Increased Concentrations of Cell-Free Plasma ... - Clinical Chemistry

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The Heartmate (Thoratec) left ventricular assist device (LVAD) used in this study is an implantable pump that connects the apex of the left ventricle and the.


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differential catalytic activity of the encoded proteins. J Biol Chem 1997;272: 10004 –12. 22. Wang Y, Spitz MR, Schabath MB, Ali-Osman F, Mata H, Wu X. Association between glutathione S-transferase p1 polymorphisms and lung cancer risk in Caucasians: a case-control study. Lung Cancer 2003;40:25–32.

DOI: 10.1373/clinchem.2004.034058

Increased Concentrations of Cell-Free Plasma DNA after Exhaustive Exercise, Johanna Atamaniuk,1* Claudia Vidotto,1 Harald Tschan,2 Norbert Bachl,2 Karl M. Stuhlmeier,3 and Mathias M. Mu¨ ller1 (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; * address 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 [email protected]) 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 exerciseinduced 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 ␮L of plasma by use of a DNA Isolation reagent set (Roche), according to the described protocol. After isolation, DNA was eluted in 50 ␮L of elution buffer. Staining with Vistra Green and measurement of cell-free plasma DNA were done on a LightCycler realtime 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/␮L. Calibrator or sample (5 ␮L) 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 ␮L 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/␮L or recalculated to genome equivalents. Repeated measurements of 500 pg/␮L 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) ␮g/L (maximum, 87 ␮g/L). Immediately after the race, serum myoglobin was significantly increased

Clinical Chemistry 50, No. 9, 2004

[maximum, 2503 ␮g/L; mean (SD), 533.48 (540.45) ␮g/L; P ⬍0.0001]. After a 2-h recovery period, mean (SD) serum myoglobin was 631.08 (500.86) ␮g/L, and the maximum was 1984 ␮g/L (P ⬍0.001). In the resting state, mean (SD) cell-free plasma DNA was 18.01 (2.80) pg/␮L (maximum, 27.4 pg/␮L; Fig. 1). Immediately after the race, plasma DNA was significantly increased [maximum, 702.4 pg/␮L; mean (SD), 334.4 (139.41) pg/␮L; P ⬍0.0001]. Interestingly, by 2 h after the race, cell-free plasma DNA had returned to baseline [maximum, 112.3 pg/␮L; mean (SD), 30.44 (18.99); P ⬍0.0001]. 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

Fig. 1. Cell-free plasma DNA concentrations in 25 half-marathon runners measured at rest (Plasma DNA 1), immediately after a race (Plasma DNA 2), and 2 h after the race (Plasma DNA 3). Boxes indicate the 25th–75th percentiles; error bars indicate the maximum and minimum concentrations; 䡺, median.


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 –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 crosslinks. 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


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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.

We thank Camelia Mot (MD), Alireza Karimi, and Christine Pollaschek for their excellent technical assistance. References 1. Phaneuf S, Leeuwenburgh C. Apoptosis and exercise [Review]. Med Sci Sports Exerc 2001;33:393– 6. 2. Park EM, Shigenaga MK, Degan P, Korn TS, Kitzler JW, Wehr CM, et al. Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column. Proc Natl Acad Sci U S A 1992;89:3375–9. 3. Mars M, Govender S, Weston A, Naicker V, Chuturgoon A. High intensity exercise: a cause of lymphocyte apoptosis? Biochem Biophys Res Commun 1998;249:366 –70. 4. Goodman C, Henry G, Dawson B, Gillam I, Beilby J, Ching S, et al. Biochemical and ultrastructural indices of muscle damage after a twenty-one kilometre run. Aust J Sci Med Sport 1997;29:95– 8. 5. Holly JL. Glutathione as an antioxidant—part II.⫽112 (accessed October 23, 2003). 6. Chevion S, Moran DS, Heled Y, Shani Y, Regev G, Abbou B, et al. Plasma antioxidant status and cell injury after severe physical exercise. Proc Natl Acad Sci U S A 2003;100:5119 –23. 7. Fehrenbach E, Northoff H. Free radicals, exercise, apoptosis, and heat shock proteins. Exerc Immunol Rev 2001;7:66 – 89. 8. Waring WS, Convery A, Mishra V, Shenkin A, Webb DJ, Maxwell SR. Uric acid reduces exercise-induced oxidative stress in healthy adults. Clin Sci (Lond) 2003;105:425–30. 9. Staub M. [Uric acid as a scavenger in oxidative stress]. Orv Hetil 1999;140: 275–9. 10. Child RB, Wilkinson DM, Fallowfield JL, Donnelly AE. Elevated serum antioxidant capacity and plasma malondialdehyde concentration in response to a simulated half-marathon run. Med Sci Sports Exerc 1998;30: 1603–7. 11. Waring WS, Webb DJ, Maxwell SR. Systemic uric acid administration increases serum antioxidant capacity in healthy volunteers. J Cardiovasc Pharmacol 2001;38:365–71. 12. Conley KE, Ordway GA, Richardson RS. Deciphering the mysteries of myoglobin in striated muscle. Acta Physiol Scand 2000;168:623–34. 13. Primeau AJ, Adhihetty PJ, Hood DA. Apoptosis in heart and skeletal muscle. Can J Appl Physiol 2002;27:349 –95. 14. Lui YY, Dennis YM. Circulating DNA in plasma and serum: biology, preanalytical issues and diagnostic applications [Review]. Clin Chem Lab Med 2002;40:962– 8. 15. Rainer TH, Lo YM, Chan LY, Lit LC, Cocks RA. Derivation of a prediction rule for posttraumatic organ failure using plasma DNA and other variables. Ann N Y Acad Sci 2001;945:211–20. 16. Lam NY, Rainer TH, Chan LY, Joynt GM, Lo YM. Time course of early and late changes in plasma DNA in trauma patients. Clin Chem 2003;49:1286 –91. 17. Anker P, Lefort F, Vasioukhin V, Lyautey J, Lederrey C, Chen XQ, et al. K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer. Gastroenterology 1997;112:1114 –20. 18. Hibi K, Robinson CR, Booker S, Wu L, Hamilton SR, Sidransky D, et al. Molecular detection of genetic alterations in the serum of colorectal cancer patients. Cancer Res 1998;58:1405–7. 19. Chiang PW, Beer DG, Wei WL, Orringer MB, Kurnit DM. Detection of erbB-2 amplifications in tumors and sera from esophageal carcinoma patients. Clin Cancer Res 1999;5:1381– 6. 20. Capone RB, Pai SI, Koch WM, Gillison ML, Danish HN, Westra WH, et al. Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV-associated head and neck squamous cell carcinoma. Clin Cancer Res 2000;6:4171–5.

21. Lo YM. Quantitative analysis of Epstein-Barr virus DNA in plasma and serum: applications to tumor detection and monitoring [Review]. Ann N Y Acad Sci 2001;945:68 –72. DOI: 10.1373/clinchem.2004.034553

B-Type Natriuretic Peptide (BNP) and N-Terminal proBNP in Patients with End-Stage Heart Failure Supported by a Left Ventricular Assist Device, Hans Kemperman,1* Mery van den Berg,1 Hans Kirkels,2 and Nicolaas de Jonge2 (1 Department of Clinical Chemistry and 2 Heart Lung Center Utrecht, University Medical Center Utrecht, Utrecht, The Netherlands; * address correspondence to this author at: UMC Utrecht, Department of Clinical Chemistry, Room G03.550, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands; fax 31-30-2505418, e-mail [email protected]) B-Type natriuretic peptide (BNP) is a cardiac neurohormone synthesized in the cardiac ventricles. It is released as a preproBNP peptide of 134 amino acids and is cleaved into proBNP (108 amino acids) and a signal peptide of 26 amino acids. ProBNP is subsequently cleaved into BNP (32 amino acids) and the inactive N-terminal proBNP peptide (NT-proBNP; 76 amino acids). The release of BNP into the circulation is directly proportional to the ventricular expansion and volume overload of the ventricles and therefore reflects the decompensated state of the ventricles (1 ). The effects of BNP—vasodilatation, natriuresis, and diuresis—lead to some improvement of the loading conditions of the failing heart. Although BNP is the active neurohormone, both BNP and NT-proBNP have been described as useful markers for the diagnosis and exclusion of congestive heart failure (2, 3 ), and plasma concentrations correlate with the functional classification of patients according to the New York Heart Association (NYHA) (2, 4 ). Therapy guided by BNP leads to a reduction of total cardiovascular events and delayed time to a new event when compared with clinically guided treatment (5 ). In the Heart Lung Center of the University Medical Center Utrecht, The Netherlands, each year ⬃25 patients with end-stage heart failure receive a donor heart. Because of a lack of donor organs, which has led to increased waiting periods, mechanical assist devices to bridge the period to transplantation are frequently used for the sickest patients. The Heartmate (Thoratec) left ventricular assist device (LVAD) used in this study is an implantable pump that connects the apex of the left ventricle and the base of the aorta (6 ). Previously, others have studied plasma BNP concentrations in patients supported by ventricular assist devices. Sodian et al. (7 ) showed that BNP concentrations decreased in 21 patients with end-stage heart failure after implantation of a ventricular assist device. Furthermore, they suggested that an early decrease in BNP might be an

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