Detection of Diuretics in Urine During Sports Events in Taiwan

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1 ORIGINAL ARTICLE Diuretics abuse in sports Detection of Diuretics in Urine During Sports Events in Taiwan Ying-Lung Tseng, Min-Hua Shieh, Chien-Tsong Lin, Fang-Hsin Kuo Institute of Pharmacology and Toxicology and Doping Control Center, Tzu Chi University, Hualien, Taiwan ABSTRACT Objective: This report presents analytical procedures for detecting banned diuretics in urine of athletes and details the incidences of violations that occurred in the doping control process in Taiwan from 1998 to Materials and Methods: For screening, urinary samples first underwent acidic and basic extractions before being analyzed by high-performance liquid chromatography (HPLC). Subsequently, if a sample was found to be suspicious for a banned substance, analysis by gas chromatography-mass spectrometry (GC-MS) was undertaken for further confirmation. Results: Of the 5,817 urine samples tested, 12 samples (0.2%) were found to contain banned diuretics, including hydrochlorothiazide (n = 8) and furosemide (n = 4). Among positive cases, sports classified by weight accounted for approximately 70% of the positive cases, in which taekwondo accounted for 33% (n = 4) and weightlifting for 25% (n = 3). Conclusions: The results demonstrate that the abuse of diuretics was primarily occurred in sports with weight classes in Taiwan. (Tzu Chi Med J 2004; 16:69-77) Key words: doping control, diuretics, HPLC, GC-MS INTRODUCTION Clinically, diuretics are used for the treatment of hypertension, congestive heart failure, nephrogenic diabetes insipidus, hypercalciuria, glaucoma, and edema, among other diseases [1,2]. General side effects resulting from diuretic use may lead to fatigue, drowsiness, muscle cramps or soreness, and a sensation of numbness, tingling, or prickling; serious side effects, caused by potent diuretics, may lead to changes in electrolyte balance and impairment of the body's thermoregulation system [3,4]. In sports, athletes misuse diuretics for several reasons: (1) to reduce body weight in order to qualify for a lower weight-class (such as in boxing, weightlifting, and judo), (2) to reduce the urinary concentration of other prohibited substances to avoid a positive doping result, and (3) to overcome fluid retention caused by the use of anabolic steroids [5-8]. Therefore, the International Olympic Committee (IOC) banned the use of diuretics in sports in 1988 [6]. From the 1968 to 2000 Olympic Games, 59 violations (0.5%) were reported out of 13,105 samples tested. Among the positive cases, diuretics (14%) ranked third behind anabolic steroids (54%) and stimulants (29%). In Taiwan, 4,220 urine samples were tested in sports from 1998 to 2001, among which 61 positive cases (1.4%) were found. Among the positive cases, diuretics also ranked third at 18%, behind stimulants (56%) and anabolic steroids (26%) (unpublished data). In this study, we present our test results in athletes who were either tested during a competition or out-ofcompetition under doping control regulations in Taiwan from 1998 to The analytical procedures carried out by HPLC and GC-MS are described. MATERIALS AND METHODS Received: July 23, 2003, Revised: August 11, 2003, Accepted: September 1, 2003 Address reprint requests and correspondence to: Associate Professor Ying-Lung Tseng, Doping Control Center, Tzu Chi University, 701, Section 3, Chung Yang Road, Hualien, Taiwan Tzu Chi Med J No. 2! SV

2 Y. L. Tseng, M. H. Shieh, C. T. Lin, et al Chemicals, solvents, and reagents All reagents were of analytical grade. Di-sodium hydrogen phosphate, lead acetate-3-hydrate, aluminum acetate, potassium carbonate, sodium hydrogen carbonate, methyl iodide, potassium dihydrogen phosphate, and phosphoric acid were purchased from.. Riedel-de Haen (Wunstorfer, Str. 40 Seelze, Germany). Acetone, acetonitrile, ethyl acetate, and methanol were from Mallinckrodt (St. Louis, MO, USA). Purified water was from a Milli-Q system (Millipore, Bedford, MA, USA). Standards Prohibited diuretics listed in the Olympic Movement Anti-Doping Code of the IOC are analyzed in our routine sport drug testing program [9]. Reference standards including amiloride HCl, acetazolamide, chlorothiazide, hydrochlorothiazide, pemoline, triamterene, hydroflumethiazide, chlorthalidone, dichlorphenamide, furosemide, trichlormethiazide, ethacrynic acid, bumetanide, indapamide, benzthiazide, probenecid, cyclothiazide, bendroflumethiazide, and phenazine (internal standard), were purchased from Sigma (St. Louis, MO, USA); spironolactone was from Aldrich (Milwaukee, WI, USA); metolazone was from RBI (Natick, MA, USA); and polythiazide was from USP (Rockville, MD, USA). Urinary samples Urine samples (n = 5817; 3263 males and 2554 females) were obtained from 2 primary sources between 1998 and 2002: (1) in-competition tests such as the National Games, National Collegiate Games, and National High and Middle School Games; and (2) out-of-competition tests when athletes were training for major international sport events. Urine samples were collected by certified doping control officials and were split into bottle A (50 ml) and bottle B (25 ml). Upon arrival, bottle A was analyzed first, and bottle B was stored at -20 C for reanalysis in the event that the bottle A sample was presumptively positive for certain prohibited substance(s). Specific gravity and ph for each urine sample were measured and recorded. Sample preparation High-performance liquid chromatography screening The sample extraction procedure was primarily based on the method developed by Cooper et al [6]. Depending upon the specific gravity of the urine, either 2.5 ml or 5 ml of urine was analyzed. Each sample underwent acid and basic extractions before being pooled. For acidic extraction, an aliquot of a urine sample was supplemented with a mixture of solid KH2PO4: Na2HPO4 (98:2 w/w; ph 5-5.5) and phenazine (as an internal standard; 100 µg/ml), followed by 4 ml ethyl aceteate. The sample was agitated and centrifuged at 2000 rpm for 10 min. The organic phase was transferred to another tube, and 2 ml lead acetate was added. The mixture underwent vortexing and centrifugation as described above. The acidic fraction (4 ml) was then ready to combine with the basic fraction. For the basic extraction, the other aliquot of the urine sample was sequentially supplemented with a solid mixture of NaHCO3:K2CO3 (3:2 w/w; ph 9-9.5) and 4 ml ethyl acetate. The mixture was vortexed and centrifuged as described above. After the acidic and basic fractions were pooled, the mixture was evaporated to dryness under a stream of nitrogen gas. The sample was reconstituted with either 100 µl or 300 µl methanol depending upon its specific gravity. A sample volume of 5 µl was injected into the HPLC. Gas chromatography-mass spectrometry confirmation In the event of a presumptive positive finding after the HPLC analysis, the sample in question was evaporated to dryness under nitrogen gas. The residue was derivatized in a mixture of 150 µl acetone, 50 µl methyliodide, and 100 mg potassium carbonate at 60 C for 1 h [7]. One microliter of sample was then injected into the GC-MS. Instrumentation and conditions High-performance liquid chromatography (HPLC) screening A Perkin-Elmer HPLC system (Perkin-Elmer, Norwalk, CT, USA) equipped with an LC-235C diodearray detector, series 200 degasser, autosampler, 250 pump, and 600 series Link and Turbochom Navigator data system was used for the screening procedure. The UV detector was adjusted to wavelengths of 240 nm and 275 nm. A Zorbax SB-C18 (25 cm 4.6 mm i.d.) reverse-phase column was applied for chromatographic separation. The mobile phase was a gradient system of acetonitrile (100%) and aluminum acetate buffer (0.02 M; ph 4.5) and was adjusted as follows. The initial concentration of acetonitrile was 15% for 2 min and was gradually increased to 85% for 18 min, then hold for 2 min at 85%. The flow rate was 1 ml/min, and the column temperature was kept constant at 25 C. Gas chromatography-mass spectrometry confirmation A Hewlett-Packard HP 6890 gas chromatograph coupled with a 5972A mass selective detector (Hewlett- Packard, Palo Alto, CA, USA) was used for the analyses. An HP-5MS crosslinked 5% diphenyl and 95% TM! Tzu Chi Med J No. 2

3 Diuretics abuse in sports dimethylpolysiloxane capillary column (25 m 0.25 mm I.D µm film thickness) was used for GC separation. Helium was used as a carrier gas. The temperature of the GC injection port was maintained at 250 C, and the transfer line temperature was 300 C. The GC temperature program was operated as follows. The initial temperature of 90 C was held for 1 min, increased by 15 C/min to 240 C then increased 10 C/min to 300 C and held for 15 min. The mass spectrometer was operated in the electron impact (EI) mode at an electron ionization (EI) energy of 70 ev. Before analysis, the mass spectrometer was tuned with perflurotributylamine (PFTBA) to optimize the selected ions of m/z 69, 219, and 502. The mass spectrum was obtained by scanning from m/z 50 to 550. Limit of detection (LOD) and recovery The LOD was defined as the analyte concentration which produced a signal-to-noise ratio (S/N) of 3. The LODs of hydrochlorothiazide and furosemide were analyzed by HPLC. For the recovery study of hydrochlorothiazide and furosemide, 3 different target concentrations of hydrochlorothiazide and furosemide at 0.5, 5, and 10 µg/ml were applied, and 3 replicates for each concentration were analyzed to determine the recovery of these 2 diuretics. Criteria for positive sample reporting In our routine doping tests, the criteria used for reporting a positive case were as follows. (1) The retention time (RT) and relative retention time (RRT) of an analyte in the HPLC chromatogram were compared between a presumptive positive sample and the positive control standard; the difference in RRT between these 2 samples should not differ by more than 1%. The RRT was determined by obtaining the ratio between the retention times of each metabolite with respect to that of the internal standard. (2) A minimum of 3 diagnostic ions was compared in the full-scan mass spectra between the analyte of the presumptive positive sample and the positive control standard; the relative percent abundance of any of the ions should not differ by more than 20% to 7.99 (mean, 6.12), whereas the specific gravity ranged from to (mean, 1.017). For the 12 positive samples containing diuretics, the measurements of ph ranged from 5.36 to 7.64 (mean, 6.31) and specific gravity ranged from to (mean, 1.018). Limit of detection (LOD) and recovery of hydrochlorothiazide and furosemide The LODs of hydrochlorothiazide and furosemide analyzed by HPLC were 4.5 and 4.8 ng/ml, respectively. The recovery of the 3 target concentrations of 0.5, 5, and 10 µg/ml for hydrochlorothiazide and furosemide were found to be 59%, 72%, and 86% and 49%, 68%, and 69%, respectively (Table 1). Identification of positive samples Two steps were taken to determine a positive sample that led to a final report of doping control. (1) Initial HPLC screening: Urine samples were first screened with HPLC for possible content of a prohibited substance (Fig. 1). After HPLC analysis, the retention time (RT) and relative retention time (RRT) of each presumptive positive peak in the chromatogram were compared to the authentic standard. The RRT differences of metabolites for all comparisons between positive samples and known samples shall be less than 1%. In addition, the UV spectra of hydrochlorothiazide and furosemide detected at UV wavelengths of 275 nm and 240 nm in positive samples were also compared with the control standards to assist the determination of a presumptive positive sample. The results of the UV spectra between positive samples and control standards of hydrochlorothazide and furosemide are shown in Fig. 2. (2) GC-MS confirmation: Following the screening step, a presumptive positive sample was confirmed in full scan mode by GC-MS. A total ion chromatogram (TIC) of methyliodide-derivatized furosemide (RT, min; RRT, 1.71) and hydrochlorothiazide (RT, min; RRT, 1.86) from an unknown sample is shown in Fig. 3. Electron ionization (EI) mass spectra for these substances were analyzed and compared with the reference standards (Fig. 4). Each compound was RESULTS Urinary ph and specific gravity Prior to the analysis, the ph and specific gravity of each urine sample were measured with a ph meter (Mettler-Toledo MP120) and a refractometer (Atago URC-NE, Atago Co., Tokyo, Japan), respectively. Among the 5817 samples tested, the ph ranged from Table 1. Recovery of Hydrochlorothiazide and Furosemide Amount Hydrochlorothiazide Furosemide (µg/ml) % Recovery %CV* % Recovery %CV *: %CV indicates the percent of coefficient of variance Tzu Chi Med J No. 2! TN

4 Y. L. Tseng, M. H. Shieh, C. T. Lin, et al Response [AU] Response [AU] Response [AU] C A B D Response [AU] Time (min) Fig. 1. Chromatograms of representative positive urine samples of hydrochlorothiazide (RT, 8.37 min) (A) and furosemide (RT, min) (B), authentic reference standards for hydrochlorothiazide and furosemide (C), and blank urine (D). Blue arrow, hydrochlorothiazide; brown arrow, furosemide. A Nanometers B C Nanometers D Fig. 2. Two-dimensional UV spectra detected at UV wavelengths of 275 nm and 240 nm from HPLC. (A) Hydrochlorothiazide from an unknown positive sample; (B) a positive control standard of hydro-chlorothiazide; (C) furosemide from an unknown positive sample; (D) a positive control standard of furosemide Nanometers Nanometers TO! Tzu Chi Med J No. 2

5 Diuretics abuse in sports Abundance ( 1000) 280- A Abundance ( 1000) Abundance ( 1000) B C Abundance ( 1000) D Time (min) Fig. 3. Total ion chromatograms of methyl derivatives of diuretics. (A) One unknown sample containing suspected furosemide at an RT of min (RRT, 1.71; IS, 9.43 min). (B) One unknown sample containing suspected hydrochlorothiazde at an RT of min (RRT, 1.86; IS = 9.41 min). (C) Standard control containing furosemide at an RT of min (RRT, 1.71; IS = 9.45 min) and hydrochlorothiazide at an RT of min (RRT, 1.87). (D) Blank urine (IS, 9.42 min). further analyzed by comparing the percent abundance of selected diagnostic ions, i.e., m/z 353, 310, 288, and 218 for hydrochlorothiazide and m/z 81, 96, 339, 372, and 374 for furosemide, between the unknown sample and the control standard. Table 2 shows the relative abundances of diagnostic ions from 1 representative positive sample of hydrochlorothiazide and furosemide. The relative abundance ratios of diagnostic ions between the positive unknown sample and the positive control standard were from 3.3 to 9.3 for hydrochlorothiazide and from 2.1 to 7.4 for furosemide (Table 2). Incidence of diuretics abuse in sports Of the 5826 samples tested from 1998 to 2002, 12 samples (0.2%) were found to contain IOC-prohibited diuretics, i.e., hydrochlorothiazide (n = 8; 67%) and furosemide (n = 4; 33%) (Table 3). Among these positive cases, taekwondo accounted for 4 cases, followed by 3 cases in weightlifting, 2 cases in volleyball, and 1 case each in swimming, wrestling, and bowling. With regard to the gender of positive cases, slightly more female athletes (n = 7) were found than male athletes (n = 5). Tzu Chi Med J No. 2! TP

6 Y. L. Tseng, M. H. Shieh, C. T. Lin, et al Table 2. m/z Selected Diagnostic Ions of Hydrochlorothiazide and Furosemide From 1 Representative Positive Sample and the Corresponding Positive Control Standard After Methyl Derivatization Positive sample Control sample Ion abundance Percent abundance Ion abundance Percent abundance Abs. rel. abundance Hydrochlorothiazide , , , , , , , , Furosemide , , ,619, ,264, , , , , ,689, ,361, : Abs. rel. abundance, absolute relative abundance Table 3. Distributions of Positive Samples with Respect to Drug Name, Sport, and Gender 310 M Sport Furosemide Hydrochlorothiazide M F M F n Taekwondo Weightlifting Volleyball Swimming Wrestling Bowling M: male; F: female; n: number of positive samples DISCUSSION 53 Fig Representative mass spectra of methyliodidederivatized diuretics in positive samples. (A) Hydrochlorothiazide and (B) furosemide. M + denotes a molecular ion M This paper describes procedure for detecting diuretics in the urine of athletes and reports the incidence of diuretics violations in sports analyzed in this laboratory from 1998 to In dope testing, urinary ph and specific gravity are routinely measured for the following reasons: (1) if the urine density is too low (< 1.005), physiologically a substance may not concentrate enough to be detectable [10]; (2) if the ph is too high, some basic substances, e.g., amphetamine, may be excreted in very low concentrations and escape detection [11]. In this study, we found that the ph and specific gravity in the urine of the athletes who tested positive for diuretics ranged from 5.36 to 7.64 (mean, 6.31) and from to (mean, 1.018), respectively. The results from recovery studies show that at a low TQ! Tzu Chi Med J No. 2

7 Diuretics abuse in sports concentration (0.5 µg/ml), the recovery was approximately 50% for furosemide and 60% for hydrochlorothiazide. At 5 and 10 µg/ml, the recoveries were 68% and 69%, respectively, for furosemide, and 72% and 86%, respectively, for hydrochlorothiazide (see Table 1). These results are in agreement with a report of Cooper et al [6], although 15 µg/ml was used as the only target concentration in their study. In this study, after the samples were derivatized with methyliodide, tetramethylhydrochlorothiazide was formed with ion m/z 353 (100%) as the molecular ion (M + ) and had an intensity similar to the characteristic ion m/z 310 (97%); the other characteristic ions were ion m/z 218 (61.1%) and ion m/z 288 (51.2%) [8,12]. The methylation of furosemide, however, resulted in trimethylated furosemide. Under this condition, the base peak, ion m/z 81 (100%), representing the furan ring, had the highest intensity, followed by the molecular ion m/z 372 (34.5%), ion m/z 374 (13.9%), ion m/z 96 (8.6%), and ion m/z 339 (7.5%). The relative percent abundances, ranged from 3.3% to 9.3% for hydrochlorothiazide and 2.1% to 7.4% for furosemide, both less than 20%, indicating that the confirmation of these 2 drugs was acceptable. Of the 5817 urine samples from tested athletes, 12 positive cases (0.2%) were found to contain hydrochlorothiazide (n = 8) and furosemide (n = 4). The incidence rate of diuretics ranked third, behind stimulants and anabolic steroids, in doping tests in Taiwan over the past 5 years (unpublished results). In comparison with international testing results, diuretic violations (n = 16; 0.2%) also ranked third during the Olympic Games from 1976 to 2000 where 10,359 samples were tested. However, one other report ranked diuretics the fourth in the commonly abused drug group, behind anabolic steroids, stimulants, and narcotics [13]. Of the 2 diuretics found in this report, hydrochlorothiazide has been grouped as a thiazide diuretic and furosemide as a "loop" diuretic. These 2 diuretics have distinct chemical structures (see Fig. 4). Hydrochlorothiazide has the structure of 1,2,4-bensothiadiazine-7- sulphonamide 1,1-dioxide with a presence of chlorine at the 6th position and is hydrogenated at the 2nd and 3rd positions [8]. Furosemide contains a sulphonamide group with carboxylic function and a furan ring. Since diuretics with carboxylic functions and sulphonamidegroups show poor chromatographic properties, their derivatization with methyliodide showed excellent chromatographic results [7]. Based on the present report, hydrochlorothiazide and furosemide are the 2 most popular drugs abused in sports among diuretics. Although the exact reasons why athletes abuse these drugs are unknown, we postulate several reasons as follows: (1) therapeutically, these 2 drugs may be used more extensively than others; (2) a greater number of pharmacological studies related to the use in sports have been carried out on these 2 drugs [14]; (3) they possess high efficacy for the rapid reduction of body weight and elimination of doping agents [15,16]; and (4) they are more accessible in local pharmacies in Taiwan. With regard to the sports that were related to diuretics violations in this study, taekwondo accounted for the highest incidence rate (33%), followed by weightlifting (25%), and volleyball (17%). Swimming, wrestling, and bowling accounted for 8% (1 instance) each. Taekwondo, weightlifting, and wrestling are sports classified by body weight and accounted for 67% of the positive cases. In contrast, volleyball, swimming, and bowling are sports without weight classes and accounted for 33% of the violations. On the basis of these findings, athletes in sports with weight classes were most likely to abuse diuretics for the following possibly reasons: (1) to reduce body weight in order to qualify for a lower weight class; (2) to reduce the urinary concentration of other prohibited substances to avoid a positive doping result; and (3) a combination of reasons (1) and (2). It has also been indicated that since sports such as wrestling, boxing, powerlifting, judo and weightlifting feature levels of competition determined by weight, this often encourages athletes to attempt to reduced their body weights in order to qualify for an established weight class just prior to competition [4]. In the 2000 Sydney Olympic Games, 3 weightlifters and 1 wrestler were tested positive for furosemide. Bodybuilders also use diuretics to achieve greater muscle definition and to appear "cut" [17]. One Chinese female swimmer was expelled from the 1998 World Championships and suspended for 2 years after testing positive for a diuretic [18]. In 1 study, diuretics use was found to be the greatest among volleyball players, followed by softball and basketball players [19]. In the present study, 2 cases of volleyball violations were found. The use of diuretics in sports without weight classifications is primarily for drug elimination purposes and/or for weight loss such as in gymnastics and ballet [4]. The use of diuretics in sports can produce serious consequences. In 1992, bodybuilder Mohammed Benaziza died suddenly of cardiac arrest due to the use of diuretics, just days after placing fourth in the Mr. Olympia competition [20]. CONCLUSIONS Tzu Chi Med J No. 2! TR

8 Y. L. Tseng, M. H. Shieh, C. T. Lin, et al The use of drugs to enhance physical performance has been a practice common to sports competitions. Although diuretics do not function as performance-enhancing drugs, they are relatively popular in some sports for weight reduction and to avoid a positive doping result during athletic competitions. This study demonstrates that the abuse of diuretics among athletes in Taiwan occurred primarily in sports where weight categories are involved, such as taekwondo and weightlifting. Hydrochlorothiazide and furosemide are the 2 commonly abused diuretics. REFERENCES 1. Cafruny EJ: Diuretics: Drugs that increase excretion of water and electrolytes. In: Wingard LB, Brody TM, Larner J, Schwartz A, eds. Human Pharmacology: Molecularto-Clinical. Mosby Year Book 1991, pp Gerber JG, Nies AS: Antihypertensive agents and the drug therapy of hypertension. In: Goodman Gilman A, Rall TW, Nies AS, Taylor P, eds. Goodman & Gilman's: The pharmacological basis of therapeutics. McGTAW- HILL 1991, pp Reents S: Sport and exercise pharmacology. Human Kinetics 2000, pp Armstrong LE: Diuretics. In: Bahrke MS, Yesalis CE, editors. Performance-enhancing substances in sport and exercise. Human Kinetics 2002, pp Verroken M: Drug use and abuse in sport. In: Mottram DR, ed. Drugs in sport. E & FN SPON 1996, pp Cooper SF, Masse R, Dugal R: Comprehensive screening procedure for diuretics in urine by high-performance liquid chromatography. J Chromatogr B 1989; 489: Schanzer W: The problem of diuretics in doping control. In: Bellotti P, Benzi G, Ljungqvist A, eds. International athletic foundation world symposium on doping in sport - official proceedings. I.A.F. International Athletic Foundation 1987, pp Tsoutsoulova-Draganova A: Investigation on products of thiazide diuretics in human urine. In: Donike M, Geyer H, Gotzmann A, Mareck-Engelke U, Rauth S, eds.12th Cologne Workshop on Dope Analysis. SPORT und.. BUCH Strauβ, Koln 1995, pp IOC: List of Prohibited Classes of Substances and Prohibited Methods. International Olympic Committee, Lausanne, Switzerland, Alonso JM: Sampling procedures. In: I. Gyulai (Chief editor): Doping is cheating. International Amateur Athletic Federation 1st World Anti-Doping Seminar, Heusenstamm, Germany 1994, Multiprint-Monaco 1995, pp Donike M, Rauth S, Sample B: Excretion of ephedrine and endogenous steroids under conditions of controlled water intake and of water diuresis. In: Donike M, Geyer H, Gotzmann A, Mareck-Engelke U, Rauth S, eds. 10th Cologne Workshop on Dope Analysis. SPORT und.. BUCH Strauβ, Koln 1993, pp Nava-Ocampo AA, Velazquez-Armenta EY, Reyes- Perez H, Ramirez-Lopez E, Ponce-Monter H: Simplified method to quantify furosemide in urine by high-performance liquid chromatography and ultraviolet detection. J Chromatogr B 1999; 730: Benzi G: Pharmacoepidemiology of the drugs used in sports as doping agents. Pharmacol Res 1994; 29: Saugy M, Meuwly P, Munafo A, Rivier L: Rapid highperformance liquid chromatographic determination with fluorescence detection of furosemide in human body fluids and its confirmation by gas chromatography-mass spectrometry. J Chromatogr B 1991; 564: Delbeke FT, Debackere M: The influence of diuretics on the excretion and metabolism of doping agents part IV - caffeine. Biopharm Drug Dispos 1988; 9: Delbeke FT, Debackere M: The influence of diuretics on the excretion and metabolism of doping agents - V. Dimefline. J Pharm Biomed Anal 1991; 9: Hickson JF, Johnson TE, Lee W, Sidor RJ: Nutrition and the precontest preparations of a male bodybuilder. J Am Diet Assoc 1990; 90: Dickey C, Helmstaedt K, Nordland R, Hayden T: The real scandal. Newsweek Feb. 15, 1999, pp Matin M, Schlabach G, Shibinki K: The use of nonprescription weight loss products among female basketball, softball, and volleyball athletes from NCAA Division I institutions: Issues and concerns. J Athletic Training 1998; 44: Vest DW: Wrestling looks to bodybuilding. Wrestling USA 1993; 29: TS! Tzu Chi Med J No. 2

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