Alternate methods to prevent protease use as a masking agent in sport
2010; Elsevier BV; Volume: 13; Issue: 5 Linguagem: Inglês
10.1016/j.jsams.2010.02.003
ISSN1440-2440
AutoresFabián Sanchís-Gomar, Vladimir E. Martínez-Bello, Ana Lúcia Rosa Nascimento, Mari Carmen Gómez‐Cabrera, José Viña,
Tópico(s)Doping in Sports
ResumoThe World Anti-doping Agency (WADA) has recently updated the 2010 prohibited list.1WADA The 2010 prohibited list of substances international standard.2010www.wada-ama.orgGoogle Scholar Among the main modifications made to the rules and in the section entitled prohibited methods (chemical and physical manipulations), the code literally states: "Tampering, or attempting to tamper, in order to alter the integrity and validity of Samples collected during Doping Controls is prohibited. These include but are not limited to catheterisation, urine substitution and/or adulteration (e.g. proteases)". This modification in the prohibited list reflects the anti-doping authorities' concern about the use of proteases to manipulate urine samples. There is evidence showing that proteases are being used by athletes as masking agents to defeat the erythropoietin (EPO) urine test.2Elliott S. Erythropoiesis-stimulating agents and other methods to enhance oxygen transport.Br J Pharmacol. 2008; 154: 529-541Crossref PubMed Scopus (83) Google Scholar This became evident when undetectable EPO profiles in ±15% of EPO tests were observed.3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar, 4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar Although some samples have low levels of eEPO (endogenous EPO) because of high hematocrit concentrations, other samples may have been manipulated. Small quantities of proteases can degrade EPO in urine after only a short period of time and protease addition to a sample during the collection process could therefore destroy both eEPO and rHuEPO (recombinant human EPO), giving a negative urine test result.2Elliott S. Erythropoiesis-stimulating agents and other methods to enhance oxygen transport.Br J Pharmacol. 2008; 154: 529-541Crossref PubMed Scopus (83) Google Scholar, 3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google ScholarA number of strategies have been suggested by different authors to avoid using proteases to alter urine samples, such as (1) direct tests for protease activity in urine samples2Elliott S. Erythropoiesis-stimulating agents and other methods to enhance oxygen transport.Br J Pharmacol. 2008; 154: 529-541Crossref PubMed Scopus (83) Google Scholar, 4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar, 5Thomas A. Kohler M. Walpurgis K. Schänzer W. Thevis M. Proteolysis and autolysis of proteases and the detection of degradation products in doping control.Drug Test Anal. 2009; 1: 81-86Crossref PubMed Scopus (20) Google Scholar; (2) optimisation of the timing of urine collection: preference should be given to early morning samples, which are the most concentrated. Therefore, out-of-competition tests should be recommended3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar; (3) in the case of undetectable samples, endogenous urine albumin may be directly used as a protein marker to determine whether a protease has been added to the urine sample3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar; (4) protease inhibitors, antibiotics, and antimycotic substances should be systematically included in urine anti-doping collection procedures3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar, 6Tsivou M. Dimopoulou H.A. Leontiou I.P. Georgakopoulos D.G. Koupparis M.A. Atta-Politou J. et al.Stabilization of human urine doping control samples: III. Recombinant human erythropoietin.Clin Chim Acta. 2009; PubMed Google Scholar; (5) all parties involved in the urine collection procedure should wash their hands thoroughly before starting and should wear disposable gloves during the sample collection.We consider that some of these strategies have weak points and in our opinion the anti-doping authorities should take other suggestions into account to stop the use of proteases in sports.We agree with Lamon et al.3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar who recommend an increase in the number of out-of-competition tests as well as the increase of morning sample collections. However, we believe the strategy of washing hands and use of latex gloves by the athlete, although necessary, could prove useless. It has been reported that some athletes introduce proteases into the urethra before delivering a doping control urine sample.4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar Thus, hand washing and use of gloves would have no effect.Regarding the analysis of endogenous urine albumin as a protein marker to determine the use of proteases,3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar it should be stated that albuminuria is not a physiological process. It can appear in association with some pathologies7de Jong P.E. Gansevoort R.T. Bakker S.J. Macroalbuminuria and microalbuminuria: do both predict renal and cardiovascular events with similar strength?.J Nephrol. 2007; 20: 375-380PubMed Google Scholar, 8Halimi J.M. Hadjadj S. Aboyans V. Allaert F.A. Artigou J.Y. Beaufils M. et al.Microalbuminuria and urinary albumin excretion: French clinical practice guidelines.Diabetes Metab. 2007; 33: 303-309Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar and it has also been described after a bout of exhaustive physical exercise.9Alvarez C. Mir J. Obaya S. Fragoso M. Hematuria and microalbuminuria after a 100 kilometer race.Am J Sports Med. 1987; 15: 609-611Crossref PubMed Scopus (8) Google Scholar, 10Beullens M. Delanghe J.R. Bollen M. False-positive detection of recombinant human erythropoietin in urine following strenuous physical exercise.Blood. 2006; 107: 4711-4713Crossref PubMed Scopus (52) Google Scholar, 11Moinuddin I. Leehey D.J. A comparison of aerobic exercise and resistance training in patients with and without chronic kidney disease.Adv Chronic Kidney Dis. 2008; 15: 83-96Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Strenuous exercise can enhance protein excretion in urine by increasing glomerular permeability and decreasing tubular reabsorption.12Poortmans J.R. Brauman H. Staroukine M. Verniory A. Decaestecker C. Leclercq R. Indirect evidence of glomerular/tubular mixed-type postexercise proteinuria in healthy humans.Am J Physiol. 1988; 254: F277-283PubMed Google Scholar, 13Poortmans J.R. Labilloy D. The influence of work intensity on postexercise proteinuria.Eur J Appl Physiol Occup Physiol. 1988; 57: 260-263Crossref PubMed Scopus (64) Google Scholar, 14Lasne F. Martin L. Martin J.A. de Ceaurriz J. Detection of continuous erythropoietin receptor activator in blood and urine in anti-doping control.Haematologica. 2009; 94: 888-890Crossref PubMed Scopus (37) Google Scholar However, the use of albumin as a marker could be inconclusive.Regarding the use of protease inhibitors, antibiotics, and antimycotic substances, a great number of proteases and protease cocktails exist15Barrett A.J. Rawlings R.D. Woessner J.F. Neil R.D. The handbook of proteolytic enzymes.2nd ed. Academic Press, 2003Google Scholar making it difficult to inhibit all of them completely. Moreover, we consider that before allowing the use of protease inhibitors in anti-doping tests, a study should be performed to check the possible effects that these substances would have on other metabolites when a sample is analysed.An alternative method is the detection of proteases in athletes' urine samples.4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar It has been demonstrated that concentrations around 15 μg/mL of proteases can be detected.4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar, 5Thomas A. Kohler M. Walpurgis K. Schänzer W. Thevis M. Proteolysis and autolysis of proteases and the detection of degradation products in doping control.Drug Test Anal. 2009; 1: 81-86Crossref PubMed Scopus (20) Google Scholar However, a quantity of 500 μg of trypsin (5 μg/mL in a urine sample of 100 mL) could destroy all the eEPO and rHuEPO existent in the urine sample without raising suspicion.3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar Moreover, not all proteases can be detected. Different authors have determined the content of bacillolysin, chymotrypsin, subtilisin, trypsin, and papain in urine.4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar, 5Thomas A. Kohler M. Walpurgis K. Schänzer W. Thevis M. Proteolysis and autolysis of proteases and the detection of degradation products in doping control.Drug Test Anal. 2009; 1: 81-86Crossref PubMed Scopus (20) Google Scholar New methodologies should be implemented to detect other proteases potentially used by athletes to mask doping.The anti-doping authorities currently face a challenge regarding the detection of urine samples manipulation through the use of proteases. We believe that a viable, relatively simple, quick to implement and inexpensive solution which would stop the use of proteases in sports could be to make the athlete (if suspected) give two consecutive samples in two different containers: the first being a small amount (for instance 20 mL) which would be discarded (or kept for comparison) and would serve to clean the urethra; and the second (of approximately 100 mL) that would be processed as a valid sample and divided into sample A and sample B.Even so, some of the proteases in the urethra could remain after the "cleansing process", however, this procedure would also serve as a deterrent because the athlete would lose the confidence proteases provide by testing negative.In conclusion, the fight against rHuEPO detection in urine samples is complex and it must go on. We hope that the two consecutive sample method can be a step towards this aim. The World Anti-doping Agency (WADA) has recently updated the 2010 prohibited list.1WADA The 2010 prohibited list of substances international standard.2010www.wada-ama.orgGoogle Scholar Among the main modifications made to the rules and in the section entitled prohibited methods (chemical and physical manipulations), the code literally states: "Tampering, or attempting to tamper, in order to alter the integrity and validity of Samples collected during Doping Controls is prohibited. These include but are not limited to catheterisation, urine substitution and/or adulteration (e.g. proteases)". This modification in the prohibited list reflects the anti-doping authorities' concern about the use of proteases to manipulate urine samples. There is evidence showing that proteases are being used by athletes as masking agents to defeat the erythropoietin (EPO) urine test.2Elliott S. Erythropoiesis-stimulating agents and other methods to enhance oxygen transport.Br J Pharmacol. 2008; 154: 529-541Crossref PubMed Scopus (83) Google Scholar This became evident when undetectable EPO profiles in ±15% of EPO tests were observed.3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar, 4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar Although some samples have low levels of eEPO (endogenous EPO) because of high hematocrit concentrations, other samples may have been manipulated. Small quantities of proteases can degrade EPO in urine after only a short period of time and protease addition to a sample during the collection process could therefore destroy both eEPO and rHuEPO (recombinant human EPO), giving a negative urine test result.2Elliott S. Erythropoiesis-stimulating agents and other methods to enhance oxygen transport.Br J Pharmacol. 2008; 154: 529-541Crossref PubMed Scopus (83) Google Scholar, 3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar A number of strategies have been suggested by different authors to avoid using proteases to alter urine samples, such as (1) direct tests for protease activity in urine samples2Elliott S. Erythropoiesis-stimulating agents and other methods to enhance oxygen transport.Br J Pharmacol. 2008; 154: 529-541Crossref PubMed Scopus (83) Google Scholar, 4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar, 5Thomas A. Kohler M. Walpurgis K. Schänzer W. Thevis M. Proteolysis and autolysis of proteases and the detection of degradation products in doping control.Drug Test Anal. 2009; 1: 81-86Crossref PubMed Scopus (20) Google Scholar; (2) optimisation of the timing of urine collection: preference should be given to early morning samples, which are the most concentrated. Therefore, out-of-competition tests should be recommended3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar; (3) in the case of undetectable samples, endogenous urine albumin may be directly used as a protein marker to determine whether a protease has been added to the urine sample3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar; (4) protease inhibitors, antibiotics, and antimycotic substances should be systematically included in urine anti-doping collection procedures3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar, 6Tsivou M. Dimopoulou H.A. Leontiou I.P. Georgakopoulos D.G. Koupparis M.A. Atta-Politou J. et al.Stabilization of human urine doping control samples: III. Recombinant human erythropoietin.Clin Chim Acta. 2009; PubMed Google Scholar; (5) all parties involved in the urine collection procedure should wash their hands thoroughly before starting and should wear disposable gloves during the sample collection. We consider that some of these strategies have weak points and in our opinion the anti-doping authorities should take other suggestions into account to stop the use of proteases in sports. We agree with Lamon et al.3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar who recommend an increase in the number of out-of-competition tests as well as the increase of morning sample collections. However, we believe the strategy of washing hands and use of latex gloves by the athlete, although necessary, could prove useless. It has been reported that some athletes introduce proteases into the urethra before delivering a doping control urine sample.4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar Thus, hand washing and use of gloves would have no effect. Regarding the analysis of endogenous urine albumin as a protein marker to determine the use of proteases,3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar it should be stated that albuminuria is not a physiological process. It can appear in association with some pathologies7de Jong P.E. Gansevoort R.T. Bakker S.J. Macroalbuminuria and microalbuminuria: do both predict renal and cardiovascular events with similar strength?.J Nephrol. 2007; 20: 375-380PubMed Google Scholar, 8Halimi J.M. Hadjadj S. Aboyans V. Allaert F.A. Artigou J.Y. Beaufils M. et al.Microalbuminuria and urinary albumin excretion: French clinical practice guidelines.Diabetes Metab. 2007; 33: 303-309Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar and it has also been described after a bout of exhaustive physical exercise.9Alvarez C. Mir J. Obaya S. Fragoso M. Hematuria and microalbuminuria after a 100 kilometer race.Am J Sports Med. 1987; 15: 609-611Crossref PubMed Scopus (8) Google Scholar, 10Beullens M. Delanghe J.R. Bollen M. False-positive detection of recombinant human erythropoietin in urine following strenuous physical exercise.Blood. 2006; 107: 4711-4713Crossref PubMed Scopus (52) Google Scholar, 11Moinuddin I. Leehey D.J. A comparison of aerobic exercise and resistance training in patients with and without chronic kidney disease.Adv Chronic Kidney Dis. 2008; 15: 83-96Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar Strenuous exercise can enhance protein excretion in urine by increasing glomerular permeability and decreasing tubular reabsorption.12Poortmans J.R. Brauman H. Staroukine M. Verniory A. Decaestecker C. Leclercq R. Indirect evidence of glomerular/tubular mixed-type postexercise proteinuria in healthy humans.Am J Physiol. 1988; 254: F277-283PubMed Google Scholar, 13Poortmans J.R. Labilloy D. The influence of work intensity on postexercise proteinuria.Eur J Appl Physiol Occup Physiol. 1988; 57: 260-263Crossref PubMed Scopus (64) Google Scholar, 14Lasne F. Martin L. Martin J.A. de Ceaurriz J. Detection of continuous erythropoietin receptor activator in blood and urine in anti-doping control.Haematologica. 2009; 94: 888-890Crossref PubMed Scopus (37) Google Scholar However, the use of albumin as a marker could be inconclusive. Regarding the use of protease inhibitors, antibiotics, and antimycotic substances, a great number of proteases and protease cocktails exist15Barrett A.J. Rawlings R.D. Woessner J.F. Neil R.D. The handbook of proteolytic enzymes.2nd ed. Academic Press, 2003Google Scholar making it difficult to inhibit all of them completely. Moreover, we consider that before allowing the use of protease inhibitors in anti-doping tests, a study should be performed to check the possible effects that these substances would have on other metabolites when a sample is analysed. An alternative method is the detection of proteases in athletes' urine samples.4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar It has been demonstrated that concentrations around 15 μg/mL of proteases can be detected.4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar, 5Thomas A. Kohler M. Walpurgis K. Schänzer W. Thevis M. Proteolysis and autolysis of proteases and the detection of degradation products in doping control.Drug Test Anal. 2009; 1: 81-86Crossref PubMed Scopus (20) Google Scholar However, a quantity of 500 μg of trypsin (5 μg/mL in a urine sample of 100 mL) could destroy all the eEPO and rHuEPO existent in the urine sample without raising suspicion.3Lamon S. Robinson N. Sottas P.E. Henry H. Kamber M. Mangin P. et al.Possible origins of undetectable EPO in urine samples.Clin Chim Acta. 2007; 385: 61-66Crossref PubMed Scopus (39) Google Scholar Moreover, not all proteases can be detected. Different authors have determined the content of bacillolysin, chymotrypsin, subtilisin, trypsin, and papain in urine.4Thevis M. Maurer J. Kohler M. Geyer H. Schänzer W. Proteases in doping control analysis.Int J Sports Med. 2007; 28: 545-549Crossref PubMed Scopus (40) Google Scholar, 5Thomas A. Kohler M. Walpurgis K. Schänzer W. Thevis M. Proteolysis and autolysis of proteases and the detection of degradation products in doping control.Drug Test Anal. 2009; 1: 81-86Crossref PubMed Scopus (20) Google Scholar New methodologies should be implemented to detect other proteases potentially used by athletes to mask doping. The anti-doping authorities currently face a challenge regarding the detection of urine samples manipulation through the use of proteases. We believe that a viable, relatively simple, quick to implement and inexpensive solution which would stop the use of proteases in sports could be to make the athlete (if suspected) give two consecutive samples in two different containers: the first being a small amount (for instance 20 mL) which would be discarded (or kept for comparison) and would serve to clean the urethra; and the second (of approximately 100 mL) that would be processed as a valid sample and divided into sample A and sample B. Even so, some of the proteases in the urethra could remain after the "cleansing process", however, this procedure would also serve as a deterrent because the athlete would lose the confidence proteases provide by testing negative. In conclusion, the fight against rHuEPO detection in urine samples is complex and it must go on. We hope that the two consecutive sample method can be a step towards this aim.
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