Role of Maspin, CK17 and Ki-67 Immunophenotyping in Diagnosing of Pancreatic Ductal Adenocarcinoma in Endoscopic Ultrasound-Guided Fine Needle Aspiration Cytology.

BACKGROUND AND STUDY AIM: One of the problems in diagnosing pancreatic ductal adenocarcinoma (PDAC) is differentiation between PDAC cells and benign pancreatic tissue cells in cytologic samples. This study aimed to evaluate the usefulness of Maspin, CK17 and Ki-67 immunocytochemistry (ICC) in differentiation between these two groups of cells. MATERIALS AND METHODS: This retrospective study was carried on 80 cases of PDAC and 25 cell blocks of benign pancreatic tissue cells as a control group for evaluation of Maspin, CK17 and Ki-67 ICC. PDAC cases were sampled by endoscopic ultrasound guided fine needle aspiration cytology (EUS-FNAC), while cell blocks of control group were aspirated from benign pancreatic tissues that were obtained from the pancreatic surgically resected specimens. Immunostaining patterns, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of each antibody as well as possible antibody combined panels of these markers in differentiation between the two groups were evaluated. RESULTS: Positive immunoreactivity for Maspin, CK17 and Ki-67 were 92.5%, 80% and 72.5% in PDAC cases, respectively. In contrast to PDAC cases, all the cell blocks of benign pancreatic tissue cells were negative for these markers. Regarding different panels, combined use of Maspin, CK17 and Ki-67 together as a triple test (at least one of them is positive) achieved the highest sensitivity of 98.8%, specificity of 100%, PPV of 100%, NPV of 96.2% and accuracy of 99% in the differentiation between PDAC and benign pancreatic tissue. CONCLUSION: Employing this short panel [Maspin, CK17 and Ki-67] is helpful for better differentiation between PDAC and benign pancreatic tissue.

Liquid chromatography-diode array-mass spectrometric analysis of amino and mercapto compounds coupled with chloroimino derivatization reagent.

A new derivatization reagent, N-(naphthalen-1-yl)-2-oxopropanehydrazonoyl chloride (UOSA54), was prepared and coupled with four drugs, bearing primary amino, secondary amino or mercapto functional groups. Glucosamine sulfate (GLU), cysteine (CYS), captopril (CAP), and vildagliptin (VIL), were used as representative reactive analytes. The prepared reagent was successfully coupled with the targeted analytes in the presence of triethylamine (TEA) as hydrochloride acceptor and acetonitrile as solvent. The resulting reaction products were separated by high-performance liquid chromatography and monitored simultaneously by diode array and triple quad mass spectrometry detectors. Enhanced DAD and electrospray ionization-MS (ESI-MS) responses were observed for the derivatized products. Complete derivatization of VIL was achieved after heating at 65 ± 3 °C for 4 min, while other analytes were derivatized instantaneously at room temperature. Both, the ESI-ionization suppression, due to the excess reagent, and matrix effect, due to co-eluted biogenic plasma constituents, were negligible. The derivatized GLU, CYS, CAP, and VIL showed a maximum absorption wavelength at 376, 417, 340, and 376 nm, with MS-limit of quantification value of 250.0, 2.0, 2.5, and 3.0 pg/µL, respectively. The relative ESI-MS response of UOSA54 derivatization products was within the range of 0.6-4.1 compared with dansylated products. The method was optimized and validated for optimal reaction product stability, sensitivity, linearity, range, precision, and accuracy. The percentage recovery was exceeding 97.2%, with an RSD value of less than 4.0%. The limit of quantification of targeted analytes was ranged from 80.0 to 0.7 pg/µL.

Fatal liver failure after therapeutic doses of paracetamol in a patient with Duchenne muscular dystrophy and atypical pharmacogenetic profile of drug-metabolizing enzymes.

Paracetamol has a good safety profile, but pharmacogenetic differences in drug-metabolizing enzymes may have an impact on its risk of hepatotoxicity. We present a case of fatal acute liver failure (ALF) after therapeutic doses of paracetamol in a patient with Duchenne muscular dystrophy, where pharmacogenetic screening was conducted. This 30-year-old man was electively admitted for a tracheostomy. A total of 14.5 g paracetamol was given over four days. He developed a severe ALF and died 11 days after admission. Pharmacogenetic screening showed absent CYP2D6 metabolism and increased CYP1A2 activity, which may have increased the formation of toxic intermediate metabolite, N-acetyl-p-benzo-quinone imine (NAPQI). He also had decreased function of UGT2B15, which increases the amount of paracetamol available for metabolism to NAPQI. Having a reduced muscle mass and thus a reduced glutathione levels to detoxify produced NAPQI may add to the risk of toxicity. This case may indicate that pharmacogenetic variability is of potential relevance for the risk of paracetamol-induced hepatotoxicity in patients with neuromuscular diseases. Further studies should investigate if pharmacogenetic screening could be a tool to detect potentially increased risk of hepatotoxicity in these patients at therapeutic doses of paracetamol and hence provide information for selection of analgesic treatment.

Novel approach for evaluating pharmaceuticals toxicity using Daphnia model: analysis of the mode of cytochrome P450-generated metabolite action after acetaminophen exposure.

Because of its widespread use, the pharmaceutical acetaminophen (APAP) is frequently detected in aquatic environments. APAP can have serious physiological effects, such as reduced reproduction, low growth rates, and abnormal behavior, in aquatic organisms. However, the methods available for evaluation of the aquatic toxicity of APAP are of limited usefulness. The present study aimed to develop reliable and sensitive markers for evaluation of APAP toxicity using Daphnia as a model organism. We focused on N-acetyl-p-benzoquinoneimine (NAPQI) production from APAP via cytochrome P450 metabolism because NAPQI causes APAP toxicity. Daphnia magna were exposed to APAP (0, 50, or 100 mg/L for 12 h or 24 h), and the total metabolites were extracted and analyzed for NAPQI. Direct detection of NAPQI was difficult because of its high reactivity, and its peak was close to that for APAP. Therefore, we tried to identify molecular and biochemical indicators associated with NAPQI generation, elimination, and its interactions with macromolecules. We identified changes in CYP370A13 gene expression, glutathione depletion, inhibition of thioredoxin reductase activity, and production of reactive oxygen species as indicators of D. magna exposure to APAP. These indicators could be used to develop sensitive and accurate techniques to evaluate the environmental toxicity of APAP.

Determination of strobilurin fungicides in cotton seed by combination of acetonitrile extraction and dispersive liquid-liquid microextraction coupled with gas chromatography.

The simultaneous determination of four strobilurin fungicides (picoxystrobin, kresoxim-methyl, trifloxystrobin, and azoxystrobin) in cotton seed by combining acetonitrile extraction and dispersive liquid-liquid microextraction was developed prior to GC with electron capture detection. Several factors, including the type and volume of the extraction and dispersive solvents, extraction condition and time, and salt addition, were optimized. The analytes were extracted with acetonitrile from cotton seed and the clean-up was carried out by primary secondary amine. Afterwards, 60 µL of n-hexane/toluene (1:1, v/v) with a lower density than water was mixed with 1 mL of the acetonitrile extract, then the mixture was injected into 7 mL of distilled water. A 0.1 mL pipette was used to collect a few microliters of n-hexane/toluene from the top of the aqueous solution. The enrichment factors of the analytes ranged from 36 to 67. The LODs were in the range of 0.1 × 10(-3) -2 × 10(-3) mg/kg. The relative recoveries varied from 87.7 to 95.2% with RSDs of 4.1-8.5% for the four fungicides. The good performance of the method, compared with the conventional pretreatments, has demonstrated it is suitable for determining low concentrations of strobilurin fungicide residues in cotton seed.

First report on the detection of pectenotoxin groups in Chinese shellfish by LC-MS/MS.

Chinese shellfish samples were harvested from different locations along the Chinese coast. These shellfish were analyzed by liquid chromatography in combination with mass spectrometry to detect the following toxins: okadaic acid (OA), dinophysistoxins (DTXs), petenotoxins (PTXs), azaspiracids (AZAs), yessotoxins (YTXs), spirlides (SPXs) and gymnodimines (GYM). The results revealed the lipophilic toxin profiles varied with shellfish sampling locations. In addition to OA, GYM and YTX derivatives, PTX-2 and its derivatives were found for the first time in the following Chinese shellfish: Crassostrea gigas, Mactra chinensis and Mytilus galloprovincialis. The presence of GYM, YTXs, OA and PTXs in Chinese shellfish collected from regions where no previous record of DSP-neutral toxic compounds was reported. Serious efforts should therefore be made to conduct a phycotoxin monitoring program to detect the presence of lipophilic toxins in biological materials of marine origin, which may ensure that Chinese seafood products do not present a health risk. With respect to suspected carcinogenicity, further research on the distribution and concentrations of toxic compounds are needed, in order to carry out long-term risk assessments, particularly sub-acute and chronic toxicity tests associated with of lower doses.

Determination of tebuconazole, trifloxystrobin and its metabolite in fruit and vegetables by a Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method using gas chromatography with a nitrogen-phosphorus detector and ion trap mass spectrometry.

A new analytical method using QuEChERS procedure by gas chromatography with a nitrogen-phosphorus detector (GC-NPD) and ion trap mass spectrometry (GC-IT-MS) for the quantitative determination of tebuconazole, trifloxystrobin and its metabolite trifloxystrobin acid has been developed and validated. The analytes were extracted from five fruit and vegetable matrices using acetonitrile and subsequently cleaned up using primary secondary amine (PSA) or octadecylsilane (C18) as sorbent prior to GC analysis. The present methods provided sufficient sensitivity as reflected by the values of limit of detection (LOD) and limit of quantification (LOQ) of 0.4-7 and 1.2-20 µg/kg for GC-IT-MS/MS and GC-NPD. The recoveries were, on average, 68-117 and 68-121%, respectively, for three compounds by GC-NPD and GC-IT-MS/MS with intra-day precision achieved with an RSD of 2.7-19.1%. The inter-day precision was better than 15.1% as determined by GC-NPD. The QuEChERS procedure, by using two sorbents (PSA and C18) and the matrix-matched standards, gave satisfactory recoveries and RSD values in different matrices. IT-MS acquisition provided higher specificity and selectivity for pesticides and better limit of detection and quantification. However, the repeatability and precision of NPD method were better compared with IT-MS.

Quantitative determination of gymnodimine-A by high performance liquid chromatography in contaminated clams from Tunisia coastline.

Quantitative determination by high performance liquid chromatography (HPLC) was performed for gymnodimine-A (GYM-A), a phycotoxin responsible for the contamination of Tunisian clams. This study demonstrates a rapid and reproducible HPLC-ultraviolet (UV) method for extraction, detection and quantification of GYM-A in toxic clams. The extraction of GYM-A from the digestive gland of clams in acetone, subsequent clean-up with diethyl ether and extraction with dichloromethane is the more valid protocol. Chromatography analyses were performed using a gradient of acetonitrile-water (10:90 to 90:10), containing trifluoroacetic acid (0.1%) for 20 min at 1 mL/min rate with a C18 column. Recovery rates exceeded 96%, and limits of detection and quantification were 5 ng/mL and 8 ng/g digestive gland, respectively. Repeatability and reproducibility were tested for various samples containing different levels of GYM-A. A significant correlation was observed between toxicity level of samples and the determined amount of GYM-A. Also, the persistence of GYM-A in contaminated clams from Boughrara lagoon was demonstrated. The kinetics discharge study of GYM-A in controlled medium, during 1 month, showed that the process of depuration was biphasic with an exponential discharge of 75% of the total amount of sequestered GYM-A during the first 12 days followed by a slow discharge (>10%) for the subsequent days up to the seventeenth day. This is the first time that a quantitative study of GYM-A in clams from Tunisian coasts is performed through the development of a new method for detection and quantify of this phycotoxin. We found HPLC-UV a reliable and suitable alternative to the mouse bioassay.

Enhanced NMR signal detection of imino protons in RNA molecules containing 3' dangling nucleotides.

We present a method for improving the quality of nuclear magnetic resonance (NMR) spectra involving exchangeable protons near the base of the stem of RNA hairpin molecules. NMR spectra of five different RNA hairpins were compared. These hairpins consisted of a native RNA structure and four molecules each having different unpaired, or dangling, nucleotides at the 3' end. NMR experiments were acquired in water for each construct and the quality of the imino proton spectral regions were examined. The imino resonances near the base of the stem of the wild type RNA structure were not observed due to breathing motions. However, a significant increase in spectral quality for molecules with dangling 3' adenosine or guanosine nucleotides was observed, with imino protons detected in these constructs that were not observed in the wild type construct. A modest improvement in spectral quality was seen for the construct with a 3' unpaired uridine, whereas no significant improvement was observed for a 3' unpaired cytidine. This improvement in NMR spectral quality mirrors the increased thermodynamic stability observed for 3' unpaired nucleotides which is dependant on the stacking interactions of these nucleotides against the base of the stem. The use of a dangling 3' adenosine nucleotide represents an easy method to significantly improve the quality of NMR spectra of RNA molecules.

Removal of famoxadone, fluquinconazole and trifloxystrobin residues in red wines: effects of clarification and filtration processes.

The effects of six clarification agents [egg albumin, blood albumin, bentonite + gelatine, charcoal, polyvinylpolypyrrolidine (PVPP) and silica gel] on the removal of residues of three fungicides (famoxadone, fluquinconazole and trifloxystrobin) applied directly to a racked red wine, elaborated from Monastrell variety grapes from the D.O. Region of Jumilla (Murcia, Spain) were studied. The clarified wines were filtered with 0.45 microm nylon filters to determine the influence of this winemaking process in the disappearance of fungicide residues. Analytical determination of fluquinconazole and trifloxystrobin was performed by gas chromatography with electron captor detector (ECD), while that of famoxadone using an HPLC equipped with a diode array detector (DAD). Generally, trifloxystrobin is the fungicide that is the lowest persistent one in wines, except in the egg albumin study whereas, the most persistent one is fluquinconazole. The elimination depends on the nature of the active ingredient, though the water stability in the presence of light within it has more influence than the solubility and polarity of the product itself. The most effective clarifying agents were the charcoal and PVPP. The silica gel and bentonite plus gelatine were not enough to reduce considerably the residual contents in the wine clarified with them. In general terms, filtration is not an effective step in the elimination of wine residues. The greatest removal after filtration is obtained in wines clarified with egg albumine and bentonite plus gelatine, and the lowest in those clarified with PVPP.

Rapid gas chromatographic method for the determination of famoxadone, trifloxystrobin and fenhexamid residues in tomato, grape and wine samples.

Trifloxystrobin, fenhexamid and famoxadone belong to the generation of fungicides acting against a broad spectrum of fungi and widely used in Integrated Pest Management strategies in different agricultural crops but mainly in viticulture. In the present work, a gas chromatographic (GC) method for their determination was developed and validated on tomato, grape and wine matrices. The method was based on a simple one step liquid-liquid microextraction with cyclohexane/dichloromethane (9+1, v/v) and determination of fungicides by gas chromatography with nitrogen phosphorous (NP-) and electron capture (EC-) detection, and ion trap mass spectrometry (ITMS) for confirmation. The method was validated by recovery experiments, assessment of matrix effect and calculation of the associated uncertainty. Recoveries for GC-NPD and GC-ECD were found in the range of 81-102% with RSD <12%, while matrix-matched calibration solutions were imposed for quantification. LOQs ranged from 0.005 to 0.05 mg/kg and 0.01 to 0.10 mg/kg for the GC-ECD and GC-NPD, respectively, depending on the sensitivity of each compound with trifloxystrobin being the most sensitive. The expanded uncertainty, calculated for a sample concentration of 0.10 mg/kg, ranged from 4.8 to 13% for the GC-ECD and from 5.4 to 29% for the GC-NPD. The concentration levels for famoxadone residues found in tomato and grape samples from field experiments were clearly below the EU established MRL values, thus causing no problems in terms of food safety.

Determination by gas chromatography/mass spectrometry of p-phenylenediamine in hair dyes after conversion to an imine derivative.

In this paper we describe an analytical method for the determination of p-phenylenediamine (PPDA) in hair dyes. In the adopted methodology the analyte is transformed into the corresponding imine derivative by treatment with benzaldehyde, and then analyzed by gas chromatography (GC) combined to mass spectrometry (MS), operating in SIM conditions. The direct and simultaneous chemical derivatization of the two amino functions of the analyte with benzaldehyde enhances the instrumental responses enabling the use of a sensitive and accurate method. Concentrations of PPDA in a set of commercial hair coloring creams are determined making use of N-benzylidene-4-methylbenzene-amine as a very stable internal standard which is easily prepared by condensation of 4-methylbenzene-amine with benzaldehyde. The regression calibration curves for PPDA in hair dyes are linear within 0.1 +/- 25 mg/ml with 0.99 as a typical correlation coefficient.

Cyanide trapping of iminium ion reactive intermediates followed by detection and structure identification using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Secondary and tertiary alicyclic amines are widely found in pharmaceuticals and environmental compounds. The formation of iminium ions as reactive intermediates in the metabolic activation of alicyclic amines has previously been investigated in radiometric assays where radiolabeled cyanide is typically employed. In this paper, we report a relatively high throughput LC-MS/MS method for the detection of the nonradiolabeled cyanide adduct formed in rat or human liver microsomal incubations via constant neutral loss scan followed by structural characterization using product ion scan on a triple quadrupole mass spectrometer. A total of 14 alicyclic amine compounds were investigated with the cyanide trapping LC-MS/MS screen and also with the glutathione (GSH) trapping screen, a well-established and commonly employed technique for reactive metabolite screening. Our results are found to be in general agreement with the previous metabolism reports for these compounds, demonstrating the effectiveness, speed, and simplicity of the cyanide trapping LC-MS/MS method to study the iminium ion intermediates from alicyclic amines and its complementarities to GSH trapping method for reactive metabolite screenings.

Preparacao e analise estrutural de iminas derivadas de dextranos oxidados e antimalaricos / Preparation and structural analysis derivative imines of oxidized dextrans and antimalarial

Com o objetivo de obter pro-farmacos potencialmente ativos em malaria, especialmente em malaria resistente, preparam-se, empregando-se o metodo da latenciacao, derivados de dextranos oxidados--MM 60.000-90.000, 70.000 e 500.000 -- com agentes antimalaricos. Sintetizaram-se iminas derivadas dos polimeros oxidados contendo um unico farmaco -- sulfas, sulfona -- e em associacao -- sulfas ou sulfona, e pirimetamina. Os compostos obtidos foram submetidos as analises espectrometricas no IV, UV de 'ANTPOT. 1'H RMN e de 'ANTPOT. 13'C RMN. Essas analises indicaram que os compostos que seguem foram obtidos: dextrano oxidado I (DOI) e dapsona (63), sulfadiazina (64), sulfametoxidiazina (65); dextrano oxidado II (DOII) e dapsona (67), sulfametoxazol, pH 6,0(71), sulfametoxipirida (72), sulfamonometoxina (73), dextrano oxidado III (DOIII) e dapsona (75), dextrano oxidado MM 70.000 (T70III) e sulfisoxazol); derivados de dextrano oxidado I (DOI) em associacao de pirimetamina e dapsona 1:1(91), de dextrano oxidado III em associacao de pirimetamina e dapsona, proporcao equimolar(95), de dextrano oxidado MM 70.000 (T70III) em associacao de pirimetamina e sulfametoxidiazina (99), de dextrano oxidado MM 70.000 (T70III) em associacao de pirimetamina e sulfametoxipiridazina (100) e ), de dextrano oxidado MM 500.000 (T500III) em associacao de pirimetamina e sulfametoxipiridazina (107). Com o objetivo de quantificar o farmaco no pro-farmaco, efetuaram-se analises quantitativas no UV com DOIDDS (63), DOISDZ (64), DOISMDZ (65), DOIISDX (69), DOIISMXZ pH 4,2 (70); DOIISMXZ pH 6,0 (71), DOIISMMX (73) e T70IIISFSXZ (80) mostraram graus de substituicao diversos. Dois desses compostos --derivados de dapsona e de sulfadiazina, foram submetidos a ensaios de liberacao in vitro, em valores diferentes de pH, apresentaram liberacao prolongada

Mechanism of protection of ebselen against paracetamol-induced toxicity in rat hepatocytes.

The protective effect of ebselen (PZ 51), an anti-inflammatory agent, on paracetamol-induced (1 mM) cytotoxicity in hepatocytes freshly isolated from beta-naphthoflavone-pretreated rats was studied. At a concentration of 50 microM added simultaneously with paracetamol, ebselen prevented paracetamol-induced leakage of lactate dehydrogenase (LDH) almost completely and lipid peroxidation (LPO) and depletion of glutathione (GSH) substantially. These protective effects were even more pronounced at 100 microM concentration of ebselen. When added to the hepatocytes 1 hr before paracetamol, 50 microM of ebselen also prevented LDH leakage, LPO and GSH depletion. Reverse addition of paracetamol and ebselen did not result in protection. Simultaneous incubation of 100 microM ebselen and paracetamol inhibited GSH conjugation of paracetamol by more than 50%, however, without any effect on glucuronidation and sulfation of paracetamol. Ebselen was shown not to react directly with paracetamol nor to inhibit cytochrome P450 activity measured as 7-ethoxycoumarin O-deethylase (ECD) activity in the hepatocytes. At mixing, synthetic ebselen selenol and synthetic N-acetyl-p-benzoquinone imine (NAPQI) were shown to form paracetamol and ebselen diselenide. No indication was found for the formation of an ebselen-paracetamol conjugate upon reacting synthetic NAPQI and synthetic ebselen selenol. Reduction of NAPQI, the reactive metabolite of paracetamol, by ebselen selenol is discussed in terms of the mechanism of cytoprotection.

Electron acceptance at photosystem II in an oxygen-evolving photosystem II preparation: clear discrimination of two sites of electron acceptance for quinones and quinonediimines associated with a photosystem II preparation.

Oxygen-producing electron transport reactions of a photosystem II preparation are described. The preparation has six major peptides with apparent molecular weights of 36,000, 31,000, 28,000, 27,000, 25,000, and 21,000. Sucrose density gradient centrifugation indicates that the preparation is more homogeneous and more dense than control thylakoid membranes. The preparation photoreduces a number of known photosystem II oxidants including the Class I acceptor, ferricyanide; the Class II acceptor, 2,6-dichloroindophenol; and the Class III acceptor, 2,6-dichlorobenzoquinone. However, quinonediimines such as phenylenediimine are not reduced, suggesting that these substances are reduced at sites in the thylakoid membrane which are not found in the photosystem II preparation. All the oxygen-producing reactions are sensitive to inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethylurea.