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- Accuracy in niacin quantification in food matrices, by an RP-HPLC methodPublication . Flores, Cristina; Dias, M. Graça; Santos, MarianaAim: This work intended to evaluate accuracy of niacin quantification in food matrices, by an RP-HPLC method, in order to validate it for four different food groups: legumes, grains, dairy products and fish. Material and methods: Analytical methodology The analytical method was a RP-HPLC with fluorometric detection, based on EN 15652:2009. Samples (1 to 10 g) were submitted to an acid hydrolysis with 0.1 mol/l HCl for 30 min in an autoclave at 121ºC. After filtration, 50 µl of the solutions, were injected and separated at 37 ºC on a HPLC system. The stationary phase was the Fortis C18 5 µm (150 x 4,6 mm) column and the mobile phase consisted of 0.07 mol/l phosphate buffer, 0.075 mol/l hydrogen peroxide and 5x10-6 mol/l copper sulphate, at a flow rate of 1 ml/min. Excitation and emission wavelengths were 322 nm and 380 nm, respectively. Quantification was made with five external standards. Results were expressed in mg/100g. Accuracy evaluation To evaluate precision, at least one matrix of each food group was chosen: roasted peanuts, canned green peas, wheat bran, powder milk and canned tuna. Method trueness was assessed by testing five different reference materials from FAPAS and NIST. Each of the FAPAS samples 2160, 2166, 2172, 2176 (Breakfast Cereals) were tested once over a period of 14 months and, NIST 1849 (Infant/Adult Formula) was tested twice in the same period. In the precision study, the five matrices were tested in triplicate in four different days. Repeatability and intermediate precision standard deviations (Sr and SPi) were calculated, for each matrix, trough ANOVA (p < 0.05). Repeatability and Intermediate precision limits; r and Pi were calculated using the following formulas: r=2.8*S_r and Pi=2.8*S_Pi . Trueness was evaluated from the z-scores obtained. Results: The values of niacin concentration obtained were: peanuts – 13.4 mg/100 g, green peas – 0.59 mg/100 g, wheat bran – 18.2 mg/100 g, powder milk – 0.73 mg/100 g and tuna – 9.5 mg/100 g. The Sr and SPi (mg/100g) were respectively 0.52 / 1.25 (peanuts), 0.02 / 0.05 (green peas), 0.23 / 0.78 (wheat bran), 0.02 / 0.06 (powder milk) and 0.24 / 0.60 (tuna). The r and Pi values obtained ranged from 0.05 to 1.46 for r and 0.14 to 3.50for Pi. All the │z-scores│ obtained for FAPAS samples were < 1 and, the │z-scores│ obtained for NIST reference material were both < 2. Conclusion: The repeatability data obtained in this study, for niacin in different food matrices were in accordance with the ones presented in the method standard EN 15652:2009. Trueness evaluation using breakfast cereals and formulas revealed a good method/laboratory performance. Accuracy evaluation, using representative matrices and niacin contents, showed the method suitable to its purpose and fit to be submitted to accreditation by an external entity.
- Determination of the atherogenic profile improves patient stratificatioPublication . Manaças, Ana Rita; Jamal, Salima; Alves, Ana Catarina; Rato, Quitéria; Bourbon, MafaldaIntrodution: Several charcteristics link small dense low density lipoprotein (sdLDL) subfractions to atherosclerosis, including: long residence time in plasma, enhanced oxidizability and increased permeability through the endothelial barrier. These particles contribute to an increased atherogenesis and cardiovascular (CV) risk. It is known that smaller and denser subparticles are more atherogenic than the large rand buoyant, so it’s important to quantify and know the type of sdLDL present in an individual in order to access cardiovascular risk. The main aim of this study was to analyze the atherogenic profile of subjects from a population sample.
- Genotype/phenotype differences in childhood and adulthoodPublication . Alves, Ana Catarina; Medeiros, Ana Margarida; Bourbon, Mafalda
- In silico classification of LDLR variants leads to misdiagnosis of FH patients – validation of a novel criteriaPublication . Alves, Ana Catarina; Medeiros, Ana Margarida; Bourbon, MafaldaIntrodution: Familial Hypercholesterolaemia (FH) is a genetic disorder most commonly caused by mutations in LDLR gene. More than 1300 different mutations on LDLR gene have been described as causing FH but, as has been previously shown, the simple finding of a variation in the coding sequence of the LDLR does not determine the actual cause of FH. Functional assessment is necessary to prove pathogenicity. The aim of this study was to compare results of in silico and in vitro functional analysis of 40 alterations in LDLR and with that validate an in silico model.
- Molecular basis of Autism Spectrum Disorder: towards a Systems Medicine approachPublication . Moura Vicente, Astrid
- Antimicrobial susceptibility of Salmonella enterica isolates from healthy breeder and broiler flocks in PortugalPublication . Clemente, Lurdes; Correia, Ivone; Themudo, Patrícia; Neto, Isabel; Caniça, Manuela; Bernardo, FernandoThree hundred and thirty-three isolates representing 40 different serotypes of Salmonella enterica, recovered from environmental and faecal samples of breeder and broiler flocks from 2009 to 2011, were studied. Antimicrobial susceptibility was determined by measuring the minimal inhibitory concentration of 11 antimicrobials using the agar dilution method. Salmonella Havana, S. Enteritidis and S. Mbandaka were the most common serotypes isolated from broiler flocks, while S. Enteritidis was the common isolate from breeder flocks. The frequency of non-wild-type Salmonella isolates (those with decreased susceptibility to the different antimicrobials) varied according to serotype. S. Mbandaka in broilers and S. Enteritidis in both breeders and broilers showed higher frequencies of reduced susceptibility to quinolones, but clinical resistance towards ciprofloxacin was not observed. Reduced susceptibility to sulfamethoxazole, tetracycline, ampicillin and streptomycin were common in Salmonella Typhimurium isolates. Two isolates of S. Havana from broilers were resistant to cefotaxime and phenotypically categorised as extended-spectrum β-lactamase producers. The results presented in this study provide useful data on the antimicrobial susceptibility of different Salmonella serotypes and highlight the high diversity of multi-drug resistance patterns present.
- Characterization of inorganic contaminants present in rice varieties consumed in PortugalPublication . Coelho, Inês; Gueifão, Sandra; Vacchina, Veronique; Donard, Olivier; Castanheira, IsabelRecent data shows that Portugal has a rice consume of 15.8 kg/capita/year, the largest in Europe. The Sado estuary, located in the West Coast of Portugal, is a major area for rice production in Portugal. Although this area is partially classified as a natural reserve, it is also threatened by many sources of anthropogenic pressure and runoff from agriculture grounds, originating a moderately contaminated estuary in some hot-spot areas. The present work aims at evaluating the level of inorganic contaminants present in different rice varieties including whole grain, white rice, husk and bran. A total of 33 samples were obtained from Sado region and from distribution stores. Contents in manganese, copper, zinc, vanadium, chromium, cobalt, nickel, arsenic, selenium, strontium, cadmium and lead, were evaluated using an ICP-MS. Samples with the highest content of arsenic, were analyzed for arsenic speciation by coupling an HPLC to the ICP-MS. Samples were analyzed in compliance with the internal quality criteria established by the laboratory. In most samples levels of lead, cadmium, cobalt, and vanadium were below or near to the LOQ. Arsenic content varies between 98 and 250 µg/kg. Speciation data, showed that the species present were mainly arsenite (AsIII), representing about 80% of total arsenic, and dimethylarsinic acid (DMA). After comparing this data with other analytical results used in risk assessment studies we can infer that inorganic contaminants in samples are not a matter of concern. This study also shows the need of powerful analytical techniques with very low detection limits to measure inorganic contaminants in foods.
- Second worldwide proficiency study on variable number of tandem repeats typing of Mycobacterium tuberculosis complexPublication . de Beer, J.L.; Ködmön, C.; van Ingen, J.; Supply, P.; van Soolingen, D.; Global Network for the Molecular Surveillance of Tuberculosis 2010BACKGROUND: The quality of variable number of tandem repeats (VNTR) typing of Mycobacterium tuberculosis was first investigated in 2009 in 37 laboratories worldwide. The results revealed an inter- and intra-laboratory reproducibility of respectively 60% and 72%. These data spurred an improvement in laboratory-specific assays and global standardisation of VNTR typing. OBJECTIVE: To measure the effects of the technical improvements and increased standardisation, a test panel consisting of 30 M. tuberculosis complex DNA samples was distributed for VNTR typing in 41 participating laboratories from 36 countries. RESULTS: The inter- and intra-laboratory reproducibil- ity increased overall to respectively 78% and 88%. The 33 laboratories that participated in both the first and second proficiency studies improved their inter- and intra-laboratory reproducibility from 62% and 72% to respectively 79% and 88%. The largest improvement in reproducibility was detected in 10 laboratories that use an in-house polymerase chain reaction technique and perform amplicon sizing using gel electrophoresis. Detailed error analysis revealed a reduction in the number of systematic errors, sample exchange events and non-amplifiable loci. CONCLUSION: This second worldwide proficiency study indicates a substantial increase in the reproduc- ibility of VNTR typing of M. tuberculosis. This will contribute to a more meaningful interpretation of molecular epidemiological and phylogenetic studies on the M. tuberculosis complex.
- Advantages and versatility of fluorescence-based methodology to characterize the functionality of LDLR and class mutation assignmentPublication . Benito-Vicente, A.; Etxebarria, A.; Alves, A.C.; Bourbon, M.; Martin, C.INTRODUCTION: Familial hypercholesterolemia (FH) is a common autosomal dominant disease with a frequency of 1:500 individuals in its heterozygous form. The genetic basis of FH is mostly mutations within the LDLR gene. Assessing the pathogenicity of LDLR variants is particularly important to give a patient a definitive diagnosis of FH. Current studies of LDLR activity ex vivo are based on the analysis of 125I-labeled lipoproteins or fluorescent-labeled LDL. The main purpose of this study was to compare the effectiveness of these two methods to assess LDLR functionality in order to validate a functional assay to analyse LDLR mutations.