Post-mortem investigation into a death involving doping agents: The case of a body builder.

INTRODUCTION: A young male was found dead on the bed of a hotel room. He was expected to take part in a bodybuilding competition the day after. During the site inspection, drugs of different types were found. The next day, an autopsy was performed. The evidence of cardiomegaly with organ congestion involving lung, liver, kidneys, adrenal glands, spleen and brain was confirmed by both the autoptic and the histopathological exam. However, the cause of death needed to be investigated. METHODS: A thorough toxicological investigation was undertaken by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS) and liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on samples of urine, blood and hair. RESULTS AND DISCUSSION: Clenbuterol, a long-acting selective beta2 agonist, was found in both blood (1 ng/ml) and urine (1 ng/ml), and evidence of its use was provided by the analysis of the 3-cm hair (25 pg/mg). The main metabolite of drostanolone (2 alpha-methyl-androsterone), an anabolic steroid, was found in the urine (202 ng/ml), where an increased ratio of testosterone/epitestosterone (T/E = 11) emerged. Due to the results of the hair analysis, a long-term use of various anabolic steroids was supposed. The integrated analysis of the results and the absence of other possible causes (such as trauma or cardiac conduction anomalies) led to the identification of the abuse of doping substances as the underlying cause of death. CONCLUSION: Hair analysis has proven to be crucial in identifying drug misuse and the contributing cause of death.

Hair analysis to discriminate voluntary doping vs inadvertent ingestion of the aromatase inhibitor letrozole.

Letrozole is an aromatase inhibitor, used to treat postmenopausal women with hormone receptor-positive or unknown advanced breast cancer. It is prohibited in sport because it is used together with androgen anabolizing steroids to avoid their adverse effects. In the case of an adverse analytical finding, it may be important to distinguish between repetitive use due to voluntary administration and occasional use, possibly due to involuntary intake. With the objective to identify the dose capable of producing a positive hair test, and to apply these results to the scenarios of inadvertent letrozole ingestion by an athlete, this study investigates the urinary excretion and incorporation into hair of single doses of letrozole. Seven subjects were recruited for an excretion study of letrozole and its metabolite bis(4-cyanophenyl) methanol (M1) in urine, after the consumption of 0.62 mg, 1.25 mg, and 2.5 mg of letrozole, and to investigate the incorporation in hair after ingestion of 0.62 mg and 2.5 mg of letrozole. Urine and hair samples were also obtained from two women in chronic therapy. Urinary concentrations of letrozole and its metabolite M1 were lower in subjects administered once with 0.62 mg, 1.25 mg, or 2.5 mg letrozole than in women in regular therapy with 2.5 mg/day. In hair collected after a single dosage, concentrations of 16-60 pg/mg were detected while in women in chronic therapy concentrations were higher than 160 pg/mg all along the hair shaft. Hair analysis turned to be a promising possibility for the discrimination of letrozole repetitive use vs occasional/inadvertent administration.

Multiple incidence of the prescription diuretic hydrochlorothiazide in compounded nutritional supplements.

Diuretic agents are prohibited in sports in- and out-of-competition according to the regulations of the World Anti-Doping Agency (WADA) because of their possible masking effects on other doping agents in urine samples, and their ability to produce fast acute weight losses. Despite previous studies reported adverse analytical findings (AAFs) resulting from contaminations at ppm level (µg/g) of medicinal products, and recommended to introduce reporting limits for diuretics in doping controls, these are not adopted in analyses performed by WADA-accredited laboratories. We report the case of an athlete with two AAFs for hydrochlorothiazide (HCTZ) at low urinary concentrations (<10 ng/mL), who declared the use of nutritional supplements prepared in a compounding pharmacy. His nutritional supplements were analyzed revealing HCTZ presence in different concentrations, at the ppm level (µg/g and ng/mL). With the aim of testing the plausibility of the observed urinary HCTZ concentrations with the nutritional supplement ingestion, a urinary excretion study with three healthy volunteers was performed. HCTZ-contaminated powder (6.4 µg/g of HCTZ) was administered to each subject in different dosages, reproducing the possible ingestion pattern occurred. Urine specimens were collected before and after ingestion of the powder, up to 24 hours, and underwent liquid-liquid extraction and liquid chromatography-tandem mass spectrometry determination. Post-administration specimens were found to contain HCTZ at concentrations of 5-230 ng/mL, which supported the accidental inadvertent intake of the prohibited substance by the athlete. This study makes the argument that the introduction of reporting limits for diuretics are warranted in doping control samples, in order to protect against inadvertent AAFs due to contaminated products.

Mechanisms involved in the antinociception caused by melatonin in mice.

The present study assesses the antinociceptive effect of melatonin in chemical behavioral models of nociception and investigates some of the mechanisms underlying this effect. Melatonin administered by intraperitoneal (i.p., 10-100 mg/kg), intracerebroventricular (i.c.v., 250-500 pmol/site) and intraplantar (i.pl., 30-100 ng/i.pl.) routes, reduced in a dose-dependent manner the nociception caused by i.pl. injection of glutamate (10 micromol/paw), with mean ID50 values of 32.6 mg/kg, 200 pmol/site and 59 ng/i.pl., respectively. Furthermore, melatonin in the dose range of 10-100 mg/kg, i.p., reduced the neurogenic pain caused by i.pl. injection of capsaicin (5.2 nmol/paw) with inhibition of 48 +/- 4%. The antinociceptive effect of melatonin (100 mg/kg, i.p.) on glutamate-induced nociception was completely prevented by the pretreatment of animals with naloxone (a nonselective opioid receptor antagonist, 1 mg/kg, i.p.), ketanserin (a preferential 5-HT2A receptor antagonist, 1 mg/kg, i.p.), sulpiride (a D2 receptor antagonist, 50 mg/kg, i.p.), L-arginine (a precursor of nitric oxide, 600 mg/kg, i.p.), yohimbine (an alpha2-adrenoceptor antagonist, 0.15 mg/kg, i.p.) and luzindole (a preferential MT2 receptor antagonist, 10 mg/kg, i.p.), but was not affected by the pretreatment with D-arginine (an inactive isomer of L-arginine, 600 mg/kg, i.p.), prazosin (an alpha1-adrenoceptor antagonist, 0.15 mg/kg, i.p.) or after bilateral adrenalectomy. Collectively, present results suggest that melatonin produces peripheral and central antinociception when assessed on capsaicin- or glutamate-induced pain in mice through mechanisms that are likely mediated by interaction with plasma membrane-bound melatonin receptors and modulated by opioid, serotonergic (5-HT2A receptors), dopaminergic (D2-receptors), adrenergic (alpha2-adrenoceptors) systems as well as the L-arginine-nitric oxide pathway.

Melatonin exerts an antidepressant-like effect in the tail suspension test in mice: evidence for involvement of N-methyl-D-aspartate receptors and the L-arginine-nitric oxide pathway.

This study investigated the effect of melatonin in the mouse tail suspension test (TST), and the contribution of N-methyl-D-aspartate (NMDA) receptors and the L-arginine-nitric oxide (NO) pathway to its antidepressant-like effect. The immobility time in the TST was reduced by melatonin given either by intraperitoneal (0.1-30 mg/kg) or intracerebroventricular (0.001-0.1 nmol/site) route. The anti-immobility effect of melatonin (1 mg/kg, intraperitoneal, i.p.) was prevented by pre-treatment with guanosine 5'-monophosphate (GMP), ascorbic acid, L-arginine or S-nitroso-N-acetyl-penicillamine, but not with D-arginine. Pre-treatment with melatonin (100 mg/kg, i.p.) prevented the anti-immobility effect of MK-801, ketamine or zinc chloride, but did not alter the effect of imipramine. Furthermore, a sub-effective dose of melatonin (0.001 mg/kg, i.p.) produced a synergistic antidepressant-like effect with MK-801, ketamine, zinc chloride and imipramine in the TST. Taken together these data indicate that the effect of melatonin in the TST seems to be mediated through an interaction with NMDA receptors and the L-arginine-NO pathway.