Development of enzymic time-temperature integrators with rapid detection for evaluation of continuous HTST pasteurization processes

The assessment of the thermal process impact in terms of food safety and quality is of great importance for process evaluation and design. This can be accomplished from the analysis of the residence time and temperature distributions coupled with the kinetics of thermal change, or from the use of a proper time-temperature integrator (TTI) as indicator of safety and quality. The objective of this work was to develop and test enzymic TTIs with rapid detection for the evaluation of continuous HTST pasteurization processes (70-85 degrees C, 10-60 s) of low-viscosity liquid foods, such as milk and juices. Enzymes peroxidase, lactoperoxidase and alkaline phosphatase in phosphate buffer were tested and activity was determined with commercial reflectometric strips. Discontinuous thermal treatments at various time-temperature combinations were performed in order to adjust a first order kinetic model of a two-component system. The measured time-temperature history was considered instead of assuming isothermal conditions. Experiments with slow heating and cooling were used to validate the adjusted model. Only the alkaline phosphatase TTI showed potential to be used for the evaluation of pasteurization processes. The choice was based on the obtained z-values of the thermostable and thermolabile fractions, on the cost and on the validation tests. (C) 2012 Elsevier Ltd. All rights reserved.

Einfluss gedächtnisrelevanter Prozesse auf die Glutamat- und GABA-Freisetzung aus hippocampalen Primärkulturzellen

Titel: Einfluss gedächtnisrelevanter Prozesse auf die Glutamat- und GABA-Freisetzung aus hippocampalen Primärkulturzellen In der vorliegenden Arbeit wurde ein biochemisches Testsystem etabliert, mit dem es möglich ist, die Freisetzung der Aminosäure-Neurotransmitter Glutamat und GABA aus neuronalem Gewebe auf dem Vielzellniveau zu untersuchen. Der qualitative und quantitative Nachweis der beiden Neurotransmitter erfolgte mit Hilfe der Reversed-Phase-Hochleistungsflüssigkeits-Chromatographie mit Fluoreszenzdetektion. Mit dem Testsystem wurden zwei Untersuchungsreihen durchgeführt: 1.) Es wurde der Einfluss des nAChR-Agonisten Nikotin und des allosterisch an nAChR wirkenden Liganden Galanthamin auf die Glutamat- und GABA-Freisetzung aus Zellen serumfreier hippocampaler Primärkulturen untersucht. Es konnte gezeigt werden, dass der für einzelne hippocampale Zellen beschriebene positiv modulatorische Effekt von Nikotin auf die glutamaterge und GABAerge Neurotransmission auch auf dem Vielzellniveau über die Neurotransmitterfreisetzung nachweisbar ist. Desweiteren konnte erstmals gezeigt werden, dass die Nikotin-modulierte Glutamat- und GABA-Freisetzung durch den allosterisch wirkenden nAChR-Liganden Galanthamin signifikant beeinflusst wird. 2.) Es wurde der Einfluss einer LTP-ähnlichen Glutamatpotenzierung auf die GABA-Freisetzung aus serumfreien hippocampalen Primärkulturen untersucht. Es konnte gezeigt werden, dass hippocampale Neuronen in potenziertem (= vorstimuliertem) Zustand auf einen zweiten Glutamat-Stimulus mit einer verringerten GABA-Freisetzung reagieren. Dieser Effekt wird im Wesentlichen über die ionotropen Glutamatrezeptoren vermittelt.

Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection

The identification of 15N-labeled 3-nitrotyrosine (NTyr) by gas chromatography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15N-L-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction, O-deacetylation, and dithionite reduction to produce an analyte containing N-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of protein-bound NTyr in normal rat plasma to be approximately 0-1 residues per 10(6) Tyr with a detection limit of 0.5 per 10(7) Tyr when > 100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an approximately 6-fold higher level of protein-bound NTyr compared with control cells and cells treated with N(G)-monomethyl-L-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to approximately 13 residues per 10(6) Tyr, an approximately 40-fold elevation compared with plasma protein of untreated rats; cotreatment with N(G)-monomethyl-L-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NO(X) exposure such as inflammation and smoking.

(Table S2) Cell concentrations of individual and combined Alexandrium spp. and phylogenetic groups as obtained by qPCR assay and Utermöhl counts, as well as particulate PSP toxin concentrations from discrete water depths

Molecular methods provide promising tools for routine detection and quantification of toxic microalgae in plankton samples. To this end, novel TaqMan minor groove binding probes and primers targeting the small (SSU) or large (LSU) ribosomal subunit (rRNA) were developed for two species of the marine dinoflagellate genus Alexandrium (A. minutum, A. tamutum) and for three groups/ribotypes of the A. tamarense species complex: Group I/North American (NA), Group II/Mediterranean (ME) and Group III/Western European (WE). Primers and probes for real-time quantitative PCR (qPCR) were species-specific and highly efficient when tested in qPCR assays for cross-validation with pure DNA from cultured Alexandrium strains. Suitability of the qPCR assays as molecular tools for the detection and estimation of relative cell abundances of Alexandrium species and groups was evaluated from samples of natural plankton assemblages along the Scottish east coast. The results were compared with inverted microscope cell counts (Utermöhl technique) of Alexandrium spp. and associated paralytic shellfish poisoning (PSP) toxin concentrations. The qPCR assays indicated that A. tamarense (Group I) and A. tamutum were the most abundant Alexandrium taxa and both were highly positively correlated with PSP toxin content of plankton samples. Cells of A. tamarense (Group III) were present at nearly all stations but in low abundance. Alexandrium minutum and A. tamarense (Group II) cells were not detected in any of the samples, thereby arguing for their absence from the specific North Sea region, at least at the time of the survey. The sympatric occurrence of A. tamarense Group I and Group III gives further support to the hypothesis that the groups/ribotypes of the A. tamarense species complex are cryptic species rather than variants belonging to the same species.

Relationship between homocysteine and cardiorespiratory fitness is sex-dependent

Elevated plasma homocysteine is recognized as an independent risk factor for cardiovascular disease. Recently, there have been conflicting reports of the relationship between physical activity and homocysteine. A more objective measure of physical activity is cardiorespiratory fitness; however, its relationship with homocysteine has yet to be investigated. The aim of this study was to determine the relationship between cardiorespiratory fitness and plasma homocysteine. Cross-sectional associations between cardiorespiratory fitness (VO(2)max) and plasma homocysteine were examined in 49 men and 11 women. A submaximal bicycle test was used to determine VO(2)max and plasma homocysteine was measured using high performance liquid chromatography with fluorescence detection. Dietary analysis determined B vitamin intake. There was a significant inverse relationship between plasma homocysteine concentration and VO(2)max in women (r = -0.81, P = 0.003) but not in men (r = -0.09, P = 0.95). There were no significant relationships between plasma homocysteine and age, BMI, body fat, total cholesterol, and LDL cholesterol. In summary, elevated cardiorespiratory fitness is associated with decreased plasma homocysteine concentrations in women. (C) 2004 Elsevier Inc. All rights reserved.

Measurement of H2S in vivo and in vitro by the monobromobimane method

The gasotransmitter hydrogen sulfide (H2S) is known as an important regulator in several physiological and pathological responses. Among the challenges facing the field is the accurate and reliable measurement of hydrogen sulfide bioavailability. We have reported an approach to discretely measure sulfide and sulfide pools using the monobromobimane (MBB) method coupled with reversed phase high-performance liquid chromatography (RP-HPLC). The method involves the derivatization of sulfide with excess MBB under precise reaction conditions at room temperature to form sulfide dibimane (SDB). The resultant fluorescent SDB is analyzed by RP-HPLC using fluorescence detection with the limit of detection for SDB (2 nM). Care must be taken to avoid conditions that may confound H2S measurement with this method. Overall, RP-HPLC with fluorescence detection of SDB is a useful and powerful tool to measure biological sulfide levels.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Casca de arroz como adsorvente para extração de micotoxinas em cebola

A produção mundial de arroz chega a 80 milhões de toneladas ao ano, considerando que as cascas representam 20% deste valor, anualmente são geradas cerca de 1.162.000 toneladas desse rejeito. Há alguns anos, esse material era descartado no ambiente, atualmente as leis de proteção ambiental, demandaram na preocupação com resíduos de casca de arroz (FOLETO, 2005). Segundo Mayer (2006) a casca leva aproximadamente 5 anos para se decompor e exala um volume elevado de metano, um dos gases responsáveis pelo efeito estufa. Visando proteger a integridade do meio ambiente, estão sendo buscadas alternativas para reduzir os impactos ambientais do descarte e recuperar os investimentos na cultura do grão. Devido seu alto teor de silício a casca de arroz, é matéria-prima de grande interesse para aplicação em vários ramos: indústria eletrônica, cerâmica e na agricultura e também pode ser utilizada como fonte energética e ser aplicadas como adsorvente, em análises químicas (FOLETO, 2005; ROSA, 2009). Neste trabalho o objetivo foi padronizar método para a determinação das aflatoxinas B1, B2, G1, G2 e ocratoxiana A em cebola, empregando a técnica de extração, Dispersão da Matriz em Fase Sólida (MSPD), tendo a casca de arroz como adsorvente, de forma a possibilitar a determinação dos contaminantes empregando cromatografia de camada delgada de alta eficiência (HPTLC) e/ou cromatografia líquida de alta eficiência acoplada a detector de fluorescência (HPLC-FD). A cebola (Allium cepa L.) foi a matriz escolhida devido sua importância econômica, ela é a terceira hortaliça mais importante economicamente no Brasil, depois do tomate e da batata. O país está entre os dez maiores produtores do mundo, sendo que na safra de 2010 a produção foi de 1.548.146 toneladas. Dentre os estados que se destacam pela sua produção estão: Santa Catarina, São Paulo e Rio Grande do Sul. No entanto, a cebola assim como os demais alimentos é suscetível à contaminação fúngica e se entre a microbiota estiverem espécies toxigênicas pode ocorrer à produção de micotoxinas. O método foi validado avaliando-se curva analítica, linearidade, limites de detecção e quantificação, precisão (repetitividade e precisão intermediária) e exatidão (recuperação) para cada tipo de determinação cromatográfica. Para HPTLC, os limites de detecção variaram entre 0,33-5µg Kg-1 e os de quantificação entre 1-15µg Kg-1 Para o método HPLC-FD os limites de detecção variaram entre 0,003– 0,26 µg Kg-1 e os de quantificação 0,03 – 2,6 µg Kg-1 . As recuperações para o método HPTLC variaram entre 76- 95% e para HPLC-FD variaram entre 72-88%. O método desenvolvido foi aplicado para verificar a ocorrência de micotoxinas em 14 amostras de cebola. A contaminação com aflatoxinas foi verificada em 43% das amostras analisadas. O nível máximo encontrado foi de 90 µg Kg-1 para aflatoxina B2 em uma amostra de cebola crioula, com o defeito de mancha negra.

Tempo de vida útil de propelentes base-simples

The purpose of this work was to determine the safe shelf life of single-base propellants. The kinetic parameters relative to the consumption of the stabilizer diphenylamine (DPA) added to the propellant were determined as a function of the storage and ageing time. High Performance Liquid Chromatography (HPLC) with spectrophotometric detection was used to determine the DPA percentage before and after the artificial ageing at 60, 70 and 80 ºC. The experimental data were very well adjusted to a pseudo-first order kinetic model and the respective kinetic constants are 8.0-10-3 day-1 (60 ºC); 1.9-10-2 day-1 (70 ºC); 1.2-10-1 day-1 (80 ºC). The activation energy was calculated as 130 kJ mol-1 and the half-time for depletion of the DPA at the hypothetical temperature of 40 ºC of storage was estimated as being 6 years.

Bioequivalence and pharmacokinetics of two zidovudine formulations in healthy Brazilian volunteers: An open-label, randomized, single-dose, two-way crossover study

Background: Zidovudine is a thymidine nucleoside reverse transcriptase inhibitor with activity against HIV type 1. Some (similar to 8) generic formulations of zidovudine are available in Brazil; however, based on a literature search, information concerning their bioavailability and pharmacokinetic properties in the Brazilian population has not been reported. Objective: The aim of this study was to compare the bioavailability and pharmacokinetic properties of 2 capsule formulations of zidovudine 100 mg in healthy Brazilian volunteers. Methods: This open-label, randomized, 2-way crossover study utilized a 1-week washout period between doses. Blood samples were collected for 8 hours after a single dose of zidovudine 100-mg test (Zidovudina, Fundaqdo para o Remedio Popular, Sao Paulo, Brazil) or reference formulation (Retrovir (R), GlaxoSmithKline, Philadelphia, Pennsylvania). Plasma zidovudine concentrations were determined using a validated high-performance liquid chromatography method with ultraviolet detection at 265 nm. C-max, T-max, AUC(0-t), AUC(0-infinity), t(1/2), and the elimination constant (k(e)) were determined using noncompartmental analysis. The formulations were considered bioequivalent if the 90% CIS for C-max, AUC(0-t), and AUC(0-infinity) fell within the interval of 80 % to 125 %, the regulatory definition set by the US Food and Drug Administration (FDA). Results: Twenty-four healthy volunteers (12 males, 12 females; mean age, 27 years; weight, 60 kg; height, 167 cm) were enrolled and completed the study. The 90% CIs of the treatment ratios for the logarithmic-transformed values of C-max, AUC(0-t), and AUC(0-infinity) were 80.0% to 113.6%, 93.9% to 109.7%, and 93.6% to 110.1 %, respectively. The values for the test and reference formulations were within the FDA bioequivalence definition intervals of 80% to 125%. Conclusions: In this small study in healthy subjects, no statistically significant differences in C-max, AUC(0-t), and AUC(0-)infinity were found between the test and reference formulations of zidovudine 100-mg capsules. The 90% CIs for the mean ratio values for the test and reference formulations of AUC(0-t), AUC(0-infinity), and C-max indicated that the reported data were entirely within the bioequivalence acceptance range proposed by the FDA of 80% to 125% (using log-transformed data).

Enzymatic determination of choline in milk using a FIA system with potentiometric detection

The development of a FIA system for the determination of total choline content in several types of milk is described. The samples were submitted to hydrochloric acid digestion before injection into the system and passed through an enzymatic reactor containing choline oxidase immobilised on glass beads. This enzymatic reaction releases hydrogen peroxide which then reacts with a solution of iodide. The decrease in the concentration of iodide ion is quantified using an iodide ion selective tubular electrode based on a homogeneous crystalline membrane. Validation of the results obtained with this system was performed by comparison with results from a method described in the literature and applied to the determination of total choline in milks. The relative deviation was always < 5%. The repeatability of the method developed was assessed by calculation of the relative standard deviation (RSD) for 12 consecutive injections of one sample. The RSD obtained was < 0.6%.

Development of an FIA system with amperometric detection for determination of bentazone in estuarine waters

On the basis of its electrochemical behaviour a new flow-injection analysis (FIA) method with amperometric detection has been developed for quantification of the herbicide bentazone (BTZ) in estuarine waters. Standard solutions and samples (200 µL) were injected into a water carrier stream and both pH and ionic strength were automatically adjusted inside the manifold. Optimization of critical FIA conditions indicated that the best analytical results were obtained at an oxidation potential of 1.10 V, pH 4.5, and an overall flow-rate of 2.4 mL min–1. Analysis of real samples was performed by means of calibration curves over the concentration range 2.5x10–6 to 5.0x10–5 mol L–1, and results were compared with those obtained by use of an independent method (HPLC). The accuracy of the amperometric determinations was ascertained; errors relative to the comparison method were below 4% and sampling rates were approximately 100 samples h–1. The repeatability of the proposed method was calculated by assessing the relative standard deviation (%) of ten consecutive determinations of one sample; the value obtained was 2.1%.

Validation of a single-extraction procedure for sequential analysis of vitamin E, cholesterol, fatty acids, and total fat in seafood

Food lipid major components are usually analyzed by individual methodologies using diverse extractive procedures for each class. A simple and fast extractive procedure was devised for the sequential analysis of vitamin E, cholesterol, fatty acids, and total fat estimation in seafood, reducing analyses time and organic solvent consumption. Several liquid/liquid-based extractive methodologies using chlorinated and non-chlorinated organic solvents were tested. The extract obtained is used for vitamin E quantification (normal-phase HPLC with fluorescence detection), total cholesterol (normal-phase HPLC with UV detection), fatty acid profile, and total fat estimation (GC-FID), all accomplished in <40 min. The final methodology presents an adequate linearity range and sensitivity for tocopherol and cholesterol, with intra- and inter-day precisions (RSD) from 3 to 11 % for all the components. The developed methodology was applied to diverse seafood samples with positive outcomes, making it a very attractive technique for routine analyses in standard equipped laboratories in the food quality control field.

Adenovirus respiratory infection: significant increase in diagnosis using PCR comparing with antigen detection and culture methods

Adenovirus (AdV) respiratory infections are usually described as being associated with high mortality rates. Laboratory diagnosis is essential for the establishment of the appropriate therapy, and for guiding the implementation of preventive measures in order to prevent the spread of the infection. Aiming to analyze the sensitivity and specificity of the laboratorial diagnosis methods available, we compared antigen detection by indirect immunofluorescence assay (IF), and a specific nested polymerase chain reaction (PCR), to detect AdV in respiratory samples collected from patients admitted to hospital with acute respiratory disease. Positive samples were inoculated into a cell culture to confirm the results. We analyzed 381 samples from the nasopharyngeal aspirates collected during the year 2008; of these, 2.6% tested were positive for adenovirus through IF and 10% through PCR; positive isolation was obtained in 40% and 26% of these cases, respectively. Most infected patients were children under six months of age, and despite of the fact that a significant number of patients required intensive care, the mortality rate was low (5%). In conclusion, molecular methods were found to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection; their introduction permitted a significant increase in diagnoses of adenovirus infections.