Introduction: 

Circulating, cell-free DNA (cirDNA) characterizes double-stranded, cell-unbound DNA fragments in blood plasma. Under normal conditions the concentration of cirDNA remains low whereas cirDNA levels increase under various pathological conditions like cancer, autoimmune diseases, stroke, sepsis and inflammation or after exhaustive short-term exercise. DNA release can be induced by apoptosis, necrosis or e.g. by an active release via microvesicles such as exosomes. Due to their vehicle function for proteins and RNA the release of DNA via microvesicles is conceivable. The aim of this study was to elucidate the mechanisms underlying the release of cirDNA during exhaustive exercise and to assess the involvement of microvesicles for exercise-dependent increases in cirDNA levels.

Methods: 

We measured cirDNA levels in isolated human blood plasma and in microvesicles. Venous blood samples were collected from a healthy volunteer before, immediately after and 90min after an exhaustive incremental bicycle test. Microvesicles were isolated from blood plasma by differential centrifugation followed by filtration and specific microvesicle markers were determined by Western blot analysis. Mitochondrial and nuclear DNA concentrations were measured by quantitative real-time PCR (qPCR) in the various fractions of the blood plasma before and after treatment with DNase.

Results: 

Upon exhaustive exercise nuclear cirDNA and microvesicles increased more than 4-fold in plasma. In contrast, we could not determine a significant increase in mitochondrial DNA. Before and after exercise only a spurious amount of the total cirDNA was located in the microvesicles.

Conclusion: 

Our results indicated that even though cirDNA and microvesicles are both released during exercise, most of the released cirDNA is not located within microvesicles. It rather appeared that the major part of circulating, cell free DNA existed unbound in blood plasma. Furthermore, the steady mitochondrial DNA level in plasma most likely excludes apoptosis as main mechanism of DNA release following exhaustive exercise.