Browsing by Author "Oliveira, Susana"
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- Determination of potential childhood asthma biomarkers using a powerful methodology based on microextraction by packed sorbent combined with ultra-high pressure liquid chromatography. Eicosanoids as case studyPublication . Berenguer, Pedro H.; Camacho, Irene C.; Câmara, Rita; Oliveira, Susana; Câmara, José S.Leukotrienes and prostaglandins are arachidonic acid bioactive derived eicosanoids and key mediators of bronchial inflammation and response modulation in the airways contributing to the pathophysiology of asthma. An easy-to-use ultra-high pressure liquid chromatography (UHPLC)-based strategy was developed to characterize biomarkers of lipid peroxidation: leukotrienes E (LTE4) and B4 (LTB4) and 11β-prostaglandin F2α (11βPGF2α), present in urine of asthmatic patients (N = 27) and healthy individuals (N = 17). A semi-automatic eVol®-microextraction by packed sorbent (MEPS) was used to isolate the target analytes. Several experimental parameters with influence on the extraction efficiency and on the chromatographic resolution, were evaluated and optimized. The method was fully validated under optimal extraction (R-AX sorbent, 3 conditioning-equilibration cycles with 250 μL of ACN-water at 0.1% FA, 10 extract-discard cycles of 250 μL of sample at a pH of 5.1, elution with 2 times 50 μL of MeOH and concentration of the eluate until half of its volume) and chromatographic conditions (14-min analysis at a flow rate of 300 μL min-1 in an UHPLC-PDA equipped with a BEH C18 column), according to IUPAC guidelines. The findings indicated good recoveries (>95%) in addition to excellent extraction efficiency (>95%) at three concentration levels (low mid and high) with precision (RSDs) less than 11%. The lack-of-fit test, goodness-of-fit test and Mandel's fitting test, revealed good linearity within the concentration range. Good selectivity and sensitivity were achieved with a limits of detection ranging from 0.04 μg L-1 for LTB4 to 1.12 μg L-1 for 11βPGF2α, and limits of quantification from 0.10 μg L-1 for the LTB4 to 2.11 μg L-1 for 11βPGF2α. The successful application of the fully validated method shows that, on average, the asthmatic patients had significantly higher concentrations of 11βPGF2α (112.96 μg L-1vs 62.56 μg L-1 in normal controls), LTE4 (1.27 μg L-1vs 0.89 μg L-1 in normal controls), and LTB4 (1.39 μg L-1vs 0.76 μg L-1 in normal controls). The results suggest the potential of the target eicosanoids on asthma diagnosis, however, a larger and more extensive study will be necessary to confirm the data obtained and to guarantee a greater robustness to the approach.
- Distribution of polymorphisms IL4 -590 C/T and IL4 RP2 in the human populations of Madeira, Azores, Portugal, Cape Verde and Guinea-BissauPublication . Berenguer, Anabela G.; Câmara, Rita A.; Brehm, António D.; Oliveira, Susana; Fernandes, Ana T.The IL4 gene is located on chromosome 5q23.3-31.2. Polymorphisms within this cytokine gene, like the derivative allele T of IL4-590, have been reported as being associated to elevated IgE serum levels and asthma. In the present work, the allelic and genotypic frequency of the IL4-590 and IL4 RP2 polymorphisms was carried out in 599 individuals from Madeira, Azores, Portugal mainland, Cape Verde and Guinea-Bissau and in a sample of 101 asthmatics from Madeira population. In all populations the polymorphisms were in LD and presented a significant dissimilar allelic and genotypic distribution (p<0.05) except between mainland Portugal and Madeira when compared to Azores. Significant differences regarding both loci were found between Madeira population and the group of asthmatics. Genotype 183183TT frequency is higher for African populations while 253253CC prevails in Caucasian populations. The existence of a Hardy-Weinberg Disequilibrium in Guinea-Bissau population not observed in neutral markers leads to the hypothesis of natural selection occurring in these loci probably associated to a rapid population growth an hypothesis strengthened by neutral STRs D5S818 and CSF1PO gene diversity.
- Genetic polymorphisms and asthma: findings from a case–control study in the Madeira island populationPublication . Berenguer, Anabela; Fernandes, Ana; Oliveira, Susana; Rodrigues, Mariana; Ornelas, Pedro; Romeira, Diogo; Serrão, Tânia; Rosa, Alexandra; Câmara, RitaBackground: Asthma is a complex disease influenced by multiple genetic and environmental factors. While Madeira has the highest prevalence of asthma in Portugal (14.6%), the effect of both genetic and environmental factors in this population has never been assessed. We categorized 98 asthma patients according to the Global Initiative for Asthma (GINA) guidelines, established their sensitization profile, and measured their forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) indexes. Selected single nucleotide polymorphisms (SNPs) were analysed as potential markers for asthma susceptibility and severity in the interleukin 4 (IL4), interleukin 13 (IL13), beta-2-adrenergic receptor (ADRB2), a disintegrin and metalloprotease 33 (ADAM33), gasdermin-like (GSDML) and the signal transducer and activator of transcription 6 (STAT6) genes comparatively to a population reference set. Results: Although mites are the major source of allergic sensitization, no significant difference was found amongst asthma severity categories. IL4-590*CT/TT and IL4-RP2*253183/183183 were found to predict the risk (2-fold) and severity (3 to 4-fold) of asthma and were associated with a lower FEV1 index. ADRB2-c.16*AG is a risk factor (3.5-fold), while genotype GSDML-236*TT was protective (4-fold) for moderate-severe asthma. ADAM33-V4*C was associated to asthma and mild asthma by the transmission disequilibrium test (TDT). Finally, ADAM33-V4*CC and STAT6-21*TT were associated with higher sensitization (mean wheal size ≥10 mm) to house dust (1.4-fold) and storage mite (7.8-fold). Conclusion: In Madeira, IL4-590C/T, IL4-RP2 253/183, GSDML-236C/T and ADAM33-V4C/G SNPs are important risk factors for asthma susceptibility and severity, with implications for asthma healthcare management.