Multi-Residue Determination of Eight Anabolic Steroids by GC-MS in Muscle Tissues from Pigs

A gas chromatography-mass spectrometry （GC-MS） method to determine eight anabolic steroids （diethylstilbestrol, methyltestosterone, norethindrone, 17α-ethynylestradiol, estradiol, 6α-methyl-17α-hydroxy-progesterone, estradiol benzoate, and chlormadinone acetate） was developed. Muscle samples were extracted with liquid-liquid extraction and clean-up was performed in two steps, the extracts obtained were derivatized with heptafluorbutyric （HBF） anhydride and analyzed by GC-MS. In the above method, the linear scope was 2.5-50 μg kg^-1. The range of the recoveries was 78.5-148% for diethylstilbestrol, 70.8-109% for methyltestosterone, 69.8-87.2% for norethindrone, 67.7-120% for 17α-ethynylestradiol, 82.8-103% for estradiol, 70.3-99.2% for 6α-methyl-17α-hydroxy-progesterone, 73.0-104% for estradiol benzoate, and 72.9- 91.8% for chlormadinone acetate. The range of the coefficients of variation within batches was 0.4-12%; the range of the coefficients of variation between batches was 6.4-11%. The limit of detections and the detection capability were 0.99 and 3.30 μg kg^-1 for diethylstilbestrol, 1.05 and 3.50 μg kg^-1 for methyltestosterone, 1.19 and 3.97 μg kg^-1 for norethindrone, 0.94 and 3.13 μg kg^-1 for 17α-ethynylestradiol, 1.45 and 4.83 μg kg^-1 for estradiol, 1.56 and 5.20 μg kg^-1 for 6α-methyl-17α- hydroxy-progesterone, 1.92 and 6.40 μg kg^-1 for estradiol benzoate, and 2.41 and 8.03 μg kg^-1 for chlormadinone acetate, respectively. These results showed that the method was widely available, accurate, and sensitive.

GAS CHROMATOGRAPHIC-MASS SPECTROMETRIC ANALYSIS OF ANABOLIC STEROIDS IN HUMAN URINE

A new GC/MS method for detection and identification of 19 anabolic steroids in human urine is presented.The procedure involves adsorption and isolation on a macroporous XAD-2 resin,enzymatic hydrolysis,alkaline extraction,derivatization,GC separation and MS detec- tion.Gas chromatographic-mass spectrometric data illustrate artifacts arising from enzymatic hydrolysis of steroid glucuronides and the structural characterization of their metabolites. Using this method,metabolic studies of these steroids in human urine were made after their ingestion by normal and healthy male volunteers.This method was proven to be suitable for large-scale routine analysis of anabolic steroids and was used successfully in passing the doping control test held by the Medical Commission of the International Olympic Committee.

Specific alterations of regional myocardial work in strength-trained athletes using anabolic androgenic steroids compared to athletes with genetic hypertrophic cardiomyopathy

Background:Strength-trained athletes using anabolic androgenic steroids(AAS)have left ventricular(LV)hypertrophy and myocardial fibrosis that can lead to sudden cardiac death.A similar feature was described in athletes with hypertrophic cardiomyopathy(HCM),which complicates the diagnosis for clinicians.In this context,we aimed to compare the LV function of the 2 populations by measuring global and regional strain and myocardial work using speckle-tracking imaging.Methods:Twenty-four strength-trained asymptomatic athletes using AAS(AAS-Athletes),22 athletes diagnosed with HCM(HCM-Athletes),and 20 healthy control athletes(Ctrl-Athletes)underwent a resting echocardiography to assess LV function.We evaluated LV global and regional strains and myocardial work,with an evaluation of the constructive work(CW),wasted work,and work efficiency(WE).Results:Compared to Ctrl-Athletes,both AAS-Athletes and HCM-Athletes had a thicker interventricular septum,with maj ored values in HCM-Athletes.LV strain was reduced in AAS-Athletes and even more in HCM-Athletes.Consequently,global WE was significantly diminished in both AAS and HCM-Athletes(93%±2%in Ctrl-Athletes,90%±4%in AAS-Athletes,and 90%±5%in HCM-Athletes(mean±SD);p<0.05).Constructive work and WE regional analysis showed specific alterations,with the basal septal segments preferentially affected in AAS-Athletes,and both septal and apical segments affected in HCM-Athletes.Conclusion:The regional evaluation of myocardial work reported specific alterations of the major LV hypertrophy induced by the regular use of AAS compared to the LV hypertrophy due to HCM.This finding could help clinicians to differentiate between these 2 forms of pathological hypertrophy.

Physiological and pharmacological basis for the ergogenic effects of growth hormone in elite sports

Growth Hormone （GH） is an important and powerful metabolic hormone that is secreted in a pulsatile pattern from cells in the anterior pituitary, influenced by several normal and pathophysiological conditions. Human GH was first isolated in the 1950s and human derived cadaveric GH was initially used to treat patients with GH deficiency. However, synthetic recombinant GH has been widely available since the mid-1980s and the advent of this recombi- nant GH boosted the abuse of GH as a doping agent. Doping with GH is a well-known problem among elite athletes and among people training at gyms, but is forbidden for both medical and ethical reasons. It is mainly the anabolic and, to some extent, the lipolytic effects of GH that is valued by its users. Even though GH＇ s rumour as an effective ergogenic drug among athletes, the effectiveness of GH as a single doping agent has been questioned during the last few years. There is a lack of scientific evidence that GH in supraphysiological doses has additional effects on muscle exercise performance other than those obtained from optimised training and diet itself. However, there might be synergistic effects if GH is combined with, for example, anabolic steroids, and GH seems to have positive effect on collagen synthesis. Regardless of whether or not GH doping is effective, there is a need for a reliable test method to detect GH doping. Several issues have made the development of a method for detecting GH doping complicated but a method has been presented and used in the Olympics in Athens and Turin. A problem with the method used, is the short time span （24-36 hours） from the last GH administration during which the test effectively can reveal doping. Therefore, out-of-competition testing will be crucial. However, work with different approaches to develop an alternative, reliable test is ongoing.

Sudden cardiac death in anabolic androgenic steroids abuse: case report and literature review

Anabolic androgenic steroids (AAS) have several adverse effects on the cardiovascular system that may lead to a sudden cardiac death (SCD).We herein report a case involving a 24-year-old male,AAS abuser with intramuscular delivery in the 6 months before,who suffered a cardiorespiratory arrest at home's bathtub when returning from New Year's party.A forensic autopsy was performed according to the guidelines of the Association for European Cardiovascular Pathology (AECVP).The body showed hypertrophy of skeletal musculature,with low amount of subcutaneous fat and no signs of injury (body mass index,BMh 26.8 kg/m^(2)).On internal examination,there were multiorgan congestion,acute pulmonary edema,and cardiomegaly (420 g) with severe coronary atherosclerosis and superimposed acute occlusive thrombosis at the left main trunk and left anterior descendant.Areas of scarring were located at the intersection between the posterior wall and the posterior third of the septum (postero-septal).At histology,acute myocardial infarction at the anterior third of the septum and the anterior wall,and subacute myocardial infarction at apical septum and apical posterior wall were detected.Other findings were small intramyocardial vessel disease and myocytes hypertrophy.Chemicotoxicological analysis in blood showed ethanol ((0.90 ±0.05) g/L),stanazolol (11.31 μg/L),nandrolone (2.05μg/L) and testosterone (<1.00μg/L).When confronted with a sudden death in a young athlete we must pay attention to the physical phenotype that may suggest AAS abuse and perform a detailed examination of the heart.Chemicotoxicological analysis is a key to establish the relationship between SCD and AAS abuse.

The reversibility of sperm quality after discontinuing nandrolone decanoate in adult male rats

Aim： To investigate the reversibility of the effect of nandrolone decanoate （ND） on sperm parameters after discontinuing the drug. Methods： Three groups of rats received peanut oil （control）, low and high doses of ND for 14 weeks, respectively. Each group was divided into subgroups A and B, in which rats were killed 14 and 28 weeks after initiating the injection, respectively. Results： Sperm count and motile sperm fraction were decreased in the subgroups A and B that received low and high doses of ND in comparison with the controls A and B, respectively. The sperm count and motile sperm fraction increased in the subgroups B that received low and high doses of ND in comparison with their corresponding subgroups A. The number of normal morphology sperm was decreased significantly in subgroups A receiving low and high doses of ND in comparison with the control subgroup A. However, this parameter was not decreased in subgroups B receiving low and high doses in comparison with the control subgroup B. The normal sperm morphology did not show any significant differences in the subgroups B in comparison with their corresponding subgroups A. Conclusion： The 14-week injection of low and high doses of ND decreases sperm quality and quantity in rats. These parameters were improved after discontinuing ND, but not recovered completely even when they are left untreated for 14 weeks.

Recovery of spermatogenesis following testosterone replacement therapy or anabolic-androgenic steroid use

The use of testosterone replacement therapy （TRT） for hypogonadism continues to rise, particularly in younger men who may wish to remain fertile. Concurrently, awareness of a more pervasive use of anabolic-androgenic steroids （AAS） within the general population has been appreciated. Both TRT and AAS can suppress the hypothalamic-pituitary-gonadal （HPG） axis resulting in diminution of spermatogenesis. Therefore, it is important that clinicians recognize previous TRT or AAS use in patients presenting for infertility treatment. Cessation of TRT or AAS use may result in spontaneous recovery of normal spermatogenesis in a reasonable number of patients if allowed sufficient time for recovery. However, some patients may not recover normal spermatogenesis or tolerate waiting for spontaneous recovery. In such cases, clinicians must be aware of the pathophysiologic derangements of the HPG axis related to TRT or AAS use and the pharmacologic agents available to reverse them. The available agents include injectable gonadotropins, selective estrogen receptor modulators, and aromatase inhibitors, but their off-label use is poorly described in the literature, potentially creating a knowledge gap for the clinician. Reviewing their use clinically for the treatment of hypogonadotropic hypogonadism and other HPG axis abnormalities can familiarize the clinician with the manner in which they can be used to recover spermatogenesis after TRT or AAS use.

Selective Androgen Receptor Modulators:An Emerging Liver Toxin

Selective androgen receptor modulators(SARMs)are a class of nonsteroidal drugs that are favored over anabolic androgenic steroids(AASs)for their tissue-selectivity and improved side-effect profile.These drugs have been evaluated for treatment of various diseases including muscle-wasting disorders,osteoporosis,and breast cancer.Despite lacking approval for therapeutic use,SARMs are widely used recreationally as performance enhancing drugs by bodybuilders and athletes.In recent years,cases of drug-induced liver injury(DILI)secondary to SARMs have begun to emerge,but little is known regarding their hepatotoxicity.In this review,we provide current knowledge regarding DILI from SARMs.A literature search was conducted regarding SARMs and liver injury to evaluate relevant cases and information.SARMs have been associated with a cholestatic syndrome congruent with that of DILI from AASs,and it consists of a bland cholestasis in which there is minimal bile duct injury,inflammation,or necrosis.Patients present with an insidious onset of jaundice with marked hyperbilirubinemia and mild hepatic enzyme elevations.No clear treatment exists,although patients typically show improvement with cessation of the offending SARM.Given the novelty of these drugs,further study is necessary to understand diagnosis,management,and complications of SARM-related DILI.

Dietary rumen-protected L-arginine or N-carbamylglutamate enhances placental amino acid transport and suppresses angiogenesis and steroid anabolism in underfed pregnant ewes

This study aimed to investigate the effects of dietary supplementation of underfed Hu ewes from d 35 to110 of gestation with either rumen-protected L-arginine(RP-Arg)or N-carbamylglutamate(NCG)on placental amino acid(AA)transport,angiogenic gene expression,and steroid anabolism.On d 35 of gestation,32 Hu ewes carrying twin fetuses were randomly divided into four treatment groups,each consisting of eight ewes,and were fed the following diets:A diet providing 100%of NRC’s nutrient requirements for pregnant ewes(CON);A diet providing 50%of NRC’s nutrient requirements for pregnant ewes(RES);RES diet plus 5 g/d NCG(RES+NCG);or RES diet plus 20 g/d RP-Arg(RES+ARG).On the d 110 of pregnancy,blood samples were taken from the mother,and samples were collected from type A cotyledons(COT;the fetal portions of the placenta).The levels of 17β-estradiol and progesterone in the maternal serum and both the capillary area density(CAD)and capillary surface density(CSD)in type A COT were decreased in response to Arg or NCG supplementation when compared to the RES group.The concentrations of arginine,leucine,putrescine and spermidine in type A COT were higher(P<0.05)in the RES+ARG or RES+NCG group than in the RES group.The mRNA expression levels of inducible nitric oxide synthase(iNOS)and solute carrier family 15,member 1(SLC15A1)were increased(P<0.05)while those of progesterone receptor(PGR)and fibroblast growth factor 2(FGF2)were decreased in type A COT by supplementation with either NCG or RP-Arg compared to the RES group.The results suggest that providing underfed pregnant ewes from d 35 to 110 of gestation with a diet supplemented with NCG or RP-Arg improves placental AA transport,and reduces the expression of angiogenic growth factor genes and steroid anabolism,leading to better fetal development.