Elucidation of metabolic changes in HFD-ApoE–/– model by SP6 peptide: A flow injection analysis magnetic resonance mass spectrometry (FIA-MRMS) study

Significance of the Topic

Cardiovascular diseases remain a leading cause of morbidity and mortality, and atherosclerosis underlies many of these conditions. Natural peptides offer a complementary strategy to conventional pharmacology, with potential advantages in safety and specificity. Metabolomics, particularly when combined with high-resolution mass spectrometry, enables comprehensive profiling of small molecules and lipids, shedding light on molecular pathways modulated by therapeutic interventions.

Objectives and Overview of the Study

This work aimed to assess the anti-atherosclerotic potential of a novel decameric peptide (SP6) derived from Spirulina platensis in a high-fat diet ApoE knockout mouse model. Using an untargeted metabolomics and lipidomics approach based on flow injection analysis magnetic resonance mass spectrometry (FIA-MRMS), the study sought to identify metabolic alterations induced by SP6 treatment and to highlight key biomarkers and pathways involved in disease progression and prevention.

Methodology and Instrumentation

Animal Model and Treatment

• ApoE–/– mice were fed normal chow for ten weeks, then switched to a high-fat Western diet for one week
• Subsequently, mice received daily oral gavage of either saline (control, n = 4) or SP6 peptide at 5 mg/kg (treated, n = 5) for four weeks
• At study end, blood was collected via cardiac puncture and plasma isolated

Sample Preparation

• Polar metabolites and lipids were extracted using the Matyash protocol, enabling phase separation of aqueous and organic fractions

FIA-MRMS Analysis and Data Processing

• Direct-infusion nanospray ionization was performed on a TriVersa NanoMate autosampler with NanoESI chips
• Mass spectra were acquired on a 7 T solariX XR MRMS in broadband mode over m/z ranges 90–800 and 150–1500 for polar and lipid extracts, respectively
• Typical acquisition comprised 32 scans at 2 million data points per scan, achieving mass accuracy below 0.3 ppm
• Data were processed in MetaboScape using smartFormula, isotopic fine structure, and database matching against HMDB and LIPIDMAPS with 0.2–1 ppm tolerance

Main Results and Discussion

Multivariate Analysis

• PLS-DA of plasma polar and lipid extracts demonstrated clear separation between SP6-treated and control groups
• Variable importance in projection (VIP) scores identified the top 15 discriminant metabolites in each extract

Key Metabolic Modulations

• Tricarboxylic acid cycle intermediates (citric acid, malic acid) and alpha-hydroxy acids (hydroxybutyric acid) showed increased levels following SP6 treatment, suggesting enhanced mitochondrial function and energy metabolism
• Arylsulfates (p-cresol sulfate, indoxyl sulfate) were downregulated, indicating a shift away from pro-atherogenic gut-derived toxins
• Lipidomics revealed normalization of lysophosphatidylcholine species (LPC 14:0 to LPC 22:6), sphingomyelins (SM 34:1, SM 36:1, SM 42:2), and ether phospholipids, reflecting improved membrane stability and reduced inflammatory signaling
• Acylcarnitines (CAR 16:0, CAR 18:1) were modulated, pointing to corrected fatty acid oxidation pathways

These changes collectively indicate that SP6 peptide mitigates key molecular events in atherogenesis, including inflammation, lipid dysregulation, and mitochondrial impairment.

Benefits and Practical Applications

• FIA-MRMS aXelerate workflow offers high throughput analysis (<2 min per sample), excellent repeatability (CV < 12 %), and ultrahigh mass accuracy for reliable metabolite annotation
• Untargeted profiling enables comprehensive assessment of peptide effects on diverse biochemical classes without chromatographic separation
• The rapid screening capability supports large-cohort studies and early-stage evaluation of bioactive compounds in drug discovery pipelines

Future Trends and Potential Applications

• Integration of targeted MS/MS validation and isotopic labeling to confirm pathway fluxes modulated by SP6 and related peptides
• Combination with proteomics and transcriptomics for multi-omics characterization of cardiovascular interventions
• Translation to clinical trials to evaluate SP6 safety, pharmacokinetics, and efficacy in human subjects
• Development of MRMS-based diagnostic metabolite panels for early detection and monitoring of atherosclerotic disease

Conclusion

This study demonstrates that the Spirulina-derived SP6 peptide exerts significant anti-atherosclerotic effects in a high-fat diet ApoE knockout mouse model. Leveraging a rapid FIA-MRMS metabolomics approach, the work revealed modulation of critical metabolic and lipid pathways associated with disease progression. The established workflow and observed biomarker changes pave the way for large-scale studies and the eventual clinical translation of SP6 as a preventive therapy against atherosclerosis.

References

1. Carrizzo E et al 2019 A gastro-intestinal digest-derived decameric peptide from Spirulina platensis reduces hypertension and atherosclerotic markers in ApoE–/– mice Hypertension AHA
2. Matyash V et al 2008 Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics Journal of Lipid Research