Don H. Catlin

Improved method of detection of testosterone abuse by gas chromatography/combustion/isotope ratio mass spectrometry analysis of urinary steroids

The current approach to detection of doping with testosterone is based on measuring the testosterone to epitestosterone ratio (T/E) in urine by gas chromatography/mass spectrometry. The median T/E for healthy males who have not used T is about 1.0. In a single urine, a T/E lower than six leads to a negative report even though it does not exclude T administration. A value greater than six indicates possible T administration or a naturally elevated ratio. It has been shown previously that the carbon isotope ratio of urinary T changes after T administration. In this study a potential confirmation method for T abuse was optimized. Gas chromatography/combustion/carbon isotope ratio mass spectrometry (GC/C/IRMS) was used to analyze two T precursors (cholesterol and 5-androsten-3 beta, 17 beta-diol) and two T metabolites (5 alpha- and 5 beta-androstane-3 alpha, 17 beta-diol) in addition to T itself in each of 25 blind urines collected from eight healthy men before, during or after T administration. The carbon isotope ratios of T and the metabolites were lower after T administration. The relationships among the variables were studied using multivariate analysis and beginning with principal components analysis; cluster analysis revealed that the data are composed of two clusters, and classified the samples obtained after T administration in one cluster and the remainder in the other; discriminant analysis correctly identified T users. The measurement of carbon isotope ratios of urinary androgens is comparable to the T/E > 6 test and continues to show promise for resolving cases where doping with T is suspected.

Analysis of over-the-counter dietary supplements.

ObjectiveTo determine if steroids containing over-the-counter (OTC) dietary supplements conform to the labeling requirements of the 1994 Dietary Supplement Health and Education Act (DSHEA). Design12 brands of OTC supplements containing 8 different steroids were randomly selected for purchase in stores that cater to athletes. There are two androstenediones (4- and 5-androstene-3,17-dione), two androstenediols (4- and 5-androstene-3&bgr;, 17&bgr;-diol), and 4 more are 19-nor cogeners (19-nor-4- and 5-androstene-3,17-dione and 19-nor-4- and 5-androstene-3&bgr;, 17&bgr;-diol). Main Outcome Measures12 brands of OTC anabolic–androgenic supplements were analyzed by high-pressure liquid chromatography. ResultsWe found that 11 of 12 brands tested did not meet the labeling requirements set out in the 1994 Dietary Supplement Health and Education Act. One brand contained 10 mg of testosterone, a controlled steroid, another contained 77% more than the label stated, and 11 of 12 contained less than the amount stated on the label. ConclusionsThese mislabeling problems show that the labels of the dietary steroid supplements studied herein cannot be trusted for content and purity information. In addition, many sport organizations prohibit OTC steroids; thus, athletes who use them are at risk for positive urine test results. In this article we provide the details of the analyses, a summary of the steroids by name and structure, and information on the nature of the positive test results. Athletes and their physicians need this information because of the potential medical consequences and positive urine test results.

Screening urine for exogenous testosterone by isotope ratio mass spectrometric analysis of one pregnanediol and two androstanediols

We propose a new screening method for testosterone (T) doping in sport. The current method for detecting T administration is based on finding a T to epitestosterone ratio (T/E) in urine that exceeds six. The difficulties with T/E are that T administration does not always result in a T/E>6 and that a rare individual will have T/E>6 in the absence of T administration. Our previous studies reveal that carbon isotope ratio helps to determine the origin of the urinary T because the values for T and its metabolites decrease after the administration of exogenous T. In this study, we present a rapid and efficient screening sample preparation method based on three successive liquid-solid extractions, deconjugation with E. coli beta-glucuronidase after the first extraction, acetylation after the second extraction, and a final extraction of the acetates. The 13C/12C of two T metabolites (5beta-androstane-3alpha,17beta-diol and 5alpha-androstane-3alpha,17beta-diol) and one pregnanediol as endogenous reference (5beta-pregnane-3alpha,20alpha-diol) was measured by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) on 10 ml of urine collected from 10 healthy men before and after T administration. Following T administration, the 13 C/12C of 5beta-androstane-3alpha,17beta-diol diacetate and 5alpha-androstane-3alpha,17beta-diol diacetate declined significantly from -26.2 per thousand to -30.8 per thousand and from -25.2 per thousand to -29.9 per thousand, respectively and the 13C/12C of 5beta-pregnane-3alpha,20alpha-diol diacetate was unchanged. In addition, the ratio of androstanediols to pregnanediol increased in the post-T urines.

Psychological moods and subjectively perceived behavioral and somatic changes accompanying anabolic-androgenic steroid use.

To assess physiological and psychological states ac companying anabolic-androgenic steroid use, male weight lifters 1) were interviewed regarding their phys ical training and the patterns and effects of any drug use; 2) completed a written physical and medical history questionnaire, a Profile of Mood States questionnaire, and the Buss-Durkee Hostility Inventory; and 3) were physically examined, including a blood sample and uri nalysis. Subjects were divided into current anabolic- androgenic steroid users (N = 12), previous users (N = 14), and nonusers (N = 24). Current and previous users reported the following changes associated with ana bolic-androgenic steroid use: increases in enthusiasm, aggression, and irritability; changes in insomnia, muscle size, muscle strength and density; faster recovery from workouts and injuries; and changes in libido. We were unable to confirm these interview and physical and medical history questionnaire responses using stand ardized and well-accepted psychological inventories. There were no significant differences among groups for any Profile of Moods factor, total mood disturbance, total Buss-Durkee Hostility Inventory score, or any subscale. For current users, there were no significant correlations between either total weekly drug dose or length of time on the current cycle of anabolic-andro genic steroids and any individual scale of the Profile of Mood States, Buss-Durkee Hostility Inventory, Profile of Mood States total mood disturbance, or composite Buss-Durkee Hostility Inventory score. Furthermore, anabolic-androgenic steroid users did not differ in their responses on these inventories from nonusers or from general population norms. The presence of subjectively perceived, anabolic-androgenic steroid-associated be havioral and somatic changes in the absence of signif icant differences in standard psychological inventory responses illustrates the complexity of these relation ships. Our data suggest that while perceived or actual psychological changes may occur in anabolic-andro genic steroid users, either the effects are too subtle or the inventories used were insensitive for detecting them.

Left ventricular function is not impaired in weight-lifters who use anabolic steroids

Recent reports suggest that anabolic steroid use might deleteriously affect left ventricular function. To examine this possibility, the present study measured left ventricular size and function with use of Doppler echocardiographic techniques in 23 weight lifters: 12 who were currently using anabolic steroids and 11 who reported that they had never used these drugs. Drug users had administered anabolic steroids to themselves for at least three cycles over the past year. All studies were interpreted by blind review and group assignment was confirmed by urine testing. Average age, years of exercise training and body weight, as well as heart rate and blood pressure at rest were similar in both groups. Cardiac dimensions (mean +/- SD) including left ventricular diastolic cavity diameter (57 +/- 3 vs. 56 +/- 5 mm), septal thickness (10 +/- 2 vs. 9 +/- 1 mm), posterior wall thickness (8 +/- 1 vs. 8 +/- 1 mm) and myocardial mass (149 +/- 27 vs. 135 +/- 21 g) did not differ between the anabolic steroid users and nonusers, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of Recombinant Human Erythropoietin in Urine by Isoelectric Focusing

BACKGROUND Doping with erythropoietic proteins such as recombinant human erythropoietin (rHuEPO) and darbepoetin alfa is a serious issue in sport. There is little information on the time course of detection of rHuEPO in urine and on methods to evaluate electrophoresis-based data. METHODS We used a recently described isoelectric focusing method for detecting rHuEPO and endogenous EPO in urine obtained from individuals treated with placebo or epoetin alfa. The latter was administered subcutaneously at 50 IU/kg on days 0, 2, 4, 7, 9, 11, 14, 16, and 18. Blood and urine samples were collected during the morning of study days -3, 0, 2, 4, 7, 9, 11, 14, 16, and 18 and on days 2, 3, 4, and 7 postadministration. We developed visual and numerical (two-band ratio) techniques to evaluate the electropherograms for the presence of rHuEPO. RESULTS Compared with the placebo group, the epoetin alfa-treated group responded with increases in hematocrit, reticulocytes, macrocytes, serum EPO, and serum soluble transferrin receptor. The electropherograms showed that the pattern of bands arising from urinary rHuEPO is different from that of endogenous urinary EPO. Both the two-band ratio and the visual technique detected rHuEPO in all 14 epoetin alfa-treated individuals 3 days after the last dose. On the 7th day after the last dose, both techniques detected rHuEPO in approximately one-half of the participants. rHuEPO was not detected in the placebo-treated individuals. CONCLUSIONS The isoelectric focusing method detects rHuEPO in most urine samples collected 3 days after nine doses of epoetin alfa. The numerical two-band ratio was equivalent to a visual method for detecting rHuEPO in urine.

Side Effects of Anabolic Steroids in Weight-Trained Men

In brief: This study documents the patterns of use of anabolic steroids in 32 body builders and power lifters. These subjects and seven control athletes who had never used steroids were interviewed and underwent a medical examination including 28 diagnostic blood tests and a urinalysis. Gynecomastia was detected in 4 of the 20 current users and 1 of the other 19 subjects. Sixty-seven percent of users reported temporary changes in libido, and 56% reported a temporary increase in irritability or aggressive behavior. Physicians should tell patients about the possible adverse side effects of such agents.

Liquid chromatography-tandem mass spectrometry assay for human serum testosterone and trideuterated testosterone.

A liquid chromatography tandem mass spectrometry assay for serum testosterone (T) and trideuterated testosterone (d(3)T) was developed in order to support clinical research studies that determine the pharmacokinetics, production rate, and clearance of testosterone by administration of trideuterated testosterone. After adding 19-nortestosterone as the internal standard (I.S.), sodium acetate buffer, and ether, to a serum aliquot, the mixture was shaken and centrifuged, and the ether was dried. The extract was reconstituted in methanol and 15 microl was injected into a liquid chromatograph equipped with an autosampler and Applied Biosystems-Sciex API 300 triple quadrupole mass spectrometer operated in the positive ion mode. T, d(3)T, and I.S. were monitored with transitions m/z 289 to m/z 97, m/z 292 to m/z 97, and m/z 275 to m/z 109, respectively. The two calibration curves were linear over the entire measurement range of 0-20 ng/ml for T and 0-2.0 ng/ml for d(3)T. The LOQs for T and d(3)T were 0.5 ng/ml and 0.05 ng/ml. The recoveries for T and d(3)T were 91.5 and 96.4%. For T at 1.25 ng/ml and 4.0 ng/ml, the intra-day precision (RSD, %) was 3.9 and 4.3% and intra-day accuracy 0.01 and 4.5%, respectively. The inter-day precision at these levels was 5.3 and 5.4% and inter-day accuracy was 1.9 and 0.3%. For d(3)T at 0.125 ng/ml and 0.4 ng/ml, the intra-day precision (RSD, %) was 2.8 and 8.3% and intra-day accuracy was 1.8 and 5.6%. The inter-day precision at these levels was 10.0 and 7.6% and inter-day accuracy was 5.7 and 3.4%. The concentrations of T in the 38 healthy subjects ranged from 2.5 to 14.0 ng/ml (mean 6.2 ng/ml).

A rapid screening assay for measuring urinary androsterone and etiocholanolone ?13C (?) values by gas chromatography/combustion/isotope ratio mass spectrometry

A gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) method is described and validated for measurement of delta(13)C values of the acetate derivatives of urinary etiocholanolone and androsterone. The analysis was performed with only 2 mL of urine. The sample preparation consisted of deconjugation with beta-glucuronidase, solid phase extraction, and derivatization with acetic anhydride and pyridine. The within-assay precision of two quality control (QC) urine samples ranged from 0.5 to 2.1 CV%. The between-assay precision in the same QC urines ranged from 1.7 to 3.4 CV%. Administration of testosterone enanthate to a subject resulted in a 6 per thousand decrease in delta(13)C values from -25 per thousand (baseline) to -31 per thousand. Two weeks after testosterone administration was discontinued, the delta(13)C values remained abnormally low while the urine testosterone/epitestosterone (T/E) ratio returned to less than 6. This relatively simple method is useful for rapidly screening a large number of urine samples, including those with T/E <6.

β-endorphin: Pharmacologic and behavioral activity in cats after low intravenous doses

Abstract Synthetic human beta-endorphin (βh-EP), an opiate-like peptide, produced characteristic responses immediately after it was intravenously (iv) administered to 7 unrestrained male cats. Doses of 50 μg/kg resulted in dose-dependent high frequency (> 1/sec) licking and diminished locomotion. At higher doses (100–500 μg/kg) the responses included vomiting and relaxation of the nictitating membrane. The responses were blocked by iv naloxone hydrochloride (1.0 mg/kg) administered 10 min prior to βh-EP. When administered after βh-EP naloxone reversed the established response. These results indicate that low iv doses of βh-EP are active in the cat, and that the effects are mediated by opiate receptors.