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Technical Briefs |
1 Institute of Laboratory Diagnostics, Kaiser Franz Josef Hospital, Vienna, Austria;2 Institute of Sports Science, Department of Sports and Exercise Physiology, University of Vienna, Vienna, Austria;3 Ludwig Boltzmann Institute for Rheumatology, Vienna, Austria;
aaddress correspondence to this author at: Institute of Laboratory Diagnostics, Kaiser Franz Josef Hospital, Kundratstrasse 3, A-1100 Vienna, Austria; fax 43-60191-3309, e-mail johanna.atamaniuk{at}wienkav.at
Physical exercise leads to temporary ischemia in muscles, followed by increased oxygen supply during recovery as a result of reperfusion. It is thought that the sudden influx of oxygen causes a calcium overload in cells, leading to an influx of inflammatory cells into reperfused tissue. This leads to the generation of reactive oxygen radicals and subsequent oxidative damage to DNA, proteins, and lipids. For example, increased oxidant production in the mitochondria of muscles during acute exercise, followed by reoxygenation, was shown to cause cellular damage (1). In addition, exercise may cause transient muscle damage, characterized by muscle soreness, muscle fiber disarrangement, muscle protein release into plasma, an acute immune response, and decreased muscle performance (2).
Regional ATP depletion during reperfusion, disruptions in calcium homeostasis, and the presence of oxygen free radicals have all been implicated in the etiology of muscle fiber damage and necrosis. Furthermore, postexercise lymphocytopenia (2) is well documented and attributed to the exit of lymphocytes from the vascular compartment (3). Other studies have reported exercise-induced DNA damage in leukocytes and raised the question of a possible link to apoptosis (3). This effect is thought to be caused by reactive oxygen species, which are released from peripheral monocytes.
The aim of this study was to investigate the effects of physical activity, in particular muscle damage and oxidative stress, during and after a half-marathon race and the subsequent recovery period; we also wanted to measure whether oxidative stress attributable to reoxygenation may be a relevant factor in cellular damage. Cell-free plasma DNA concentrations were used as a sensitive tool for quantification of cellular damage and compared with the conventional measurements of myoglobin and uric acid in blood samples.
In this study, blood samples from half-marathon runners were taken before the race, immediately after the race, and 2 h after the race.
We tested a group of 25 healthy half-marathon runners (12 males and 13 females; age range, 28–56 years). The participants had been training at least 2–8 h per week for 2 years. Blood samples (EDTA plasma and serum) were taken before the race, immediately after the race, and 2 h after the race. These timing intervals were designated as groups 1, 2, and 3. After centrifugation, plasma and serum were separated and immediately frozen at –20 °C until further processing.
DNA was isolated from 800 µL of plasma by use of a DNA Isolation reagent set (Roche), according to the described protocol.
After isolation, DNA was eluted in 50 µL of elution buffer. Staining with Vistra Green and measurement of cell-free plasma DNA were done on a LightCycler real-time PCR system (Roche) by fluorescent signal detection according to the following protocol: Human placental DNA calibrators (Sigma) were dissolved in Tris-EDTA buffer (1 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 8.0; Sigma) to create a calibration curve ranging from 100 to 5000 pg/µL. Calibrator or sample (5 µL) was pipetted into precooled capillary tubes. Vistra Green nucleic acid gel stain (stock solution in dimethyl sulfoxide; Amersham Bioscience) was diluted 1:1000 in Tris-EDTA buffer, and 5 µL of this dilution was added to each sample or calibrator. After centrifugation (0.8g for 30 s), the emitted fluorescent signals from samples and calibrators were measured in the LightCycler at 530 nm. The cell-free plasma DNA concentrations were reported in pg/µL or recalculated to genome equivalents. Repeated measurements of 500 pg/µL DNA calibrators yielded an analytical imprecision (CV) of 3.1%.
For myoglobin measurements, we used an immunofluorescence assay (Brahms; Kryptor Analytical System); the assay imprecision (CV) was 2.8% (mean concentration, 46.1 mg/L). Serum uric acid was measured using an enzymatic colorimetric method (Roche, Germany); the assay imprecision (CV) was 1.2% (mean concentration, 42.6 mg/L).
Data were analyzed with STATISTICA for Windows, Ver. 6.0. Descriptive data are reported as the mean (SD). Statistical significance was determined by the Wilcoxon matched-pairs test for nonparametric variables. Statistical significance was defined as P <0.05.
The maximum serum uric acid in the resting state was 69 mg/L [mean (SD), 43.6 (10.6) mg/L]. Immediately after the race, uric acid was significantly increased [maximum, 84 mg/L; mean (SD), 53.5 (12.5) mg/L; P <0.0001], and it remained significantly increased 2 h after the race [maximum, 83 mg/L; mean (SD), 54.7 (12.4) mg/L; P for comparison with baseline <0.0001].
Before the race, the mean (SD) serum myoglobin was 44.3 (12.59) µg/L (maximum, 87 µg/L). Immediately after the race, serum myoglobin was significantly increased [maximum, 2503 µg/L; mean (SD), 533.48 (540.45) µg/L; P <0.0001]. After a 2-h recovery period, mean (SD) serum myoglobin was 631.08 (500.86) µg/L, and the maximum was 1984 µg/L (P <0.001).
In the resting state, mean (SD) cell-free plasma DNA was 18.01 (2.80) pg/µL (maximum, 27.4 pg/µL; Fig. 1
). Immediately after the race, plasma DNA was significantly increased [maximum, 702.4 pg/µL; mean (SD), 334.4 (139.41) pg/µL; P <0.0001]. Interestingly, by 2 h after the race, cell-free plasma DNA had returned to baseline [maximum, 112.3 pg/µL; mean (SD), 30.44 (18.99); P <0.0001].
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Increased oxidant production in mitochondria of muscles during acute exercise, followed by reoxygenation, has been shown to cause cellular damage, which may be reflected by increased serum myoglobin concentrations (4). Originating within the mitochondria of aerobic cells is a steady supply of oxygen free radicals, unavoidably generated from the process that uses oxygen to make ATP, the energy storage molecule in the body (5). Consequently, increased electron reflux through the rapidly respiring mitochondria in the active muscle may lead to enhanced electron leakage and production of reactive oxygen species (6).
Free radicals arising from the metabolism or from environmental sources interact continuously in biological systems. Oxidants and antioxidants must therefore be kept in balance to minimize molecular, cellular, and tissue damage. Evidence is accumulating that free radicals have important functions in the signaling network of cells, including induction of growth and apoptosis, and as killing tools of immunocompetent cells (7). During exercise, oxygenation throughout the entire body is greatly increased. For example, muscle oxygen use during strenuous exercise can increase to as high as 100–200 times above that at rest (6). In addition, in working muscles and in tissues that undergo ischemia reperfusion, excessive reactive oxygen species may be generated during and after physical exercise (6).
In our study, we used assays for uric acid, myoglobin, and plasma cell-free DNA to determine the effects of strenuous exercise for the following reasons:
It is known that uric acid possesses free-radical-scavenging properties (8)(9)(10). Increased uric acid concentrations are associated with increased serum antioxidant capacity and reduced oxidative stress and are found during acute physical exercise in healthy individuals (11).
The role of myoglobin as an O2 carrier depends on the reversible binding of O2, which again depends on PO2 and may lead to storage of O2, buffering of PO2 in the cell to prevent mitochondrial anoxia, and to parallel diffusion of O2 (12). The myoglobin content in skeletal muscle increases in response to hypoxic conditions. Furthermore, skeletal muscle is unique in that it is multinucleated. Evidence suggests that skeletal muscle can undergo individual myonuclear apoptosis as well as complete cell death (13).
Excessive stress can induce DNA damage in the form of oxidized nucleosides, strand breaks, or DNA cross-links. Possible consequences of DNA damage are defective repair, apoptosis, and necrosis (6). Defective repair may lead to DNA sequence alterations and possibly to the development of cancer or, in case of mitochondrial DNA, to metabolic dysfunction (7). Nonspecific DNA repair enzymes excise damaged DNA lesions to release deoxynucleotides (3). In addition, base-specific repair glycosylases excise the corresponding bases (2). Deoxynucleotides are enzymatically hydrolyzed to stable deoxynucleosides, and these products are transported through the blood and excreted in urine. The origins of cell-free plasma DNA are still unclear, but it has been found in many cases where apoptosis or necrosis is involved, suggesting that such events are the main source for its presence.
The measurement of circulating DNA has been used as a prognostic tool in the posttreatment monitoring of transplant patients and to assess the prognoses for trauma patients (14). It has been shown that within 15 min to 3 h after major bodily injury, circulating plasma DNA concentrations in the peripheral blood of trauma patients are significantly increased, e.g., in patients who develop posttraumatic organ failure and multiple organ dysfunction syndrome, compared with individuals who do not develop these complications (15). A previous study showed that increased plasma DNA concentrations persist for days after the injury, especially in patients with multiple organ dysfunction syndrome (16).
Tumor-derived genetic alterations in DNA fragments have been detected in plasma and serum, e.g., oncogene mutations (17)(18), oncogene amplifications (19), and tumor-related viral DNA (20)(21). Cell-free plasma DNA originating from tumor cells suggests that their origin is the necrosis of malignant cells.
In this study the significant increase in cell-free plasma DNA immediately after exhaustive exercise and its disappearance within 2 h after the race suggests that cell-free plasma DNA could possibly be an important tool for monitoring and quantification of cellular damage. Experiments are ongoing in our laboratories to determine whether apoptotic or necrotic events are responsible for the observed phenomenon. Although it seems likely that the source of cell-free plasma DNA is the skeletal muscle cell, further investigations are needed to determine whether other cell types are involved as well.
Acknowledgments
We thank Camelia Mot (MD), Alireza Karimi, and Christine Pollaschek for their excellent technical assistance.
References
The following articles in journals at HighWire Press have cited this article:
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  M. P. Langford, T. B. Redens, N. R. Harris, S. Lee, S. K. Jain, S. Reddy, and R. McVie Plasma Levels of Cell-Free Apoptotic DNA Ladders and Gamma-Glutamyltranspeptidase (GGT) in Diabetic Children Experimental Biology and Medicine, October 1, 2007; 232(9): 1160 - 1169. [Abstract] [Full Text] [PDF]
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 I. G. Fatouros, A. Destouni, K. Margonis, A. Z. Jamurtas, C. Vrettou, D. Kouretas, G. Mastorakos, A. Mitrakou, K. Taxildaris, E. Kanavakis, et al. Cell-Free Plasma DNA as a Novel Marker of Aseptic Inflammation Severity Related to Exercise Overtraining Clin. Chem., September 1, 2006; 52(9): 1820 - 1824. [Abstract] [Full Text] [PDF]
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J. Atamaniuk, K. Ruzicka, K. M. Stuhlmeier, A. Karimi, M. Eigner, and M. M. Mueller Cell-Free Plasma DNA: A Marker for Apoptosis during Hemodialysis. Clin. Chem., March 1, 2006; 52(3): 523 - 526. [Abstract] [Full Text] [PDF]
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