New sensing materials of molecularly-imprinted polymers for the selective recognition of Chlortetracycline

As a result of the stressful conditions in aquaculture facilities there is a high risk of bacterial infections among cultured fish. Chlortetracycline (CTC) is one of the antimicrobials used to solve this problem. It is a broad spectrum antibacterial active against a wide range of Gram-positive and Gram-negative bacteria. Numerous analytical methods for screening, identifying, and quantifying CTC in animal products have been developed over the years. An alternative and advantageous method should rely on expeditious and efficient procedures providing highly specific and sensitive measurements in food samples. Ion-selective electrodes (ISEs) could meet these criteria. The only ISE reported in literature for this purpose used traditional electro-active materials. A selectivity enhancement could however be achieved after improving the analyte recognition by molecularly imprinted polymers (MIPs). Several MIP particles were synthesized and used as electro-active materials. ISEs based in methacrylic acid monomers showed the best analytical performance according to slope (62.5 and 68.6 mV/decade) and detection limit (4.1 × 10−5 and 5.5 × 10−5 mol L−1). The electrodes displayed good selectivity. The ISEs are not affected by pH changes ranging from 2.5 to 13. The sensors were successfully applied to the analysis of serum, urine and fish samples.

Elétrodo de pasta de carbono molecularmente impresso para deteção voltamétrica de Furazolidona

A furazolidona é uma substância ativa do medicamento Giarlam que contém um espetro anti-bacteriano relativamente amplo e que é frequentemente usado para tratar certas doenças bacterianas e protozoárias no homem. A maioria dos fármacos exige uma dosagem que garanta os níveis de segurança e eficácia de atuação. A necessidade de dosear os medicamentos e os seus metabólitos exige o desenvolvimento constante de métodos analíticos eficientes. Neste trabalho desenvolveu-se um novo sensor eletroquímico para a deteção da furazolidona, baseado num elétrodo de pasta de carbono modificado com um polímero molecularmente impresso. A procura de novos materiais que permitam uma melhor seletividade e sensibilidade aos sistemas de deteção é especialmente importante no desenvolvimento de métodos analíticos. Os polímeros molecularmente impressos enquadram-se nesse perfil e o seu uso tem vindo a ser cada vez mais frequente como ferramenta importante em química analítica. Assim, sintetizou-se um polímero com cavidades seletivas para a Furazolidona. Este polímero foi, misturado com grafite e perafina de modo a produzir uma pasta de carbono. Uma seringa de plástico foi usada como suporte da pasta de carbono. O comportamento eletroquímico do sensor foi avaliado e diversas condições de utilização foram estudadas e otimizadas. O sensor apresenta um comportamento linear entre a intensidade do pico e a concentração numa gama de concentrações entre 1 e 100 μM, um limite de deteção de 1 μM e uma precisão (repetibilidade) inferior a 7%. A aplicabilidade do sensor fabricado em amostras complexas foi avaliada pela deteção do fármaco em amostras de urina.

Determination of permethrin and its metabolite in brains and urines of rats by GC-ECD and GC-MS

Synthetic pyrethroids are pesticides derived from naturally occurring pyrethrins, taken from pyrethrum of dried Chrysanthemum flowers. Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Many pyrethroids can significantly harm the nervous system. Permethrin could be one of the factors involved in the onset of neurodegenerative diseases. The present study aims to evaluate in brain, the effect that can induce the exposure to permethrin, during early life of female rats (from 6 to 21 days of life). Therefore, have been examined the concentrations of permethrin and its main metabolite (3-PBA) in the brain and urine in female rats sacrificed the day after and 14 days after treating. The different concentrations of permethrin and 3-PBA (after 24h and after 14 days in the end of treatment) were obtained using two different methods. The evaluation of permethrin by liquid-liquid extraction and GC-ECD was performed. The levels of the 3-PBA (in urine and brains) were obtained by SPE procedure and GC-MS using the 2- PBA as internal standard.

Desenvolvimento de um biossensor para Ácido Glutâmico

A presente dissertação tem com objetivo o desenvolvimento de um biossensor com base nos polímeros de impressão molecular para a deteção de uma molécula alvo, o ácido glutâmico que é convertido em glutamina pela glutamina sintetase, recorrendo à potenciometria. Nas células neoplásicas a glutamina não é sintetizada podendo-se considerar que o ácido glutâmico é um potencial agente anti-cancro. A técnica de impressão molécular utilizada foi a polimerização em bulk, combinando a acrilamida e a bis acrilamida com o ácido glutâmico. Para se verificar se a resposta potenciométrica obtida era de facto da molécula alvo foram preparados em paralelo com os sensores, materiais de controlo, ou seja, moléculas sem impressão molécular (NIP). Para se controlar a constituíção química dos vários sensores nomeadamente, do NIP e do polímero de impressão molecular (MIP) antes e após a remoção bem como a molécula foram realizados estudos de Espetroscopia de Infravermelhos de Transformada de Fourier (FTIR), Scanning electron microscope (SEM) e Espetroscopia de Raios X por dispersão em energia (EDS). Os materiais desenvolvidos foram aplicados em várias membranas que diferiam umas das outras, sendo seletivas ao ião. A avaliação das características gerais das membranas baseou-se na análise das curvas de calibração, conseguidas em meios com pHs diferentes, comparando os vários elétrodos. O pH 5 foi o que apresentou melhor resultado, associado a uma membrana que continha um aditivo, o p-tetra-octilphenol, e com o sensor com percentagem de 3%. Posto isto, testou-se em material biológico, urina, com as melhores características quer em termos de sensibilidade (18,32mV/década) quer em termos de linearidade (1,6x10-6 a 1,48x10-3 mol/L). Verificou-se ainda que aplicando iões interferentes na solução, estes não interferem nesta, podendo ser aplicados na amostra sem que haja alteração na resposta potenciométrica. O elétrodo é capaz de distinguir o ácido glutâmico dos restantes iões presentes na solução.

Urinary Tract Effects of HPSE2 Mutations

Urofacial syndrome (UFS) is an autosomal recessive congenital disease featuring grimacing and incomplete bladder emptying. Mutations of HPSE2, encoding heparanase 2, a heparanase 1 inhibitor, occur in UFS, but knowledge about the HPSE2 mutation spectrum is limited. Here, seven UFS kindreds with HPSE2 mutations are presented, including one with deleted asparagine 254, suggesting a role for this amino acid, which is conserved in vertebrate orthologs. HPSE2 mutations were absent in 23 non-neurogenic neurogenic bladder probands and, of 439 families with nonsyndromic vesicoureteric reflux, only one carried a putative pathogenic HPSE2 variant. Homozygous Hpse2 mutant mouse bladders contained urine more often than did wild-type organs, phenocopying human UFS. Pelvic ganglia neural cell bodies contained heparanase 1, heparanase 2, and leucine-rich repeats and immunoglobulin-like domains-2 (LRIG2), which is mutated in certain UFS families. In conclusion, heparanase 2 is an autonomic neural protein implicated in bladder emptying, but HPSE2 variants are uncommon in urinary diseases resembling UFS.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.

Exposição profissional ao chumbo : utilização do doseamento do ácido delta-aminolevulínico urinário na vigilância médica dos trabalhadores

RESUMO - O ácido δ-aminolevulínico presente na urina (ALA-U) é generalizadamente aceite como um indicador adequado para a vigilância de saúde de trabalhadores expostos a chumbo. A necessidade ou não de recurso a colheitas de urina de 24 horas para um correcto doseamento desse metabolito tem, entretanto, suscitado algumas dúvidas. Num estudo abrangendo 45 indivíduos (28 dos quais profissionalmente expostos a chumbo inorgânico) efectuou-se o doseamento do ALA em urinas colhidas durante 24 horas e em urina de colheita única. Confirmou-se a existência de uma boa correlação entre o ALA urinário e a plumbemia e que o tipo de colheita de urina efectuada não influencia significativamente essa associação. Contudo, pela apreciação das variações interindividuais registadas, parece aconselhável que seja privilegiado o doseamento em urinas de 24 horas.

Pharmacokinetics and pharmacogenomics of once-daily raltegravir and atazanavir in healthy volunteers.

Atazanavir inhibits UDP-glucuronyl-transferase-1A1 (UGT1A1), which metabolizes raltegravir, but the magnitude of steady-state inhibition and role of the UGT1A1 genotype are unknown. Sufficient inhibition could lead to reduced-dose and -cost raltegravir regimens. Nineteen healthy volunteers, age 24 to 51 years, took raltegravir 400 mg twice daily (arm A) and 400 mg plus atazanavir 400 mg once daily (arm B), separated by ?3 days, in a crossover design. After 1 week on each regimen, raltegravir and raltegravir-glucuronide plasma and urine concentrations were measured by liquid chromatography-tandem mass spectrometry in multiple samples obtained over 12 h (arm A) or 24 h (arm B) and analyzed by noncompartmental methods. UGT1A1 promoter variants were detected with a commercially available kit and published primers. The primary outcome was the ratio of plasma raltegravir C(tau), or concentration at the end of the dosing interval, for arm B (24 h) versus arm A (12 h). The arm B-to-arm A geometric mean ratios (95% confidence interval, P value) for plasma raltegravir C(tau), area under the concentration-time curve from 0 to 12 h (AUC(0-12)), and raltegravir-glucuronide/raltegravir AUC(0-12) were 0.38 (0.22 to 0.65, 0.001), 1.32 (0.62 to 2.81, 0.45), and 0.47 (0.38 to 0.59, <0.001), respectively. Nine volunteers were heterozygous and one was homozygous for a UGT1A1 reduction-of-function allele, but these were not associated with metabolite formation. Although atazanavir significantly reduced the formation of the glucuronide metabolite, its steady-state boosting of plasma raltegravir did not render the C(tau) with a once-daily raltegravir dose of 400 mg similar to the C(tau) with the standard twice-daily dose. UGT1A1 promoter variants did not significantly influence this interaction.

Genome-wide association study of metabolic traits reveals novel gene-metabolite-disease links.

Metabolic traits are molecular phenotypes that can drive clinical phenotypes and may predict disease progression. Here, we report results from a metabolome- and genome-wide association study on (1)H-NMR urine metabolic profiles. The study was conducted within an untargeted approach, employing a novel method for compound identification. From our discovery cohort of 835 Caucasian individuals who participated in the CoLaus study, we identified 139 suggestively significant (P<5×10(-8)) and independent associations between single nucleotide polymorphisms (SNP) and metabolome features. Fifty-six of these associations replicated in the TasteSensomics cohort, comprising 601 individuals from São Paulo of vastly diverse ethnic background. They correspond to eleven gene-metabolite associations, six of which had been previously identified in the urine metabolome and three in the serum metabolome. Our key novel findings are the associations of two SNPs with NMR spectral signatures pointing to fucose (rs492602, P = 6.9×10(-44)) and lysine (rs8101881, P = 1.2×10(-33)), respectively. Fine-mapping of the first locus pinpointed the FUT2 gene, which encodes a fucosyltransferase enzyme and has previously been associated with Crohn's disease. This implicates fucose as a potential prognostic disease marker, for which there is already published evidence from a mouse model. The second SNP lies within the SLC7A9 gene, rare mutations of which have been linked to severe kidney damage. The replication of previous associations and our new discoveries demonstrate the potential of untargeted metabolomics GWAS to robustly identify molecular disease markers.

Is the formula of Traub still up to date in antemortem blood glucose level estimation?

According to the hypothesis of Traub, also known as the 'formula of Traub', postmortem values of glucose and lactate found in the cerebrospinal fluid or vitreous humor are considered indicators of antemortem blood glucose levels. However, because the lactate concentration increases in the vitreous and cerebrospinal fluid after death, some authors postulated that using the sum value to estimate antemortem blood glucose levels could lead to an overestimation of the cases of glucose metabolic disorders with fatal outcomes, such as diabetic ketoacidosis. The aim of our study, performed on 470 consecutive forensic cases, was to ascertain the advantages of the sum value to estimate antemortem blood glucose concentrations and, consequently, to rule out fatal diabetic ketoacidosis as the cause of death. Other biochemical parameters, such as blood 3-beta-hydroxybutyrate, acetoacetate, acetone, glycated haemoglobin and urine glucose levels, were also determined. In addition, postmortem native CT scan, autopsy, histology, neuropathology and toxicology were performed to confirm diabetic ketoacidosis as the cause of death. According to our results, the sum value does not add any further information for the estimation of antemortem blood glucose concentration. The vitreous glucose concentration appears to be the most reliable marker to estimate antemortem hyperglycaemia and, along with the determination of other biochemical markers (such as blood acetone and 3-beta-hydroxybutyrate, urine glucose and glycated haemoglobin), to confirm diabetic ketoacidosis as the cause of death.

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples.

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.

Morphogenesis of the ureterovesical junction:a histologic and microanatomic study in the rat.

OBJECTIVES: To investigate the development of the ureterovesical junction in rats. METHODS: A total of 110 albino rats (50 prenatal and 60 newborn) with a gestation of 21 days were studied at the age of 17 days after conception until 5 days after birth. The lower urinary tract was microdissected. Microphotography (110 animals), histologic examination (44 animals), and scanning electron microscopy (66 animals) of the ureterovesical junction were performed. Urea and creatinine from the amniotic fluid of 20 fetuses and from the urine of 10 neonates were measured. RESULTS: At day 17 after conception, separate penetration of the mesonephric duct and ureter into the wall of the urogenital sinus was observed. Continuity between the lumen of the ureter and the urogenital sinus was established on day 19 after conception. The straight passage of the intramural ureter into the urogenital sinus at day 17 after conception changed to the definitive L-shape with a vertical entry into the bladder on day 5 after birth. In the distal ureter, the change of the mesenchymal tissue into immature smooth muscle was first observed at birth, and the muscle became mature on the fifth postnatal day. At birth, Waldeyer's sheath was recognized. The creatinine and urea levels were stable prenatally (average 22.4 micromol/L and 6.88 mmol/L, respectively) and rose significantly postnatally (average 133 micromol/L and 32.65 mmol/L, respectively). CONCLUSIONS: The attachment of the ureter to the urogenital sinus and later to the bladder, the modification of its passage, and its mobility within Waldeyer's sheath may be essential in preventing vesicoureteral reflux. The production of urine and its flow does not seem to be the trigger of ureteral smooth muscle formation.

Anti-doping testing at the 2008 European football championship.

Big sports events like the 2008 European Football Championship are a challenge for anti-doping activities, particularly when the sports event is hosted by two different countries and there are two laboratories accredited by the World Anti-Doping Agency. This challenges the logistics of sample collection as well as the chemical analyses, which must be carried out timeously. The following paper discusses the handling of whereabouts information for each athlete and the therapeutic use exemption system, experiences in sample collection and transportation of blood and urine samples, and the results of the chemical analysis in two different accredited laboratories. An overview of the analytical results of blood profiling and growth hormone testing in comparison with the distribution of the normal population is also presented.

β-Arrestin2 influences the response to methadone in opioid-dependent patients.

β-Arrestin2 (ARRB2) is a component of the G-protein-coupled receptor complex and is involved in μ-opioid and dopamine D(2) receptor signaling, two central processes in methadone signal transduction. We analyzed 238 patients in methadone maintenance treatment (MMT) and identified a haplotype block (rs34230287, rs3786047, rs1045280 and rs2036657) spanning almost the entire ARRB2 locus. Although none of these single nucleotide polymorphisms (SNPs) leads to a change in amino-acid sequence, we found that for all the SNPs analyzed, with exception of rs34230287, homozygosity for the variant allele confers a nonresponding phenotype (n=73; rs1045280C and rs2036657G: OR=3.1, 95% CI=1.5-6.3, P=0.004; rs3786047A: OR=2.5, 95% CI=1.2-5.1, P=0.02) also illustrated by a 12-fold shorter period of negative urine screening (P=0.01). The ARRB2 genotype may thus contribute to the interindividual variability in the response to MMT and help to predict response to treatment.

Detection of testosterone administration based on the carbon isotope ratio profiling of endogenous steroids: international reference populations of professional soccer players.

BACKGROUND AND OBJECTIVES: The determination of the carbon isotope ratio in androgen metabolites has been previously shown to be a reliable, direct method to detect testosterone misuse in the context of antidoping testing. Here, the variability in the 13C/12C ratios in urinary steroids in a widely heterogeneous cohort of professional soccer players residing in different countries (Argentina, Italy, Japan, South Africa, Switzerland and Uganda) is examined. METHODS: Carbon isotope ratios of selected androgens in urine specimens were determined using gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS). RESULTS: Urinary steroids in Italian and Swiss populations were found to be enriched in 13C relative to other groups, reflecting higher consumption of C3 plants in these two countries. Importantly, detection criteria based on the difference in the carbon isotope ratio of androsterone and pregnanediol for each population were found to be well below the established threshold value for positive cases. CONCLUSIONS: The results obtained with the tested diet groups highlight the importance of adapting the criteria if one wishes to increase the sensitivity of exogenous testosterone detection. In addition, confirmatory tests might be rendered more efficient by combining isotope ratio mass spectrometry with refined interpretation criteria for positivity and subject-based profiling of steroids.