Anabolic steroids and cardiovascular risk: A national population-based cohort study.

BACKGROUND: Non-therapeutic use of anabolic androgenic steroids (AAS) has been associated with various adverse effects; one of the most serious being direct cardiovascular effects with unknown long-term consequences. Therefore, large studies of the association between AAS and cardiovascular outcomes are warranted. We investigated cardiovascular morbidity and mortality in individuals who tested positive for AAS. METHODS AND RESULTS: Between 2002 and 2009, a total of 2013 men were enrolled in a cohort on the date of their first AAS test. Mortality and morbidity after cohort entry was retrieved from national registries. Of the 2013 individuals, 409 (20%) tested positive for AAS. These men had twice the cardiovascular morbidity and mortality rate as those with negative tests (adjusted hazard ratio (aHR) 2.0; 95% confidence interval (CI) 1.2-3.3). Compared to the Swedish population, all tested men had an increased risk of premature death from all causes (standardized mortality ratio for AAS-positive: 19.3, 95% CI 12.4-30.0; for AAS-negative: 8.3, 95% CI 6.1-11.0). CONCLUSION: Non-therapeutic exposure to AAS appears to be an independent risk factor for cardiovascular morbidity and premature death.

SULT2A1 Gene Copy Number Variation is Associated with Urinary Excretion Rate of Steroid Sulfates.

Human cytosolic sulfotransferases (SULT) 2A1 is the main enzyme involved in the sulfate conjugation of dehydroepiandrosterone, a weak androgen, and the main androgen precursor, whereas estrogens are mainly conjugated by SULT1A1. Here we have identified a copy number variation (CNV) polymorphism in the SULT2A1 gene in a Swedish population including healthy men (N = 30). Moreover, the CNV of SULT1A1 and SULT2A1 was further characterized in relation to urinary levels of androgen sulfate metabolites before and after an intramuscular dose of 500 mg testosterone enanthate. Individuals expressing two or more CNVs excrete 80 and 40% higher levels of DHEAS (p = 0.02) and androsteroneS (p = 0.01), respectively as compared to individuals with one gene copy. The mean area under the urine concentration time-curve from time 0 (prior to the administration of 500 mg testosterone) to 15 days post dose values were 80% higher for DHEAS (p = 0.046) and testosteroneS (p = 0.019) in individuals with two and three SULT2A1 gene copies as compared to individuals with one gene copy. The SULT1A1 CNV on the other hand did not affect the sulfation activity toward the androgens. In conclusion our results indicate that functional CNV polymorphisms in SULT2A1 and SULT1A1 are common in a Swedish population and that SULT2A1 CNV is associated with the urinary concentrations of androgen sulfate metabolites.

Implication of Human UGT2B7, 2B15, and 2B17 in 19-Norandrosterone Metabolism.

Nandrolone (19-nortestosterone) is an anabolic androgenic steroid commonly abused for doping purposes. Nandrolone is mainly metabolized in the liver into 19-norandrosterone prior to glucuronidation and excretion through urine over an extended period of time. Several UGTs (i.e., UGT2B7, UGT2B15, and UGT2B17) are thought to be the major enzymes responsible for conjugation of androgens in human. An in vitro study using recombinant enzymes expressed in insect cells showed that UGT1A4 and UGT2B7 are the two main enzymes responsible of 19-norandrosterone glucuronidation. However, the identity of the enzyme involved in nandrolone metabolism in vivo together with their relative contribution and regulation remain unknown. Inhibition assays using human liver microsomes (HLM) incubated with 19-norandrosterone and selective inhibitors confirmed that UGT2B7 and UGT2B15 are involved in 19-norandrosterone glucuronidation, since the presence of the specific UGT2B7 and UGT2B15 inhibitors gemfibrozil and valproic acid inhibited the 19-norandrosterone glucuronidation by 35 and 45%, respectively. HLM were genotyped for UGT2B15 D85Y, UGT2B7 H268Y, and the UGT2B17 deletion polymorphism. The glucuronidation activity on 19-norandrosterone was significantly higher in UGT2B15 DD than in the other UGT2B15 genotypes (p < 0.05). Moreover, human liver cancer HepG2 cells were exposed to androgens to determine if the transcriptional activity of the genes of interest was affected. Only UGT2B7 mRNA expression was significantly increased (1.8-folds) after incubation with nandrolone decanoate. These results show that the UGT2B7 and UGT2B15 are involved in 19-norandrosterone glucuronidation and that the UGT2B15 polymorphism (D85Y) is the only UGT genetic variation that influences the glucuronidation activity. This could partly explain the inter-individual variation in 19-norandrosterone excretion.

Non-steroidal anti-inflammatory drugs do not influence the urinary testosterone/epitestosterone glucuronide ratio.

The UDP Glucuronosyl Transferase (UGT) enzymes are important in the pharmacokinetics, and conjugation, of a variety of drugs including non-steroidal anti-inflammatory drugs (NSAIDs) as well as anabolic androgenic steroids (AAS). Testosterone glucuronidation capacity is strongly associated with a deletion polymorphism in the UGT2B17 gene. As the use of high doses of NSAIDs has been observed in athletes there is a risk for a drug-drug interaction that may influence the doping tests for AAS. In vitro studies show inhibitory potential on UGT2B7, 2B15, and 2B17 enzymes by NSAIDs. The aim of this study was to investigate if concomitant use of NSAIDs and a single dose of testosterone enanthate would affect the excretion rate of testosterone and epitestosterone glucuronide (TG and EG) as well as the T/E ratio, thereby affecting the outcome of the testosterone doping test. The study was designed as an open, randomized, cross-over study with subjects being their own control. The 23 male healthy volunteers, with either two, one or no allele (ins/ins, ins/del, or del/del) of the UGT2B17 gene, received the maximum recommended dose of NSAID (Ibuprofen or Diclofenac) for 6 days. On day three, 500 mg of testosterone enanthate was administered. Spot urine samples were collected for 17 days. After a wash-out period of 4 months the volunteers received 500 mg testosterone enanthate only, with subsequent spot urine collection for 14 days. The glucuronides of testosterone and epitestosterone were quantified. NSAIDs did not affect the excretion of TG or EG before the administration of testosterone. The concomitant use of NSAIDs and testosterone slightly increased the TG excretion while the EG excretion was less suppressed compared to testosterone use only. The effects of the NSAIDs on the TG and EG excretion did not differ between the UGT2B17 genotype groups. In conclusion, the outcome of testosterone doping tests does not seem to be affected by the use of NSAIDs.

Patients with anaemia can shift from kidney to liver production of erythropoietin as shown by glycoform analysis.

The primary production site of erythropoietin (EPO) is shifted from the liver to the kidney shortly after birth. Under conditions of lost or reduced kidney production, it is valuable to measure the production capacity of the liver. However, there is a lack of urine or serum based methods that can distinguish endogenous EPO produced in different cell types. Here is presented a method based on chromatographic interaction with the lectin wheat germ agglutinin (WGA) that can distinguish presumably liver-produced EPO, found in anaemic patients receiving epoetin and darbepoetin, from kidney-produced EPO found in healthy individuals. All the tested samples from haemodialysis patients with end-stage renal disease showed a presence of liver EPO. In some samples, the liver-produced EPO made up 90-100% of total EPO at a concentration of 8-10 ng/L in urine, which indicates that the liver has a quite high production capacity, although not adequate for the degree of anaemia. This glycoform analysis has made it possible to affirm that some anaemic patients can increase their liver-production of EPO. The use of such a method can give better insight into the regulation of non-renal endogenous EPO production, a potential source of EPO intended to replace administration of exogenous EPO.

Testosterone challenge and androgen receptor activity in relation to UGT2B17 genotypes.

BACKGROUND: We investigated the androgen receptor (AR) bioluminescense response in serum and urine before and after testosterone challenge in different genotypes of the UGT2B17 enzyme, which catalyses testosterone glucuronidation. MATERIAL AND METHODS: The androgen receptor activity was determined using a yeast-based bioluminescence assay. The androgens were analysed using LC-MS/MS, and the individuals were genotyped for UGT2B17 deletion polymorphism using real-time polymerase chain reaction. RESULTS: The serum concentrations of testosterone and dihydrotestosterone (DHT) were markedly elevated on days 2 and 4 and were still above baseline on day 15 after a dose of 500 mg testosterone enanthate. The androgenic activity in serum increased in parallel and correlated with the hormone concentrations and remained above baseline on day 15. The urinary androgenic activity increased 4-5-fold and was closely related to the unconjugated testosterone and independent of the UGT2B17 genotype. CONCLUSIONS: The AR assay may serve as a complement to the urinary testosterone/epitestosterone (T/E) doping test, because this is profoundly influenced by the UGT2B17 deletion polymorphism. It may also be useful for detection of other illicit androgens in sports, or in the society, or for monitoring and diagnostics of androgen-related disorders.

Biomarkers of coagulation, inflammation, and angiogenesis are independently associated with preeclampsia.

PROBLEM: Although preeclampsia has been associated with inflammation, coagulation, and angiogenesis, their correlation and relative contribution are unknown. METHOD OF STUDY: About 114 women with preeclampsia, 31 with early onset (EOP) and 83 with late onset preeclampsia (LOP), and 100 normal pregnant controls were included. A broad panel of 32 biomarkers reflecting coagulation, inflammation, and angiogenesis was analyzed. RESULTS: Preeclampsia was associated with decreased antithrombin, IL-4 and placental growth factor levels and with increased C3a, pentraxin-3, and sFlt-1 levels, with more marked differences in the EOP group. The Th1-associated chemokines CXCL10 and CXCL11 were significantly higher in the preeclampsia and EOP group than in controls, respectively. No correlations between the biomarkers were found in preeclampsia. Multivariate logistic regression tests confirmed the results. CONCLUSIONS: Cytokines, chemokines and complement activation seem to be part of a Th1-like inflammatory reaction in preeclampsia, most pronounced in EOP, where chemokines may be more useful than cytokines as biomarkers. Biomarkers were not correlated suggesting partly independent or in time separated mechanisms.

Anabolic androgenic steroids in police cases in Sweden 1999-2009.

Anabolic Androgenic Steroids (AAS) are considered drugs of abuse and are controlled substances in Sweden since 1999. Traditionally AAS have been used by elite athletes to enhance performance, but in recent years it has become an increasing problem outside elite sport among athletes, bodybuilders and criminals. Use of AAS is associated with psychiatric side effects such as aggression, depression and violent behavior. Supraphysiological doses and long term use can cause serious physical harm such as cardiovascular toxicity and even premature death. We investigated and evaluated the drug analytical findings in forensic cases from suspected perpetrators in cases from the police where a screening for AAS was requested to get information about the prevalence of AAS use and the occurrence of poly-drug abuse. The study was based on samples submitted from the police authorities to the Department of Forensic Toxicology in Sweden during the period 1999-2009. Urines were analyzed by methods based on GC-MS and LC-MS-MS. We also analyzed the prevalence of AAS use at the prison and probation services. A total number of 12,141 urine samples (6362 police cases and 5779 inmates) were analyzed and 33.5% of the cases from the police and 11.5% of the inmates were tested positive for AAS. The users of AAS were mainly in 99.2% men with a mean age of 26.2±6.2 years whereas the women were 29.5±6.5 years old. The most frequently used AAS was nandrolone followed by testosterone and methandienone. Other illicit and licit drugs were detected in 60% of the cases from the police, strongly indicating a frequent poly-drug abuse among users of AAS.

Rapid detection of erythropoiesis-stimulating agents in urine and serum.

A rapid and easy-to-use test kit, EPO WGA MAIIA, which can be used for distinguishing various endogenous human erythropoietins (hEPOs) and several recombinant hEPO and EPO analogues, has been evaluated. The test is based on chromatographic separation of the glycosylated isoforms of EPO using wheat germ agglutinin (WGA) and a sensitive immunoassay using anti-EPO carbon black nanostrings and image scanning for quantification. All of the reactions take place along the porous layer of a lateral flow microcolumn containing WGA and anti-EPO zones. The presence of molecules resembling hEPOs, such as Mircera, was detected by the aberrant affinity interaction with the antibody zone on the strip. It was possible to distinguish nine recombinant hEPOs expressed in hamster and human cell lines, as well as Aranesp and Mircera, from endogenous urine hEPO. The required amount of EPO in the samples, a few picograms, is very low compared with other methods for EPO isoform identification. This EPO isoform determination method opens the possibility to monitor recombinant EPO therapy for clinical research and seems to be a valuable candidate to the arsenal of EPO doping control tests.

Long term perturbation of endocrine parameters and cholesterol metabolism after discontinued abuse of anabolic androgenic steroids.

AIMS: To study the long-term impact of anabolic androgenic steroid (AAS) abuse on the cholesterol profile, and the potential to suppress endocrine activity in men working out at gym facilities. To study the relation between urinary biomarkers for testosterone and nandrolone abuse and the UGT2B17 genotype and time profile. EXPERIMENTAL DESIGN: Subjects (N = 56) were recruited through Anti-Doping Hot-Line. Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), plasma levels of low density lipoprotein (LDL), high density lipoprotein (HDL) and urinary steroid profile were regularly measured for a period of up to one year after cessation of intramuscular AAS abuse. RESULTS AND DISCUSSION: A sustained suppression of LH, and FSH was observed for several months. The nandrolone urinary biomarker 19-NA was detectable several months after the last nandrolone intake and was correlated to the levels of LH and FSH. Testosterone abuse on the other hand was detectable only for a few weeks, and some of the testosterone abusers did not test positive due to a genetic deletion polymorphism of the UGT2B17. Significantly increased levels of HDL and decreased levels of LDL were observed for 6-months after cessation of AAS abuse. CONCLUSION: Some individuals had a sustained suppression of LH and FSH for a period of 1 year whereas the cholesterol profile was normalized within 6 month. The long term consequences of these findings remain to be established.

Androgen sulfation in healthy UDP-glucuronosyl transferase 2B17 enzyme-deficient men.

CONTEXT: The conspicuous interindividual differences in metabolism and urinary excretion of testosterone and its metabolites make it challenging to reveal testosterone doping. The variation in testosterone glucuronide excretion is strongly associated with a deletion polymorphism in the uridine diphosphate-glucuronosyltranferase (UGT) 2B17 gene. OBJECTIVE: The objective of the study was to identify additional biomarkers to detect testosterone abuse and to elucidate alternative pathways for testosterone elimination in individuals devoid of the UGT2B17 enzyme. For this purpose a new ultraperformance liquid chromatographic tandem mass spectrometric method for simultaneous determination of 10 different sulfo- and glucuronide-conjugated steroids was developed. PARTICIPANTS: Fifty-four healthy male volunteers with two, one, or no allele (ins/ins, ins/del, or del/del) of the UGT2B17 gene participated in the study. INTERVENTION: Intervention included a single im dose of 500 mg testosterone enanthate. MAIN OUTCOME MEASURES: Urinary sulfo- and glucuronide-conjugated steroids were measured. RESULTS: Testosterone sulfate levels decreased in all individuals after the dose. The individual differences in the excretion of all sulfated metabolites were large. Thus, these metabolites will not serve as appropriate biomarkers for testosterone abuse. However, androsterone glucuronide excretion increased in all of our study subjects after the testosterone dose. Etiocholanolone sulfate was excreted at significantly higher levels in UGT2B17 del/del individuals. CONCLUSION: We propose that the androsterone glucuronide to epitestosterone glucuronide ratio may serve as a complementary biomarker to reveal testosterone abuse.

Rapid affinity purification of erythropoietin from biological samples using disposable monoliths.

Identification of post-translational modifications of proteins in biological samples often requires access to preanalytical purification and concentration methods. In the purification step high or low molecular weight substances can be removed by size exclusion filters, and high abundant proteins can be removed, or low abundant proteins can be enriched, by specific capturing tools. In this paper is described the experience and results obtained with a recently emerged and easy-to-use affinity purification kit for enrichment of the low amounts of EPO found in urine and plasma specimens. The kit can be used as a pre-step in the EPO doping control procedure, as an alternative to the commonly used ultrafiltration, for detecting aberrantly glycosylated isoforms. The commercially available affinity purification kit contains small disposable anti-EPO monolith columns (6 µL volume, Ø7 mm, length 0.15 mm) together with all required buffers. A 24-channel vacuum manifold was used for simultaneous processing of samples. The column concentrated EPO from 20 mL urine down to 55 µL eluate with a concentration factor of 240 times, while roughly 99.7% of non-relevant urine proteins were removed. The recoveries of Neorecormon (epoetin beta), and the EPO analogues Aranesp and Mircera applied to buffer were high, 76%, 67% and 57%, respectively. The recovery of endogenous EPO from human urine was 65%. High recoveries were also obtained when purifying human, mouse and equine EPO from serum, and human EPO from cerebrospinal fluid. Evaluation with the accredited EPO doping control method based on isoelectric focusing (IEF) showed that the affinity purification procedure did not change the isoform distribution for rhEPO, Aranesp, Mircera or endogenous EPO. The kit should be particularly useful for applications in which it is essential to avoid carry-over effects, a problem commonly encountered with conventional particle-based affinity columns. The encouraging results with EPO propose that similar affinity monoliths, with the appropriate antibodies, should constitute useful tools for general applications in sample preparation, not only for doping control of EPO and other hormones such as growth hormone and insulin but also for the study of post-translational modifications of other low abundance proteins in biological and clinical research, and for sample preparation prior to in vitro diagnostics.

Substantial advantage of a combined Bayesian and genotyping approach in testosterone doping tests.

Testosterone abuse is conventionally assessed by the urinary testosterone/epitestosterone (T/E) ratio, levels above 4.0 being considered suspicious. A deletion polymorphism in the gene coding for UGT2B17 is strongly associated with reduced testosterone glucuronide (TG) levels in urine. Many of the individuals devoid of the gene would not reach a T/E ratio of 4.0 after testosterone intake. Future test programs will most likely shift from population based- to individual-based T/E cut-off ratios using Bayesian inference. A longitudinal analysis is dependent on an individual's true negative baseline T/E ratio. The aim was to investigate whether it is possible to increase the sensitivity and specificity of the T/E test by addition of UGT2B17 genotype information in a Bayesian framework. A single intramuscular dose of 500mg testosterone enanthate was given to 55 healthy male volunteers with either two, one or no allele (ins/ins, ins/del or del/del) of the UGT2B17 gene. Urinary excretion of TG and the T/E ratio was measured during 15 days. The Bayesian analysis was conducted to calculate the individual T/E cut-off ratio. When adding the genotype information, the program returned lower individual cut-off ratios in all del/del subjects increasing the sensitivity of the test considerably. It will be difficult, if not impossible, to discriminate between a true negative baseline T/E value and a false negative one without knowledge of the UGT2B17 genotype. UGT2B17 genotype information is crucial, both to decide which initial cut-off ratio to use for an individual, and for increasing the sensitivity of the Bayesian analysis.

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.

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.

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.

Criminality among individuals testing positive for the presence of anabolic androgenic steroids.

CONTEXT: Observations suggest that the use of anabolic androgenic steroids (AAS) may trigger uncontrolled, violent rage. Other observations indicate that certain groups of criminals may use AAS with the intention of being capable of committing crime more efficiently. OBJECTIVE: To examine the proposed association between the use of AAS and criminality. DESIGN: A controlled retrospective cohort study of registered criminal activity among individuals tested for AAS use during the period of January 1, 1995, to December 31, 2001. SETTING: All individuals in Sweden who were tested for AAS use during this period. These individuals were referred for testing from both inpatient and outpatient clinics as well as from centers for treatment of substance abuse. PARTICIPANTS: Individuals testing positive for AAS (n=241), with those testing negative for AAS during the same period (n=1199) serving as the control group. MAIN OUTCOME MEASURES: The ratios (expressed as relative risk [RR]) of the incidences of several categories of crime in the 2 study groups. RESULTS: The risk of having been convicted for a weapons offense or fraud was higher among individuals testing positive for AAS than among those testing negative (RR, 2.090 and 1.511, respectively; 95% confidence interval [CI], 1.589-2.749 and 1.208-1.891, respectively) whereas there were no significant differences with respect to violent crimes (RR, 1.116; 95% CI, 0.981-1.269) or crimes against property (RR, 0.942; 95% CI, 0.850-1.044). When patients referred from substance abuse centers were excluded, a lower risk for crimes against property was observed for the individuals who tested positive for AAS (RR, 0.761; 95% CI, 0.649-0.893) and the risk for fraud in the 2 groups was equalized (RR, 1.117; 95% CI, 0.764-1.635). The increased risk for a weapons offense among the individuals testing positive for AAS remained virtually unchanged. CONCLUSIONS: In addition to the impulsive violent behavior previously shown to be related to AAS use, such use might also be associated with an antisocial lifestyle involving various types of criminality. However, the existence and nature of this possible association remain unclear and call for further investigation.

Morbidity and mortality in patients testing positively for the presence of anabolic androgenic steroids in connection with receiving medical care. A controlled retrospective cohort study.

Observations by health-care professionals suggest that the use of anabolic androgenic steroids (AAS) may be associated with lethal complications, but this has not yet been confirmed by controlled epidemiological studies. Here, we investigated the diagnoses (in the Swedish patient care records) and mortality rate among patients who tested positively for the presence of AAS (n = 248) in connection with receiving medical care. Patients who had tested negatively (n = 1215) were used for comparison. The proportions of patients who had received institutionalized care for substance abuse, psychiatric disorder or central thoracic pain were significantly higher in the AAS-positive subjects (RR = 2.2, 95% CI = 1.2-4.2; RR = 2.1, 95% CI = 1.4-3.2 and RR = 3.5, 95% CI = 1.1-10.9, respectively). Furthermore, unspecified convulsions were highly over-represented in the AAS-positive group (RR = 53.9, 95% CI = 7.0-415.7) and one of these patients died during a seizure. The standardized mortality ratios (SMR) in the AAS-positive patients and -negative patients were 20.43 (95% CI = 10.56-35.70) and 6.02 (95% CI = 3.77-9.12), respectively. The relatively higher SMR in the AAS-positive patients was observed irrespective of what type clinic had referred the patients for AAS testing. In conclusion, use of AAS appears to be an indicator of increased risk for premature death in several categories of patients. However, the nature of the association between AAS and premature death remains unclear and additional research on this question is urgently required.