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00005768-200404000-0000600005768_2004_36_588_nissen_profiling_4article< 98_0_16_5 >Medicine & Science in Sports & Exercise©2004The American College of Sports MedicineVolume 36(4)April 2004pp 588-593Serum sTfR Levels May Indicate Charge Profiling of Urinary r-hEPO in Doping Control[BASIC SCIENCES: Original Investigations]NISSEN-LIE, GRO; BIRKELAND, KÅRE; HEMMERSBACH, PETER; SKIBELI, VENKEHormone Laboratory, Section for Doping Analyses, Aker University Hospital, Oslo, NORWAYAddress for correspondence: Gro Nissen-Lie, Section for Doping Analysis, Hormone Laboratory, Aker University Hospital, 0514 Oslo, Norway; E-mail: for publication August 2003.Accepted for publication November 2003.ABSTRACTNISSEN-LIE, G., K. BIRKELAND, P. HEMMERSBACH, and V. SKIBELI. Serum sTfR Levels May Indicate Charge Profiling of Urinary r-hEPO in Doping Control. Med. Sci. Sports Exerc., Vol. 36, No. 4, pp. 588–593, 2004.Purpose: The aim of the study was to demonstrate whether changes in the charge pattern of urinary human erythropoietin (u-hEPO) from well-trained athletes before, during and after controlled administration of recombinant human EPO (r-hEPO) could be related to altered levels of hemoglobin (Hb), hematocrit (Hct), soluble transferrin receptor (sTfR) and maximal oxygen uptake (V̇O2max).Methods: Urinary samples from athletes in an EPO-receiving group and a control group were collected before, during and after r-hEPO administration. The samples were analyzed with respect to the charge pattern of hEPO by iso-electric focusing (IEF).Results: The charge of the u-hEPO variants shifted from an acidic to a more basic pattern after initiating r-hEPO administration. This shift appeared together with increased levels of sTfR, and appeared before increased levels of Hb, Hct and V̇O2max. Until three days after the last injection, the IEF profiles were similar to the charge profile of r-hEPO. Thereafter the levels of sTfR decreased and the charge profiles of the hEPO variants gradually became more acidic. In contrast, the levels of Hb, Hct and V̇O2max remained elevated for an extended period of time.Conclusion: A significant correlation was found between the relative amount of basic u-hEPO variants and the relative levels of sTfR, demonstrating that the relative levels of sTfR may be used as a marker to select urinary samples for further analysis of r-hEPO by IEF in routine doping control.Human EPO is a glycoprotein primarily produced in the kidneys (7). The hormone is the main regulator of erythropoiesis (12). Human EPO consists of a single 165-amino acid polypeptide chain with three N-glycosylation sites and one O-glycosylation site. The average carbohydrate content is approximately 40% (12). Like other glycoproteins, EPO exhibits considerably heterogeneity, and a spectrum of variants is present in both serum and urine (14,26,28,29). These glycoform populations have been shown to be cell and species as well as polypeptide and site specific (21).The human EPO gene was first cloned in 1985 (11,16) and r-hEPO has been available as a drug since 1988. The misuse of r-hEPO in sport has been suspected for many yr, and in 2000 the existence of r-hEPO in urine from an athlete was demonstrated for the first time (14). Studies have shown that the use of r-hEPO promote enhanced performance capacity in athletes (2,3,6). The International Olympic Committee (IOC) prohibited the use of r-hEPO in 1990. However, at that time no method was available for the detection of r-hEPO neither in blood nor urine. Both indirect and direct methods have been explored to detect misuse of r-hEPO in sports. Wide et al. first reported a direct detection of r-hEPO in both serum and urine in 1995 (29), clearly demonstrating that r-hEPO was less negatively charged than endogenous EPO in healthy individuals. By analyzing the sugar part of hEPO Skibeli et al. demonstrated significant differences between human serum EPO and r-hEPO (26). A method for the direct detection of r-hEPO in urine was introduced for routine doping control in 2001 (14).Because hematologic parameters are quite stable over time in healthy individuals, these variables have been used to show an abnormal altered erythropoiesis that forms the basis for the indirect methods of detecting EPO abuse (4,5,9,22). On the other hand, many conditions, among them high altitude, may also influence erythropoiesis (1). Therefore, direct detection of r-hEPO in the urine is to prefer as the final proof of r-hEPO abuse. As the IEF urinary method is rather expensive and time consuming, limiting the number of samples for analysis, the need for selecting interesting samples for determining r-hEPO is apparent. At present, the concentration of Hb and the amount of reticulocytes are the most frequently used variables in the screening for athletes to be tested with the direct urinary method.In the present investigation samples from well-trained athletes treated with r-hEPO were monitored with respect to the charge pattern of r-hEPO before, during and after r-hEPO administration. These results were compared to the corresponding levels of sTfR, Hb, Hct and V̇O2max. To our knowledge this is the first time such studies have been performed.MATERIALS AND METHODSAdministration of r-hEPO.From a placebo-controlled clinical trial with the administration of r-hEPO previously published (3), we selected five subjects that received r-hEPO and three subjects in the placebo control group for the present study. The well-trained male athletes received 5000 U r-hEPO (NeoRecormon, Boehringer Mannheim GmbH, Mannheim, Germany) 3× wk−1 for 1 month (181–232 U·kg−1·wk−1) (3). The subjects, including the placebo-treated athletes, were informed in writing and verbally, and gave written informed consent to participate. The study was approved by the Regional Ethics Committee of the Norwegian Research Council for Science and Humanities and the Norwegian Medicines Control Authority.Blood and urine sampling.Blood and urine samples were collected immediately before start of treatment, 3× wk−1 during the treatment period, daily for the first 5 d after the end of treatment, and subsequently 2× wk−1 during the rest of the posttreatment period (3).Measurement of physiological variables.EDTA tubes were used for collection of blood for measurement of Hb concentration and Hct with a Sysmex K-100 Cell Counter (Toa Medical Electronic Comp. Ltd, Kobe, Japan). Serum was made from 5 mL blood samples, and sTfR concentration was measured with an immunoenzymometric method (Orion Diagnostica, Espoo, Finland). Further details of these measurements and description of maximal oxygen uptake have been described previously (3).Urine sample preparation.Twenty milliliters of urine were reduced to a volume of 50 μL using several steps of filtration and centrifugation as described by Lasne et al. (15).Isoelectric focusing (IEF).Charge pattern of hEPO in the concentrated urine were analyzed by IEF according to Lasne (13). Twenty microliters of the urine sample were saturated with urea (Amersham Biosciences, United Kingdom) before IEF.Immunoblotting.Proteins from the IEF gel were blotted onto a polyvinylidene difluoride membrane (PVDF) according to Lasne (13). A mouse antihuman EPO antibody (Clone AE7A5, R&D Systems, Oxford, United Kingdom) was used as the primary antibody. EPO were visualized with a chemiluminescent substrate (Covalight, Covalab, Lyon, France) and the signals detected in a CCD (Charge Coupled Device) camera (LAS 1000 Plus, Fuji film, Stockholm, Sweden).Estimation of the content of basic variants.The chemiluminescent signals, representing the amount of EPO blotted onto the membranes, were transformed into intensity profiles by means of the software Aida 2.43. (Raytest, Straubenhardt, Germany). The percent basic EPO variants calculations are defining the amount of EPO in the urinary sample that is corresponding to the mobility of a standard preparation of r-hEPO relative to the total amount of EPO as described by Pascual et al. (23)Statistics.Values for correlation and linear regression analyses were related to the levels of day 1 in the experiment (defined as 1), representing the amount of basic variants and physiological variables before the r-hEPO administration was initiated. Coefficients of correlation were calculated according to Pearson assuming normal distribution of data. Regression analysis was performed according to Passing and Bablok (24). P-values <0.05 were considered significant.RESULTSCharge patterns of r-hEPO in urine from athletes.Analyses by IEF of urinary samples from five well-trained athletes on r-hEPO administration, demonstrated that the charge pattern of EPO became more basic after 3 d of treatment. Ten days posttreatment, the charge pattern resumed to the original shape. Figure 1A depicts the IEF pattern of urinary EPO from one athlete. On day 1 (before start of treatment with r-hEPO), 10–15 detectable urinary EPO variants were present in the acidic part of the pH gradient. During the r-hEPO administration the charge pattern of hEPO gradually changed, and bands in the more basic part of the pH gradient showed increased intensity, while they faded away in the acidic part (Fig. 1A). After 2 wk of treatment the charge pattern of EPO in urine from the athletes compared closely to the pattern of r-hEPO, including 4–5 major bands. This pattern remained during the rest of the treatment period. After cessation of treatment the pattern remained for 3 d and subsequently the intensity of the more basic variants decreased and acidic variants increased again. Ten to 14 d posttreatment the EPO variants had resumed the original endogenous pattern. Figure 1B shows the calculated percent basic EPO variants represented in Figure 1A. The first day of the trial period (day 1) the calculated amount of basic variants for the five athletes varied between 25% and 40%. After approximately 3 wk, the amount of basic urinary variants had reached an amount of basic variants between 88% and 95%. Three days posttreatment (day P3) the amount of basic variants was decreased (between 80% and 93%). Two weeks posttreatment, the amount of basic variants had resumed the normal levels for all athletes.FIGURE 1— Charge patterns of urinary hEPO from one athlete during controlled administration of r-hEPO. A. Demonstrates the separation of urinary hEPO variants by IEF followed by immunoblotting. A mouse antihuman EPO antibody (Clone AE7A5, R&D Systems, Oxford, United Kingdom) was used as the primary antibody. Erythropoietin BRP (Batch No.1) from Council of Europe European Pharmacopoeia Commission (equal amounts of epoetin alfa and beta) was used as a standard. B. Demonstrates the flow of percent basic forms in urine from the athlete during the administration study, calculated on the basis of the pattern in A. One to 19 indicate the number of days after the initiation of r-hEPO administration, and P1–14 indicate the number of days after the cessation of r-hEPO treatment.The urinary charge pattern of EPO from athletes receiving placebo was analyzed on IEF. The charge distribution of urinary EPO from one athlete is depicted in Figs. 2A and B. The charge analyses of urinary hEPO showed a pattern stretching towards the acidic part of the pH gradient corresponding to the endogenous hEPO pattern throughout the whole trial period. The calculated values of basic variants through the trial period varied from 32% to 47%, all demonstrating an endogenous hEPO charge pattern.FIGURE 2— Charge patterns of urinary hEPO in one athlete receiving placebo. A. Shows the separation of urinary hEPO variants obtained from one athlete in the placebo group by IEF followed by immunoblotting. A mouse antihuman EPO antibody (Clone AE7A5, R&D Systems, Oxford, United Kingdom) was used as the primary antibody. Erythropoietin BRP (Batch No.1) from Council of Europe European Pharmacopoeia Commission (equal amounts of epoetin alfa and beta) was used as a standard. B. Shows the relative amount of basic variants in the placebo urines. One to 29 indicate the number of days after the initiation of r-hEPO administration, and P1–28 indicate the number of days after the secession of r-hEPO treatment.Figure 3A depicts the mean values of basic variants from all five athletes. Endogenous hEPO (day 1) revealed proportions of basic forms with a mean value of 32%. Nine days after the start of the r-hEPO administration (day 9) the amount of basic variants had increased to 80%. The maximum amounts of basic bands were observed on day P1 with a mean value of 94%, and above 90% for all the athletes. Three days after the end of treatment (day P3) the amount of basic forms was 89%. Seven days after the end of the r-hEPO administration (day P7), all urines contained hEPO with less than 70% basic forms with a mean value of 59%.FIGURE 3— Calculated levels of more basic variants compared to the measured levels of sTfR and Hb during controlled administration of r-hEPO. Values are mean with SE 1–28 indicate the number of days after the initiation of r-hEPO administration and P1–14 indicate the number of days after the cessation of r-hEPO treatment. Significant differences are marked with: *P < 0.02, #P < 0.05.The intensity profiles in Figure 4 represent the intensity of the different variants of urinary hEPO from one athlete before, during and after treatment with r-hEPO. The figure clearly demonstrates the shift from more basic profiles during r-hEPO administration and the subsequent return to the endogenous charge pattern after the end of r-hEPO administration. Three days after r-hEPO administration (day 3) the profiles showed an increase in the intensity of the peaks revealing the more basic urinary hEPO variants. Twenty-two days after start of r-hEPO treatment (day 22) the profile corresponded to the profile of r-hEPO. Posttreatment (P3 and P5) the intensity of the peaks corresponding to the more basic variants decreased in contrast to the increasing peaks representing the acidic variants. Ten days postadministration (day P10) the urinary hEPO profile had resumed the endogenous profile pattern.FIGURE 4– Shift in the charge pattern of hEPO from one athlete shown as intensity profiles during administration of r-hEPO. The intensity profiles from a CCD camera were obtained by analyzing the hEPO pattern from IEF by the software Aida 2.43. One to 22 indicate the number of days after the initiation of r-hEPO administration, and P1–10 indicate the number of days after the cessation of r-hEPO treatment.Comparison between the direct analysis of urinary hEPO and indirect variables of erythropoiesis.The content of basic hEPO variants in urine from five athletes was compared to the levels of several indirect variables related to erythropoiesis. The serum levels of sTfR increased rapidly after the initiation of r-hEPO treatment and remained elevated during the treatment period, similar to the levels of basic EPO variants, and until 1 wk after cessation of treatment (Fig. 3). Correlation and linear regression analysis of the relationship between the relative values of basic variants and sTfR levels showed a significant correlation with a correlation coefficient of 0.65 (P < 0.0001) (Fig. 5). This increased sTfR level appeared when elevated amount of more basic forms were present in the urine. The blood levels of Hct and Hb, as well as the V̇O2max, rose gradually during 4 wk of r-hEPO treatment. After the end of r-hEPO administration V̇O2max stayed elevated for up to 3 wk and similarly, the levels of Hct and Hb gradually decreased and had still not reached the normal levels 4 wk posttreatment (3). This was in contrast to the urinary basic variants of EPO that decreased significantly more rapidly and were normalized after approximately 10–14 d (Fig. 3). Figure 3 demonstrates how the levels of Hb and the amount of basic EPO variants were regulated differently throughout the course of the experiment. Correlation and linear regression analysis of the relationship between the relative values of basic variants and relative values of Hb, Hct and V̇O2max, showed no association between these variables.FIGURE 5— The relationship between the urinary charge pattern of hEPO in urine and levels of sTfR in plasma during controlled administration of r-hEPO. The plotted values were set relative to the levels of day 1 in the experiment. The regression analyses were done according to Passing and Bablok (24).DISCUSSIONTo our knowledge this is the first time that the charge profile of urinary hEPO have been monitored and compared to changes in hematological variables during administration of r-hEPO to well-trained athletes.The observed shift in the charge pattern of urinary hEPO after the start of r-hEPO administration reflects the increased amounts of excreted r-hEPO and a suppressed endogenous hEPO production. As a consequence the acidity of the charge pattern decreased, demonstrating increased similarity to the more basic profile of r-hEPO. A shift to more basic charge profiles of urinary EPO after treatment with r-hEPO was previously shown in other studies (14,15,28,29). Wide (28,29) demonstrated a modification of hEPO patterns to a less acidic charge pattern after r-hEPO treatment in both serum and urine. Lasne (14) confirmed the charge discrepancy between natural u-hEPO and r-hEPO observed by Wide (29) and showed for the first time a charge pattern of r-hEPO in urine from athletes suspected of r-hEPO doping. In the present study the relative amount of basic urinary hEPO variants remained elevated during the treatment period. However, some faint acidic bands were visible in the lanes, stretching towards the acidic end of the pH-gradient beyond the r-hEPO bands. These may be due to endogenous variants not fully suppressed, or alternatively be caused by a charge modification of r-hEPO during renal clearance as has previously been shown for s-hEPO demonstrating an approximately 30% change to more acidic forms in the urine (20). The present findings were supported by Wide et al. who reported a significant acidification of excreted r-hEPO (29). However, in a recent report Lasne et al. (15) indicated the appearance of only the recombinant forms in urine from an individual injected several times with r-hEPO. After the cessation of r-hEPO treatment, the charge pattern of hEPO became more acidic, gradually normalizing, as less r-hEPO was secreted into the urine. Wide et al. (29) demonstrated a charge shift back to the normal pattern 2 d posttreatment, while in the current study the charge profile 2 d after cessation of treatment was still corresponding to r-hEPO. This might be explained by the higher amount of r-hEPO administrated in the present study.The observed minor charge pattern variations of u-hEPO from the athletes in the placebo group were probably due to natural modifications of endogenous hEPO. Wide et al. (28) reported that the charge of serum hEPO varies during the day, as shown by less negatively charged hEPO in samples collected in the evening when compared with samples collected in the morning.IEF analysis of urinary hEPO and r-hEPO have led to different conclusions (10,14,15,27). The acidic charge pattern of u-hEPO demonstrated in the present study was also shown by Lasne (14,15). The acidic IEF pattern of u-hEPO is difficult to explain by the present knowledge of the molecular structure of both natural and r-hEPO. Further structural investigations have to be performed to elucidate the nature of the charge discrepancy between endogenous and r-hEPO.The positive correlation between the relative amount of basic variants of urinary hEPO and the relative levels of sTfR in serum, shown in the present study, suggests the feasibility of using this variable as an indicator for shifted urinary charge pattern. sTfR is a strong indicator of increased erythropoiesis (2,3,8,22). Nevertheless, the use of increased levels of sTfR as proof for r-hEPO misuse were found to be of limited value by Birkeland et al. (3) due to large variations of basal levels in the population, and also to effects related to high altitude, training status, ethnicity and other physiological and pathological states. Nicolaidis et al. reported that the intra-individual day-to-day variation in levels of sTfR might be as large as 23% (19). Furthermore, quantification of sTfR is assay dependent, as several methods report different normal ranges (30), rendering the use of measured levels of sTfR as proof of r-hEPO misuse even more uncertain. We therefore suggest that selecting samples for r-hEPO determination in urine could be based on a shift in sTfR levels relative to a method based reference value.Improved endurance capacity has in sport medicine been related to increased Hb concentration leading to increased oxygen delivery. Previous studies demonstrated that V̇O2max levels remained elevated after 3 to 4 wk of r-hEPO administration (3,22,25). As a consequence, neither the relative levels of sTfR in serum nor the amount of basic hEPO variants in urine actually reflect the whole period with increased oxygen delivery resulting from r-hEPO abuse.The use of repeated monitoring of blood parameters in athletes has been suggested (17). A significant change in the Hb levels, indicating an elevated erythropoiesis, should subsequently lead to a further investigation of the athlete. The lack of significant correlation between the changes of Hb levels and changes in the amount of basic variants indicates that monitoring of Hb is not the best variable to select samples for further analyses of r-hEPO in urine.In the present study the increase in the levels of Hb and Hct were found to be delayed compared to the increased amount of urinary basic hEPO variants detected during r-hEPO treatment. On the other hand, the increase in basic variants corresponds with the increased levels of sTfR in this period. The more rapid response in sTfR after r-hEPO administration, as compared to Hb, was previously described by Nakanishi et al. (18). Four weeks posttreatment, the Hb and Hct levels were still significantly elevated in contrast to the amount of basic variants in urine. Because the Hb level, both in the start of EPO administration and postadministration, was significantly delayed compared to basic EPO variants and the level of sTfR, its use to predict the detection of r-hEPO intake by IEF is limited. However, by using the relative levels of sTfR to select samples the likelihood to detect r-hEPO in the urine would be much higher because of a positive correlation between the course of sTfR and urinary basic EPO. The urine test performed in the laboratory today is expensive and time-consuming. A more precise screening of blood samples increase the possibility of selecting the correct samples for further spotting of r-hEPO by the existing IEF method, decreasing the high costs, and hopefully leading to more efficient detection of EPO abuse.“Second generation” blood tests to detect r-hEPO abuse that include levels of Hb, EPO, and sTfR have recently been developed with increased sensitivity compared to the “first generation” models (9). One of these models may be in better agreement with the elevated amount of basic EPO variants than the models previously proposed (22). This ON-hes model makes it possible to select samples without the need of defined baseline values. Our results indicate that sTfR measurements alone may be sufficient for sample selection for r-hEPO determination.The present, as well as previous results, underline the importance in choosing the right timing for doping control to disclose r-hEPO misuse. It is of utmost importance to favor urinary EPO-tests out-of-competition in the period before big events. Moreover, blood parameters that correlate with basic EPO variants in urine during the whole course of r-hEPO treatment are most suitable for selecting samples for further analysis of urinary EPO. The significant correlation between the relative amounts of sTfR and basic variants reveal the usefulness including the levels of sTfR in longitudinal personal profiles to screen for urine samples to be tested for r-hEPO by the IEF method used in routine doping control.The study was supported by: The Norwegian Research Council, the Norwegian Department for Cultural Affairs and the International Olympic Committee. We thank Mette Borgen for skilful technical help. We are also grateful to Dr. Peter Torjesen for valuable comments.REFERENCES1. Ashenden, M. J., C. J. Gore, R. Parisotto, K. Sharpe, W. G. Hopkins, and A. G. Hahn. 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BIRKELAND, KÅRE; HEMMERSBACH, PETER; SKIBELI, VENKEBASIC SCIENCES: Original Investigations436InternalMedicine & Science in Sports & Exercise10.1249/mss.0b013e31811e9d55200739101742-1747OCT 2007Detection of Darbepoetin Alfa Misuse in Urine and Blood: A Preliminary InvestigationMORKEBERG, J; LUNDBY, C; NISSEN-LIE, G; NIELSEN, TK; HEMMERSBACH, P; DAMSGAARD, R