Objectives Exosomes are small lipid bilayer vesicles that are defined by their endocytic origin and size range of 30–140 nm. They are constantly produced by different cell types, by both healthy and abnormal cells, and can be isolated from almost all body fluids. Little information exists in isolating exosomes from plasma due to the complexity of its content and the presence of contaminating plasma proteins. Design and methods We carried-out liquid chromatography-mass spectrometry (LC-MS/MS) analyses of plasma-derived vesicles from 4 healthy donors obtained by 2 coupled methodologies: Ultracentrifugation (UC) coupled with size-exclusion chromatography (SEC) to isolate and subsequently enrich exosomes. We compared the proteins detected by UC alone and UC coupled with SEC. Results In the coupled UC + SEC methodology we found 52.25% more proteins enriched in exosomes as CD9, Annexins, YWHAZ (14-3-3 family) and others, than by using UC alone. There is also a reduction of 98.8% of contaminating plasma proteins by coupling UC and SEC in comparison to using UC alone. Conclusions We conclude that exosomes can be successfully isolated from plasma using a very simple combination of standard methods, which could largely improve the proteomics profiling of plasma exosomes.
Background 5-Fluorouracil (5-FU) is frequently used for the treatment of gastrointestinal tumors. The pharmacological effect of 5-FU is influenced by genetic polymorphisms as well as differently dosed regimens. Currently, 5-FU is generally administered as a continuous infusion via an implanted port system using a body surface area (BSA)-based dose calculation. In order to optimize treatment, the area under the curve (AUC) can be estimated to allow for individual dose adjustment. A 5-FU AUC range between 20 and 30 [mg×h×L] is recommended. The aim of the current study was to assess if blood for AUC analysis could also be drawn at the side where the port system had been placed. Methods We collected EDTA blood samples of patients receiving infusional 5-FU simultaneously from different sampling points (right/left cubital vein). 5-FU concentrations were measured in a steady-state equilibrium based on nanoparticle immunoassay (My5-FU; Saladax). Results A total of 39 patients took part in this study. About half of the patients did not reach the target 5-FU concentration window (37% were under- and 16% of the patients were overdosed). Calculated median AUC was 23.3 for the right arm (range 5.8–59.4) and a median of 23.4 for the left arm (range 5.3–61.0). AUC values showed no difference between right compared to left arms (p=0.99). Conclusions In all, these results confirm that a high percentage of patients are not treated with 5-FU doses reaching suggested AUC levels of 20–30. The location of venepuncture, however, had no impact on the results of plasma 5-FU concentration.
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