Fatty Acid Profiles Based on Their Locality in Urinary Extracellular Vesicles
DOI:
https://doi.org/10.11113/jomalisc.v4.91Keywords:
extracellular vesicles, exosomes, fatty acids, fatty acid, methyl estersAbstract
The use of fatty acids in extracellular vesicles (EVs) has been largely understudied, despite their selective characteristics which hold potential as disease biomarkers. EVs are nanosized vesicles secreted by human cells and play key roles in intercellular communication. Although lipid profiling of EVs has shown promise in differentiating between healthy and diseased individuals, fatty acids specifically remain underexplored. Due to their structural simplicity and high selectivity, this study focuses on optimizing the isolation of urinary EVs and analyzing their fatty acid content using gas chromatography-mass spectrometry (GC-MS). The goal is to identify fatty acid profiles in EVs based on the locality of urine samples (control and luminal space) and evaluate their potential as practical indicators for disease detection. Optimal EV isolation was achieved using a 2-cycle ultracentrifugation process, yielding vesicles with an average size of 131.87 nm and significantly higher protein concentration (2.970 mg/mL, p < 0.05). A total of 14 fatty acid methyl esters (FAMEs) were detected, with 12 shared across all localities and four (C13, C15, C18, and C30) found to vary by sample origin. Tetracosanoic acid methyl ester was the most abundant (84.05%), while tetradecanoic acid methyl ester was the least (11.79%). These results support urinary EV fatty acid profiling as a promising non-invasive biomarker strategy.
References
Aitekenov, S., Gaipov, A., & Bukasov, R. (2021). Review: Detection and quantification of proteins in human urine. Talanta, 223(1), 121718. https://doi.org/10.1016/j.talanta.2020.121718
Burgess, D. J., Duffy, E., Etzler, F., & Hickey, A. J. (2004). Particle size analysis: AAPS workshop report, cosponsored by the Food and Drug Administration and the United States Pharmacopeia. The AAPS Journal, 6(3), 20. https://doi.org/10.1208/aapsj060320
Chiu, H.-H., & Kuo, C.-H. (2020). Gas chromatography-mass spectrometry-based analytical strategies for fatty acid analysis in biological samples. Journal of Food and Drug Analysis, 28(1), 60–73. https://doi.org/10.1016/j.jfda.2019.10.003
De Girolamo, A., Lippolis, V., & Pascale, M. (2022). Overview of recent liquid chromatography mass spectrometry-based methods for natural toxins detection in food products. Toxins, 14(5), 50328. https://doi.org/10.3390/toxins14050328
Erdbrügger, U., Blijdorp, C. J., Bijnsdorp, I. V., Borràs, F. E., Burger, D., Bussolati, B., Byrd, J. B., et al. (2021). Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles. Journal of Extracellular Vesicles, 10(7), e12093. https://doi.org/10.1002/jev2.12093
Fitts, C. A., Ji, N., Li, Y., & Tan, C. (2019). Exploiting exosomes in cancer liquid biopsies and drug delivery. Advanced Healthcare Materials, 8(6), e1801268. https://doi.org/10.1002/adhm.201801268
Hadizadeh, N., Bagheri, D., Shamsara, M., Hamblin, M. R., Farmany, A., Xu, M., Liang, Z., Razi, F., & Hashemi, E. (2022). Extracellular vesicles biogenesis, isolation, manipulation and genetic engineering for potential in vitro and in vivo therapeutics: An overview. Frontiers in Bioengineering and Biotechnology, 10, 1019821. https://doi.org/10.3389/fbioe.2022.1019821
Han, P., Bartold, P. M., & Ivanovski, S. (2022). The emerging role of small extracellular vesicles in saliva and gingival crevicular fluid as diagnostics for periodontitis. Journal of Periodontal Research, 57(1), 219–231. https://doi.org/10.1111/jre.12950
Haney, M. J., Klyachko, N. L., Zhao, Y., Gupta, R., Plotnikova, E. G., He, Z., Patel, T., Piroyan, A., Sokolsky, M., Kabanov, A. V., & Batrakova, E. V. (2015). Exosomes as drug delivery vehicles for Parkinson's disease therapy. Journal of Controlled Release, 207, 18–30. https://doi.org/10.1016/j.jconrel.2015.03.033
Hwang, H. S., Kim, H., Han, G., Lee, J. W., Kim, K., Kwon, I. C., Yang, Y., & Kim, S. H. (2021). Extracellular vesicles as potential therapeutics for inflammatory diseases. International Journal of Molecular Sciences, 22(11), 5487. https://doi.org/10.3390/ijms22115487
Jalaludin, I., Nguyen, H. Q., Jang, K. S., Lee, J., Lubman, D. M., & Kim, J. (2023). Matrix-assisted laser desorption/ionization-Fourier-transform ion cyclotron resonance-mass spectrometry analysis of exosomal lipids from human serum. Rapid Communications in Mass Spectrometry, 37(2), e9427. https://doi.org/10.1002/rcm.9427
Kim, T. K. (2015). T test as a parametric statistic. Korean Journal of Anesthesiology, 68(6), 540–546. https://doi.org/10.4097/kjae.2015.68.6.540
Lu, M., DiBernardo, E., Parks, E., Fox, H., Zheng, S.-Y., & Wayne, E. (2021). The role of extracellular vesicles in the pathogenesis and treatment of autoimmune disorders. Frontiers in Immunology, 12, 566299. https://doi.org/10.3389/fimmu.2021.566299
Mező, E., Hartmann-Balogh, F., Madarászné Horváth, I., Bufa, A., Marosvölgyi, T., Kocsis, B., & Makszin, L. (2022). Effect of culture conditions on fatty acid profiles of bacteria and lipopolysaccharides of the genus Pseudomonas—GC-MS analysis on ionic liquid-based column. Molecules, 27(20), 6930. https://doi.org/10.3390/molecules27206930
Ostermann, A. I., Müller, M., Willenberg, I., & Schebb, N. H. (2014). Determining the fatty acid composition in plasma and tissues as fatty acid methyl esters using gas chromatography – A comparison of different derivatization and extraction procedures. Prostaglandins, Leukotrienes and Essential Fatty Acids, 91(6), 235–241. https://doi.org/10.1016/j.plefa.2014.10.002
Park, S., Jalaludin, I., Hwang, H., Ko, M., Adelipour, M., Hwan, M., Cho, N., Kim, K. K., Lubman, D. M., & Kim, J. (2023). Size-exclusion chromatography for the characterization of urinary extracellular vesicles. Journal of Chromatography B, 1228, 123828. https://doi.org/10.1016/j.jchromb.2023.123828
Puhm, F., Boilard, E., & Machlus, K. R. (2021). Platelet extracellular vesicles: Beyond the blood. Arteriosclerosis, Thrombosis, and Vascular Biology, 41(1), 87–96. https://doi.org/10.1161/atvbaha.120.314644
Saini, R. K., Prasad, P., Shang, X., & Keum, Y. S. (2021). Advances in lipid extraction methods – A review. International Journal of Molecular Sciences, 22(24), 13643. https://doi.org/10.3390/ijms222413643
Salimon, J., Omar, T., & Salih, N. (2014). Comparison of two derivatization methods for the analysis of fatty acids and trans fatty acids in bakery products using gas chromatography. The Scientific World Journal, 2014, A906407. https://doi.org/10.1155/2014/906407
Shehadul Islam, M., Aryasomayajula, A., & Selvaganapathy, P. R. (2017). A review on macroscale and microscale cell lysis methods. Micromachines, 8(3), 83. https://doi.org/10.3390/mi8030083
Street, J. M., Koritzinsky, E. H., Glispie, D. M., & Yuen, P. S. T. (2017). Urine exosome isolation and characterization. In J.-C. Gautier (Ed.), Drug Safety Evaluation: Methods and Protocols (pp. 413–423). Springer. https://doi.org/10.1007/978-1-4939-7172-5_23
Tan, B., Zhang, Y., Zhang, T., He, J., Luo, X., Bian, X., Wu, J., Zou, C., Wang, Y., & Fu, L. (2020). Identifying potential serum biomarkers of breast cancer through targeted free fatty acid profiles screening based on a GC-MS platform. Biomedical Chromatography, 34(10), e4922. https://doi.org/10.1002/bmc.4922
Thomas, C. E., Sexton, W., Benson, K., Sutphen, R., & Koomen, J. (2010). Urine collection and processing for protein biomarker discovery and quantification. Cancer Epidemiology, Biomarkers & Prevention, 19(4), 953–959. https://doi.org/10.1158/1055-9965.EPI-10-0069
Walsh, M. C., McLoughlin, G. A., Roche, H. M., Ferguson, J. F., Drevon, C. A., Saris, W. H., Lovegrove, J. A., Risérus, U., López-Miranda, J., Defoort, C., Kieć-Wilk, B., Brennan, L., & Gibney, M. J. (2014). Impact of geographical region on urinary metabolomic and plasma fatty acid profiles in subjects with the metabolic syndrome across Europe: The LIPGENE study. British Journal of Nutrition, 111(3), 424–431. https://doi.org/10.1017/S0007114513002602
Welsh, J. A., van der Pol, E., Bettin, B. A., Carter, D. R. F., Hendrix, A., Lenassi, M., Langlois, M. A., Llorente, A., van de Nes, A. S., Nieuwland, R., Tang, V., Wang, L., Witwer, K. W., & Jones, J. C. (2020). Towards defining reference materials for measuring extracellular vesicle refractive index, epitope abundance, size and concentration. Journal of Extracellular Vesicles, 9(1), 1816641. https://doi.org/10.1080/20013078.2020.1816641
Witwer, K. W., Buzas, E. I., Bemis, L. T., Bora, A., Lasser, C., Lotvall, J., Nolte-‘t Hoen, E. N., Piper, M. G., Sivaraman, S., Skog, J., Thery, C., Wauben, M. H., & Hochberg, F. (2013). Standardization of sample collection, isolation and analysis methods in extracellular vesicle research. Journal of Extracellular Vesicles, 2, 20360. https://doi.org/10.3402/jev.v2i0.20360
Woith, E., Fuhrmann, G., & Melzig, M. F. (2019). Extracellular vesicles – Connecting kingdoms. International Journal of Molecular Sciences, 20(22), 5695. https://doi.org/10.3390/ijms20225695
Wu, H. H. L., Possell, M., Nguyen, L. T., Peng, W., Pollock, C. A., & Saad, S. (2024). Evaluation of urinary volatile organic compounds as a novel metabolomic biomarker to assess chronic kidney disease progression. BMC Nephrology, 25(1), 352. https://doi.org/10.1186/s12882-024-03819-0
