Circulating, cell-free DNA as a marker for exercise load in intermittent sports.

BACKGROUND: Attempts to establish a biomarker reflecting individual player load in intermittent sports such as football have failed so far. Increases in circulating DNA (cfDNA) have been demonstrated in various endurance sports settings. While it has been proposed that cfDNA could be a suitable marker for player load in intermittent sports, the effects on cfDNA of repeated sprinting as an essential feature in intermittent sports are unknown. For the first time, we assessed both alterations of cfDNA due to repeated maximal sprints and due to a professional football game. METHODS: Nine participants were subjected to a standardised sprint training session with cross-over design of five maximal sprints of 40 meters with either "short" (1 minute) or "long" pauses (5 minutes). Capillary cfDNA and lactate were measured after every sprint and venous cfDNA before and after each series of sprints. Moreover, capillary cfDNA and lactate values were taken in 23 professional football players before and after incremental exercise testing, during the course of a training week at rest (baseline) and in all 17 enrolled players following a season game. RESULTS: Lactate and venous cfDNA increased more pronounced during "short" compared to "long" (1.4-fold, p = 0.032 and 1.7-fold, p = 0.016) and cfDNA correlated significantly with lactate (r = 0.69; p<0.001). Incremental exercise testing increased cfDNA 7.0-fold (p<0.001). The season game increased cfDNA 22.7-fold (p<0.0001), while lactate showed a 2.0-fold (p = 0.09) increase compared to baseline. Fold-changes in cfDNA correlated with distance covered during game (spearman's r = 0.87, p = 0.0012), while no correlation between lactate and the tracking data could be found. DISCUSSION: We show for the first time that cfDNA could be an objective marker for distance covered in elite intermittent sports. In contrast to the potential of more established blood-based markers like IL-6, CK, or CRP, cfDNA shows by far the strongest fold-change and a high correlation with a particular load related aspect in professional football.

Acute effects of strength exercises and effects of regular strength training on cell free DNA concentrations in blood plasma.

Creatine kinase (CK) is a marker for muscle cell damage with limited potential as marker for training load in strength training. Recent exercise studies identified cell free DNA (cfDNA) as a marker for aseptic inflammation and cell damage. Here we overserved in a pilot study the acute effects during strength exercise and chronic effects of regular strength training on cfDNA concentrations over a period of four weeks in three training groups applying conservation training (CT) at 60% of the 1 repetition maximum, high intensity-low repetition training (HT) at 90% of the 1 repetition maximum and differential training (DT) at 60% of the 1 repetition maximum. EDTA-plasma samples were collected before every training session, and on the first and last training day repeatedly after every set of exercises. CfDNA increased significantly by 1.62-fold (mean (±SD) before first exercise: 8.31 (2.84) ng/ml, after last exercise 13.48 (4.12) ng/ml) across all groups within a single training session (p<0.001). The increase was 1.77-fold higher (mean (±SD) before first exercise: 12.23 (6.29) ng/ml, after last exercise 17.73 (11.24) ng/ml) in HT compared to CT (mean (±SD) before first exercise: 6.79 (1.28) ng/ml, after last exercise 10.05 (2.89) ng/ml) (p = 0.01). DNA size analysis suggested predominant release of short, mononucleosomal DNA-fragments in the acute exercise setting, while we detected an increase of mostly longer, polynucleosomal cfDNA-fragments at rest before the training session only at day two with a subsequent return to baseline (p<0.001). In contrast, training procedures did not cause any alterations in CK. Our results suggest that during strength exercise short-fragmented cfDNA is released, reflecting a fast, aseptic inflammatory response, while elevation of longer fragments at baseline on day two seemed to reflect mild cellular damage due to a novel training regime. We critically discuss the implications of our findings for future evaluations of cfDNA as a marker for training load in strength training.

Voluntary distance running prevents TNF-mediated liver injury in mice through alterations of the intrahepatic immune milieu.

Physical activity confers a broad spectrum of health benefits. Beyond the obvious role in metabolically driven diseases, the role of physical activity in acute liver injury is poorly explored. To study the role of physical activity in acute liver injury, a novel model of voluntary distance running in mice was developed and mice were subjected to acute liver injury induced by N-galactosamine (GalN) and lipopolysaccharide (LPS). Analyses included histological stains, immunoblotting, qRT-PCR and FACS analysis. Voluntary distance running increased to an average of 10.3 km/day after a learning curve. Running lead to a decrease in the absolute numbers of intrahepatic CD4+ T and B lymphocytes and macrophages after 7 weeks. In parallel, hepatic mRNA expression of inflammatory cytokines including IL-6 and IL-1beta, TGF-beta and monocyte chemoattractant protein-1 (MCP-1/CCL2) were suppressed, while TNF-α was not affected by exercise. Likewise, expression of the macrophage-specific antigen F4/80 was downregulated 1.6-fold from exercise. Notably, acute liver injury from GaIN/LPS was significantly blunted following 7 weeks of voluntary exercise as determined by liver histology, a 84.6% reduction of alanine aminotransferase (P<0.01) and a 54.6% reduction of aspartate aminotransferase (P<0.05) compared with sedentary mice. Additionally, proinflammatory cytokines, activation of caspase 3 and JNK were significantly lower, while antiapoptotic protein A20 increased. Voluntary distance running alters the intrahepatic immune phenotype producing an environment that is less susceptible to acute liver injury.

Exploring the Potential of Cell-Free-DNA Measurements After an Exhaustive Cycle-Ergometer Test as a Marker for Performance-Related Parameters.

PURPOSE: Intensive exercise is known to be accompanied by a rapid release of cell-free DNA (cfDNA). The physiological significance of cfDNA release for performance diagnostics has not been studied. The authors analyzed the release of cfDNA during bicycle exercise and its correlation with physiological parameters. METHODS: Eleven male athletes performed an incremental cycling test. Venous blood was collected before and immediately after exercise and after 90 min of recovery. Since the amount of cfDNA is influenced by preanalytical parameters like DNA extraction and quantification method, the authors applied different measurement approaches based on quantitative real-time polymerase chain reaction. They compared a direct measurement procedure not requiring cfDNA extraction for a short (L1PA290) and a long fragment (L1PA2222) and a procedure for extracted cfDNA for a short (LTR570) and long fragment (LTR5323) with primers targeting the repetitive sequences L1PA2 and LTR5 in both assays, respectively. RESULTS: With the exception of LTR5323, the procedures revealed significant increases of cfDNA postexercise, whereas the direct approach showed lower interindividual variance in cfDNA values. When linking cfDNA levels to parameters of exercise performance the authors observed that, especially, the measurement based on L1PA2222 correlated significantly with exercise markers. These correlations were similar to the relationship of the performance markers among themselves. CONCLUSIONS: cfDNA is a possible physiological marker to assess and predict exercise performance in athletes. In addition, the results indicate that using cfDNA as a marker in exercise physiology requires careful selection of a suitable measurement technique, whether it is eluted DNA or directly quantified.

Physical exercise induces rapid release of small extracellular vesicles into the circulation.

Cells secrete extracellular vesicles (EVs) by default and in response to diverse stimuli for the purpose of cell communication and tissue homeostasis. EVs are present in all body fluids including peripheral blood, and their appearance correlates with specific physiological and pathological conditions. Here, we show that physical activity is associated with the release of nano-sized EVs into the circulation. Healthy individuals were subjected to an incremental exercise protocol of cycling or running until exhaustion, and EVs were isolated from blood plasma samples taken before, immediately after and 90 min after exercise. Small EVs with the size of 100-130 nm, that carried proteins characteristic of exosomes, were significantly increased immediately after cycling exercise and declined again within 90 min at rest. In response to treadmill running, elevation of small EVs was moderate but appeared more sustained. To delineate EV release kinetics, plasma samples were additionally taken at the end of each increment of the cycling exercise protocol. Release of small EVs into the circulation was initiated in an early phase of exercise, before the individual anaerobic threshold, which is marked by the rise of lactate. Taken together, our study revealed that exercise triggers a rapid release of EVs with the characteristic size of exosomes into the circulation, initiated in the aerobic phase of exercise. We hypothesize that EVs released during physical activity may participate in cell communication during exercise-mediated adaptation processes that involve signalling across tissues and organs.

Release of bulk cell free DNA during physical exercise occurs independent of extracellular vesicles.

PURPOSE: Strenuous exercise induces a rapid and transient elevation of cell free DNA (cfDNA) concentration in blood plasma. The detection of cfDNA in the presence of plasma nucleases could indicate an association of cfDNA with protective vesicular structures. Several cell types release extracellular vesicles (EVs), including exosomes and shedding microvesicles, which are known to mediate the exchange of proteins and nucleic acids (largely RNA) between cells. Here, we assessed whether EVs play a role in the exercise-dependent release of cfDNA in blood plasma. METHODS: Venous blood collected from healthy volunteers before and after incremental treadmill exercise was separated into vesicular (EV) and soluble fractions. Nuclear and mitochondrial DNA content in plasma supernatants and EV fractions was determined by quantitative real-time PCR (qPCR). RESULTS: We show that the majority of cfDNA is located in the plasma supernatants. Only minute amounts of DNA were observed in the EV-associated fractions including microvesicles and exosomes. Nuclear and mitochondrial DNA species differ in terms of their quantities in the several plasma fractions. CONCLUSIONS: Our results indicate that cfDNA liberated in response to acute physical exercise is not released by vesicular means and circulates in a soluble form in blood plasma which could indicate different biological functions exerted by cfDNA and EVs. The different nature of DNA species in plasma has major implications for the preparation of plasma and other bodily fluids prior to analysis.

Exercise-induced increases in cell free DNA in human plasma originate predominantly from cells of the haematopoietic lineage.

The role of cell free DNA (cfDNA) has been intensively discussed under various pathological conditions and after acute bouts of exercise. To date, there is still no conclusive evidence concerning the cellular origin of cfDNA and the entire mechanism leading to elevated cfDNA concentrations in human plasma and serum. Here, we investigated the cellular origin of cfDNA in sex-mismatched haematopoietic stem cell transplantation (HSCT) and liver transplantation (LT) patients by determining the relative proportion of Y-chromosomal to total nuclear cfDNA. Total nuclear cfDNA and Y-chromosomal cfDNA concentrations were determined in blood plasma before and after an incremental exercise test via quantitative real-time PCR (qPCR). Female HSCT patients showed high proportions of Y-chromosomal cfDNA. Both total nuclear and Y-chromosomal cfDNA increased significantly and in a highly correlated fashion due to exercise. In male HSCT patients with female donors less than 10% of the cfDNA was of Y-chromosomal origin at any point in time and even though the total amount of cfDNA increased during exercise, no increases in Y-chromosomal DNA could be detected. The percentage of Y-chromosomal cfDNA in female LT patients with male donors was very low and levels remained unchanged during exercise. This indicates that cells not derived from the bone marrow, in this case transplanted liver cells, represented only a minor fraction of cfDNA in blood plasma and were not released during acute physical exercise. Even though many physiological conditions may be altered in transplant patients versus healthy people, our results strongly suggest that cells from the haematopoietic lineage are the main source of cfDNA released during acute bouts of exercise.

Correlation between cell free DNA levels and medical evaluation of disease progression in systemic lupus erythematosus patients.

High levels of cell free DNA (cfDNA) in human blood plasma have been described in patients with autoimmune diseases. The aim of this study was to determine the levels of cfDNA in systemic lupus erythematosus (SLE) patients and to assess fluctuations of cfDNA concentrations compared to the course of disease progression under standard treatment. Therefore, nuclear cfDNA concentrations in plasma were measured in 59 SLE patients and 59 healthy controls. Follow-up blood plasma was collected from 27 of the 59 SLE patients. Patients were characterised by clinical parameters (antinuclear antibodies (ANA), anti-dsDNA-antibodies, C3, C4, and CRP), SLE disease activity index (SLEDAI) and medical therapy. Our results showed that cfDNA concentrations were significantly higher in SLE patients compared to healthy individuals. Levels of cfDNA assessed in serial samples correlated significantly with the medical evaluation of disease activity in SLE patients. Our results could implicate cfDNA as a global marker for disease activity.

Direct quantification of cell-free, circulating DNA from unpurified plasma.

Cell-free DNA (cfDNA) in body tissues or fluids is extensively investigated in clinical medicine and other research fields. In this article we provide a direct quantitative real-time PCR (qPCR) as a sensitive tool for the measurement of cfDNA from plasma without previous DNA extraction, which is known to be accompanied by a reduction of DNA yield. The primer sets were designed to amplify a 90 and 222 bp multi-locus L1PA2 sequence. In the first module, cfDNA concentrations in unpurified plasma were compared to cfDNA concentrations in the eluate and the flow-through of the QIAamp DNA Blood Mini Kit and in the eluate of a phenol-chloroform isoamyl (PCI) based DNA extraction, to elucidate the DNA losses during extraction. The analyses revealed 2.79-fold higher cfDNA concentrations in unpurified plasma compared to the eluate of the QIAamp DNA Blood Mini Kit, while 36.7% of the total cfDNA were found in the flow-through. The PCI procedure only performed well on samples with high cfDNA concentrations, showing 87.4% of the concentrations measured in plasma. The DNA integrity strongly depended on the sample treatment. Further qualitative analyses indicated differing fractions of cfDNA fragment lengths in the eluate of both extraction methods. In the second module, cfDNA concentrations in the plasma of 74 coronary heart disease patients were compared to cfDNA concentrations of 74 healthy controls, using the direct L1PA2 qPCR for cfDNA quantification. The patient collective showed significantly higher cfDNA levels (mean (SD) 20.1 (23.8) ng/ml; range 5.1-183.0 ng/ml) compared to the healthy controls (9.7 (4.2) ng/ml; range 1.6-23.7 ng/ml). With our direct qPCR, we recommend a simple, economic and sensitive procedure for the quantification of cfDNA concentrations from plasma that might find broad applicability, if cfDNA became an established marker in the assessment of pathophysiological conditions.