Release of bulk cell free DNA during physical exercise occurs independent of extracellular vesicles.
Author(s): Helmig S, Frühbeis C, Krämer-Albers EM, Simon P, Tug S
Publication: Eur J Appl Physiol, 2015, Vol. 115(11), Page 2271-80
PubMed ID: 26126838 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effects of exercise on cell-free nuclear and mitochondrial DNA, and microvesicle (MV) levels in plasma fractions. Whole blood lactate levels and cell counts were also accessed pre- and post-exercise.
Conclusion of Paper
While >90% of the plasma nuclear DNA was non-vesicle bound, 48.39% of mitochondrial DNA was observed in the initial centrifugation (10,000 x g) pellet pellet, which contained large MVs, apoptotic bodies and cell debris including mitochondrial fragments. Exercise caused (i) an immediate 7.6-10.1 fold increase in cell-free nuclear DNA in all fractions, (ii) a decrease in MVs in the pellet from initial centrifugation (10,000 x g), (iii) a slight increase in MVs in the pellet from the second centrifugation (100,000 x g), which contained exosomes and smaller MVs), (iv) an increase in lactate levels, and (v) an increase in erythrocyte, platelet, monocyte and lymphocyte counts. Conversely, exercise had little effect on levels of cell-free mitochondrial DNA. Significant effects 90 minutes post-exercise were limited to elevated lactate levels in whole blood.
Studies
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Study Purpose
The purpose of this study was to determine the effects of exercise on cell-free nuclear and mitochondrial DNA levels and MVs in plasma fractions, as well as lactate levels and cell counts in whole blood. Blood was collected from 5 healthy men into K3EDTA tubes before exercise, and 0 min, 10 min, 30 min and 90 min after completing a treadmill test. Plasma was obtained by initial centrifugation at 1600 x g, centrifuged at 10,000 x g, filtration, then third centrifugation at 100,000 x g. All supernatants and pellets were saved. The pellets were resuspended in DNAse buffer with or without DNAse. Plasma, resuspended pellets and supernatants were all stored at -20°C for 1 week before analysis. DNA was extracted from all specimens using the QIAamp Circulating Nucleic Acid Kit and quantified by real-time PCR. Additionally, lactate was measured by an autoanalyzer in capillary blood from the ear lobe collected before exercise and 0, 3, 5, 10, 30 and 90 min after exercise. Complete blood counts were performed on fresh venous blood before, immediately following and 90 minutes after exercise.
Summary of Findings:
Greater than 90% of plasma nuclear DNA was non-vesicle bound. Of the remaining nuclear DNA, 4.6% was located in the pellet from the initial centrifugation (10,000 x g; comprised of large MVs, apoptotic bodies and cell debris) and 3.94% was located in the pellet from the second centrifugation (100,000 x g; comprised of exosomes and smaller MVs). In contrast, 48.39% of mitochondrial DNA was observed in the pellet from the initial centrifugation (10,000 x g), but only 0.12% in the pellet from the second centrifugation (100,000 x g).
While exercise caused a 20.5-fold increase in the levels of cell-free nuclear DNA in the pellet from the second centrifugation (100,000 x g), only a 9.1-fold increase in cell-free nuclear DNA was observed when the pellet was then washed with DNAse to remove surface bound DNA. Conversely, comparable increases (7.6-10.1-fold) were observed post-exercise in plasma and other plasma fractions. All cell-free nuclear DNA levels retuned to baseline 90 minutes after exercise. There was no effect of exercise on levels of cell-free mitochondrial DNA. The EV marker protein Flotilin 1 was detected in pellets from both centrifugations. The pellet from the initial 10,000 x g centrifugation contained large MVs, apoptotic bodies and cell debris), while the pellet from the second 100,000 x g centrifugation contained exosomes and smaller MVs). Compared to pre-exercise levels, Flotilin 1 levels post-exercise were lower in the pellet obtained from 10,000 x g centrifugation (larger MVs and cell debris) and slightly higher in the pellet from the 100,000 x g centrifugation (smaller MVs and exosomes), indicating an overall decrease in MVs in plasma following exercise. Compared to blood obtained pre-exercise, blood obtained immediately post-exercise had higher levels of lactate (p<0.001) and higher erythrocyte (p<0.001), platelet (p<0.05), monocyte (p<0.05) and lymphocyte (p<0.001) counts, although 90 min after exercise only lactate levels were significantly higher (p<0.05).
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform DNA Real-time qPCR Cell count/volume Hematology/ auto analyzer Protein Western blot Small molecule Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Preaquisition Diagnosis/ patient condition Pre-exercise
Post-exercise
Biospecimen Acquisition Time of biospecimen collection Pre-exercise
0 min post-exercise
3 min post-exercise
5 min post-exercise
10 min post-exercise
30 min post-exercise
90 min post-exercise
Analyte Extraction and Purification Nucleic acid digestion DNAse treated pellet
Untreated pellet
Real-time qPCR Specific Targeted nucleic acid Human endogenous retrovirus group K (HERVK) family long terminal repeat 5
85 bp fragment of mitochondrial DNA
Biospecimen Aliquots and Components Centrifugation Different number of centrifugation steps compared