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. 2019 Oct;1(10):624-638.
doi: 10.1096/fba.2019-00030. Epub 2019 Aug 24.

Lipidomic analysis of urinary exosomes from hereditary α-tryptasemia patients and healthy volunteers

Sarah C Glover  1 , Mohammad-Zaman Nouri  2 , Kubra M Tuna  3 , Lybil B Mendoza Alvarez  4 , Lisa K Ryan  1 , James F Shirley  1 , Ying Tang  1 , Nancy D Denslow  2 , Abdel A Alli  3
Affiliations

Lipidomic analysis of urinary exosomes from hereditary α-tryptasemia patients and healthy volunteers

Sarah C Glover et al. FASEB Bioadv. 2019 Oct.

Abstract

Exosomes are nano-sized vesicles that are involved in various biological processes including cell differentiation, proliferation, signaling, and intercellular communication. Urinary exosomes were isolated from a cohort of hereditary α-tryptasemia (HαT) patients and from healthy volunteers. There was a greater number of exosomes isolated from the urine in the HαT group compared to the control volunteers. Here, we investigated the differences in both lipid classes and lipid species within urinary exosomes of the two groups. Lipids were extracted from urinary exosomes and subjected to liquid chromatography mass spectrometry using a targeted approach. Various molecular species of glycerophospholipids, glycerolipids, and sterols were significantly reduced in HαT patients. Out of a possible 1127 lipids, 521 lipid species were detected, and relative quantities were calculated. Sixty-four lipids were significantly reduced in urinary exosomes of HαT patients compared to controls. All significantly reduced sphingolipids and most of the phospholipids were saturated or mono-unsaturated lipids. These results suggest exosome secretion is augmented in HαT patients and the lipids within these exosomes may be involved in various biological processes. The unique lipid composition of urinary exosomes from HαT patients will contribute to our understanding of the biochemistry of this disease.

Keywords: glycerolipids; glycerophospholipids; hereditary α-tryptasemia; lipidomics; sterols; urinary exosomes.

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Conflict of interest statement

Conflict of Interest Statement There are no conflicts of interest to declare from any of the authors

Figures

Figure 1
Figure 1
CD117 expression in patients with hereditary alpha tryptasemia compared to systemic mastocytosis. Panel A and C are representative hematoxylin and eosin (H&E) and CD117 stains of duodenum of a patient with systemic mastocytosis. Panel B and D are representative H&E and CD117 from duodenum of a patient with hereditary alpha tryptasemia (HαT). Mast cells are clustered, expanded in number, and spindle shaped in systemic mastocytosis. In HαT, CD117 positive mast cells are expanded in the lamina propria with slightly clustering and are increased in cell size. CD117 positive cells stain brown. Slides stained for CD117 are counterstained with hematoxylin. Bar length = 50 µm
Figure 2
Figure 2
CD117 and tryptase expression in patients with hereditary alpha tryptasemia compared to irritable bowel syndrome. Panel A and B are representative CD117 and tryptase stains of duodenum of a patient with irritable bowel syndrome (IBS). Panel C and D are representative CD117 and tryptase from duodenum of a patient with hereditary alpha tryptasemia (HαT). In contrast with IBS, there are increased numbers of lamina propria mast cells in HαT as well as increased amount of tryptase release into the surrounding tissue. CD117 and tryptase are in brown. Slides are counterstained with hematoxylin. Bar length = 60 µm
Figure 3
Figure 3
Characterization of urinary exosomes from patients with HαT compared to healthy volunteers. (A), Representative nanoparticle tracking analysis showing concentration and size of the urinary exosomes isolated from HαT patients or healthy volunteers (ctrl). (B), Summary bar graphs of exosome concentration and size from the two groups. *represents statistical significance (P < .05)
Figure 4
Figure 4
Characterization of urinary exosomes from HαT patients and healthy volunteers by Western blot analysis. Panel A shows Western blots for four extracellular vesicle markers. Thirty micrograms of exosomal lysate was resolved by SDS‐PAGE before the proteins were transferred to nitrocellulose membranes in order to probe for multiple exosomal markers by western blot using specific antibodies against flotillin‐1, annexin A2, Tsg101, and caveolin‐1. There were no observable differences in caveolin‐1 protein expression between control (CTRL) and HαT samples. Panel B shows the densitometry for the Tsg101 Western blot normalized to the caveolin‐1 Western blot. * indicates significance at P < .05
Figure 5
Figure 5
Relative quantity of glycerophospholipids in freshly isolated urinary exosomes from HαT patients and healthy volunteers. Panel A, B, and C represent significantly changed phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylglycerol (PG), respectively. Data are mean ± SE of relative quantity (pg/µL). * and ** indicate significant at 5% and 1% probability levels, respectively
Figure 6
Figure 6
Relative quantity of phosphatidylethanolamines in freshly isolated urinary exosomes from HαT patients and healthy volunteers. Panel A, B and C represent significantly changed phosphatidylethanolamine (PE) and PE‐P, PE‐O and lysophosphatidylethanolamine (LPE), respectively. Data are mean ± SE of relative quantity (pg/µL). * and ** indicate significance at P < .05 and < .01, respectively
Figure 7
Figure 7
Relative quantity of glycerolipids in freshly isolated urinary exosomes from HαT patients and healthy volunteers. Panel A represents various significantly changed triacylglycerol (TAG) and Panel B, diacylglycerol (DAG) species. Data are mean ± SE of relative quantity (pg/µL), * and ** indicate significance at P < .05 and < .01, respectively
Figure 8
Figure 8
Relative quantity of sphingolipids in freshly isolated urinary exosomes from HαT patients and healthy volunteers. Graphs A, B and C represent Sphingomyelin (SM), ceramide (CER) and dihydroceramide (DCER), respectively. Data are mean ± SE of relative quantity (pg/µL). * indicate significance at P < .05
Figure 9
Figure 9
Relative quantity of cholesterol ester in freshly isolated urinary exosomes from HαT patients and healthy volunteers. Data are mean ± SE of relative quantity (pg/µL). * and ** indicate significant at 5% and 1% probability levels, respectively
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