Mats Garle

Use of doping agents, particularly anabolic steroids, in sports and society

The use of doping agents, particularly anabolic androgenic steroids (AAS), has changed from being a problem restricted to sports to one of public-health concern. We review the prevalence of misuse, the evidence that some drugs improve performance in sport, their side-effects, and the long-term consequences of AAS misuse for society at large. There is substantial under-reporting of the side-effects of AAS to health authorities. We describe neuropsychiatric side-effects of AAS and their possible neurobiological correlates, with particular emphasis on violent behaviour. Analytical methods and laboratories accredited by the World Anti-Doping Agency can detect the misuse of all doping agents; although the analysis of testosterone requires special techniques, and recently discovered interethnic differences in testosterone excretion should be taken into account. The prevention of misuse of doping agents should include random doping analyses, medical follow-ups, pedagogic interventions, tougher legislation against possession of AAS, and longer disqualifications of athletes who use AAS.

Doping Test Results Dependent on Genotype of Uridine Diphospho-Glucuronosyl Transferase 2B17, the Major Enzyme for Testosterone Glucuronidation

CONTEXT Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. The large variation in testosterone glucuronide (TG) excretion and its strong association with a deletion polymorphism in the uridine diphospho-glucuronosyl transferase (UGT) 2B17 gene challenge the accuracy of the T/E ratio test. OBJECTIVE Our objective was to investigate whether genotype-based cutoff values will improve the sensitivity and specificity of the test. DESIGN This was an open three-armed comparative study. PARTICIPANTS A total of 55 healthy male volunteers with either two, one, or no allele [insertion/insertion, insertion/deletion, or deletion/deletion (del/del)] of the UGT2B17 gene was included in the study. INTERVENTION A single im dose of 500 mg testosterone enanthate was administered. MAIN OUTCOME MEASURES Urinary excretion of TG after dose and the T/E ratio during 15 d were calculated. RESULTS The degree and rate of increase in the TG excretion rate were highly dependent on the UGT2B17 genotype with a 20-fold higher average maximum increase in the insertion/insertion group compared with the del/del group. Of the del/del subjects, 40% never reached the T/E ratio of 4.0 on any of the 15 d after the dose. When differentiated cutoff levels for the del/del (1.0) and the other genotypes (6.0) were applied, the sensitivity increased substantially for the del/del group, and false positives in the other genotypes were eliminated. CONCLUSIONS Consideration of the genetic variation in disposition of androgens will improve the sensitivity and specificity of the testosterone doping test. This is of interest not only for combating androgen doping in sports, but also for detecting and preventing androgen abuse in society.

A high-throughput multicomponent screening method for diuretics, masking agents, central nervous system (CNS) stimulants and opiates in human urine by UPLC-MS/MS

A simple and rapid multicomponent screening method of 130 substances for direct injections of urine samples has been developed. The fully automated method based on ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS) is used for three different classes of doping agents: diuretics, central nervous system stimulants (CNS stimulants) and opiates. The samples are diluted with buffer containing internal standards (IS) by a pipetting robot system into 96-well plates. Samples are injected on a reversed phase sub 2-microm particle column connected to a fast polarity switching and rapid scanning tandem mass spectrometer with an electrospray interface. The software used to evaluate the results produced reports containing a small-sized window for each component and a data table list with flags to indicate any adverse analytical findings in the sample. The report can also be processed automatically using an application software, which interpret the data and indicate if there is a suspicious sample. One 96-well plate can be analyzed within 16 h.

The anti-doping hot-line, a means to capture the abuse of doping agents in the Swedish society and a new service function in clinical pharmacology

With the support of the Swedish National Institute of Health a national information service was started in 1993 aiming to capture the abuse of doping agents in the general public. It was organized as a telephone service, called the Anti-Doping Hot-Line, from our department and managed by trained nurses co-operating with clinical pharmacologists. Important information collected about all callers (anonymous) was: date of call, its origin, category of caller, doping experience and main question being asked. Abusers were asked about their age, sex, affiliation, abused drug(s), duration of abuse, habit of administration and adverse reactions (ADRs). Between October 1993 and December 2000 25,835 calls were received with a peak during spring and autumn. Most calls (12,400) came from non-abusers, 60% being males. Callers connected with gyms represented the largest group (30%). Most calls about specific drugs concerned anabolic androgenic steroids (AAS). Other drugs or products included ephedrine, clenbuterol and creatine. The most commonly abused anabolic steroids were testosterone, nandrolone-decanoate, methandienone and stanozolol. The ten most commonly reported ADRs of AAS were aggressiveness (835), depression (829), acne (770), gynecomastia (637), anxiousness (637), potency problems (413), testicular atrophy (404), sleep disorders (328), fluid retention (318) and mood disturbances (302). Female side effects included menstruation disturbances, hair growth in the face, lower voice and enlarged clitoris. During the period 1996–200, totally 4339 persons reported about 10,800 side effects. This figure should be compared with the very low number of ADRs (27) reported by prescribers to the Swedish ADR committee during the same period. Abuse of doping agents appears to be a new public health problem that needs detection, medical care and prevention.

A gas chromatographic method for the quantitative determination of nortriptyline and some of its metabolites in human plasma and urine

Abstract A gas chromatographic method has been developed which enables accurate and specific quantitative determinations in plasma and urine of the tricyclic antidepressant drug nortriptyline and some of its desmethylated or hydroxylated metabolites and their conjugates formed in vivo . The compounds are extracted as bases into hexane (the conjugated metabolites after hydrolysis) and, after a clean-up procedure, derivatized with heptafluorobutyric anhydride. The heptafluorobutyryl derivatives are separated on a gas chromatograph equipped with an electron capture detector. Accurate determination are possible in concentrations down to 10 ng/ml.

Plasma disappearance of transplacentally transferred diphenylhydantoin in the newborn studied by mass fragmentography

With the intention of studying the fate of transplacentally transferred diphenylhydantoin (DPH, phenytoin) in the newborn infant, a mass fragmentographic analytic method has been developed, in which deuterium‐labeled DPH is used as internal standard. The analytic procedure permits precise determinations of DPH in 100 µl plasma samples in concentrations down to 0.01 µg per milliliter. Seven newborn infants of epileptic mothers treated with DPH throughout the pregnancy were studied during the first 4 to 9 days of life. The rate of plasma disappearance of DPH in the newborn infants was of the same order of magnitude as previously reported in adults. The initial parts of the plasma concentration curves indicated zero‐order disappearance of DPH. Nursing did not seem to affect the rapid plasma level decline in the babies until extremely low concentrations (0.1 p.g per milliliter) were reached. The arr,wunts of unchanged drug excreted in the urine were inSignificant. It is therefore concluded that babies born by mothers treated chronically with DPH are able to metabolize this drug effectively immediately after birth.

Biomarkers of Coagulation, Inflammation, and Angiogenesis are Independently Associated with Preeclampsia

Although preeclampsia has been associated with inflammation, coagulation, and angiogenesis, their correlation and relative contribution are unknown.

Serum and urinary markers of exogenous testosterone administration.

In an attempt to find optimal markers of exogenous testosterone (T) administration in male athletes, a number of compounds were measured in 11 healthy men before and after 3, 6 and 9 months of weekly administration of 250 mg i.m. T enanthate and in age-matched untreated controls. The following variables were measured in serum: T, 17 alpha-hydroxyprogesterone (17-OHP), sex hormone-binding globulin (SHBG), estradiol-17 beta, estrone (free + conjugated) and luteinizing hormone (LH). The following variables were measured in urine: T glucuronide (urinary T), epitestosterone glucuronide (urinary epiT), estrone (free + conjugated) and LH. Serum T, serum T/17-OHP ratio, serum T/LH ratio, serum T/SHBG ratio, serum and urinary estrogens, urinary T/creatinine-, T/epiT- and T/LH ratios increased whereas serum 17-OHP, SHBG and LH and urinary epiT/creatinine- and LH/creatinine-ratios decreased significantly during treatment. Levels above the upper reference limit were found in all subjects at 3, 6 and 9 months for serum T/17-OHP and serum and urinary T/LH ratios and at 6 months for the urinary T/epiT ratio. Levels below the lower reference limit were found in all subjects at 3, 6 and 9 months for serum LH and the urinary LH/creatinine ratio, at 3 months for the urinary epiT/creatinine ratio and at 9 months for serum 17-OHP. No other variable showed abnormal values in all subjects at the same occasion. Despite significant changes during treatment, steroid concentrations as such are poor indicators of T doping. Serum and urinary LH levels, T/LH ratios and serum T/17-OHP ratios seem to be the most reliable markers of exogenous T administration in males.

Determination of aglycones of ginsenosides in ginseng preparations sold in Sweden and in urine samples from Swedish athletes consuming ginseng

Recently developed gas chromatographic and gas chromatographic-mass spectrometric methods were used to characterize 17 different commercial ginseng preparations sold in Sweden. The contents of total ginsenosides per capsule or per tablet varied from 2.1 to 13.3 mg. Unlike the other preparations, a red ginseng and three liquid ginseng preparations (after releasing the sugar moieties from ginsenosides) were shown also to contain significant amounts of 20-epimers of 20(S)-protopanaxadiol and 20(S)-protopanaxatriol as well as their corresponding 24,25-hydrated compounds. In addition to the genuine and artificial sapogenins mentioned above, two epimeric pairs of prosapogenines (ginsenoside Rg3 and 20(S)-Rg3, ginsenoside Rh1 and 20(R)-Rh1) were also found in the liquid formulations. These results suggest that hydrolysis, epimerization and hydration in the side-chain of the aglycone moiety of ginsenosides may occur in the liquid formulations under weak acidic conditions (pH 3.0-3.5 with 9-10% of alcohol at room temperature). The new method was also used to determine the aglycones of ginsenosides in urine samples from Swedish athletes stating that they had consumed ginseng preparations within 10 days before urine collection. Out of the 65 samples analysed, 60 were found to contain 20(S)-protopanaxatriol. The concentrations of 20(S)-protopanaxatriol ginsenosides varied from 2 to 35 ng ml-1 urine. This is the first demonstration of uptake of ginsenosides in humans after oral administration of ginseng preparations.

Increased urinary testosterone/epitestosterone ratios found in Swedish athletes in connection with a national control program Evaluation of 28 cases

In connection with a national anti-doping control program, including analysis of 8946 urine samples, 28 athletes were found to have delivered samples free from xenobiotic anabolic steroids but with an increased testosterone/epitestosterone (T/E) ratio (> 6). Unannounced testing of the above athletes produced 2-4 additional urine samples during the next 2-3 months. A low degree of variation of the T/E ratio, with a C.V. below 30% was found in 17 of the subjects whereas 10 had a C.V. varying from 31% to 43%. One subject with a high urinary T/E ratio (10.5) had a C.V. of this ratio of 126% and also an extremely high ratio between testosterone and LH in urine. It has been reported that non-users of testosterone have T/E ratios fluctuating around a mean with a C.V. that will not exceed 30%. We found that administration of testosterone to seven healthy volunteers resulted in urinary T/E ratios that varied with a C.V. ranging from 67% to 130% during the following 4 weeks. It is concluded that among the above 28 cases, only one can be regarded as a clear case of testosterone doping. Although the vast majority of Swedish athletes have urinary T/E ratios below six, there is a subfraction with a constant higher ratio, possibly due to genetic factors.