Search PubMed⌕ Search

Biomedical subjects

M Barroso

Publications and source records attributed to M Barroso.

At least 19 recordsLinked to original sources
PubMed2006

Determination of parathion in biological fluids by means of direct solid-phase microextraction.

A new and simple procedure for the determination of parathion in human whole blood and urine using direct immersion (DI) solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS) is presented. This technique was developed using only 100 microL of sample, and ethion was used as internal standard (IS). A 65-microm Carbowax/divinylbenzene (CW/DVB) SPME fibre was selected for sampling, and the main parameters affecting the SPME process such as extraction temperature, adsorption and desorption time, salt addition, agitation and pH effect were optimized to enhance the sensitivity of the method. This optimization was also performed to allow the qualitative determination of parathion's main metabolite, paraoxon, in blood. The limits of detection and quantitation for parathion were 3 and 10 ng/mL for urine and 25 and 50 ng/mL for blood, respectively. For paraoxon, the limit of detection was 50 ng/mL in blood. The method showed linearity between the LOQ and 50 microg/mL for both matrices, with correlation coefficients ranging from 0.9954 to 0.9999. Precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The mean absolute recoveries were 35.1% for urine and 6.7% for blood. Other parameters such as dilution of sample and stability were also validated. Its simplicity and the fact that only 100 microL of sample is required to accomplish the analysis make this method useful in forensic toxicology laboratories to determine this compound in intoxications, and it can be considered an alternative to other methods normally used for the determination of this compound in biological media.

Humans↗
PubMed2006

Analytical approach to determine ticlopidine in post-mortem blood.

A new and sensitive method to determine ticlopidine in whole human blood using proadifen as the internal standard (IS) is described. The analyte and IS were extracted by solid-phase extraction using Oasis HLB cartridges, and the extracts were analyzed by gas chromatography-electron impact ionisation-mass spectrometry (GC/EI-MS). Calibration curves were established daily in spiked blood samples using a non-linear calibration model, between 0.01 and 4.5 microg/mL. The correlation coefficients were higher than 0.995. Precision and accuracy fulfilled the internationally accepted criteria (coefficients of variation were less than 9%, and the measured concentrations were within +/-7% of the true value). Limits of detection and quantitation were respectively, 3 and 10 ng/mL. No interfering substances were detected by analysis of 10 blank blood samples of different origin. Mean recovery, calculated at three concentration levels, was 71%. Because of its simplicity and speed, the proposed method can be applied in the determination of this inhibitor of platelet aggregation in whole blood samples, and is suitable for application in toxicology routine analysis.

Chromatography, Gas↗
PubMed2006

Determination of quinalphos in blood and urine by direct solid-phase microextraction combined with gas chromatography-mass spectrometry.

A new method based on direct solid-phase microextraction (DI-SPME) followed by gas chromatography-mass spectrometry was developed for the purpose of determining quinalphos in blood and urine. Two types of coated fibre have been assayed and compared: carbowax/divinylbenzene (CW/DVB 65 microm) and polydimethylsiloxane (PDMS 100 microm). The main parameters affecting the SPME process such as temperature, salt addition, pH, stirring and adsorption/desorption time profiles were optimized to enhance the sensitivity of the procedure. The method was developed using only 100 microL of blood and urine. Limits of detection of the method for blood and urine matrices were, respectively, 10 and 2 ng/mL. Linearity was established over concentration ranges from 0.05 to 50 microg/mL for blood, and 0.01 to 50 microg/mL for urine, with regression coefficients ranging between 0.9991 and 0.9999. Intra- and interday precision values were less than 13%, and accuracy was within +/-15% of the nominal concentration for all studied levels in both matrices. Absolute recoveries were 14 and 26% for blood and urine, respectively.

Adsorption↗
PubMed2006

Solid-phase microextraction for gas chromatographic/mass spectrometric analysis of dimethoate in human biological samples.

A new, simple and rapid procedure for the determination of dimethoate in urine and blood samples was developed using direct immersion solid-phase microextraction and gas chromatography/mass spectrometry. This technique required only 0.1 mL of sample, and ethion was used as internal standard. Two types of coated fibre were compared (100 microm polydimethylsiloxane, and 65 microm Carbowax/divinylbenzene). Other parameters, such as extraction temperature, adsorption and desorption time, salt addition, agitation and pH, were optimized to enhance the sensitivity of the method. Limits of detection (LODs) and quantitation (LOQs) were 50 and 100 ng/mL for urine and 200 and 500 ng/mL for blood, respectively. The method was found to be linear between the LOQ and 40 microg/mL for urine, and between the LOQ and 50 microg/mL for blood, with correlation coefficients ranging from 0.9923-0.9996. Precision (intra- and interday) and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The mean absolute recoveries of dimethoate were 1.24 and 0.50% for urine and blood, respectively. Because of its simplicity and the fact that small volumes of sample are used, the described method can be successfully used in the diagnosis of poisoning by this pesticide, namely in those situations where the sample volume is limited, as frequently occurs in forensic toxicology.

Dimethoate↗
PubMed2005

Application of solid phase microextraction to the determination of strychnine in blood.

A simple and rapid method based on solid phase microextraction (SPME) via direct immersion followed by gas chromatography coupled with electron impact ionization/mass spectrometry (GC/EI-MS) was developed for the determination of strychnine in blood. Papaverine was used as internal standard (I.S.). Two types of fibre coating were tested, 100 microm polydimethylsiloxane and 65 microm Carbowax/Divinylbenzene, the latter giving higher recoveries of the compound. The main factors affecting the SPME process, such as sample dilution (1:10), adsorption and desorption times (20 and 10 min, respectively), carry-over effect (not observed), pH and salt addition (no modifications on pH or salt concentration) were optimized. The procedure was validated in terms of linearity (r(2)=0.9992 for concentrations ranging from 0.10 to 5.00 microg/mL), intra and interday precision (0.93 and 4.62%, respectively at 0.50 microg/mL; 3.33 and 8.06%, respectively at 2.50 microg/mL), sensitivity (6.83 and 8.91 ng/mL for LOD and LOQ, respectively) and extraction recovery (0.54 and 0.39% at 0.50 and 2.50 microg/mL, respectively). The developed procedure was found suitable for forensic investigations and was considered a good alternative to the liquid-liquid extraction methods normally used for the determination of this compound in biological media.

Adsorption↗
PubMed2003

A fatal forensic intoxication with fenarimol: analysis by HPLC/DAD/MSD.

Fenarimol (Rubigan) is a pyrimidine ergosterol biosynthesis inhibitor used as a systemic fungicide. The authors present a fatal fenarimol intoxication case analysed in the Forensic Toxicology Service of the National Institute of Legal Medicine. The results were used to compare two different HPLC techniques, regarding selectivity and sensitivity: an HPLC system with a diode array detector (DAD) and an HPLC system with a DAD and a mass spectrometry detector (MSD) with an electrospray interface. All biological samples were submitted to a solid-phase extraction procedure. The detection and quantification limits of fenarimol, linearity, precision and accuracy were evaluated. The fenarimol concentration levels determined were of 89.0 mg/ml in gastric contents, 1.9 mg/g in liver and 0.4 mg/g in kidney. Blood was not available at autopsy. No published data related to fenarimol self-poisoning were found, so it was not possible to interpret the results obtained by comparison with toxic/lethal levels.

Chromatography, High Pressure Liquid↗
PubMed2001

Laparoscopic pericystectomy for liver hydatid cysts.

BACKGROUND: The laparoscopic approach for managing of liver echinococcosis is a controversial issue because of scarce experience worldwide. The aim of this report is to describe the technical details of our laparoscopic method and present our results. METHODS: Consecutive cases of liver echinococcosis managed by laparoscopic surgery are reported. Thoracic x-ray and abdominal ultrasound had been performed previously. The following aspects were considered as selection criteria: unique cyst located in segments III, IV, V, VI, and VIII; diameter less than 7 cm; and no evidence of infection or calcification. An evacuating puncture was performed, germinative membrane removed, and pericystectomy performed, which extirpated the pericystic structure with the surrounding liver parenchyma. Specimens were removed in a plastic bag through one of the ports. Surgical morbidity, hospital stay, time until return to work, and evidences of hydatid recurrence were measured. RESULTS: Surgery was performed on eight patients (5 women and 3 men) with a mean age of 44.9 years (range, 22-83 years) who had a liver hydatid cyst with a mean diameter of 6.6 cm (range, 5-7 cm). During a mean follow-up period of 30 months (range, 23-44 months), no morbidity or hydatid recurrence were verified. Hospital stay was 2 days in all cases, and return to work was within 15 days. CONCLUSION: This laparoscopic technique, applied with selective criteria, can be a useful alternative for treating patients with liver hydatidosis because its results are comparable with those for open surgery studies involving similar follow-up time.

Adult↗
PubMed1999

The EF-hand Ca(2+)-binding protein p22 associates with microtubules in an N-myristoylation-dependent manner.

Proteins containing the EF-hand Ca(2+)-binding motif, such as calmodulin and calcineurin B, function as regulators of various cellular processes. Here we focus on p22, an N-myristoylated, widely expressed EF-hand Ca(2+)-binding protein conserved throughout evolution, which was shown previously to be required for membrane traffic. Immunofluorescence studies show that p22 distributes along microtubules during interphase and mitosis in various cell lines. Moreover, we report that p22 associates with the microtubule cytoskeleton indirectly via a cytosolic microtubule-binding factor. Gel filtration studies indicate that the p22-microtubule-binding activity behaves as a 70- to 30-kDa globular protein. Our results indicate that p22 associates with microtubules via a novel N-myristoylation-dependent mechanism that does not involve classic microtubule-associated proteins and motor proteins. The association of p22 with microtubules requires the N-myristoylation of p22 but does not involve p22's Ca(2+)-binding activity, suggesting that the p22-microtubule association and the role of p22 in membrane traffic are functionally related, because N-myristoylation is required for both events. Therefore, p22 is an excellent candidate for a protein that can mediate interactions between the microtubule cytoskeleton and membrane traffic.

Amino Acid Sequence↗
PubMed1997

Experimentally induced changes in the endocytic traffic of P-glycoprotein alter drug resistance of cancer cells.

The MDR-1 gene product, plasma membrane glycoprotein or P-glycoprotein (PGP), has been shown to confer drug resistance to cancer cells by acting as an energy-dependent drug-efflux pump. We have examined the endocytic traffic of PGP in human multidrug-resistant cells and tested whether the traffic and the steady-state intracellular localization of PGP can be experimentally modulated. Here we show that 1) under steady state approximately 70% of cellular PGP is on the surface whereas approximately 30% is intracellular, 2) surface PGP undergoes constitutive endocytosis and recycling, 3) endocytosis of PGP involves clathrin and adaptin complex 2-dependent mechanism, and 4) PGP cycles through a Rab5-responsive endosomal compartment. Biochemical (such as antibody crosslinking of PGP or treatment of cells with chloroquine) and molecular (such as overexpression of Rab5) treatments were used to modulate the endocytic/ recycling traffic of PGP. Such treatments resulted in the redistribution of PGP from the cell surface to intracellular compartments. Cells with such "mislocalized" PGP showed a decrease in multidrug resistance, suggesting that clinically relevant strategies can be attempted by modulating PGP's temporal and spatial distribution within cancer cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Critical amino-terminal segments in insertion of rat liver cytochrome P450 3A1 into the endoplasmic reticulum membrane.

An in vitro transcription-translation assay was used to study the membrane topology of rat liver cytochrome P450 3A1. N-terminus deletion mutants were constructed to assess the importance of N-terminal regions in the stable incorporation of the protein into the microsomal membranes. Wild-type nascent cytochrome P450 bound to microsomes as an integral membrane protein through its hydrophobic N-terminal segments, uncleaved by signal peptidase. Deletion of the most N-terminal hydrophobic segment (positions 7-26) had a dramatic effect on endoplasmic reticulum membrane integration. Confirming the essential role of this stretch in P450 3A1 membrane targeting, proteolysis-resistant membrane-associated peptides were observed in all the in vitro translated mutants containing that segment. It is concluded that the membrane topogenesis of P450 3A1 is determined mainly by the amino-terminal hydrophobic segment.

Amino Acid Sequence↗
PubMed1995

Transcytosis-associated protein (TAP)/p115 is a general fusion factor required for binding of vesicles to acceptor membranes.

Transcytosis-associated protein (TAP) is found on transytotic vesicles (TCVs) and is required for their fusion with the target membrane. We developed a cell-free assay capable of differentiating targeting/binding of TCVs to membrane from later fusion events. We found that TAP mediates stable association of TCVs with the target membrane. The sequence of rat liver TAP (959-amino acid open reading frame) encodes a protein that contains (i) an N-terminal region (amino acids 1-649), (ii) an internal region with several coiled-coil stretches (amino acids 650-930), and (iii) a C-terminal acidic region (amino acids 931-959). Comparisons between TAP and other sequences indicate that TAP is identical to p115, a protein involved in cis to medial Golgi transport, and homologous to Uso1p, a yeast protein involved in endoplasmic reticulum to Golgi transport. Our findings suggest that TAP/p115/Usop1 is a general factor acting within the secretory and endocytic pathways to bind transport vesicles prior to membrane fusion.

Adenosine Triphosphate↗
PubMed1994

Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome.

We have used temperature and nocodazole blocks in an in vivo basolateral to apical transcytosis assay to dissociate the early transcytotic steps occurring during the formation of transcytotic vesicles and their microtubule-dependent translocation into the apical region, from the late steps when transcytotic cargo is delivered into the apical media. We found that polarized MDCK cells transfected with rabbit polymeric IgA receptor (pIgA-R) internalize basolaterally added pIgA-R ligand ([Fab]2 fragment of IgG against the receptor's ectodomain) at 17 degrees C but do not deliver it to the apical PM. Instead, the ligand accumulates in an apically localized transcytotic compartment, distal to the basolateral endosome and the microtubule-requiring translocation step. We have characterized this compartment and show that it is distinct from basolateral transferrin recycling endosomes, basolateral early endosomes or late endosomes or lysosomes. The apical transcytotic compartment colocalizes with the compartment containing apically recycling membrane markers (ricin and apically internalized pIgA-R ligand) but is distinct from the compartment receiving apically internalized fluid phase marker (BSA). This compartment is an intermediate station of the overall pathway since transcytotic ligand can exit the compartment and be released into the apical medium when cells preloaded at 17 degrees C are subsequently incubated at 37 degrees C. We have used this system to examine the effect of Brefeldin A (BFA) and the involvement of trimeric GTPases in the late (post apical transcytotic compartment) steps of the transcytotic pathway. We found that addition of BFA or cholera toxin, a known activator of Gs alpha, to cells preloaded with transcytotic ligand at 17 degrees C significantly inhibits the exit of ligand from the apical transcytotic compartment. General structure and function of the apical endosome are not affected since neither BFA nor cholera toxin inhibit the recycling of apically internalized membrane markers (ricin and pIgA-R ligand) from the same compartment. The data suggest that transcytosis connects the "membrane-sorting" sub-domain of the basolateral endosome with a homologous sub-domain of the apical endosome and that exit of transcytosing cargo from the apical endosome is controlled by a BFA and trimeric G protein sensitive mechanism, distinct from that used for recycling of apically internalized proteins (ricin or pIgA-R).

Animals↗
PubMed1991

Pilonidal disease: 25 cases treated by the Dufourmentel technique.

Twenty-five patients with pilonidal disease were treated by the Dufourmentel technique between 1984 and 1989. In this series, 18 patients (72 percent) were women and 7 (28 percent) were men (mean age, 24 years); hospital stay averaged 4 days. There were no recurrences and no reports of surgical wound infection. Diagnosis was confirmed histologically in all cases.

Adolescent↗
PubMed1990

Modulation of P-450 IIC7 and IIIA1,2 mRNA in pre-neoplastic liver. Effect of promotion by phenobarbital.

P-450 IIC7 and IIIA2 mRNAs are constitutively expressed in the hepatic tissue under developmental control. Both forms--as well as IIIA1, 90% homologous to IIIA2 mRNA--display positive modulation by phenobarbital a prototype inducer of the liver monooxygenases and a strong promoter of experimental chemical hepatocarcinogenesis. In the present work the variations in the concentration of these P-450 mRNA were studied in rats submitted to the hepatocarcinogenic protocol of Solt and Farber. We demonstrate that a decrease in the relative concentrations of P-450 IIC7 and IIIA1, 2 mRNA is set up along the tumor promotion stage. Animals--starting the experimental carcinogenic protocol at pubertal age--show a partial inhibition of the physiological expression of P-450 IIIA1,2 mRNA associated to male sex maturation. Administration of phenobarbital results in an acceleration of the pre-neoplastic process which is concomitant with an induction of P-450 IIC7 as well as IIIA1,2 at the earlier promotion stages. P-450 mRNA concentration markedly decreases as the preneoplastic process develops. While an impaired P-450 IIIA1,2 mRNA relative abundance is observed, an inversion of the modulation of P-450 IIC7 as well as of the male phenotype marker alpha-2u-globulin mRNA arises as the tumor promotion stage progresses, both mRNA becoming repressed in response to phenobarbital.

Alpha-Globulins↗
PubMed1990

Effect of rat developmental stage at initiation on the expression of biochemical markers during liver tumor promotion.

The phenotypic response of rat liver to a carcinogenic protocol involving initiation/selection and promotion with and without phenobarbital (PB) feeding was studied in pubertal and adult male rats. Considering the early presence of preneoplastic nodular areas, it appeared that pubertal rats, initiated at 6-7 weeks, presented a higher susceptibility to the protocol than adult rats initiated at 9-10 weeks. Altered liver phenotype was characterized by: (1) gamma-glutamyl-transpeptidase (GGT) and glutathione S-transferase (GST) activities; (2) the expression of two forms of cytochrome P-450; de novo PB-inducible P-450 II B 1,2 and P-450 II C 7 normally expressed in 45-day-old rats and PB-inducible, and (3) the expression of albumin and alpha-fetoprotein cDNAs. In the absence of PB, the susceptibility of pubertal rat liver to hepatocarcinogenesis was related to a special metabolic phenotype enriched in GGT and GST activities by comparison with the quasi-normal expression of both P-450s. Adult rat liver presented a less altered pattern closer to that of noninitiated rat liver. During PB promotion, the loss of PB inducibility of P-450 II C 7 in pubertal rat liver suggested that the hormonal status of the animals could interact with initiation to modulate specific gene expression. The late phase of PB promotion revealed the loss of highly differentiated functions (P-450s, albumin), whereas enzymatic markers associated with preneoplastic foci showed a persistent high expression.

Animals↗
PubMed1990

[Modulation of mRNAs P450 and hepatocarcinogenesis promotion].

The adaptive response of the liver to phenobarbital is characterized by a strong cell hypertrophy and coordinate induction of specific P450 forms (IIB1, 2; IIC7, IIIA1). The pattern of active mRNA is significantly changed, demonstrating the establishment of PB phenotype. Employed as a promoting agent in experimental hepatocarcinogenesis, PB triggers a significantly different, uncoordinated response. Only P450 IIB1 is positively modulated while P450 IIC7 mRNA becomes repressed. Mechanisms underlying the differential P450 adaptative response to PB in the initiated versus non-initiated liver are discussed in the light of both the importance of epigenetic events and the possible role of P450 mono-oxygenases in hepatocarcinogenic promotion by PB.

Animals↗
PubMed1988

Expression of a constitutive form of cytochrome P450 during rat-liver development and sexual maturation.

Cytochrome P450 cDNA clone designated pP450 IGC 1 has been previously isolated from a phenobarbital-induced rat liver cDNA library, characterized and proven to correspond to a constitutive cytochrome P450 sensitive to phenobarbital stimulation. IGC 1 pDNA, as well as a unique synthetic oligonucleotide probe, were used to investigate the mRNA levels at different developmental stages (fetal, male and female rats of various ages) in parallel with the study of the expression of the liver development and male phenotype markers: albumin, alpha-fetoprotein and alpha 2u globulin mRNAs. Cytochrome P450 IGC 1 mRNA responsiveness to phenobarbital administration was studied in animals of both sexes at different developmental stages. P450 IGC 1 mRNA was not detectable in fetuses nor in male or female rat liver before sexual maturation, becoming elevated at 45 days age, increasing up to 3 months and reaching relatively higher levels in the female liver than in the male. The concentrations of P450 IGC 1 mRNA closely paralleled the increases in alpha 2u globulin mRNA in male liver, as analyzed by dot and Northern blot hybridization. However, P450 IGC 1 was markedly induced by phenobarbital already at 20 days age both in males and females while alpha 2u globulin mRNA was not inducible before sexual maturation. It is concluded that the expression of cytochrome P450 IGC 1 mRNA is associated with the sexual maturation being differently modulated in the male and in the female rat liver. This cytochrome P450 isoenzyme may play an important physiological role in sex differential steroid metabolism and susceptibility to cytotoxic and genotoxic agents.

Age Factors↗