Proficiency testing for psychoactive substances in Italy.

This paper describes the general design and main results of the Italian proficiency testing program for the analysis of psychoactive substances in urine, a long-term initiative created in 1995 on an educational basis and characterized by an innovative internet-based service for data exchange between laboratories and the organizing body. Batches of six urine samples, validated by reference laboratories, are sent every 3 months to participating laboratories, which may choose which classes of substances to test from those planned by the program panel and, within those classes, which type of analytical commitment to work on: identification of just one class (Option 1), identification of single substances (Option 2), or identification and quantification of single substances (Option 3). Comprehensive periodical reports and annual reports are provided to participants with evaluation of their performance and an annual workshop is organized to discuss technical-scientific topics related to clinical, forensic and analytical toxicology. About 200 laboratories currently participate in the program and a total of 67,059 analyses have been carried out since 1995. The mean percentage of correct results was 96.8%, with a yearly improvement of about 0.4%. The best average false positive and false negative rates were obtained for methadone (0.2% and 2.1% respectively) and cocaine (0.3% and 2.2%). The worst average false positive rates were obtained for amphetamines and opiates (3.2% and 5.0%) and worst average false negative rates for amphetamines, barbiturates and cannabinoids (17.4%, 30.7% and 19.9%).

Effects of gangliosides on the expression of autoimmune demyelination in the peripheral nervous system.

To test whether gangliosides (GA) might exert neuritogenic effects in vivo, experimental allergic neuritis (EAN) was studied clinically, neuropathologically, and immunologically in Lewis rats immunized with bovine peripheral nerve, P2 myelin protein, P2 myelin protein plus two different doses of GA, P2 with galactocerebroside (GC), and GA alone, each emulsified in adjuvant. All except the GA-treated group developed signs of EAN between days 11 and 14 after the injection. Rats immunized with P2 alone were the most severely affected. Rats given P2 plus GA and those given P2 plus GC displayed a significantly lower clinical score. Histological analysis revealed a comparable degree of inflammation of the peripheral nervous system and demyelination in the spinal nerve roots of bovine peripheral nerve- and P2-immunized rats. The P2 plus GA and P2 plus GC groups revealed similar degrees of pathology in the spinal nerve roots but the latter group stood apart from the rest in that it showed widespread peripheral nervous system changes extending distally into the sciatic nerve. Serological analysis demonstrated that P2 and GC, but not GA, elicited antibody (IgG) responses, but there was no correlation between antibody titer and clinical or histological involvement. The present data fail to support an enhancing role for gangliosides in the expression of EAN and, by extrapolation, in the Guillain-Barré syndrome, for which EAN serves as the laboratory model, and in which suggestions have been made that antibodies to GA may have pathogenetic significance.

Pharmacokinetic characterization of phosphatidylserine liposomes in the rat.

1. The plasma decay, tissue uptake and biotransformation of radiolabelled phosphatidylserine (PS) liposomes have been investigated in rats following bolus i.v. injection (2 mg kg-1). 2. PS plasma concentration showed a biexponential decay with half-lives of 0.85 and 40 min. The following interpretation of the biphasic decay is proposed: (1) The rapid initial decline is due to the irreversible uptake of PS liposomes by the mononuclear phagocyte system, as demonstrated by the almost exclusive accumulation of PS in liver and spleen. (2) The slow decay phase reflects the elimination of that fraction of PS that has been incorporated into high density plasma lipoproteins (HDL). A kinetic model has been developed to describe these phenomena and a good agreement has been observed between experimental data and theoretical values. 3. Evidence has been obtained that a large fraction of PS is hydrolyzed at the injection site, probably by phospholipase A2 and other hydrolytic enzymes released by platelets. Hydrolysis at the injection site has also been observed following intraperitoneal and intramuscular injections. 4. As shown by the comparative analysis of the biotransformation products found in tissues after administration of either [3H]-glycerol-PS or [14C]-serine-PS, parenterally administered PS follows two distinct metabolic pathways: (1) decarboxylation to phosphatidylethanolamine and (2) extensive hydrolytic degradation with release of the individual components of the molecule. These pathways probably reflect the two main mechanisms of PS uptake, incorporation into the plasma membrane and internalization by endocytosis, respectively.

Signalling mechanism in the lysophosphatidylserine-induced activation of mouse mast cells.

Lysophosphatidylserine (0.1-1 microM) elicits histamine release in isolated mouse peritoneal mast cells. The effect becomes manifest after a lag of 30 s and reaches completion in 5 min. Maximal activity is observed when serine is in L-configuration. As shown by the activity of a lysophosphatidylserine analogue lacking the OH group in C2 position of glycerol, conversion into phosphatidylserine is not required. When 32PO4-labeled mast cells are challenged 2-5 min with lysophosphatidylserine, the labeling of phosphatidate, phosphatidylinositol and phosphatidylcholine is increased. When [3H]arachidonate-labeled mast cells are used, lysophosphatidylserine increases the appearance of isotopic diacylglycerol and phosphatidate. Like the secretory response, these effects are independent of the presence of extracellular Ca2+. Incubations in the presence of [3H]glycerol show that lysophosphatidylserine does not activate the de novo synthesis of phospholipids. In agreement with a participation of phosphoinositidase C in the action of lysophosphatidylserine, we observe accumulation of inositol phosphates in [3H]inositol labeled mast cells incubated in the presence of Li+. The results suggest that lysophosphatidylserine delivers its stimulus to mast cells, by the activation of phosphoinositide-dependent signalling mechanism.

ACTH response to corticotropin releasing hormone in Cushing's disease before and after ketoconazole: in vivo and in vitro studies.

Ketoconazole has been used as a palliative treatment of Cushing's syndrome, due to its ability to lower cortisol production. We evaluated the effects of ovine Corticotropin Releasing Hormone (oCRH) 100 micrograms i.v. on ACTH and cortisol levels in 6 patients with Cushing's disease before and after treatment with ketoconazole 600 mg/day. Both hormones increased after oCRH. During ketoconazole, cortisol was lowered to normal levels and its response to oCRH was impaired. After treatment, basal ACTH showed variable changes while the response to oCRH was markedly enhanced compared to that before ketoconazole. In vitro: In a continuous perfusion system of isolated anterior pituitary cells from rats or human anterior pituitary adenoma, producing ACTH, ketoconazole 10(-5)-10(-6) M showed no inhibitory effects on both basal or lisine-vasopressin and oCRH stimulated ACTH secretion. Our findings confirm the inhibitory action of ketoconazole on basal and stimulated cortisol secretion. No inhibition of ACTH levels was observed both in vivo and in vitro.

Alpha-1 adrenergic blockade: a possible mechanism of action of dopaminergic drugs on ACTH secretion.

In some cases of Cushing's syndrome both bromocriptine and lisuride inhibit the secretion of ACTH but it is still unknown if they act at pituitary or at higher levels. In order to evaluate this aspect, we set up an in vitro perfusion system with anterior pituitary cells from rats and from human ACTH-secreting tumors. In both preparations lysine-vasopressin (LVP) and epinephrine (EPI) stimulated ACTH secretion; prazosin (alpha-1 blocker) inhibited the EPI but not the LVP-mediated stimulation. Similarly, the infusion of lisuride blocked ACTH response to EPI but not to LVP. These data may suggest that "dopaminergic" drugs could act by an adrenergic blockade and not necessarily by a dopaminergic inhibition.

Evidence for ectopic ACTH production years after bilateral adrenalectomy for Cushing's syndrome: in vivo and in vitro studies.

Three patients presented with hyperpigmentation and high plasma levels of ACTH 4-5 years after bilateral adrenalectomy for Cushing's "disease". X-rays and ct-scan of the lungs showed a small pulmonary mass in all. ACTH levels (1100-2000 pg/ml) were not suppressible by high doses hydrocortisone. A carcinoid tumor was removed in two cases and a chemodectoma in the third. Evidence for ACTH secretion by these tumors was provided by both decrease of ACTH levels after surgery and/or in vitro studies. Both carcinoid cultures showed a basal ACTH production, which was clearly increased by LVP (10(-7) M) and CRF (10(-7) M). Our findings in vitro studies suggest the presence of specific receptor sites for physiological stimuli (LVP and CRF) in tumor cell membranes.