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- Simposio 4: Grupos de Trabalho da Sociedade Portuguesa de Medicina Laboratorial - Pré-AnalíticoPublication . Faria, Ana Paula; Cardoso, AnaGrupo de trabalho: objetivo; Metodologia proposta para monitorização do desempenho; Apresentação dos resultados da monitorização dos indicadores e das auditorias realizadas -2016; Novidades: 4th EFLM-BD European Conference on Preanalytical Phase; Atividades PNAEQ - 2017.
- EQA quantification HbA1c Diabetes – Long-term and SIGMA analytical performance for Twenty one Portuguese LaboratoriesPublication . Miranda, Armandina; Silva, Susana Pereira; Reguengos, João; Requeijo, José; Correia, Helena; Cardoso, Ana; Meijer, Piet; Faria, Ana PaulaGlycated hemoglobin (HbA1c) plays a crucial role in the monitoring and diagnosis of diabetes. In Portugal 9,8% of the population has diabetes (HbA1c ≥ 6,5% or treatment with glucose-lowering medications)1. Six sigma metrics combine bias, precision, and allowable total error (Tea), and can be used for assessing the quality of the analytic phase. The main objective of this study was to apply a linear regression model for long-term evaluation of the precision and inaccuracy, and apply the sigma metric to evaluate the performance of laboratories in HbA1c quantification2.
- Statistical experimental design screening strategies for free-monomeric isocyanates determination by UPLC in materials used in cork stoppers manufacturingPublication . André, Catarina; Delgado, Inês; Castanheira, Isabel; Bordado, João; Matos, Ana SofiaA statistical experimental design was used to screen variables of the analytical procedure to quantify free monomeric isocyanates presented in polyurethane based pre-polymers in trace amounts. For this purpose, diphenylmethane-4,4’-diisocyanate (4,4'-MDI), 2,4-toluene diisocyanate (2,4-TDI) and 2,6-toluene diisocyanate (2,6-TDI) were analysed by Ultra Performance Liquid Chromatography with a Photo Diode Array detector (UPLC-PDA). A preliminary study was performed with three derivatization agents, being 1-(2-piridyl) piperazine (1,2-PP) the most suitable one. Column temperature, flow and percentage of ammonium acetate (% NH4Ac.) were the factors studied at two levels each. A sequence of experiments was planned according to a 23 full factorial design with three replicates and two repetitions. Analysis of variance (ANOVA) was applied for the identification of significant factors and interactions. Higher responses were achieved when the column temperature was 30 °C, a flow of 0.3 mL min-1 and a solvent with a percentage of ammonium acetate of 0.1 %. Figures of merit were assessed within-laboratory as a preliminary step for method validation. Similar values were obtained for TDI and MDI. Recoveries are approximately 100 %. In addition, the values of detection limits (LODs) for MDI and TDI were 0.08 and 0.11 μg mL−1, respectively, and quantification limits (LOQs) were 0.25 and 0.33 μg mL−1 for MDI and TDI, respectively. The working range was between 0.01 and 10.00 μg mL−1 for MDI and 0.01 – 4.95 μg mL−1 for TDI. These figures of merit seemed adequate to detect low amounts of free monomeric isocyanates presented in agglomerates and foams for agglomerated cork stoppers production. This data is suitable to address the optimization of an analytical method by a response surface methodology.
- Analysis of Pelagia noctiluca proteome Reveals a Red Fluorescent Protein, a Zinc Metalloproteinase and a PeroxiredoxinPublication . Frazão, Bárbara; Campos, Alexandre; Osório, Hugo; Thomas, Benjamin; Leandro, Sérgio; Teixeira, Alexandre; Vasconcelos, Vitor; Antunes, AgostinhoPelagia noctiluca is the most venomous jellyfish in the Mediterranean Sea where it forms dense blooms. Although there is several published research on this species, until now none of the works has been focused on a complete protein profile of the all body constituents of this organism. Here, we have performed a detailed proteomics characterization of the major protein components expressed by P. noctiluca. With that aim, we have considered the study of jellyfish proteins involved in defense, body constituents and metabolism, and furthered explore the significance and potential application of such bioactive molecules. P. noctiluca body proteins were separated by1D SDS-PAGE and 2DE followed by characterization by nanoLC-MS/MS and MALDI-TOF/TOF techniques. Altogether, both methods revealed 68 different proteins, including a Zinc Metalloproteinase, a Red Fluorescent Protein (RFP) and a Peroxiredoxin. These three proteins were identified for the first time in P. noctiluca. Zinc Metalloproteinase was previously reported in the venom of other jellyfish species. Besides the proteins described above, the other 65 proteins found in P. noctiluca body content were identified and associated with its clinical significance. Among all the proteins identified in this work we highlight: Zinc metalloproteinase, which has a ShK toxin domain and therefore should be implicated in the sting toxicity of P. noctiluca.; the RFP which are a very important family of proteins due to its possible application as molecular markers; and last but not least the discovery of a Peroxiredoxin in this organism makes it a new natural resource of antioxidant and anti-UV radiation agents.
- International Normalized Ratio (INR): Performance of External Quality Assessment 2016 results - PNAEQ and ECAT FoundationPublication . Correia, Helena; Silva, Susana Pereira; Miranda, Armandina; Reguengos, João; Cardoso, Ana; Brito, Cristina; Clemente, Vera; Faria, Ana Paula; Meijer, PietThe International Normalized Ration (INR) is derived from the measurement of the Prothrombin Time (PT) and the International Sensitivity Index (ISI), and is a quantitative measure of the responsiveness of individual thromboplastin reagents to the different clotting factors involved in the PT measurement. Under the INR system, all results are standardized. For example, a person taking an oral anticoagulant (ex. Coumadin) would need regularly to have a blood test to measure the INR. The INR permits patients on anticoagulants to travel and obtain comparable test results wherever they are. So it is very important to verify the laboratories performance over this test. In 2014 the Portuguese National External Quality Assessment Program (PNAEQ) has established a consortium with ECAT Foundation for INR measurement where Portuguese laboratories are included in the statistical analysis. The main objective of this study was to evaluate the performance for INR of PNAEQ participants that used two different thromboplastin reagents during 2016 and compared those with the results of ECAT participants.
- Lipid oxidation of a meat product packaged with poly (lactic acid)/clay nanocompositesPublication . Vilarinho, Fernanda; Buonocore, Giovanna; Vaz, M.; Sanches-Silva, A.Introduction: Polylactic acid or polylactide (PLA, Poly) is a biodegradable thermoplastic aliphatic polyester derived from renewable resources, such as corn starch, tapioca roots, chips or starch, or sugarcane. Biopolymer nanocomposites are of great interest to the packaging industry as they can overcome the limitations of biopolymers compared to synthetic polymers. In the last two decades, the nanocomposites have been studied intensively, once the addition of fillers such as organoclays, in particular, montmorillonite (MMT), can improve rheological, thermal and mechanical properties of the biopolymers (Jollands M. et al. 2010). The presence of MMT can lead to materials which generally exhibit great property enhancements, mainly due to its intercalation or exfoliation into the polymer chains. In this work, PLA was incorporated with 5% (w/w) Cloisite Na+ prepared through a two-step process: first extrusion of pellets and secondly melted matter was pressed. The nanocomposite was used to pack a model food (salami) in order to evaluate of the ability of the new packaging to inhibit lipid oxidation. Thiobarbituric Acid Reactive Substances (TBARS) assay was used to evaluate the lipid oxidation stage. This assay allows to measure malondialdehyde (MDA) content, which is formed in the lipid oxidation of polyunsaturated fatty acids. Material and Methods: Packaged salami was homogenized with trichloroacetic acid (10 %) in 0.02 M of orthophosphoric acid and the solution was filtered. The filtered solution was homogenized with thiobarbituric acid aqueous solution (0.02 M) and heated at 100 °C for 40 min. Solutions were cooled down and absorbance was measured at 530 nm. Results were expressed as mg MDA per kg of salami. Results and Discussion: Salami slices were packaged with a control film and with the nanocomposite and analysed at initial time and after 15, 30, 60 and 90 days of contact. Results showed that salami packaged with the nanocomposite presented lower amount of MDA after all contact periods, except after 60 days, where there were no differences between control and nanofilm. Conclusion: Although the results showed that the new nanocomposite tends to reduce the production of MDA, further studies should be carried out to confirm the inhibition of lipid oxidation, such as the peroxide index, p-anisidine value, or the monitorization of a lipid oxidation indicator like hexanal.
- Low-density lipoprotein receptor mutational analysis in diagnosis of familial hypercholesterolemiaPublication . Bourbon, M.; Alves, A.C.; Sijbrands, E.J.PURPOSE OF REVIEW: To present up to date evidence on the pathogenicity of low-density lipoprotein receptor (LDLR) variants and to propose a strategy that is suitable for implementation in the clinical work-up of familial hypercholesterolaemia. RECENT FINDINGS: More than 1800 variants have been described in the LDLR gene of patients with a clinical diagnosis of familial hypercholesterolaemia; however, less than 15% have functional evidence of pathogenicity. SUMMARY: The spectrum of variants in the LDLR identified in patients with clinical familial hypercholesterolaemia is increasing as novel variants are still being reported. However, over 50% of all LDLR variants need further evidence before they can be confirmed as mutations causing disease. Even with applying the recent American College of Medical Genetics variant classification, a large number of variants are still considered variants of unknown significance. Before obtaining an undisputable confirmation of the effect on the expression and activity of the LDLR, reporting these variants as part of a clinical diagnosis to the patient holds the risk that it might need to be withdrawn in a later stage. An investment should be made to develop functional assays to characterize LDLR variants of unknown significance for a better patient diagnosis and to prevent confusion in the physician's office.
- Frecuencia, aspectos clínicos y moleculares de la hipercolesterolemia familiar en una unidad de endocrinología de ciudad Bolívar, VenezuelaPublication . M. Lima-Martínez, Marcos; Paoli, Mariela; Vázquez-Cárdenas, Alejandra; Magaña-Torres, María Teresa; Guevara, Ornella; Muñoz, María Carolina; Medeiros, Ana; Bourbon, MafaldaObjetivo: Describir la frecuencia, los aspectos clínicos, bioquímicos y moleculares de la hipercolesterolemia familiar (HF) en sujetos que acuden a una unidad de endocrinología.
- Monitorization of hexanal as lipid oxidation indicator in a processed meat product packaged with poly(lactic acid)/clay nanocomposite filmsPublication . Vilarinho, Fernanda; Buonocore, Giovanna; Stanzione, M.A.; Vaz, M.; Sanches-Silva, A.One of the most detrimental processes in fatty foodstuffs is lipid oxidation, which occurs during production and storage, and influences food composition and safety. Through the analysis of volatile lipid oxidation products we can have an insight into the oxidation, and some volatiles, such as hexanal, which can be markers of undergoing oxidation processes. Hexanal is formed when fatty acids are oxidized and is one of many well-documented aromatic components that contributes to flavour and aroma in common food products containing fatty acids. During the last decade, the interest in polymer layered silicate (PLS) nanocomposites has rapidly increased due to their potential for enhancing physical, chemical, and mechanical properties of conventional materials. Polymer nanocomposites are represented by a polymeric matrix reinforced with nanoscale fillers, among them the most common silicate clays are represented by montmorillonite (MMT), which is naturally occurring and readily available in large quantities. The presence of MMT can lead to materials which generally exhibit great property enhancements, mainly due to its intercalation or exfoliation into the polymer chains. In this work natural MMT Cloisite Na+ was incorporated in PLA. The PLA/Cloisite® Na+ films were prepared through a two-step process. In the first step, PLA pellets were fed into a corotating laboratory twin-screw extruder at 170 °C and 50 rpm for 2 min. Subsequently, Cloisite® Na+ powder (5%, w/w) were added and mixed. After extrusion, the melted matter was then pressed with a P300P hot press at 170 °C and 100 bar to obtain the PLA/Cloisite® Na+ films. Salami slices were packaged with PLA-OMMT film and with a control film (PLA). After different storage times (0, 15, 30, 60 and 90 days), salami slices were analysed regarding their hexanal content. The hexanal derivatization was performed in a solution of 2,4-dinitrophenylhydrazine in sulfuric acid during 4 h in the dark, and the hexanal extraction was performed with n-hexane and evaporation till dryness. The residue was dissolved in methanol, filtered and analysed. The quantification of hexanal was performed by Ultra High Performance Liquid Chromatography coupled with Diode Array Detector at 365 nm, with a Pre-column AcquityTM UPLC® BEH C18 (2.1 x 5 mm, 1.7 μm particle size) and a column AcquityTM UPLC® BEH C18 (2.1 × 50 mm, 1.7 μm particle size), the mobile-phase was acetonitrile-water (75:25, v/v). The amount of hexanal in packaged salami decreased in the first 60 days of storage. In this period of time the hexanal content of the salami packaged with the PLA/Cloisite® Na+ films was lower than the salami packaged with control film, except after 15 days of storage, where there was no difference between two films. After 90 days of storage, the amount of hexanal in the samples increased, although it was higher in the samples packaged with control film (94.7 ± 6.02 μg/100g salami) than salami packaged with PLA/Cloisite® Na+ films (65.1 ± 6.12 μg/100g salami). The presence of MMT in the polymer film can reduce the lipid oxidation of processed meat products, extending their shelf life. Further studies to evaluate differences between PLA and the nanocomposite (PLA-5%Cloisite®Na+) in what regards to the mechanical and barrier properties are in progress.
- Green tea as a promising extract of active food packagingPublication . Vilarinho, Fernanda; Martins, C.; Ramos, F.; Castilho, M.C.; Vaz, M.; Sanches-Silva, A.Introduction: Tea is one of the most popular and frequently consumed beverages in the world and its consumption dates back to more than 2000 years in China and then spread to other areas including Japan and later on to Europe (Zhao et al., 2014). Green tea is produced from Camellia sinensis (L.) Kuntze leaf infusion and is well known for its pleasant flavour and is associated with positive health effects. The biological activity of green tea is related with the considerable amount of catechins and other phenolic compounds, in particular flavonols and phenolic acids, present in its composition (Zhao et al., 2014). These phenolic compounds prevent the oxidative damage through their antioxidant activity and also reduce the risk of cancer, cardiovascular and neurodegenerative diseases (Lorenzo et al., 2014). The process of oxidation is one of the most common mechanisms of degradation of foodstuffs and it can alter food texture and colour, decrease nutritional quality, develop off-odours and also produce possible toxic compounds. As a consequence, the shelf-life and commercial acceptability of the food products decrease. Currently, one of the major concerns of the consumers is the impact of food on health. In line with this, food industry is trying to substitute synthetic additives by natural compounds. These can be directly added to food or incorporated in food packaging with the aim of being controlled released throughout the product shelf life. This concept is so-called Active packaging and allows the packaging to positively interact with foods to increase food shelf-life. This interaction can be due to the intended release of compounds from packaging to the foods or to their headspace, or due to the scavenging of compounds by the packaging from the packaged foods. Due to the antioxidant capacity of green tea, its extract can be proposed as an alternative to synthetic antioxidants (Giménez et al., 2013). In fact, it has already been applied in active food packaging. Material and Methods: The present review focuses on the application of green tea extract in active packaging. In this regard, an extensive bibliographic research was carried out in order to evaluate the polymers already used to incorporate green tea extract, as well as the mechanical and barrier properties and efficiency of these packaging systems in contact with foods. Results and Discussion: The chemical composition of tea leaves on active compounds with antioxidant activity is well documented. Bioactive constituents of the tea leaves include catechin gallates such as epigallocatechin gallate and gallocatechin gallate (López de Dicastillo et al., 2011). However the levels of these compounds depend on many factors, such as the edaphoclimatic conditions and drying conditions of the Camellia sinensis leaves. Moreover the extraction and analysis methods can also have a great influence in their content. Green tea extract has already been incorporated into different polymers. In fact, most of them are edible such as proteic films from distilled dry beans (Yang et al., 2016), agar (Lacey et al., 2014), chitosan (Siripatrawan et al., 2012; Siripatrawan et al., 2010) and gelatine (Hong et al (2009). Green tea extract (GTE) can offers protection against oxidation, significantly reducing rancidity and thereby extending the shelf-life of packaged foods. Moreover the sensory analysis also demonstrated that packaged food was unaffected by GTE (Carrizo et al., 2016). According to Yang et al. (2016), the incorporation of the GTE did not alter the physical properties of the films. According to Siripatrawan et al. (2010), the incorporation of GTE improved the mechanical and water vapour barrier properties. In general, GTE provides a very positive impact in the reduction of oxidation of all types of food, from aqueous to fatty (López de Dicastillo et al., 2011), although most of the studies selected meat (e.g. pork, pork sausages, pork loins), or fish products (e.g. fillets of hake, salted sardines) to test the efficiency of the active films. Conclusion: Green tea has great potential of application in active food packaging due to its antioxidant capacity. Therefore, in the near future, is it possible that new food packaging based on GTE will arise in the market. However, more studies are require to elucidate about the concentrations of GTE that do not affect or affect positively the mechanical or barrier properties of the packaging and that are effective as oxidation inhibitors of packaged foods