Abstract
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.
Highlights
Extracellular vesicles (EVs) are the lipid-enclosed particles that can be found mainly in human body fluids, urine[1]
The data showed that thin layer liquid chromatography (TLC) band patterns obtained from urinary microvesicles and exosomes were consistent in all four independent experiments
Exosomes are originated from an invagination of endosomes that subsequently fuse with multivesicular body (MVB) and expel from the cells[5,9]
Summary
Extracellular vesicles (EVs) are the lipid-enclosed particles that can be found mainly in human body fluids, urine[1]. There are two major types of EVs, including exosomes and microvesicles, that play several important roles in cell biology and serve as potential biomarkers in various kidney diseases[3] These two types of EVs have some differential biophysical and biochemical properties, e.g., size, shape, density, and biomolecular components[4]. Exosomes are the nano-scale vesicles ranging from 30–120 nm with spherical or cup-like morphology, whereas microvesicles are in irregular shape and larger with a wide range of size up to approximately 1,500 nm (>10 times greater than that of exosomes)[4,5] Base on their biophysical and biochemical properties, current isolation protocols of exosomes and microvesicles (e.g., differential ultracentrifugation and OptiPrep kit) are based mainly on their sizes, densities and flotation velocities[6,7,8]. The identified distinct lipid species that could potentially be useful for discriminating them was validated by conventional immunoassay
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