Analytical techniques in androgen anabolic steroids (AASs) analysis for antidoping and forensic purposes.

A survey on the main analytical challenges related to the analysis of Androgen Anabolic Steroids (AASs) is reported. AASs analysis is an issue regarding antidoping analyses as well as forensic toxicology applications. This paper reports an overview of the more recent literature regarding various aspects of sample preparation, analytical techniques and interpretation of results for AASs identification in biological samples. New analytical approaches, mainly for their application to the antidoping field, are reported. The application of AASs analysis in forensic cases is also described, taking into consideration mainly the different biological samples that can be analysed for forensic purposes. Particular attention was played on the application of hair analysis as alternative biological specimen for the determination of AASs abuse.

Suicide due to oral ingestion of lidocaine: a case report and review of the literature.

The Authors describe a rare case of suicide in a 31-year-old woman, due to oral ingestion of lidocaine; the histological and toxicological findings are discussed to provide useful information to the present experience with this particular modality of death. Histological examination revealed generalized stasis. In the myocardium we observed segmentation of the myocardial cells and/or widening of intercalated discs and associated group of hypercontracted myocardial cells with "square" nuclei in line with hyperdistended ones. Non-eosinophilic bands of hypercontracted sarcomeres alternating with stretched, often apparently separated sarcomeres, small foci of paradiscal contraction band necrosis, and perivascular fibrosis were observed too. Lidocaine was detected in the subject's urine through immunoenzymatic screening. Toxicological analysis by solid-liquid extraction and gas chromatography-mass spectrometry (GC-MS) analysis, was carried out to identify and quantify the individual substances present in the biological fluids and organs. Lidocaine concentrations were as follows: blood 31 microg/mL, gastric content 2.5 g, liver 10 microg/g, kidney 12 microg/g, brain 9 microg/g, spleen 24 microg/g, lung 84 microg/g, heart 9 microg/g, urine 9 microg/mL, and bile 6 microg/mL. No other drugs or alcohol were detected. When blood lidocaine reaches toxic levels, serious toxic symptoms associated with the central nervous system and cardiac system are noted. The overdose of lidocaine produces death from ventricular fibrillation or cardiac arrest. In this case, according to macroscopic and microscopic findings, the cause of death was most likely cardiac and possibly related to ventricular fibrillation.

Markers of cardiac oxidative stress and altered morphology after intraperitoneal cocaine injection in a rat model.

This study was designed to assess the parameters of myocardial oxidative stress and related cardiac morphological changes following intraperitoneal cocaine exposure in rats. The cardiac levels of reduced glutathione(GSH), oxidised glutathione(GSSG), ascorbic acid (AA), and the production of malondialdehyde (MDA) were measured, as well as the variations of activity in the enzyme systems involved in cell antioxidant defence, glutathione peroxidase (GSH-Px), glutathione reductase (GR) and superoxide dismutase (SOD). After chronic cocaine administration for 30 days GSH was significantly depleted in the heart from 30 min (P < 0.001) to 24 h (P < 0.001) after exposure, and GSSG was increased for a similar time (P < 0.05 at 30 min and P < 0.01 at 24 h). SOD increased during the first hour (P < 0.001), GR and GSH-Px both increased from 30 min to 24 h, and these increases were statistically significant (P < 0.01 and P < 0.001 at 30 min and P < 0.01 and P < 0.001 at 24 h, respectively). The AA levels increased after 1 h (P < 0.01), remaining significantly so for 24 h (P < 0.001) and MDA increased from 30 min to 24 h, all values being highly significant (P < 0.001). The body weight was significantly (P < 0.001) reduced in both cocaine groups (40 mg/kg x 30 days and 40 mg/kg x 10 days + 60 mg/kg x 20 days). The heart weight (P < 0.01) and its percentage of the body weight (P < 0.001) were significantly higher in these two groups than in the controls. Similarly, in the noradrenaline 4 mg/ kg x 30 days group, the body weight was significantly (P < 0.001) reduced and the heart weight (P < 0.01) and its percentage of body weight (P < 0.001) were significantly higher than in the controls. In comparing the cocaine and noradrenaline experiments, the frequency and extent of cardiac lesions obtained with 40 mg/kg x 10 days + 60 mg/kg x 20 days of cocaine were similar to those with 8 mg/kg of noradrenaline at 24 h. In this experimental model, cocaine administration compromised the antioxidant defence system of the heart associated with a significant increase of heart weight and the percentage of body weight.

Immunohistochemical quantification of pulmonary mast-cells and post-mortem blood dosages of tryptase and eosinophil cationic protein in 48 heroin-related deaths.

Recent studies suggest that many fatal heroin overdoses are caused by anaphylactoid reaction. In the present study we measured tryptase and eosinophil cationic protein in post-mortem blood of 48 deaths after heroin injection. We also investigated the presence and pulmonary distribution of mast-cells using specific immunohistochemical antibody for tryptase and morphometric evaluation in those cases of heroin-related deaths. The data were compared with 44 subjects who died following head trauma and to 32 cases of fatal anaphylactic shock. In the heroin-related death cases, the measurements of serum tryptase levels and eosinophil cationic protein dosages resulted in particularly elevated concentrations compared with the trauma cases. Nevertheless, the data that our study supplies by immunohistochemical techniques indicate that when mast-cells count in the lung was determined, no definite pattern was obtained between fatal heroin overdose cases and the control groups. Furthermore, the wide range of morphine concentrations found in post-mortem blood samples suggest that the term 'overdose' is relative and does not sufficiently characterize death associated with heroin addiction. Our study confirms that elevated concentrations of serum tryptase are associated with many heroin-related deaths. At this moment to attribute the cause of these deaths to 'heroin overdose' ignores the likely causal contribution of other possible systemic reactions to the mechanism of death.

Adam (MDMA) and Eve (MDEA) misuse: an immunohistochemical study on three fatal cases.

Three fatal cases of MDMA/MDEA misuse have been examined. These referred to white males between 19 and 20 years of age, in which post-mortem toxicology showed the presence of MDMA (in one case), MDEA (in one case) and both (in one case). The clinical data were analysed and the histopathological findings were studied following immunohistochemical investigations. A complete immunohistochemical study has made it possible to demonstrate rhabdomyolysis and myoglobinuria with alterations of the organs typical of a DIC. Clinical, histopathological and toxicological data suggest that severe or fatal complications following ecstasy ingestion could be related to idiosyncratic response.

Uremia, dialysis and aluminium.

Few studies have dealt with assaying aluminium levels in different tissues of uremic patients; so far a comparison has never been made between its accumulation in the various tissues of uremic patients and controls. Aluminium levels were determined in the following biological samples: 1) 111 serum samples from hemodialysis patients and 55 serum samples from normal subjects; 2) 47 urine samples from the same dialysis patients and 45 from the controls; autopsy tissue specimens (blood, bile, brain, rib, cartilage, cranium, lung, spleen, kidney, aorta, vena cava, liver, muscle) from 12 deceased dialysis patients undergoing post-mortem diagnosis and 10 autopsy cases in which death was not associated with uremia. In living subjects, both serum and urinary levels of aluminium are significantly higher in hemodialysis patients than in controls; a significant positive correlation was found between serum and urinary levels of aluminium. In autopsy specimens, aluminium levels were higher in the dialysis group than controls for all tissues; the differences were statistically significant except in heart and urine. Tissue concentrations of aluminium in the two groups were then analysed separately both in uremic patients and controls. The highest values found in dialysis cases were in the bile, followed by blood, urine and lung; levels in the other tissues were considerably lower. In controls, the distribution was somewhat different, due to much lower levels in the liver and bile with respect to dialysis cases. Again we found surprisingly high levels in the lung. The results show that aluminium storage in uremic patients occurs in all organs and tissues, albeit to different degrees.

Quantitative and qualitative analysis of MDMA, MDEA, MA and amphetamine in urine by headspace/solid phase micro-extraction (SPME) and GC/MS.

The results of qualitative and quantitative analysis of some amphetamines and their analogs isolated from urine samples by solid phase micro-extraction with polydimethylsiloxane fibers are reported. The analytical method employed was gas-chromatography/mass spectrometry of head space samples.

Histological criteria for diagnosis of amanita phalloides poisoning.

Five fatal cases of poisoning from ingestion of Amanita phalloides, a very common mushroom in central Italy, are reported. The fact that four of the cases occurred simultaneously enabled uniform collection of clinical, pathology and toxicology data, which is presented with particular emphasis on the histological aspects. The fifth case involved a six-year-old girl, and is discussed with reference to differential diagnosis with respect to Reye's syndrome, which was the initial diagnosis, demonstrated incorrect by the histology, pathology and toxicology findings. The typical liver and kidney alterations of Amanita phalloides poisoning, consisting of massive hepatic central lobular cell necrosis and acute tubular necrosis of the kidney are described. Outside the liver, there was often general hemorrhagic diathesis and severe brain edema. Although poisoning by Amanita phalloides is rare, these cases confirm the requirement for as complete a comparison as possible between circumstantial histopathological and toxicological data for the purposes of forensic diagnosis.

Synthesis and biological evaluation of novel NK-1 tachykinin receptor antagonists: the use of cycloalkyl amino acids as a template.

In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the superimposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 A distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the pi stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed.

Pharmacodynamics of ticlopidine in man in relation to plasma and blood cell concentration.

In 6 normal volunteers given single oral doses of 250,500 and 1000 mg ticlopidine (T), the peak plasma level of unchanged drug was reached after about 2 h. There was no correlation between the plasma T level and its inhibitory effect on platelet function, expressed as % inhibition of ADP-induced aggregation. By means of HPLC and GC/MS significant concentrations of T were demonstrated in washed red cells, platelets and neutrophils, with a marked difference in the time course of the appearance of cell-associated drug. The time course of platelet-associated T very accurately fitted that of the antiaggregatory activity. After subacute oral administration (250 mg b.d. for 7 days), the maximum effect on platelet function was observed after 3 to 4 days, when a significant concentration of platelet-associated T had been reached. The pharmacological effect persisted as long as drug was detectable in platelet. An in vitro study strongly suggested that the antiaggregating effect was retained by treated washed platelets but not by treated plasma. It is suggested that the platelet compartment represents the pharmacological target of T via a specific uptake system.

Cannabinoid level in the leaves as a tool for the early discrimination of Cannabis chemiovariants.

One hundred and seventy-six plants of 22 different lots of Cannabis sativa L., grown at the Botanical Garden of Siena (Italy) were chromatographically analysed in order to define the cannabinoid content in their leaves. The content of the major cannabinoids, delta 9-tetrahydrocannabinol, cannabidiol, cannabinol and cannabichromene, determined weekly in vegetative and floral leaves enabled the determination of the chemical types of the plants, according to Turner's classification. The plants were easily distinguishable in drug, intermediate and fiber types. The cannabinoid characteristic of each type remains predominant, as compared with the other cannabinoids, throughout the whole period of growth, including the floral stage and after harvesting. On this basis, the predominant concentration of a specific cannabinoid can be used reliably for forensic application concerning drug-suspected material in very young plants.

Packed column chromatography, high-resolution gas-chromatography and high pressure liquid chromatography in comparison for the analysis of cannabis constituents.

The identification and the quantitative estimation of cannabis constituents is important for forensic purposes. High resolution gas-chromatography gives better results than gas-liquid chromatography with packed columns, as it shows a better resolution and higher number of constituents. Different quantitative values were found with the two chromatographic procedures. High-pressure liquid chromatography revealed the presence of cannabinoid acids in fresh cut influorescences of cannabis plants. The ratio acid/neutral cannabinoid may be useful in supplying information about the age of the cannabis preparations.

[Experimental chronic poisoning by mercury and alcohol: histopathologic, ultrastructural findings and toxicologic and enzymatic evaluations]. / Intossicazione cronica sperimentale da mercurio ed alcool: reperti istopatologici, ultrastrutturali e dosaggi tossicologici ed enzimatici.

Rats were treated with HgCl2 and/or alcohol per os in a standard diet as to get a chronic intoxication. Histological and ultrastructural examination of the liver, GPT, GOT, LDH enzymatic activities, and toxicological mercury evaluation were performed. Liver cell degenerative changes with focal necrosis, and structural alteration and fibrosis were demonstrated in the group of rats treated with HgCl2. The combined administration of HgCl2 and alcohol did not result in more advanced lesions, even if steatosis could be demonstrated in the liver. An increase of the GPT, GOT and LDH enzymatic activities was demonstrated in the rats of both groups but it was higher in the rats treated with HgCl2 and alcohol combined. On the contrary, the liver and kidney mercury storage was higher in the rats treated with HgCl2 alone.

Respiratory tract lesions induced in rabbits by short-term exposure to N-hexane.

New Zealand rabbits were exposed in inhalation chambers to 3,000 ppm of n-hexane 8h/d for 8 d, and the acute respiratory effects were studied by light and electron microscopy. Animals intoxicated showed morphological changes in the lung parenchyma characterized by centriacinar emphysema and scattered micro haemorrhages. Lung damage was most severe at the transition zone from terminal bronchiole to alveolar ducts. This centriacinar lesion consisted to degenerative and necrotic phenomena in bronchiolar epithelium with cellular desquamation, increased number of macrophages within proximal alveoli of alveolar ducts, increased number and size of lamellar bodies in alveolar type II cells, changes in the vascular endothelium. Focal subpleural atelectasis, alveolar and interstitial oedema were also found. The segmental distribution of the pulmonary lesions were confirmed by transmission electron microscopy.