Discrimination of urinary exosomes from microvesicles by lipidomics using thin layer liquid chromatography (TLC) coupled with MALDI-TOF mass spectrometry

Urinary extracellular vesicles (EVs), including microvesicles and exosomes, play several important roles in cell biology and serve as potential biomarkers in various kidney diseases. Although they have differential biophysical properties, specific biomarkers are required to discriminate these EVs during isolation/purification. The present study aimed to define differential lipidome profiles of urinary microvesicles vs. exosomes. Urine samples collected from eight healthy individuals were pooled and underwent lipid extraction using 2:1(v/v) chloroform/methanol. The recovered lipids were resolved by thin layer liquid chromatography (TLC) and analyzed by MALDI-TOF MS. From three and five TLC bands observed in microvesicles and exosomes, respectively, several fatty acids, glycerolipids and phospholipids were identified from both EVs without clear differential patterns. However, their sphingolipid profiles were unique. Ceramide phosphates (CerP), hexosyl sphingoid bases (HexSph), lactosyl ceramides (LacCer), mannosyl di-PI-ceramides (M(IP)2 C), sulfatides hexosyl ceramide (SHexCer) and sulfatides hexoxyl sphingoid bases (SHexSph) were detectable only in urinary exosomes, whereas phosphatidylinositol ceramides (PI-Cer) were detectable only in urinary microvesicles. The presence of CerP only in urinary exosomes was successfully validated by dot blot analysis. Our extensive lipidome analyses of urinary microvesicles vs. exosomes provide potential lipidome markers to discriminate exosomes from microvesicles and may lead to better understanding of EVs biogenesis.