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Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.

Sódar BW, Kittel Á, Pálóczi K, Vukman KV, Osteikoetxea X, Szabó-Taylor K, Németh A, Sperlágh B, Baranyai T, Giricz Z, Wiener Z, Turiák L, Drahos L, Pállinger É, Vékey K, Ferdinandy P, Falus A, Buzás EI - Sci Rep (2016)

Bottom Line: Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates.Based on biophysical properties of LDL this finding was highly unexpected.Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, 1085, Hungary.

ABSTRACT
Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

No MeSH data available.


Related in: MedlinePlus

The impact of food intake on the number of blood plasma particles within the size range of EVs.(A,B) Representative scatter plots (A) and summarized data (B) of PFP samples from healthy donors (n = 3), analyzed by FCM in fasting state, 15 min, 30 min, 90 min, 180 min and 360 min after a standard high-fat meal. The MV gate was established using MegamixTM beads (diameter: 160 nm–500 nm), and the gating was optimized with 1 μm silica beads. Note that the particle number within the MV gate significantly increased 90 min postprandially (mean + SEM, ***P < 0.001, one-way ANOVA), and remained elevated up to 6 h after food intake. (C) Representative data of TRPS analysis of fasting and 4 h postprandial human blood plasma samples (n = 3, continuous and dotted lines, respectively, *P < 0.05 Wilcoxon matched-pairs signed rank test). The samples were measured on NP200, NP400 and NP800 nanopore membranes. (D) TRPS measurement (NP200) of fasting (black bars) and 4 h postprandial (gray bars) plasma samples purified with SEC prior to TRPS analysis.
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f1: The impact of food intake on the number of blood plasma particles within the size range of EVs.(A,B) Representative scatter plots (A) and summarized data (B) of PFP samples from healthy donors (n = 3), analyzed by FCM in fasting state, 15 min, 30 min, 90 min, 180 min and 360 min after a standard high-fat meal. The MV gate was established using MegamixTM beads (diameter: 160 nm–500 nm), and the gating was optimized with 1 μm silica beads. Note that the particle number within the MV gate significantly increased 90 min postprandially (mean + SEM, ***P < 0.001, one-way ANOVA), and remained elevated up to 6 h after food intake. (C) Representative data of TRPS analysis of fasting and 4 h postprandial human blood plasma samples (n = 3, continuous and dotted lines, respectively, *P < 0.05 Wilcoxon matched-pairs signed rank test). The samples were measured on NP200, NP400 and NP800 nanopore membranes. (D) TRPS measurement (NP200) of fasting (black bars) and 4 h postprandial (gray bars) plasma samples purified with SEC prior to TRPS analysis.

Mentions: To study the effect that food intake has on the detectable particle concentration in blood plasma, platelet-free plasma (PFP) was collected from healthy individuals (n = 3) after 12 h fasting as well as at multiple time points postprandially, after a standard high-fat meal. Figure 1a,b show flow cytometry (FCM) scatter plots with a significant (up to 5×) increase in the detectable particle number within the MV gate. This increase became significant (***P < 0.001) after 90 min (Fig. 1b) and remained elevated even after 6 h. Therefore, in our subsequent experiments we chose to use 4 h postprandial PFP samples. As shown in Fig. 1c, TRPS analysis of fasting and 4 h postprandial PFPs revealed an increased postprandial particle concentration (*P < 0.05), however, without any significant alteration in the particle size (Supplementary Fig. S1). Similarly, tunable resistive pulse sensing (TRPS) analysis (a method suitable for measuring particle size and concentration) of PFPs purified on a qEVTM size exclusion chromatography (SEC) column (Fig. 1d) maintained the prominent difference between fasting and postprandial samples, although there was a general reduction in particle concentration as compared to the unpurified samples (Fig. 1c,d).


Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection.

Sódar BW, Kittel Á, Pálóczi K, Vukman KV, Osteikoetxea X, Szabó-Taylor K, Németh A, Sperlágh B, Baranyai T, Giricz Z, Wiener Z, Turiák L, Drahos L, Pállinger É, Vékey K, Ferdinandy P, Falus A, Buzás EI - Sci Rep (2016)

The impact of food intake on the number of blood plasma particles within the size range of EVs.(A,B) Representative scatter plots (A) and summarized data (B) of PFP samples from healthy donors (n = 3), analyzed by FCM in fasting state, 15 min, 30 min, 90 min, 180 min and 360 min after a standard high-fat meal. The MV gate was established using MegamixTM beads (diameter: 160 nm–500 nm), and the gating was optimized with 1 μm silica beads. Note that the particle number within the MV gate significantly increased 90 min postprandially (mean + SEM, ***P < 0.001, one-way ANOVA), and remained elevated up to 6 h after food intake. (C) Representative data of TRPS analysis of fasting and 4 h postprandial human blood plasma samples (n = 3, continuous and dotted lines, respectively, *P < 0.05 Wilcoxon matched-pairs signed rank test). The samples were measured on NP200, NP400 and NP800 nanopore membranes. (D) TRPS measurement (NP200) of fasting (black bars) and 4 h postprandial (gray bars) plasma samples purified with SEC prior to TRPS analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834552&req=5

f1: The impact of food intake on the number of blood plasma particles within the size range of EVs.(A,B) Representative scatter plots (A) and summarized data (B) of PFP samples from healthy donors (n = 3), analyzed by FCM in fasting state, 15 min, 30 min, 90 min, 180 min and 360 min after a standard high-fat meal. The MV gate was established using MegamixTM beads (diameter: 160 nm–500 nm), and the gating was optimized with 1 μm silica beads. Note that the particle number within the MV gate significantly increased 90 min postprandially (mean + SEM, ***P < 0.001, one-way ANOVA), and remained elevated up to 6 h after food intake. (C) Representative data of TRPS analysis of fasting and 4 h postprandial human blood plasma samples (n = 3, continuous and dotted lines, respectively, *P < 0.05 Wilcoxon matched-pairs signed rank test). The samples were measured on NP200, NP400 and NP800 nanopore membranes. (D) TRPS measurement (NP200) of fasting (black bars) and 4 h postprandial (gray bars) plasma samples purified with SEC prior to TRPS analysis.
Mentions: To study the effect that food intake has on the detectable particle concentration in blood plasma, platelet-free plasma (PFP) was collected from healthy individuals (n = 3) after 12 h fasting as well as at multiple time points postprandially, after a standard high-fat meal. Figure 1a,b show flow cytometry (FCM) scatter plots with a significant (up to 5×) increase in the detectable particle number within the MV gate. This increase became significant (***P < 0.001) after 90 min (Fig. 1b) and remained elevated even after 6 h. Therefore, in our subsequent experiments we chose to use 4 h postprandial PFP samples. As shown in Fig. 1c, TRPS analysis of fasting and 4 h postprandial PFPs revealed an increased postprandial particle concentration (*P < 0.05), however, without any significant alteration in the particle size (Supplementary Fig. S1). Similarly, tunable resistive pulse sensing (TRPS) analysis (a method suitable for measuring particle size and concentration) of PFPs purified on a qEVTM size exclusion chromatography (SEC) column (Fig. 1d) maintained the prominent difference between fasting and postprandial samples, although there was a general reduction in particle concentration as compared to the unpurified samples (Fig. 1c,d).

Bottom Line: Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates.Based on biophysical properties of LDL this finding was highly unexpected.Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, 1085, Hungary.

ABSTRACT
Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL.

No MeSH data available.


Related in: MedlinePlus