Automated fast procedure for the simultaneous extraction of hair sample performed with an automated workstation.

INTRODUCTION: Hair testing has a leading role in toxicology practice and even more in those aspects tightly linked to the assessment of psychoactive drug use and abuse in social life. AIM: The objective of the present study was to develop and validate an automated SPE sample-preparation step, suited for GC/MS confirmation analysis of basic drugs in hair drug control. The method was studied and optimized for quantitative determination and in a second time it was extended to real hair samples. The purpose of method validation was to ensure good reliability, reproducibility and quickness. METHODS: Janus Automated Workstation (PerkinElmer) was employed to perform SPE hair extraction, using 96-well plate SPEC MP1 acquired from Varian (Agilent Technologies). After derivatization of dried extracts, screening confirmations were performed using gas chromatography (GC) followed by mass spectrometry (MS). GC/MS data were validated following standard guidelines, but our attention was focused on three headings: samples cross-contamination, "memory effect" and extraction recovery. RESULTS: Validation requests were fully accomplished and we always obtained best results with the automated procedure. For instance, analytes mean recovery was between 70 and 90% and data analysis proved that no contamination between samples occurred. CONCLUSIONS: The automated workstation has shown good reliability (cross contamination and "memory effect" were tested and excluded), effectiveness (no false negative was detected), solvent saving (500µL/sample vs traditionally LLE 4mL/sample) and quickness (50min for 96 tests cycle).

Detection and quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in hair by GC/MS/MS in Negative Chemical Ionization mode (NCI) with a simple and rapid liquid/liquid extraction.

INTRODUCTION: The fine detection of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in hair matrix remains one of the most important topics in hair analysis. This relevance lies in the necessity to obtain evidence of effective drug consumption and dispel any doubt of environmental contamination. THCCOOH is the highest and mainly represented Δ9-THC metabolite, but its concentration in hair is very low. A sensitive method for quantitative determination of THCCOOH in hair was developed. As first step, the method was tested with different SPE/LLE conditions, but the best results were obtained with a simple ad hoc LLE extraction. The final method was fully validated, evaluating parameters like extraction recovery, linearity, specificity and sensitivity. More than one hundred hair samples were then analyzed with the validated method. Data analysis was performed so as to determine respective concentrations of the metabolite and active molecule. METHODS: Hair was washed and cut into small pieces (2-4mm). Samples (20-50mg) were spiked with deuterated internal standard (THC-d(3) and THCCOOH-d(3)) and then hydrolyzed at 90°C in 1mL of 1M NaOH for 15min. THC was isolated by a LLE basic extraction with n-hexane:ethyl acetate (9:1). Next the aqueous solution was acidified (pH 4) adding concentrated acetic acid. THCCOOH was extracted with the same mixture. Dried extracts were derivatized with pentafluoropropionic anhydride and hexafluoroisopropanol and analyzed by GC/MS/MS (Agilent 7000B triple quadrupole) in NCI mode. RESULTS: The linear range of THCCOOH is 0.1-5pg/mg, with good correlation coefficients (r(2)>0.9993). This method has great sensitivity (LOD 0.01pg/mg to LOQ 0.04pg/mg), high recovery, reproducibility and robustness. CONCLUSIONS: Based on these results, the method proved to be effective for the rapid determination of THC and THCCOOH in hair specimens.

Estimates of lifetime attributable risk of cancer after a single radiation exposure from 64-slice computed tomographic coronary angiography.

AIMS: To estimate the life attributable risk (LAR) of cancer incidence over a wide range of dose radiation exposure and a large spectrum of possible diagnostic computed tomographic coronary angiography (CTCA) scenarios. METHODS: This study included 561 consecutive patients who underwent a successful prospective ECG-gating CTCA protocol (low-dose group) 64-slice CTCA and 188 patients who underwent retrospective ECG-gating CTCA with ECG-triggered dose modulation CTCA (high-dose group). LAR was computed, given the organ equivalent dose, for all cancers in both sexes. LAR was tabulated for each decile of dose-length product by 10-year age classes, separately for each sex. RESULTS: Estimates of LAR of any cancer for an exposure at age < or =40 year were lower in males than in females for any given quantile. At age >/ or =50 years, LAR was similar between sexes only at the lowest exposure doses, whereas at higher dosage, it was, in general, higher for women. At the median age of this case series (62 years) and for a radiation exposure ranging from 1.33 to 3.81 mSv, LAR was 1 in 4329 (or 23.1 per 10(5) persons exposed) and 1 in 4629 (or 21.6 per 10(5) persons) in men and women, respectively. For an exposure ranging from 10.34 to 18.97 mSv at the same median age, the LAR of cancer incidence was 1 in 1336 (or 74.8 per 10(5) persons) in men and doubled (1 in 614 or 162.8 per 10(5) persons) in women. CONCLUSIONS: This study provided an estimate of the LAR of cancer in middle-aged patients of both sexes after a single diagnostic CTCA, providing an easy-to-read table.

Relation between coronary atherosclerotic plaques and traditional risk factors in people with no history of cardiovascular disease undergoing multi-detector computed coronary angiography.

AIMS: To prospectively investigate the prevalence of coronary artery plaques (CAP) as detected by computed tomography-based angiography in a large number of consecutive individuals with no history of coronary artery disease (CAD) or acute coronary syndrome; to evaluate whether traditional risk factors are related to prevalence of CAP and to the expected 10-year risk of first major or fatal cardiovascular event (CVE). DESIGN: Prospective, single-centre, cross-sectional study. SETTING: The division of Cardiology at Fondazione Cardiocentro Ticino Lugano, Switzerland. METHODS: We prospectively included 920 consecutive individuals with no history of CAD who underwent computed tomography coronary angiography (CTCA). Risk estimation of fatal and non-fatal CVE was assessed using Global Assessment Risk (GAR) and Systematic Coronary Risk Evaluation (SCORE), respectively. Logistic regression was used to assess the association of risk factors with the prevalence of CAP. RESULTS: CAP was found in 459 (49.9%) individuals. Older age, higher body mass index, male gender, diabetes, hypertension and dyslipidaemia all increased the likelihood of the CAP burden at univariable analysis (p<0.001). At the multivariable analysis older age, male gender, hypertension and diabetes independently increased the likelihood of CAP burden (p<0.001). An increase in likelihood of CAP was observed in the presence of one, two and three or more risk factors and with an increasing value of GAR and SCORE. Notably, about 18% of subjects with CAP did not report any traditional risk factors and among individuals without CAPs, 12% had three or more risk factors. CONCLUSIONS: A direct relation between the prevalence of CAP, number of risk factors and the related 10-year risk of CVE was found. 18% of subjects without risk factors had CAP. In these individuals CTCA may help in further optimising the risk reduction strategies on an individual basis.

Assessment of right ventricular function by strain rate imaging in chronic obstructive pulmonary disease.

The purpose of the current study was to compare right ventricular (RV) myocardial wall velocities (tissue Doppler imaging) and strain rate imaging (SRI) parameters with conventional echocardiographic indices evaluating RV function in chronic obstructive pulmonary disease (COPD) patients. In total, 39 patients with COPD and 22 healthy subjects were included in the current study. Seventeen patients had pulmonary artery pressure <35 mmHg (group I) and 22 patients had pulmonary artery pressure >35 mmHg (group II). Tissue Doppler imaging, strain and strain rate (SR) values were obtained from RV free wall (FW) and interventricular septum. Respiratory function tests were performed (forced expiratory volume in one second/vital capacity (FEV(1)/VC) and carbon monoxide diffusion lung capacity per unit of alveolar volume (D(L,CO)/V(A))). Strain/SR values were reduced in all segments of group II patients compared with group I patients and controls with lowest values at basal FW site. A significant relationship was shown between peak systolic SR at basal FW site and radionuclide RV ejection fraction. A significant relationship was shown between peak systolic SR at basal FW site and D(L,CO)/V(A) and FEV(1)/VC. In conclusion, in chronic obstructive pulmonary disease patients, strain rate imaging parameters can determine right ventricular dysfunction that is complementary to conventional echocardiographic indices and is correlated with pulmonary hypertension and respiratory function tests.

Assessment of severity of mechanical prosthetic mitral regurgitation by transoesophageal echocardiography.

OBJECTIVE: To evaluate the ability of colour Doppler transoesophageal echocardiography (TOE) to assess quantitatively prosthetic mitral valve insufficiency. METHODS: 47 patients were studied with multiplane TOE and cardiac catheterisation. Proximal jet diameter was measured as the largest diameter of the vena contracta. Regurgitant area was measured by planimetry of the largest turbulent jet during systole. Flow convergence zone was considered to be present when a localised area of increased systolic velocities was apparent on the left ventricular side of the valve prosthesis. Pulmonary vein flow velocity was measured at peak systole and diastole. RESULTS: Mean (SD) proximal jet diameter was 0.63 (0.16) cm, with good correlation with angiographic grades (r = 0.83). Mean (SD) maximum colour jet area was 7.9 (2.5) cm2 (r = 0.69) with worse correlation if a single imaging plane was used for measurements (r = 0.62). The ratio of systolic to diastolic peak pulmonary flow velocity averaged 0.7 (1.3) cm (r = -0.66) with better correlation (r = -0.71) if patients with atrial fibrillation were excluded. Mean (SD) regurgitant flow rate was 168 (135) ml/s and regurgitant orifice area was 0.56 (0.43) cm2, with good correlation with angiography (r = 0.77 and r = 0.78, respectively). CONCLUSIONS: TOE correctly identified angiographically severe prosthetic mitral regurgitation, mainly by the assessment of the flow convergence region and the proximal diameter of the regurgitant jet.

Molecular characterization of the ets-related human transcription factor ER81.

The PEA3 group is a homogeneous group of the ets transcription factor family and is composed of three known members, PEA3, ERM and ER81, which, on the amino acid (AA) level, are more than 95% identical within the DNA-binding domain (the Ets domain), more than 85% within a 32 AA domain (the acidic domain) localized in the amino-terminus and almost 50% identical overall. By screening a human kidney cDNA library with a specific probe obtained from mouse ER81, we isolated two clones of 1.6 and 1.5 kb in length encoding a 458 AA open reading frame. When compared to mouse ER81, the present human ER81 lacks the last 13 AA of the acidic domain and the 5 AA contiguous to the carboxy-terminal part of the acidic domain. Of the 458 AA of the human ER81 protein, 97% are identical to mouse ER81. Gel shift analysis indicates that the full-length human ER81 protein is able to bind specifically to an oligonucleotide containing the binding sites recognized by most of the Ets proteins. By transient expression in RK13 mammalian cells, human ER81 protein transactivated a reporter plasmid containing Ets binding sites, indicating that this molecule is a bonafide transcriptional activator, while by expression in Cos-1 transfected cells, we detected the presence of human ER81 protein in the nucleus using immunocytochemistry. In human tissues, ER81 mRNA is very highly expressed in brain, highly expressed in testis, lung and heart, moderately in spleen, small intestine, pancreas and colon, weakly in liver, prostate and thymus, very weakly in skeletal muscle, kidney and ovary and not in placenta and peripheral blood leukocytes. Analysis of human solid or haematopoietic tumour cell lines showed that most of them did not express ER81 detectably. Database searches revealed that ETV1 mRNA is highly similar to human ER81 described here, although it contains the full-length acidic domain present in mouse ER81. By screening a genomic DNA library, we characterized the intron-exon junction within the acidic domain of human ER81 and we showed that this junction corresponds to the site where the remaining AA of the acidic domain of ETV1 or mouse ER81 are inserted.

[Transcription factors of the PEA3 group in mammary cancer]. / Les facteurs de transcription du groupe PEA3 dans le cancer mammaire.

Prognosis factors such as mutated or amplified oncogenes are used in the treatment of breast cancer. We have recently shown that the members of the PEA3 group (ER81, ERM and PEA3) from the transcription factor family of the ets genes are overexpressed in breast cancer tumors arising from MMTV-neu transgenic animals. Moreover, we have shown that ER81, and in a lesser extent ERM and PEA3, are not expressed in the estrogen and/or progesterone receptor-positive mammary cancer cell lines, whereas they are expressed in the receptor negative ones. Our research interest in now focused on the role(s) of these oncogenes in the development and the regulation of breast tumors.

[Pregnancy and diabetes]. / Schwangerschaft und Diabetes

There is a mutual effect between gravidity and diabetes. Diabetes can have disadvantageous effect over gravidity, but the state of gravidity can contribute to the earlier diagnosis of diabetes. The author had 4850 cases of gravidity of 1686 women, indiscriminately, suffering from diabetes observed and on the basis of his observation he found that the manifestations of diabetes in the state of gravidity are respectively infrequent nowadays but the number of manifestation increases parallel with the fatness of women. The transitional diabetogen effect, the pathologic gravidities and the huge-embryos are, however, able to indicate the early stage of diabetes, that other methods or medical examinations could not still indicate. The birth of huge-embryos is the earliest and most characteristic praediabetic sign and it can appear 30 to 50 years before the development of diabetic trouble of metabolism. The number of pathological gravidities increases parallel with the advance of diabetic manifestation. The author emphasises on basis of his observations the importance of obstetrical and pregnancy-anamnesis in the prevention of diabetic manifestation and in the reduction of foetal losses.