+1 to A+3 peaks.
2. Extraction of experimental IFS and relative intensities.
3. Assignment of M+1 peaks from 15N, 33S and 13C; estimation of carbon count.
4. Filtering candidates based on 13C peak intensity.
5. Assignment of A+2/A+3 peaks from multiple heavy-isotope combinations.
6. Theoretical IFS generation using isotope-pattern software.
7. Comparison of experimental vs. theoretical spectra to confirm elemental compositions.

Used Instrumentation

• SolariX 7T MRMS system (FT-ICR MS) for accurate mass and IFS measurements.
• HPLC system for chromatographic separation.
• Direct-infusion source module for fraction analysis.
• Compass Isotope Pattern software for theoretical IFS simulation.
• Morris water maze apparatus for behavioral testing.

Main Results and Discussion

Initial HPLC-MRMS screening identified serum features with sub-ppm accuracy. One unknown at m/z 524.37054 (RT 16.24 min) yielded four candidate formulas, which were reduced to C26H55NO7P+ through IFS analysis. Overall, 20 metabolites associated with AD progression were definitively identified; 17 of these returned towards control levels after daily RCE administration (2.24 g/kg). Metabolic pathway analysis indicated that RCE modulated disturbances in:

• Tryptophan metabolism
• Sphingolipid metabolism
• Glycerophospholipid metabolism

The dual-mode MRMS strategy provided high-confidence identification of biomarkers and clarified how RCE exerts neuroprotective effects.

Benefits and Practical Applications

The combined accurate mass and IFS approach enhances certainty in elemental formula assignment, overcoming ambiguities common to conventional metabolomics. This methodology accelerates discovery of disease biomarkers and enables rigorous evaluation of TCM efficacy. Laboratories focused on QA/QC, pharmaceutical research, and clinical studies can adopt this workflow to improve metabolic profiling accuracy.

Future Trends and Potential Applications

Advances in MRMS instrumentation and data analysis are expected to further increase resolution and throughput. Integration with high-throughput chromatography, bioinformatics-driven pathway mapping, and large-scale clinical cohorts will expand applications to other neurodegenerative diseases and TCM formulations. Automation of IFS assignment and incorporation of machine learning models may streamline biomarker discovery across diverse fields.

Conclusion

The presented metabolomic strategy successfully identified and characterized serum biomarkers linked to AD and demonstrated that Rhodiola crenulata extract reverses key metabolic disturbances. Accurate mass and isotopic fine structure analysis via dual-mode MRMS provides a robust platform for biomarker discovery and mechanistic studies of therapeutic interventions.

References

[1] Zhang X., Jiang X., Wang X., Zhao Y., Jia L., Chen F., Yin R., Han F. A metabolomic study based on accurate mass and isotopic fine structures by dual mode combined-FT-ICR-MS to explore the effects of Rhodiola crenulata extract on Alzheimer disease in rats. Journal of Pharmaceutical and Biomedical Analysis, 2019, 166: 347–356.