Searching for Potential Markers of Glomerulopathy in Urine by HS-SPME-GC×GC TOFMS

Abstract

Volatile organic compounds (VOCs) exiting in urine are potential biomarkers of chronic kidney diseases. Headspace solid phase microextraction (HS-SPME) was applied for extraction VOCs over the urine samples. Volatile metabolites were separated and identified by means of two-dimensional gas chromatography and time of flight mass spectrometry (GC × GC TOF MS). Patients with glomerular diseases (n = 27) and healthy controls (n = 20) were recruited in the study. Different VOCs profiles were obtained from patients and control. Developed methodology offers the opportunity to examine the metabolic profile associated with glomerulopathy. Four compounds found in elevated amounts in the patients group, i.e., methyl hexadecanoate; 9-hexadecen-1-ol; 6,10-dimethyl-5,9-undecadien-2-one and 2-pentanone were proposed as markers of glomerular diseases.

Keywords: comprehensive two-dimensional gas chromatography; kidney diseases; urine analysis; volatile organic compounds.

Figure 1

Optimization of extraction parameters, including comparison of PDMS/DVB, Carboxen/PDMS and PDMS coatings for extraction of VOC (part (A,A1)), different extraction temperatures (part (B,B1)) and extraction time (part (C,C1)). The boxplots in the upper were drawn according with total peak area of extracted VOCs (subfigures A–C), while the bottom box plots represent the number of extracted VOCs (subfigures A1–C1). Optimization of extraction parameters, including comparison of Polydimethylsiloxane/Divinylbenzene (PDMS/DVB), Carboxen/PDMS (CAR/PDMS), Polydimethylsiloxane (PDMS) for extraction of VOC (part A), different extraction temperatures (part B) and extraction time (part C). The boxplots in the upper were drawn according with total peak area of extracted VOCs (subfigures A–C), while the bottom box plots represent the number of extracted VOCs (subfigures A1–C1).

Figure 2

Network analyses presenting separation between the two investigated groups based on the VOCs specific for patients with glomerular diseases (brown diamonds), VOCs common between groups (yellow diamonds) and VOCs characteristic for healthy group (green diamonds), where H 1 to 20 represents the number of healthy control and P 1 to 27 the number of patients.

Figure 3

Heat map combined with dendrogram presenting hierarchical clusters segregation according with patient and control groups, where H 1to 20 represents the number of healthy control and P 1 to 27 the number of patients.

Figure 4

Principal component analyses (PCA) used to display variation between statistically significant determined VOCs, where: 1. cyclohexanone; 2. 3-ethylcyclopentanone; 3. 3-hexanone; 4. 3-heptanone; 5. methyl hexadecanoate; 6. 9-hexadecen-1-ol; 7. 3-methyl-2-pentanone; 8. 6,10-dimethyl-5,9-undecadien-2-one; 9. 2-pentanone; 10. acetophenone; 11. 2-methoxy-4-vinylphenol; 12. 1-decanol; 13. N-acetylpyrrole; 14. 6-methylhept-5-en-2-one; 15. dimethyl sulfone; 15. dimethyl sulfone; 16. 1-tetradecanol, 17. 4-heptanone; 18. benzaldehyde; 19. 2-nonanone; 20. 5-methyl-3-hexanone; 21. dimethyl trisulfide; 22. 2-aminobenzaldehyde; 23. 3-methylcyclopentanone; 24. hexanal; 25. 1-octanol; 26. benzeneacetaldehyde; 27. 2,5-dimethylpyrazine; 28. nonanal; 29. 9-octadecen-1-ol; 30. indole; 31. theaspirane; 32. benzonitrile; 33. 2-heptanone; 34. 4-methylphenol; 35. phenol; 36. decanal; 37. 1-methyl-4-(1-methylethenyl)-benzene; 38. N-phenylformamide, 39. ethyl acetate; 40. octanal.