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Olive pomace, a heterogeneous solid waste from olive oil production, is one of the most widespread agro- industrial by-products in Mediterranean area. The amount of organic compounds with high chemical and biological oxygen demands present in olive pomace stresses the related industries to adapt adequate treatments for this by-product. Being rich in bioactive molecules, it could be considered a low-cost and renewable source of high added value compounds, such as polyphenols. Dietary plant polyphenols are the subject of increasing scientific interest because of their possible beneficial effects on human health. Recent studies suggested that action of polyphenols goes beyond the modulation of oxidative stress and can improve several physiopathological conditions. In the present study, we investigated the possible effects of polyphenols extracted from olive pomace (PEOP) and of the main single phenolic compounds present in the extract (tyrosol, apigenin and oleuropein) in protecting hepatocytes against excess fat and oxidative stress. PEOP were extracted using a high pressure- temperature agitated reactor (25 bar, 180 °C for 90 min). A mixture of ethanol:water (50:50 v/v) was used as extraction solvents resulting to total polyphenol concentration of 5.77 mgCaffeic Acid Equivalent/mL. In order to test the biological effects of the extract we used rat hepatoma FaO cells exposed to a mixture of oleate/palmitate (2:1 molar ratio) for 3 h which represent a reliable in vitro model for hepatic steatosis. After lipid-loading, hepatic cells were incubated for 24 h in the absence or in the presence of 10 µg/mL tyrosol, 13 µg/mL apigenin or 50 µg/mL oleuropein. Content of intra- and extra-cellular triglycerides (TGs), and oxidative stress-related parameters were evaluated. The preliminary results showed that PEOP ameliorated lipid accumulation and lipid-dependent oxidative unbalance showing their potential applications as therapeutic agents.

BACKGROUND: The magnitude of thrombotic risk during ovarian stimulation cycles is not known. We calculated the magnitude of thrombotic risk in a cohort of women starting a new cycle of ovarian stimulation and investigated the role of inherited and acquired thrombophilia for these events. METHODS: This is an observational study involving outpatients of a clinical research centre. Consecutive women undergoing ovarian stimulation (n=305) were enrolled. Blood samples for studying inherited and acquired thrombophilia were obtained ≥2 months after the last cycle of treatment. Odds ratios (OR) and confidence intervals (CI) were determined for markers significantly associated with thrombotic events. Blood samples were analysed for inherited and acquired causes of thrombophilia (antithrombin, protein C, protein S, antiphospholipid antibodies, the Factor V Leiden and FIIA20210 mutations, the TT677 MTHFR genotype, and homocysteine plasma levels). RESULTS: Thrombotic events were observed in 4/747 cycles of ovarian stimulation, with a prevalence of 0.5%, corresponding to 1.6 per 100 000 cycles/woman. Age ≥39 years and homocysteine plasma levels above the 97.5 percentile were significantly associated with thrombotic events during IVF [OR 15.2 (95% CI 2.0–115.0) and 14.4 (1.5–141.3) respectively]. CONCLUSIONS: Age ≥39 years and mild hyperhomocysteinaemia are strongly associated with the occurrence of thrombotic events during IVF. [ABSTRACT FROM AUTHOR]

By changing many aspects of everyday life, the COVID-19 pandemic and the social distance policies implemented to face it have affected the behaviour of people all over the world. Has the pandemic also affected people's approach towards the divine? Previous evidence suggests that prayer searches on the Internet rose during the pandemic and that people tend to rely mainly on intrinsic rather than extrinsic religiousness to cope with adversity. In the present contribution, using a set of panel random effect estimators, we compare the change in religious attendance in Italian regions before and during the pandemic. Our results suggest that there has been an increase in religiousness during the COVID-19 pandemic. Our findings are robust to several specifications of the model and to different estimators. This suggests that people derive more comfort from religious activities during hard times that are characterized by uncertainty., (© 2023. The Author(s).)

Background: Cobalamin metabolism disorders are rare, inherited diseases which cause megaloblastic anaemia and other clinical manifestations. Early diagnosis of these conditions is essential, in order to allow appropriate treatment as early as possible., Case Presentation: Here we report the case of a patient who was apparently healthy until the age of 20, when she presented with impaired renal function and normocytic anaemia. At the age of 34, when her first pregnancy resulted in an intrauterine death of a morphologically normal growth-restricted foetus, she was diagnosed with homocystinuria and methylmalonic aciduria due to cyanocobalamin C (cblC) defect, which was confirmed by molecular investigation. Consequently, hydroxocobalamin was administered to correct homocysteine plasma levels. This treatment was efficacious in lowering homocysteine plasma levels and restored anaemia and renal function. During a second pregnancy, the patient was also administered a prophylactic dose of low molecular -weight heparin. The pregnancy concluded with a full-term delivery of a healthy male., Conclusions: This case emphasises the importance of awareness and appropriate management of rare metabolic diseases during pregnancy. We suggest that women with late-onset cblC defect can have a positive pregnancy outcome if this metabolic disease is treated adequately.

Overconsumption of fats and sugars is a major cause of development of non‑alcoholic fatty liver disease (NAFLD). The main objectives of the present study were to explore the pathways sustaining the interfering metabolic effects of excess fructose and fatty acids in hepatocytes, and to clarify the mechanisms through which the nutraceutical silybin rescues the functional and metabolic alterations that are associated with the NALFD progression. Cultured hepatocytes were exposed to fructose and fatty acids, alone or in combination, to induce different grades of steatosis in vitro. Cell viability, apoptosis, free radical production, lipid content, lipid peroxidation and activity of lipogenic enzymes were assessed by spectrophotometric assays. Oxygen consumption and mitochondrial respiration parameters were measured using a Seahorse analyzer. Expression of markers for liver steatosis and dysfunction were also evaluated by reverse transcription‑quantitative polymerase chain reaction. The data revealed that fructose and fatty acid combination in vitro had a positive interference on lipogenic pathways, leading to more severe steatosis and liver dysfunction, reduced cell viability, increased apoptosis, oxidative stress and mitochondrial respiration. Hepatic cell abnormalities were almost completely alleviated by silybin treatment. These findings suggest that silybin may serve as a novel and cost‑effective dietary supplement for treatment and/or prevention of hepatosteatosis associated with NAFLD progression.

Purpose: Phenolic compounds (PC) of virgin olive oil exert several biochemical and pharmacological beneficial effects. Some dietary PC seem to prevent/improve obesity and metabolic-related disorders such as non-alcoholic fatty liver disease (NAFLD). We investigated the possible effects of PC extracted from olive pomace (PEOP) and of the main single molecules present in the extract (tyrosol, apigenin, oleuropein, p-coumaric and caffeic acid) in protecting hepatocytes and endothelial cells against triglyceride accumulation and oxidative stress., Methods: Rat hepatoma and human endothelial cells were exposed to a mixture of oleate/palmitate to mimic the condition of NAFLD and atherosclerosis, respectively. Then, cells were incubated for 24 h in the absence or in the presence of PC or PEOP. Different parameters were evaluated, such as lipid accumulation and oxidative stress-related markers., Results: In hepatic cells, expression of peroxisome proliferator-activated receptors (PPARs) and of stearoyl-CoA desaturase 1 (SCD-1) were assessed as index of lipid metabolism. In endothelial cells, expression of intercellular adhesion molecule-1 (ICAM-1), activation of nuclear factor kappa-B (NF-kB), release of nitric oxide (NO), and wound-healing rate were assessed as index of inflammation., Conclusion: PEOP extract ameliorated hepatic lipid accumulation and lipid-dependent oxidative imbalance thus showing potential applications as therapeutic agent tuning down hepatosteatosis and atherosclerosis.

Pregnancies after assisted reproductive technologies (ART) have been associated with an increased risk of venous thromboembolism (VTE). On the contrary, the magnitude of this risk in unsuccessful ART cycles (not resulting in a clinical pregnancy) has not yet been clearly defined. In this study, we evaluated the incidence of VTE in unsuccessful cycles and compared it with that recorded in successful cycles in the same study population. From a cohort of 998 women consecutively referred by local Fertility Clinics to our Atherosclerosis and Thrombosis Unit (April 2002-July 2011), we identified and included women with at least one cycle of ovarian stimulation and a negative history for VTE. Overall, 661 women undergone 1518 unsuccessful and 318 successful cycles of ovarian stimulation, respectively, were analysed. VTE events occurred in 2/1518 (1.3‰) unsuccessful cycles compared with 3/318 (9.4‰) successful cycles, (Two-tailed Fisher exact test, p = 0.04, OR 0.14, 95% CI 0.02-1.02). Both cases observed in unsuccessful cycles were isolated pulmonary embolism occurred after OHSS; no antithrombotic prophylaxis had been prescribed. At logistic regression analysis, the occurrence of successful cycle and BMI were significantly and independently associated with the occurrence of VTE with an OR of 13.94 (95% CI 1.41-137.45) and 1.23 (95% CI 1.01-1.49), respectively. VTE incidence is significantly lower in unsuccessful cycles as compared to that of successful ones. However, although rare, thrombotic risk during ovarian stimulation cannot be excluded and, when it occurs, can be life-threatening. Therefore, particular attention should be paid to these women, independently of ART outcome.

Non-alcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidity and mortality. Oxidative stress and release of pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), are major consequences of hepatic lipid overload, which can contribute to progression of NAFLD to non-alcoholic steatohepatitis (NASH). Also, mitochondria are involved in the NAFLD pathogenesis for their role in hepatic lipid metabolism. Definitive treatments for NAFLD/NASH are lacking so far. Silybin, the extract of the milk thistle seeds, has previously shown beneficial effects in NAFLD. Sequential exposure of hepatocytes to high concentrations of fatty acids (FAs) and TNFα resulted in fat overload and oxidative stress, which mimic in vitro the progression of NAFLD from simple steatosis (SS) to steatohepatitis (SH). The exposure to 50 µM silybin for 24 h reduced fat accumulation in the model of NAFLD progression. The in vitro progression of NAFLD from SS to SH resulted in reduced hepatocyte viability, increased apoptosis and oxidative stress, reduction in lipid droplet size, and up-regulation of IκB kinase β-interacting protein and adipose triglyceride lipase expressions. The direct action of silybin on SS or SH cells and the underlying mechanisms were assessed. Beneficial action of silybin was sustained by changes in expression/activity of peroxisome proliferator-activated receptors and enzymes for FA oxidation. Moreover, silybin counteracted the FA-induced mitochondrial damage by acting on complementary pathways: (i) increased the mitochondrial size and improved the mitochondrial cristae organization; (ii) stimulated mitochondrial FA oxidation; (iii) reduced basal and maximal respiration and ATP production in SH cells; (iv) stimulated ATP production in SS cells; and (v) rescued the FA-induced apoptotic signals and oxidative stress in SH cells. We provide new insights about the direct protective effects of the nutraceutic silybin on hepatocytes mimicking in vitro NAFLD progression.

Introduction: Dabigatran etexilate is given in fixed doses without coagulation monitoring for the prevention of blood clots in at risk adults. A high inter-individual variability in blood concentrations of the active metabolite of dabigatran has been reported. ABCB1 and CES1 exert an important effect in the metabolism of dabigatran etexilate and allele variants at these two loci are likely to play a pivotal role. To investigate whether screening for polymorphisms within the ABCB1 and the CES1 genes would explain a portion of the inter-individual variability in blood concentrations of the active metabolite of dabigatran., Material and Methods: In a cohort of patients who had atrial fibrillation and on anticoagulant prophylaxis with dabigatran etexilate, we investigated whether genotypes at rs4148738 (ABCB1), rs8192935 (CES1), and rs2244613 (CES1) loci would affect plasma dabigatran trough and peak concentrations., Results and Discussion: Among 92 patients (median age: 72.0years, range: 52-92) analyzed, no clinical variable or genotype was associated with a significant difference in dabigatran peak concentrations. As for trough concentrations, in addition to creatinine clearance, and sex a significant association with the CES1 SNP rs8192935 (p=0.023) was detected. The mean adjusted plasma levels were higher among patients with the CC genotype (86.3ng/dl) than in those carrying the T allele (62.1ng/dl). No significant effect was found for the ABCB1 SNP rs4148738. The CES1 SNP rs8192935 significantly influenced the dabigatran trough concentrations and carriers of the T allele showed significantly lower concentrations than did carriers of the CC genotype., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aim: To investigate in vitro the therapeutic effect and mechanisms of silybin in a cellular model of hepatic steatosis., Methods: Rat hepatoma FaO cells were loaded with lipids by exposure to 0.75 mmol/L oleate/palmitate for 3 h to mimic liver steatosis. Then, the steatotic cells were incubated for 24 h with different concentrations (25 to 100 μmol/L) of silybin as phytosome complex with vitamin E. The effects of silybin on lipid accumulation and metabolism, and on indices of oxidative stress were evaluated by absorption and fluorescence microscopy, quantitative real-time PCR, Western blot, spectrophotometric and fluorimetric assays., Results: Lipid-loading resulted in intracellular triglyceride (TG) accumulation inside lipid droplets, whose number and size increased. TG accumulation was mediated by increased levels of peroxisome proliferator-activated receptors (PPARs) and sterol regulatory element-binding protein-1c (SREBP-1c). The lipid imbalance was associated with higher production of reactive oxygen species (ROS) resulting in increased lipid peroxidation, stimulation of catalase activity and activation of nuclear factor kappa-B (NF-κB). Incubation of steatotic cells with silybin 50 μmol/L significantly reduced TG accumulation likely by promoting lipid catabolism and by inhibiting lipogenic pathways, as suggested by the changes in carnitine palmitoyltransferase 1 (CPT-1), PPAR and SREBP-1c levels. The reduction in fat accumulation exerted by silybin in the steatotic cells was associated with the improvement of the oxidative imbalance caused by lipid excess as demonstrated by the reduction in ROS content, lipid peroxidation, catalase activity and NF-κB activation., Conclusion: We demonstrated the direct anti-steatotic and anti-oxidant effects of silybin in steatotic cells, thus elucidating at a cellular level the encouraging results demonstrated in clinical and animal studies.

Excess ethanol consumption and fatty acid intake lead to a cumulative effect on liver steatosis through still unclear mechanisms. This study aimed to characterize the lipid homoeostasis alterations under the exposure of hepatocytes to ethanol alone or combined with fatty acids. FaO hepatoma cells were incubated in the absence (C) or in the presence of 100 mM ethanol (EtOH) or 0.35 mM oleate/palmitate (FFA) alone or in the combination (FFA/EtOH). Content of intra- and extra-cellular triglycerides (TAGs) and of lipid droplets (LDs), expression of lipogenic and lipolytic genes, and oxidative stress-related parameters were evaluated. Exposure to either FFAs or EtOH given separately led to steatosis which was augmented when they were combined. Our results show that FFA/EtOH: (i) increased the LD number, but reduced their size compared to separate treatments; (ii) up-regulated PPARγ and SREBP-1c and down-regulated sirtuin-1 (SIRT1); (iii) impaired FFA oxidation; (iv) did not change lipid secretion and oxidative stress. Our findings indicate that one of the major mechanisms of the metabolic interference between ethanol and fat excess is the impairment of FFA oxidation, in addition to lipogenic pathway stimulation. Interestingly, ethanol combined with FFAs led to a shift from macrovesicular to microvesicular steatosis that represents a more dangerous condition., (Copyright © 2016. Published by Elsevier Ltd.)

Adipose tissue, dietary lipids and de novo lipogenesis are sources of hepatic free fatty acids (FFAs) that are stored in lipid droplets (LDs) as triacylglycerols (TAGs). Destiny of TAGs stored in LDs is determined by LD proteomic equipment. When adipose triglyceride lipase (ATGL) localizes at LD surface the lipid mobilization is stimulated. In this work, an in vitro model of cultured rat hepatocytes mimicking a mild steatosis condition was used to investigate the direct lipid-lowering action of iodothyronines, by focusing, in particular, on LD-associated proteins, FFA oxidation and lipid secretion. Our results demonstrate that in "steatotic" hepatocytes iodothyronines reduced the lipid excess through the recruitment of ATGL on LD surface, and the modulation of the LD-associated proteins Rab18 and TIP47. As an effect of ATGL recruitment, iodothyronines stimulated the lipid mobilization from LDs then followed by the up-regulation of carnitine-palmitoyl-transferase (CPT1) expression and the stimulation of cytochrome-c oxidase (COX) activity that seems to indicate a stimulation of mitochondrial function. The lipid lowering action of iodothyronines did not depend on increased TAG secretion. On the basis of our data, ATGL could be indicated as an early mediator of the lipid-lowering action of iodothyronines able to channel hydrolyzed FFAs toward mitochondrial beta-oxidation rather than secretion.

Sporadic and familiar forms of non-histaminergic angioedema and normal C1 inhibitor encompass a group of disorders possibly caused by bradikinin. We aimed to study the subgroups of hereditary angioedema with FXII mutation (FXII-HAE), unknown genetic defect (U-HAE) and idiopathic non-histaminergic acquired angioedema (InH-AAE). We screened the F12 locus in our cohort and delineated the clinical, laboratory and genetic features. Four families carried the p.Thr309Lys mutation in F12 gene. Haplotyping confirmed the hypothesis of a common founder. Six families were affected by U-HAE and 13 patients by sporadic InH-AAE. C4 levels were significantly lower in FXII-HAE than in InH-AAE. In the FXII-HAE group, none had attacks exclusively in high estrogenic states; acute attacks were treated with icatibant. Prophylaxis with tranexamic acid reduced the attack frequency in most patients. Our study provides new data on the diagnosis, clinical features and treatment of non-histaminergic angioedema, underlying the role of the screening for F12 mutations., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Oxidative stress is implicated in pathogenesis of alcoholic liver disease (ALD). This study investigated the possible correlation among the erythrocyte indices of oxidative stress, the leukocyte panels of antioxidant proteins (metallothioneins), the serum biochemical parameters and the liver steatosis grade., Methods: A total of 118 cases including 60 alcoholic subjects and 58 controls were enrolled. All the alcoholic subjects were screened for body mass index (BMI), liver steatosis, and blood chemistry and serology. The level of oxidative stress and oxidative stress-related parameters were measured in the blood and correlated with clinical findings., Results: Alcoholic subjects showed higher BMI, moderate/severe hepatic steatosis, increase in the levels of triglycerides, cholesterol, glucose, γ-glutamyl-transpeptidase (GGT), alanine aminotransferase (ALT), bilirubin, alpha 1 and beta 2 globulins, iron and a decrease in the levels of aspartate aminotransferase (AST) and beta 1 globulin with respect to the reference values. Moreover, alcoholic subjects showed: (i) an increase in Thiobarbituric Acid Reactive Substance (TBARS) content representing a good estimation of global oxidative stress; (ii) a stimulation of the activities of the antioxidant enzymes catalase and SOD; (iii) a modulation of expression of metallothioneins, with a down-regulation of MT-1A and an up-regulation of MT-1E isoforms., Conclusions: Our data suggest that alcoholism is strongly associated with altered pattern of blood metallothioneins; this parameter combined with the score calculated by an ad hoc implemented algorithm (HePaTest) could offer a non-invasive alternative approach for evaluating alcohol-related damages and could be used in follow-up of alcoholic patients., (Copyright © 2014. Published by Elsevier Ireland Ltd.)

Background: Wolfram Syndrome type 2 (WFS2) is considered a phenotypic and genotypic variant of WFS, whose minimal criteria for diagnosis are diabetes mellitus and optic atrophy. The disease gene for WFS2 is CISD2. The clinical phenotype of WFS2 differs from WFS1 for the absence of diabetes insipidus and psychiatric disorders, and for the presence of bleeding upper intestinal ulcers and defective platelet aggregation. After the first report of consanguineous Jordanian patients, no further cases of WFS2 have been reported worldwide. We describe the first Caucasian patient affected by WFS2., Case Presentation: The proband was a 17 year-old girl. She presented diabetes mellitus, optic neuropathy, intestinal ulcers, sensorineural hearing loss, and defective platelet aggregation to ADP. Genetic testing showed a novel homozygous intragenic deletion of CISD2 in the proband. Her brother and parents carried the heterozygous mutation and were apparently healthy, although they showed subclinical defective platelet aggregation. Long runs of homozygosity analysis from SNP-array data did not show any degree of parental relationship, but the microsatellite analysis confirmed the hypothesis of a common ancestor., Conclusion: Our patient does not show optic atrophy, one of the main diagnostic criteria for WFS, but optic neuropathy. Since the "asymptomatic" optic atrophy described in Jordanian patients is not completely supported, we could suppose that the ocular pathology in Jordanian patients was probably optic neuropathy and not optic atrophy. Therefore, as optic atrophy is required as main diagnostic criteria of WFS, it might be that the so-called WFS2 could not be a subtype of WFS. In addition, we found an impaired aggregation to ADP and not to collagen as previously reported, thus it is possible that different experimental conditions or inter-patient variability can explain different results in platelet aggregation. Further clinical reports are necessary to better define the clinical spectrum of this syndrome and to re-evaluate its classification.

Kidney transplanted patients need immunosuppressant therapies. Therapeutic drug monitoring approaches produce the optimal clinical outcome is still under debate. This review details strength and limits of methods available: immunoassay and chromatography-based. Liquid chromatography with mass spectrometry detection is a major breakthrough in therapeutic drug monitoring of immunosuppressive agents and is considered as the method of choice in therapeutic drug monitoring (TDM) of immunosuppressants. Despite the initial high cost for the instrumentation, HPLC-MS is more cost effective than microparticle enzyme immunoassay. The main important features of LC-MS/MS methodology for immunosuppressive drugs are the shortened analysis time, an increased throughput, higher selectivity, specificity, and sensitivity, and low cost of analysis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A simple liquid chromatographic tandem mass spectrometry (LC-MS/MS) method has been developed for simultaneous analysis of 17 basic and one acid psychotropic drugs in human plasma. The method relies on a protein precipitation step for sample preparation and offers high sensitivity, wide linearity without interferences from endogenous matrix components. Chromatography was run on a reversed-phase column with an acetonitrile-H₂O mixture. The quantification of target compounds was performed in multiple reaction monitoring (MRM) and by switching the ionization polarity within the analytical run. A further sensitivity increase was obtained by implementing the functionality "scheduled multiple reaction monitoring" (sMRM) offered by the recent version of the software package managing the instrument. The overall injection interval was less than 5.5 min. Regression coefficients of the calibration curves and limits of quantification (LOQ) showed a good coverage of over-therapeutic, therapeutic and sub-therapeutic ranges. Recovery rates, measured as percentage of recovery of spiked plasma samples, were ≥ 94%. Precision and accuracy data have been satisfactory for a therapeutic drug monitoring (TDM) service as for managing plasma samples from patients receiving psycho-pharmacological treatment., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The accuracy of antemortem diagnosis of pulmonary embolism is within the range of just 10-30%, so representing one of the most frequent missed diagnosis in sudden, unexpected death. We describe 43 fatal cases of pulmonary embolism as confirmed by post-mortem examination. The aim of our study was to verify the systematic search for the most common genetic thrombophilias (Factor V Leiden (G1691A) and FII (G20210A) gene variants) and dating the thrombus. As a whole, 41 patients (95.3%) had at least one risk factor. Pre-existing symptoms are described just before fatal embolism in 18 (41.9%) out 43 patients. In 18 out of 43 (41.9%) it was not possible to find the thrombotic site. In 24 out of the remaining 25 cases the involvement of the deep veins of one leg was shown; in 1 case the thrombus was localised in the inferior caval vein. 10 (41.7%) were iliac vein thromboses, 7 (29.1%) femoral, 2 (8.3%) popliteal, 3 (12.6%) posterior-tibial, 1 (4.1%) anterior-tibial and 1 (4.1%) peroneal vein thromboses. In our cohort of patients, 4 (10%) out of 40 cases carried the 20210A prothrombin gene variant in heterozygosis. One (2.5%) out of 40 carried the Factor V Leiden (G1691A) gene variant in heterozygosis. Patients carrying these gene variants in homozygosis or carrying both were not present in our case-series. We strongly underline the relevance of a complete methodological approach, integrating clinical data by means of autopsy findings and histological study. On the contrary, investigating common inherited thrombophilia is not warranted., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objective: Cytochrome P450 (CYP) 2D6 enzyme is the major responsible for the metabolism of donepezil, an inhibitor of acetyl cholinesterase currently used for the symptomatic treatment of mild-to-moderate Alzheimer's disease (AD). Functional polymorphisms in the CYP2D6 gene may affect enzyme activity and thus, the metabolism of donepezil. The aim of this study was to evaluate the effect of 16 functional polymorphisms in the CYP2D6 gene on the clinical response to donepezil treatment in patients with mild-to-moderate AD., Methods: In this multicenter prospective cohort study we evaluated 57 unrelated Caucasians clinically diagnosed as AD according to the National Institute of Neurological and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association Work Group criteria. Patients were treated with donepezil (5-10 mg/daily) for 6 months. The response to donepezil treatment was evaluated at 6-month follow-up according to the National Institute for Health and Clinical Excellence requirements. The identification of 16 clinically relevant CYP2D6 gene variants was performed by a high-throughput genetic analysis., Results: Thirty-eight of 57 patients (67%) were responders and 19 patients (33%) were nonresponders to donepezil treatment. A significantly higher frequency of gene variants conferring decreased or absent enzyme activity was observed in responder than in nonresponder patients (73.68% vs. 36.84%; P=0.005). The presence of gene variants conferring decreased or absent activity of the CYP2D6 enzyme was significantly associated with a clinical response to donepezil treatment (odds ratio=6.286; 95% confidence interval=1.828-21.667)., Conclusions: Functional polymorphisms in the CYP2D6 gene can influence the clinical efficacy of donepezil. The analysis of CYP2D6 genotypes may be useful in identifying subgroups of AD patients with different clinical response to donepezil treatment.

Homocysteine is an endogenous sulphydryl aminoacid irreversibly catabolized by transsulfuration to cysteine or remethylated to methionine. Increased plasma levels of homocysteine are an independent risk factor for atherosclerosis and cardiovascular disease. Accurate and reliable quantification of this amino acid in plasma samples is essential in clinical practice to explore the presence of a hyperhomocysteinemia, for instance after an ischemic event, or to control a possible adjunctive risk factor in patients at higher risk. In this review, LC-ESI-MS/MS methods are discussed and compared with other analytical methods for plasma homocysteine. LC-ESI-MS/MS is a technique combining the physicochemical separation of liquid chromatography with the analysis of mass spectrometry. It is based on stable-isotope dilution and possesses inherent accuracy and precision. Quantitative analysis is achieved by using commercially available homocystine-d(8) as an internal standard. Taking advantage of the high sensitivity and specificity, approaches involving LC-ESI-MS/MS require less laborious sample preparation, no derivatization and produce reliable results.

6-oxo-PGF(1alpha) and TXB(2) are the metabolites of the prostanglandin PGI(2) and of the thromboxane TXA(2), respectively. PGI(2) and TXA(2) are arachidonic acid-derived compounds which regulate the blood hemostasis. Their quick metabolism leads to the 6-oxo-PGF(1alpha) and TXB(2) metabolites in plasma. In order to study on a large base the external factors influencing the hemostatic conditions, there is a need for a fast and reliable assay for quantitating these metabolites. Some methods have been published for the analysis of the arachidonic acid-derived compounds and some are dealing with mass spectrometry but nonspecifically centered on these specific compounds with a fast and cheap protocol, amenable for large-scale studies. Here we describe an analytical strategy that incorporates a two-dimensional chromatography running coupled to tandem mass spectrometry that minimizes the sample preparation and addresses the presence of the TXB(2) anomers for a robust quantitation measurement. After a protein precipitation, 100 microl of the supernatant (corresponding to 50 microl of the original plasma) was injected in a two-dimensional chromatographic system which operates an on-line clean-up and a subsequent chromatographic separation of the targeted analytes with a limit of quantitation (LOQ) of 22 pg/ml for 6-oxo-PGF(1alpha), and and a LOQ of 25 pg/ml for TXB(2)., (Copyright (c) 2009 John Wiley & Sons, Ltd.)

A dysfunctional Glanzmann Thrombasthenia (variant) is a rare bleeding disorder due to qualitative abnormalities of platelets alphaIIBbeta3 heterodimers. Dynamically conformational change of alphaIIBbeta3 is a complex mechanism that is not fully understood. For these reasons, genotyping and functional analysis of variant Glanzmann Thrombasthenia is important to elucidate the molecular basis of alphaIIBbeta3 receptor functions. In this report, we have analyzed the molecular effects of an A>T substitution leading to an amino acid change, D217>V, in the beta3 integrin gene identified in patients with variant Glanzmann Thrombasthenia. As the D217 residue is highly conserved among all seven beta integrin subunits and among beta3 integrins of different species, we tested the effect on the phenotype of the D217V mutation by cotransfecting the beta3 mutant (V217) or wild-type beta3 (D217) construct with the wild-type alphaIIb into eukaryotic Chinese hamster ovary cells. Levels of mutant alphaIIBbeta3 heterodimers on Chinese hamster ovary cell surface were lightly reduced as compared with the wild type. Functional investigation of alphaIIBbeta3 V217 on Chinese hamster ovary cell surface was carried out, as fibrinogen binding, adhesion and aggregation tests showed a substantial reduction in respect to the control sample. Our results confirm ex-vivo data and suggest that the D217 amino acid is required for alphaIIBbeta3 receptor interactions with fibrinogen.

Gene variant intron C G-42A of protein Z is significantly associated with the occurrence of fetal loss. A previously unreported sporadic missense mutation within exon 8 is described in a patient with very low protein Z levels.

Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis. In the majority of cases, the Y chromosome-specific sequences are commonly used as a fetus-specific marker with a high risk of false-negative cases. We attempted to develop a sensitive and reliable X chromosome short tandem repeat (STR) multiplex PCR amplification system that is suitable for the amplification of short-sized templates of free fetal DNA. Because of specific characteristics of fetal DNA in maternal plasma, cell-free fetal DNA is smaller than corresponding maternal DNA, and so we selected 10 X-STR loci in which the allele size was 250 bp. In addition, fetal sex was also investigated using the amelogenin gene in the same multiplex assay. Twenty-six women were enrolled in the study. Eight of 26 total fetuses analyzed were male and 18 were female. In the whole sample, X-STRs were informative with a mean of 4.84 +/- 1.43. A mean of 2.67 +/- 1.28 X-STR markers per sample (range 1-5) of paternally inherited fetal alleles were detected in pregnant women carrying a female fetus. In all cases, blind determination of fetal sex by means of the identification of amelogenin and X-STR markers was confirmed by fetal karyotyping. This study showed that this noninvasive technique is a reliable and accurate tool to investigate free fetal DNA in pregnancies within the first trimester and could be widely used in clinical research and diagnosis.

Hemophilia A is an X-linked bleeding disorder caused by widespread mutations in the human coagulation factor 8 gene. We have searched for mutations in factor 8 gene DNAs from 40 unrelated Italian patients with hemophilia A. All patients came from the same region (Calabria) and were followed-up at the same hemophilia center. Of the 40 patients, 20 (50%) had severe hemophilia A, 19 (47.5%) had moderate hemophilia A, and one (2.5%) had mild hemophilia A. All patients were first screened for the common intron 22 and intron 1 inversions. Inversion-negative samples were screened for point mutations by direct sequencing of all coding regions and intron-exon boundaries of the factor 8 gene. Mutations previously reported as causative of hemophilia A were identified in 14 of the 40 patients. These included five (12.5%) intron 22 inversions, one (2.5%) small deletion, one (2.5%) small insertion and seven (17.5%) point mutations. In all patients with moderate and mild hemophilia A, a nucleotide change in the c.1538 -18G>A in intron 10, not reported in the HAMSTeRS factor 8 mutation database (http://europium.csc.mrc.ac.uk/), was found. The G-to-A change predicts the appearance of a new acceptor splice site. We have also demonstrated that all patients share a common haplotype, suggesting that the mutation probably occurred in a single ancestor. In conclusion, we suggest that the c.1538-18G>A transition can be the putative mutation, which probably occurred in a common ancestor and then spread in neighbours, in patients with moderate-mild hemophilia A investigated in the present study.

We describe a new fully validated enantioselective LC-MS/MS method for stereospecific quantification of both the racemic forms of Warfarin (WF), Phenprocoumon and Acenocoumarol in human plasma. Measurement specificity was assessed by using different blank donor plasma samples, where no interfering reagent peak appeared at the retention time (RT) of the targeted analytes. Response was linear for all analytes. Typical linear regression coefficients have >0.99. The recoveries ranged from 98% to 118%. Determinations in 10 normal healthy individuals revealed a high reproducibility of RTs. These findings confer to the method suitability for large population studies.

A new analytical determination method of homocystine in human plasma has been developed. The method utilises liquid chromatography coupled to ionspray tandem mass spectrometry. Quantitative analysis was achieved using as an internal standard homocystine-d8. Mass spectrometer operated in the multiple reaction mode: homocystine and homocystine-d8 were detected through the transition from the precursor to the product ion (from m/z 269.3 to 90.0, and m/z 277.3 to 94.0, respectively). The method is extremely sensitive, with limit of detection in the range of 6 fmol/L. The interassay and intraassay coefficients of variation for homocystine were 6.22% and 3.4%, respectively. The accuracy for the added homocystine ranged from 85% to 110%. High specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for a rapid and reliable assay of homocystine.

Fetal DNA identification in maternal circulation has provided a new approach for non-invasive prenatal diagnosis. However, fetal DNA can persist in maternal blood long after the delivery, severely hampering this possibility. We addressed the issue of fetal DNA persistence in maternal blood. Thus, we investigated cell-free fetal DNA as a reliable approach in prenatal diagnosis of haemophilia. Forty non-pregnant women, who had had at least a male fetus, 29 control pregnant women, and 14 pregnant women, carriers of hemophilia A or B. The assessment of Y-chromosomal sequences was performed by analysing SRY and amelogenin genes using PCR-based techniques. A protocol consisting of double centrifugation at full speed followed by plasma filtration hampered the detection of Y chromosome-specific sequence in non-pregnant women. In 29 control pregnant women, blinded determination of fetal sex confirmed the specificity and sensitivity of the method applied. In 14 pregnant carriers of hemophilia, the investigation revealed a male fetus in nine pregnancies. Excluding the three cases in which a spontaneous miscarriage occurred, the sensitivity and specificity of fetal sex prediction by SRY and amelogenin gene analyses were both 100% as compared with the invasive approach and the fetal sex outcome at birth (six males and five females). Because of its high accuracy in prediction, fetal gender determination with cell-free fetal DNA in maternal plasma may be a useful tool in prenatal diagnosis of haemophilia allowing for the avoidance of invasive procedures for female fetuses.

Hyperhomocysteinemia could play a similar role in the placenta to that played in adults at risk of thrombosis. Moreover, hyperhomocysteinemia in women is described to be associated with the birth of small for gestational age (SGA) newborns, although there are discrepancies on this issue. To date, there is no biochemical marker predictive of SGA in a given pregnancy. We verified the presence of a relationship between homocysteine in amniotic fluid at mid-pregnancy and birth-weight. Amniotic fluid was obtained from 459 healthy women undergoing midtrimester amniocentesis (17.1 +/- 1.2 weeks) because of maternal age. Homocysteine levels were measured in 434 (10 twin) pregnancies. In addition, femur length (FL) and biparietal diameter (BPD) were measured. Outcome of pregnancy was recorded. 233 (53.7%) foetuses were males, 201 (46.3%) females. The mean homocysteine concentration was 1.04 +/- 0.72 microM, (95% C.I. 0.43-2.41). An univariate analysis showed the presence of an association with gestational age, FL, BPD. A multiple linear regression showed that homocysteine levels were significantly associated with FL (p < 0.001) and BPD (p = 0.011). After excluding twin pregnancies, 31 newborns (7.3%) were classified as SGA. Mean birth-weight was 2390 g in SGA, whereas it was 3360 g in 393 adequate for gestational age (AGA) newborns (p < 0.001). The adjusted mean level of homocysteine was significantly lower in AGA (1.01 microM; 95% C.I: 0.94-1.08) than that recorded in pregnancies resulting in a SGA (1.29 microM; 95% CI: 1.05-1.51; p = 0.03). In a large setting, these data provide reference values for homocysteine in amniotic fluids. Moreover, they suggest that homocysteine levels in amniotic fluids may be higher in pregnancies with a SGA newborn.

Objective: To evaluate the role of different polymorphic gene variants involved in homocysteine metabolism and plasma levels of homocysteine, folate and vitamin B12 in families from southern Italy with neural tube defects (NTDs)., Methods: Eighteen fathers, 15 NTD children and 60 women who had conceived NTD foetuses were investigated. A group of 100 adults and 43 apparently healthy children was used as control. At the time of blood draw, none were taking vitamin pills or nutritional supplements., Results: Among controls, 79 (55.2%) were heterozygous for C677T MTHFR variant and 26 (18.2%) were TT homozygous. Among the cases, 35 (61.4%) out of 57 mothers and 7 (38.9%) out of 18 fathers carried the T allele; 12 (21.1%) mothers and 2 (11.1%) fathers had the TT genotype. Four (26.7%) out of 15 probands were TT homozygous and 11 (73.3%) were heterozygous (Fisher exact test p = 0.025). No significant difference between groups was observed for the 1298C MTHFR variant and CBS haplotypes. Median homocysteine in NTD children was significantly higher (10.0 micromol/L) than that of controls (median 4.5 micromol/L, Mann-Whitney p < 0.05). Folate and B12 were not different among groups., Conclusions: The T677 MTHFR allele is significantly associated with the occurrence of NTDs; no significant association has been observed with other genetic determinants analysed. Homocysteine levels in children with NTDs are significantly higher than those of the paediatric population from the same geographical area., (2006 John Wiley & Sons, Ltd.)

This is the case of a 28-year-old woman in apparently good health, who had an ischemic stroke and significant proteinuria developed during her first attempt of in vitro fertilization. She was evaluated for inherited and acquired thrombophilia and tested positive for high titer of antiphospholipid antibodies and mild hyperhomocysteinemia. The potential thrombotic risks associated with in vitro fertilization will be discussed.

Objective: Nitric oxide is suggested to play a role in the development of preeclampsia., Methods: We studied 61 patients with gestational hypertension (GH), 77 with GH and significant proteinuria (urine protein excretion > or = 300 mg/24 h), 82 with essential hypertension (EH) and 188 normotensive women with at least one normal pregnancy., Main Outcome Measure(s): A polymorphism within the constitutive endothelial nitric oxide synthase (ecNOS) gene in various types of hypertension in pregnancy was explored., Results: Allelic and genotypic frequencies did not differ between controls and case groups. A significant difference was observed between the frequency of the rare allele in GH patients and that in EH group (chi2: 4.47, P <.04). This difference approximated the significance when GH subjects with or without proteinuria were grouped (chi2 square: 3.33; P =.068). Cigarette smoking or gravidity did not interact with the ecNOS polymorphism in identifying different types of hypertension in this setting., Conclusion: Our findings argue against an association between ecNOS polymorphism and preeclampsia and support the hypothesis for a different pathogenesis of GH in respect to EH.

Glanzmann's thrombasthenia (GT) is a genetically heterogeneous autosomal recessive syndrome associated with a bleeding tendency. To elucidate molecular basis of GT we have screened for mutations 30 GT patients. On the whole, 21 different candidate causal mutations, 17 in the alphaIIb and 4 in the beta3 gene have been found. Only two (alphaIIb Pro145Ala and IVS3(-3)-418del) have been previously reported. Nine mutations (42.9%) were likely to produce truncated proteins, whereas the remaining 12 were missense mutations that affected highly conserved residues in alphaIIb and beta3 genes. Six mutations were found in different patients suggesting a possible founder effect. The wide spectrum of expressivity, ranging from mild to severe also among patients carrying the same mutations, provided evidence for a role of different loci or circumstantial factors. In conclusion, we have identified a spectrum of unreported mutations that may be of value to unravel the role of specific regions of alphaIIb and beta3 genes.

We have investigated a 53-yr-old asymptomatic white man with decreased functional, but not immunologic, fibrinogen plasma levels together with prolonged thrombin and reptilase times, detected through routine coagulation studies prior to a surgical procedure. A new heterozygous single nucleotide deletion (C) at position Ala499 within the Aalpha-chain gene was identified, which predicted changes of the corresponding amino acids encoded by the subsequent portion of the exon V and the appearance of a premature stop codon at position 518 (Aalpha[499]Ala frameshift stop). The new dysfunctional fibrinogen, San Giovanni Rotondo variant, was confirmed in vivo by SDS-PAGE analysis of HPLC-purified fibrinogen chains. Mass spectrum examination of the abnormal HPLC-purified peak gave an estimated mass (56,088 Da) similar to that predicted by DNA analysis of the mutated Aalpha-chain gene (56,088 Da) and, after tryptic digestion, the truncated Aalpha-chain was shown only in the propositus, who also carried normal Aalpha-chain. In addition, mass spectrum analysis of the tryptic digest of the abnormal chain confirmed the presence of a new and unpaired cysteine at the last position that was predicted to form a disulfide bridge with human serum albumin. Immuno-blot analysis confirmed that fibrinogen San Giovanni Rotondo variant, but not normal fibrinogen. contained substantial amounts of albumin. Present findings confirm that truncated Aalpha-chain lacking part of the terminal domain may be incorporated into mature fibrinogen molecules and normally secreted in the bloodstream.

An automatic on-line digestion-liquid chromatography/mass spectrometry/collision-induced dissociation mass spectrometry (LC-MS/CID-MS) protocol has been developed for detection of errors in the biosynthesis of human fibrinogen, such as amino acid (AA) mismatch or incorrect post-translational modification (PTM). Using on-line digestion on an immobilized-enzyme column, the reaction time is significantly reduced (less than 20 min) and the entire approach is suitable for automation. The two-loop MS experiments (full-scan acquisition and sugar moieties monitoring by SIM) allow checking both the correct AA mapping via the peptides generated by the digestion of the PTM. Since the protocol was designed for application on a routine basis, as a proof-of-concept detection of a rare case of 'abnormal' fibrinogen has been demonstrated. The advantage of the proposed approach is exemplified by the fact that the DNA sequence information for the case investigated had not shown any evidence of the abnormality., (Copyright 2001 John Wiley & Sons, Ltd.)