Decrease in Cellular Nanovesicles Concentration in Blood of Athletes More Than 15 Hours After Marathon

Abstract

Introduction: Cellular nanovesicles (CNVs), that are shed from cells, have been recognized as promising indicators of health status. We analyzed the effect of long-distance running on concentration of CNVs, along with some standard blood parameters, in 27 athletes two days before and >15 hours after physical effort.

Methods: CNVs were isolated by repetitive centrifugation and washing of samples, and assessed by flow cytometry. Cholinesterase (ChE) and glutathione S-transferase (GST) activity were measured spectrophotometrically. Interleukin 6 (IL-6) and tumor necrosis factor-α (TNF-α) concentrations were measured using enzyme-linked immunosorbent assay (ELISA). C-reactive protein (CRP) was measured with immunoturbidimetric determination and lipidogram parameters were measured by enzymatic colorimetric assay. Flow cytometry was used for blood cell count and mean platelet volume (MPV) measurement.

Results: More than 15 hours after physical effort a decrease was found in CNVs' concentration in isolates from blood (46%; p<0.05), in ChE activity in whole blood (47%; p<0.001), in plasma (34%; p<0.01), and in erythrocyte suspension (54%; p<0.001), as well as in GST activity in erythrocyte suspension (16%; p<0.01) and in IL-6 concentration in plasma (63%; p<0.05). We found no change in GST activity in plasma and in TNF-α concentration in plasma. Correlations (>0.8; p<0.001) between CNVs' concentration and ChE activity, and GST activity, respectively, in erythrocyte suspension were found.

Conclusion: We found that >15 hours post-physical effort, CNVs' concentration was below the initial value, concomitant with other measured parameters: ChE and GST activity as well as IL-6 concentration, indicating a favorable effect of physical effort on health status. CNVs' concentration and ChE activity in isolates from peripheral blood proved to have potential as indicators of the response of the human body to inflammation after physical effort. Physical activity should be considered as an important factor in preparation of subjects for blood sampling in procedures focusing on CNV-containing diagnostic and therapeutic compounds.

Keywords: blood samples; cellular nanovesicles; inflammation process; marathon; membrane vesiculation; physical effort.

Figure 1

Gating strategy for the analysis of CNVs by FCM. 2D-plot of typical CNV isolates, prepared from blood plasma of a donor before (A) and after (B) the marathon. Gating of the CNVs and residual cells (RCs) was as indicated. The total number of events in the CNVs gate were taken.

Figure 2

Scanning electron micrograph of a typical isolate of CNVs from the blood plasma sample with characteristic shapes of particles without internal structures including spheres, tubules (black arrows), and tori (white arrows). No residual cells are observed in the figure.

Figure 3

The effect of physical effort on concentration of microparticles and measured physiological state-related parameters in blood. (A) CNVs concentration in 1 mL plasma samples two days before and >15 hours after physical effort. (B) Activity of ChE (n=24) (in mM/min/mL of sample) two days before and >15 hours after physical effort in plasma, erythrocyte suspension and whole blood samples. (C) Activity of GST (n=24) (in mM/min/mL of sample) in plasma and erythrocyte suspension samples two days before and >15 hours after physical effort. Scale on the left represents values of plasma GST activity and scale on the right represents values of erythrocyte suspension GST activity. (D) Concentration of IL-6 and TNF-α (n=26) (in pg/mL of plasma) two days before and >15 hours after physical effort. Statistical differences before and after physical effort at: * p<0.05, ** p<0.01 and *** p<0.001.