Fig 1.
Schematic explanation of PBMC processing by the UUM method (Ultrasound and Ultrafiltration Method). Using these methods, it is possible to obtain the metabolomic profile in 6 h starting from whole blood. The PBMC samples were isolated from the peripheral blood of healthy human individuals. Samples were split into an aliquot (12.5 million cells) for characterization. Finally, PBMC were extracted and the 1H-NMR metabolic profiles were determined.
Fig 2.
Comparison of the spectra obtained from the three different PBMC processing methods evaluated for metabolomic profiling.
a) Ultrasound and Ultrafiltration Method, b) Ultrasound Method, c) Folch Method. The differences in specter regions between methods are seen in red boxes.
Fig 3.
The assigned UUM 1H-NMR spectrum of PBMCs.
Metabolite assignments are indicated by the numbers 1. Valine, 2. Leucine, 3. Isoleucine, 4. Ethanol, 5. Threonine, 6. 3-Hydroxyisovalerate, 7. Lactate, 8. Alanine, 9. Acetate, 10. Glutamate, 11. Methionine, 12. Pyruvate, 13. Succinate, 14. Citrate, 15. Aspartate, 16. Sarcosine, 17. Creatine, 18. Creatinine, 19. Phenylalanine, 20. Choline, 21. O-Phosphocoline, 22. Carnitine, 23. Betaine, 24. Taurine, 25. Tyrosine, 26. Trimethylamine N-oxide, 27. Methanol, 28. Glycine, 29. Glycerol, 30. Serine, 31. Inosine, 32. GTP, 33. Xanthurenate, 34. Oxypurinol, 35. Xanthine, 36. AMP, 37. Formate, 38. 2-hydroxybutyrate, 39. Lysine, 40. Glutamine, 41. Hydroxyacetone, 42. Acetoacetate, 43. Methylacetoacetate, 44. Reduced glutathione (GSH), 45. Oxidized glutathione (GSSG), 46. Malonate, 47. Trimethylamine.
Table 1.
List and detail of the metabolites assigned in the different methods evaluated.
Table 2.
Metabolites significant for the comparisons between FM vs. UM and FM vs. UUM.
Fig 4.
A comparative repeatability and reproducibility analysis (FM vs. UM vs. UUM) for metabolites Valine, Alanine, Taurine and Inosine was carried out using the Six Sigma tool from RStudio. FM: Folch Method, UM: Ultrasound Method, UUM: Ultrasound and Ultrafiltration Method, R Chart by Method: Range chart by method (Numbers 1, 2, and 3 on the x-axis: Patients evaluated; Differences in normalized concentration values on the y-axis), Metabolite by Method: Metabolite concentration of samples by method (Methods evaluated on the x-axis; Normalized concentration values on the y-axis), CV: coefficient of variation as a percent.
Fig 5.
Comparative repeatability graphical analysis (FM vs. UM vs. UUM) for metabolites Valine, Alanine, Taurine and Inosine.
a) Three overlapping methods, b) Folch method, c) Ultrasound and ultrafiltration method, d) Ultrasound method. A duplicate spectrum, acquired for the same patient and on the same day, is shown for each method.
Fig 6.
Comparative reproducibility graphical analysis (FM vs. UM vs. UUM) for metabolites Valine, Alanine, Taurine and Inosine.
a) Three overlapping methods, b) Folch method, c) Ultrasound and ultrafiltration method, d) Ultrasound method. For each method a duplicate spectrum is shown for the same patient but processed on different days.
Fig 7.
Sensitivity comparison of the FM, UM and UUM methods with the limit of detection and limit of quantification.
Calculated by the standard deviation (LOD = 3xSD and LOQ = 10xSD) of the integrals of the three selected noise regions of the spectrum (0.5 ppm, 6.5 ppm and 9.5 ppm) for all the spectra of each method. Normalized concentration values on the y-axis.
Table 3.
General comparison of the three methods tested for the metabolomics profiling of PBMCs by NMR.