Development of a combined SEM and ICP-MS approach for the qualitative and quantitative analyses of metal nano and microparticles in food products [corrected].

An integrated approach based on the use of inductively coupled plasma mass spectrometry (ICP-MS) and scanning electron microscopy (SEM) for the qualitative and quantitative analyses of metal particles in foods was devised and validated. Different raw materials and food products, like wheat, durum wheat, wheat flour, semolina, cookies, and pasta were considered. Attention was paid to the development of sample treatment protocols for each type of sample to avoid potential artifacts such as aggregation or agglomeration. The analytical protocols developed followed by ICP-MS and SEM investigations allowed us the quantitative determination and the morphological and dimensional characterization of metal nano- and microparticles isolated from the raw materials and finished food products considered. The ICP-MS method was validated in terms of linearity (0.8-80 µg/g and 0.09-9 µg/g for Fe and Ti, respectively), quantification limits (0.73 µg/g for Fe and 0.09 µg/g for Ti), repeatability (relative standard deviation (RSD) % equal to 10% for Fe and 20% in a wheat matrix as an example), and extraction recoveries (93 ± 2-101 ± 2%). Validation of the scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS) measurements was performed working in a dimensional range from 1 to 100 µm with an estimated error in the size determination equal to 0.5 µm. ICP-MS data as well as SEM measurements showed a decrease in the concentration of metal particles from wheat to flour and from durum wheat to semolina samples, thus indicating an external contamination of grains by metal particles. These findings were confirmed by environmental SEM analysis, which allowed investigation of particles of lower dimensions. Generally, the largest number of particles was found in the case of iron and titanium, whereas particles of copper and zinc were only occasionally found without any possibility of quantifying their number.

Association of GNAS imprinting defects and deletions of chromosome 2 in two patients: clues explaining phenotypic heterogeneity in pseudohypoparathyroidism type 1B/iPPSD3.

BACKGROUND: The term pseudohypoparathyroidism (PHP) describes disorders derived from resistance to the parathyroid hormone. Albright hereditary osteodystrophy (AHO) is a disorder with several physical features that can occur alone or in association with PHP. The subtype 1B, classically associated with resistance to PTH and TSH, derives from the epigenetic dysregulation of the GNAS locus. Patients showing features of AHO were described, but no explanation for such phenotypic heterogeneity is available. An AHO-like phenotype was associated with the loss of genetic information stored in chromosome 2q37, making this genomic region an interesting object of study as it could contain modifier genes involved in the development of AHO features in patients with GNAS imprinting defects. The present study aimed to screen a series of 65 patients affected with GNAS imprinting defects, with or without signs of AHO, for the presence of 2q37 deletions in order to find genes involved in the clinical variability. RESULTS: The molecular investigations performed on our cohort of patients with GNAS imprinting defects identified two overlapping terminal deletions of the long arm of chromosome 2. The smaller deletion was of approximately 3 Mb and contained 38 genes, one or more of which is potentially involved in the clinical presentation. Patients with the deletions were both affected by a combination of the most pathognomic AHO-like features, brachydactyly, cognitive impairment and/or behavioural defects. Our results support the hypothesis that additional genetic factors besides GNAS methylation defects are involved in the development of a complex phenotype in the subgroup of patients showing signs of AHO. CONCLUSIONS: For the first time, the present work describes PHP patients with hormone resistance and AHO signs simultaneously affected by GNAS imprinting defects and 2q37 deletions. Although further studies are needed to confirm the cause of these two rare molecular alterations and to identify candidate genes, this finding provides novel interesting clues for the identification of factors involved in the still unexplained clinical variability observed in PHP1B.

Development and validation of an inductively coupled plasma mass spectrometry method with optimized microwave-assisted sample digestion for the determination of platinum at ultratrace levels in plasma and ultrafiltrate plasma.

A highly sensitive inductively coupled plasma mass spectrometry (ICP-MS) method with microwave-assisted sample digestion for the determination of total platinum in rat whole and ultrafiltrate plasma was developed and validated. A first step of this study concerned the optimization of the mineralization procedure, in order to obtain good extraction recovery (higher than 90%) and repeatability (less than 6%) and the absence of matrix effect. ICP-MS analysis was then performed using the "hot plasma/protective ion extraction" mode, achieving high sensitivity and very high signal/noise ratio. Iridium was added as internal standard. The method was then submitted to validation, performed according to the FDA Bioanalytical Validation Methods guidelines and to the Eurachem guide. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision, accuracy and stability. An instrumental LOQ of 1.9ngL(-1), corresponding to a concentration of 955ngL(-1) in matrix under the adopted conditions, was obtained, allowing the quantitative analysis of Pt ultratraces. Instrumental linearity was verified in the range 1.9-14,000ngL(-1), corresponding to a concentration range from 955ngL(-1) to 6825microgL(-1) in matrix. Accuracy was evaluated by analyzing control samples for both matrices at different concentration levels; a good agreement (<15%) was obtained. Sample stability was tested by analyzing control samples maintained for 4h at room temperature or submitted to three freezing-thawing cycles. Finally, the developed method was applied to the analysis of plasma and ultrafiltrate plasma of rats treated with oxaliplatin-base drug, thus demonstrating its reliability in pharmacokinetic studies.

Experimental design optimization for the ICP-AES determination of Li, Na, K, Al, Fe, Mn and Zn in human serum.

A chemometric approach based on experimental design and desirability functions was used to develop and validated a method for the determination of some metals of biological concern by a fast sequential ICP-AES. The elements considered are Al, Fe, Mn, Zn, Li, Na and K. The experimental design was used to investigate the effects of three instrumental most crucial parameters, such as sheath gas flow rate, pump speed and auxiliary gas flow rate. In order to improve the multielemental analysis speed, although a sequential instrument allows the use of a separate parameter set for each wavelength, regression models and desirability functions were applied to find the experimental conditions providing the highest global sensitivity. Validation was performed in terms of limits of detection (LOD), limits of quantitation (LOQ), linearity, precision and recovery. By using the 167.02 nm wavelength, aluminium LOD was 0.5 microg L(-1) while the highest LOD was found for K (65 microg L(-1)). A linear range of at least three orders of magnitude was statistically demonstrated for each element. Precision was evaluated by testing two concentration levels, and good results in terms of intra-day repeatability were obtained, with R.S.D. values lower than 4.1% at the lowest concentration level. Lacking a suitable certified reference material, trueness was estimated using the recovery rate on fortified samples. The validated method was then used in the quantification of the elements considered in a serum sample.

Liposome-entrapped GABA modifies behavioral and electrographic changes of penicillin-induced epileptic activity.

We studied Sprague-Dawley rats with spike activity and myoclonus after intraperitoneal injections of penicillin. Twenty minutes after penicillin injection, one group received a random crossover treatment by intraperitoneal GABA (gamma-aminobutyric acid) or liposome-entrapped GABA (LEG) or phosphatidylserine alone. The other group received GABA, LEG, or phosphatidylserine followed 15 minutes later by the injection of penicillin. LEG decreased or prevented the epileptic activity, whereas no significant changes were seen with either GABA or phosphatidylserine given alone. LEG may enhance penetration of GABA across the blood-brain barrier because of the carrier action of the liposomes.

Proliferative activity and steroid hormone receptor status in male breast carcinoma.

Hormonal factors have been implicated in the development of both female and male breast cancers (MBC). However, MBCs are rare and seem to have different biological behavior than those of females. The aim of this study was to evaluate proliferative activity and to establish an association with steroid hormone receptor concentration and clinicopathological parameters in MBC. Proliferative activity was assessed in 18 MBC by mitotic figure counts and immunohistochemical evaluation of MIB-1 and proliferating cell nuclear antigen (PCNA). Estrogen (ER), progesterone (PR) and androgen (AR) receptors were evaluated in serial section from the same tumor by immunohistochemistry. PCNA (range 17-73%; mean, 51.6%) and MIB-1 (range 18.5-58%; mean 38.4%) were positive correlated with the mitotic rate. High proliferative activity assessed either by mitotic index or MIB-1 expression was associated with more poorly differentiated tumors. Sixty one percent (11/18) of the tumors were ER+, 72% (13/18) PR+ and 38.5% (5/13) AR+. Proliferative activity in tumors displaying ER+/PR+ phenotype showed a tendency to be higher than in ER-/PR- tumors. This difference was statistically significant when MIB-1 expression was used as proliferation marker. An association between AR concentration and age at diagnosis was found; in the AR negative group (8/13) mean age at diagnosis was 54.4 +/- 7.3 which was significantly lower than the age of patients with AR+ tumors, 63.2 +/- 11.1 (5/13). Results presented here show that decreased androgen action (AR-) within the breast might contribute to an earlier development of MBC. Besides that, the presence of ER and PR in carcinoma cells is considered to provide a growth advantage as shown by the positive association between the phenotype (ER+/PR+) and high proliferative activity. These results add information for a better understanding of hormonal control of MBC growth and development.

Long-term observations on the clinical use of lamotrigine as add-on drug in patients with epilepsy.

We report open-label clinical observations of additional lamotrigine (LTG) in 16 adult patients with refractory epilepsy, aimed to assess the long-term efficacy and safety of LTG in clinical use. LTG was added to the current antiepileptic drug (AED) regimen at a daily dosage of 200-400 mg depending on the concomitant treatment. Ten patients completed one year's treatment and were followed up to an overall exposure ranging 15-38 months. Six patients (38% of the initial group) had a reduction of seizure frequency greater than 50% of pre-treatment baseline after one year; the further follow-up indicated some efficacy decline, since the percentage of improved patients dropped to 19% after 2 years and 13% after 3 years. The dropouts during the first year were due to seizure breakthrough (two patients), Steven-Johnson-like syndrome (one patient) and reasons unrelated to treatment (three patients); in one patient LTG treatment was stopped due to macrocytic anemia after 23 months. Other reported adverse events were dizziness, mild ataxia, diplopia and localized purpura. No other hematological or biochemical changes were noted. LTG was not associated with any significant changes in plasma concentrations of concomitant AEDs. These findings confirm the moderate efficacy and low toxicity of long-term LTG in severe epilepsy.

Vigabatrin in complex partial seizures: a long-term study.

The efficacy and safety of oral vigabatrin (VGB) as add-on therapy in the long-term treatment of poorly controlled epilepsy were evaluated in 19 patients with complex partial seizures, either with or without secondary generalization. The study was run with a single-blind, placebo-controlled, crossover design, and included 2 months of placebo and 13-15 months of treatment with VGB, at doses ranging from 1 to 4 g/day. Of the 14 patients who completed the trial, 2 were seizure free, in 5 seizure frequency dropped by more than 75% and in another 5 by more than 50% with respect to baseline. The decrease in seizure frequency in the group as a whole was significant at all observation points of the trial. Three patients were not entered into the long-term phase due to lack of improvement (an increase in seizure frequency was observed in one of them), and 2 were excluded later because improvement disappeared leading to unauthorized changes in comedication. Side effects were mild and never caused discontinuation of treatment. In conclusion, VGB showed a remarkable efficacy and safety in the long-term treatment of complex partial seizures.

Preliminary evaluation of the effect of GABA and phosphatidylserine in epileptic patients.

The effect of the combined administration of gamma-aminobutyric acid (GABA) and phosphatidylserine was evaluated in a pilot study of 42 patients with drug-resistant epilepsy. The group included patients with complex partial seizures, simple partial seizures and absence seizures. Patients with complex partial seizures and simple partial seizures showed no significant improvement; on the other hand, there was a remarkable decrease in absence seizures, linearly related to the dose of GABA and phosphatidylserine. Side effects occurred in 9 patients and were usually mild.

Processing fine needle aspirates of prostate carcinomas for standard immunocytochemical studies and in situ apoptosis detection.

A method is described for making permanent histological sections of prostate carcinoma material obtained by fine needle aspiration (FNA) under ecography guidance. Smears made from prostate aspirates were used for diagnosis and from the same patient remaining aspirates were expelled into fixative filled microcentrifuge tube. Aspirates were pelleted and further processed to paraffin blocks. Permanent histological sections were obtained and each section was defined as satisfactory when it contained about 200 intact tumor cells. We have used these tumor sections and immunocytochemistry (ICC) procedures to study molecular biological marker expression. The technique described here has proven to be easy to use and offered a fast, reliable and cost-effective method to obtain suitable samples for standard ICC and in situ apoptosis detection from FNA prostate carcinoma. The method should be equally suitable for outpatient use on other tumors in which FNA and ICC or in situ apoptosis detection is likely to be helpful.

Bcl-2 correlates with tumor ploidy and nuclear morphology in early stage prostate carcinoma. A fine needle aspiration biopsy study.

We evaluated the nuclear morphology, ploidy, bcl-2 expression and in situ apoptosis in sections of fine-needle aspiration (FNA) biopsy specimens of thirty-one randomly selected Stage B prostate carcinomas. Sections of paraffin-embedded pelleted cells obtained from FNA biopsy specimens were studied. Nuclear grade was determined according to the WHO system. Nuclear morphometry and DNA ploidy were carried out using an automated image analyzer. We used immunostaining and the TUNEL method to evaluate bcl-2 expression and in situ apoptosis. The median nuclear area increased with increasing nuclear grade. Ploidy analysis showed that 54.8% of tumors were diploid, 3.2% tetraploid and 41.9% aneuploid. Bcl-2 overexpression was found in 10 of 31 tumors. There was a significant positive correlation between bcl-2 expression and nuclear area (r(s): 0.45 p < 0.01). Nine of ten bcl-2-positive tumors had a nuclear area larger than the median of the series, and 70% of bcl-2-positive tumors were of the aneuploid type. The apoptotic index had a negative correlation with nuclear area, and the lowest indexes were found in aneuploid tumors. Bcl-2 expression showed a highly significant association with both parameters of high aggressiveness: nuclear size and aneuploidy. The combined evaluation of nuclear morphology, ploidy and cell survival parameters might better identify patients with poor prognosis among early stage prostate carcinomas diagnosed by FNA biopsies.

Vigabatrin in chronic epilepsy: a 7-year follow-up study of responder patients.

Data on efficacy and safety of vigabatrin over very protracted treatment periods are still limited. This study reports the follow up of 23 responder epileptic patients who continued vigabatrin treatment after completion of the first year, to an overall long-term exposure ranging 21-84 months (median 60; mean 58.0 +/- 24.0 sd). The seizure frequency during the follow up was compared with that at the end of the first year on vigabatrin. The rates of patients who gained a further improvement and those who deteriorated were almost identical, ranging 33-45% and 33-46% respectively at individual time points. At the trial endpoint, nine patients (39%) were improved, five (22%) were unchanged and nine (39%) showed some deterioration. All patients still had a 14-100% decrease of seizure frequency as compared with pretreatment baseline. Two patients discontinued vigabatrin for occasional reasons. No patient experienced new adverse events during the follow up after the first year on vigabatrin. No significant effects were noted on any of the routine hematologic or metabolic screening assessments. Although reduction of concomitant treatment was rarely possible, the overall number of associated antiepileptic drugs dropped from 42 at entry to 40 at the trial endpoint. These findings indicate that vigabatrin retains its efficacy and safety in responder patients for periods up to 7 years.

MRI findings in epileptic patients on vigabatrin for more than 5 years.

Although vigabatrin is a promising new antiepileptic drug, its safety has been challenged by the report of dose-dependent central nervous system myelin vacuolation in some preclinical animal studies. Since it has been shown that vacuolation is associated with specific magnetic resonance imaging (MRI) findings in rats and dogs, MRI of the brain was performed in 11 patients with complex partial seizures who had been receiving vigabatrin for 64-78 months (mean 74.0 +/- 5.0 sd) as additional treatment for epilepsy, with a cumulative exposure ranging 4200 to 9360 g. In no case did MRI show white matter changes similar to the pathological findings of microvacuolation observed in animals. These results would appear to confirm that current doses of vigabatrin do not cause myelin vacuolation in humans, even for treatment periods of longer than 5 years.

Preliminary observations on the activity of progabide, administered as monotherapy in complex partial seizures.

Progabide (PGB), a gamma-amino-butyric acid receptor agonist, was administered, according to an open-label long-term design, to 40 adult patients suffering from complex partial seizures, with or without secondary generalization, whose response to carbamazepine (CBZ) monotherapy was unsatisfactory. A reference-baseline period of two months with carbamazepine monotherapy was followed by a two-month "add-on" period where increasing doses of progabide were added without modifying the CBZ regimen; then CBZ was withdrawn over 15-60 days and patients were followed up to 12 months' progabide treatment. Twenty-seven patients completed the trial but 12 of them had to be returned to CBZ + PGB bitherapy due to an increase of seizures following CBZ withdrawal. A definite therapeutic effect could be observed in nine patients on PGB monotherapy and in six patients on CBZ + PGB bitherapy. Side-effects of clinical relevance occurred in three cases and were represented by remarkable anxiety in two patients and a rise in serum glutamic oxalo-acetic acid and pyruvic transaminases with clinical symptoms of liver dysfunction in one, with rapid recovery following progabide discontinuation. In conclusion, progabide was effective against complex partial seizures in about 40% of patients not responding satisfactorily to available antiepileptic drugs. Although the withdrawal of previous antiepileptic drugs was not possible in all patients, progabide monotherapy was sometimes more effective than CBZ monotherapy, and several patients in whom bitherapy had to be restored benefited from the association of progabide.

Clinical pharmacology of atypical antipsychotics: an update.

This review will concentrate on the clinical pharmacology, in particular pharmacodynamic data, related to atypical antipsychotics, clozapine, risperidone, paliperidone, olanzapine, que¬tiapine, amisulpride, ziprasidone, aripiprazole, asenapine, iloperidone, lurasidone and cariprazine. A summary of their acute pharmacokinetics properties are also reported. Four new second-generation antipsychotics are available: iloperidone, asenapine, lurasidone and in the next future cariprazine. Similar to ziprasidone and aripiprazole, these new agents are advisable for the lower propensity to give weight gain and metabolic abnormalities in comparison with older second-generation antipsychotics such as olanzapine or clozapine. Actually lurasidone seems to be best in terms of minimizing unwanted alterations in body weight and metabolic variables. Therapeutic drug monitoring is not strictly necessary for all of the new antipsychotic drugs because there are no unequivocal data supporting a relationship between plasma drug levels and clinical outcomes or side effects. The exception can be represented by clozapine for which plasma levels of 350-420 ng/ml are reported to be associated with an increased probability of a good clinical response. Also for olanzapine an established therapeutic range (20-50 ng/ml) is proposed to yield an optimal response and minimize side effects.

The clinical and molecular characterization of patients with dyshormonogenic congenital hypothyroidism reveals specific diagnostic clues for DUOX2 defects.

CONTEXT: Mutations in the DUOX2 gene have been associated with transient or permanent congenital hypothyroidism due to a dyshormonogenic defect. OBJECTIVE: This study aimed to verify the prevalence of DUOX2 mutations and the associated clinical features in children selected by criteria supporting a partial iodide organification defect (PIOD). PATIENTS AND METHODS: Thirty children with PIOD-like criteria were enrolled and genotyped. A detailed clinical characterization was undertaken together with the functional analysis of the DUOX2 variations and the revision of the clinical and molecular data of the literature. RESULTS: In this large selected series, the prevalence of the DUOX2 mutations was high (37%). We identified 12 missense variants, one splice site, and three frameshift DUOX2 mutations. Functional analyses showed significant impairment of H2O2 generation with five missense variants. Stop-codon mutants were shown to totally abolish DUOX2 activity by nonsense-mediated RNA decay, exon skipping, or protein truncation. DUOX2 mutations, either mono- or biallelic, were most frequently associated with permanent congenital hypothyroidism. Moreover, the present data suggested that, together with goiter and PIOD, the most significant features to select patients for the DUOX2 analysis are the low free T4 and the high TSH concentrations at the first postnatal serum sampling, despite borderline blood spot TSH. Interestingly, the analysis of previously described DUOX2 mutated cases confirmed the validity of these findings. CONCLUSIONS: The defects in the peroxide generation system are common among congenital hypothyroidism patients with PIOD. The most robust clinical parameters for selecting patients for DUOX2 analysis have been identified, and several DUOX2 variants have been functionally characterized.

Suicide attempts in schizophrenic patients: clinical variables.

INTRODUCTION: Schizophrenia is associated with a significant risk of suicide: 40-50% of schizophrenic patients report suicidal ideation at some point in their lives, and 4-13% eventually commit suicide. In order to be able to predict and prevent suicide in schizophrenic patients, it is necessary to investigate and characterise suicide victims who meet the criteria for psychotic disorders and risk factors. METHODS: The aim of this retrospective study was to verify the associations between suicide attempts (SAs) and the demographic and clinical variables of 106 patients who met the DSM-IV-TR criteria for schizophrenia. The patients were divided into two groups on the basis of the presence/absence of lifetime suicide attempts, and their main demographic and clinical characteristics were analysed and compared. RESULTS: The patients with a history of SAs frequently had a duration of untreated psychosis (DUP) of ≥1 year (chi-squared test=9.984, df=1, p=0.0016). They also showed significant associations with the presence of a depressive dimension (chi-squared test=4.439, df=1, p=0.0351), hospitalisations before SAs (chi-squared test=25.515, df=1, p <0.001), and a family history of psychiatric disorders (chi-squared test=12.668, df=2, p=0.0018) or suicidal behaviours (chi-squared test=18.241, df=2, p=0.0001). Finally, they were more frequently prescribed typical antipsychotic agents. CONCLUSIONS: The severity of psychiatric symptoms indicates a high risk of suicide in schizophrenic patients. Further prospective studies of larger samples should investigate the role of early interventions and atypical antipsychotic treatment in reducing the risk.

Aggression and psychopharmacological treatments in major psychosis and personality disorders during hospitalisation.

BACKGROUND: A number of large-scale studies have shown that there is a relationship between many psychiatric disorders and aggression or violence. As no medication is currently approved for the treatment of aggression, pharmacotherapy (often involving drug combinations) is used on a trial-and-error basis with various degrees of response. METHOD: The study involved 244 in-patients aged 19-83 years (mean 41.9 ± 11.3 SD). The Modified Overt Aggression Scale (MOAS) was used to assess any aggressive or violent behaviors occurring in the week before admission and upon discharge. Psychopathology was assessed using the Brief Psychiatric Rating Scales (BPRS). RESULTS: All of the patients showed a significant improvement (p<0.001) in mean weighted total MOAS scores at the end of the study, with no significant differences between the various drugs or combination therapies. The patients who received combination treatments including antidepressants showed a worsening in the weighted total MOAS score (18.46% ± 114.31% SD); the patients who did not receive antidepressants had an improvement (13.61% ± 257.36% SD) (p=0.0069). CONCLUSIONS: Multivariate testing of the variables age, gender, substance/alcohol abuse, the duration of hospitalisation, the administration of mood stabilisers, and the use of typical or atipical antipsychotics showed that the severity of the psychopathological picture correlated significantly with the presence of violence, whereas the effect of combined antidepressant treatment on violent behavior was only relative.

Interactions between central monoaminergic systems: dopamine-serotonin.

Concentration of dopamine and serotonin metabolites (HVA and 5-HIAA) in the CSF was evaluated before and after pharmacological treatment in 19 patients with different neuropsychiatric diseases. In every case a reciprocal modification of the two metabolites occurred after treatment. The result supports the hypothesis of a functional balance between the monoaminergic systems in the central nervous system.