Browsing by Author "Moreira, Nathalie"
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- Analysis of extracellular metabolome by HS-SPME/GC–MS: optimization and application in a pilot study to evaluate galactosamine-induced hepatotoxicityPublication . Araújo, Ana Margarida; Moreira, Nathalie; Lima, Ana Rita; Bastos, Maria de Lourdes; Carvalho, Félix; Carvalho, Márcia; Guedes de Pinho, PaulaTwo methods based on headspace solid-phase microextraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) were developed to study in vitro the volatile exometabolome, which were then further tested in a pilot study to evaluate galactosamine-induced hepatotoxicity. The analysis of volatile organic compounds (VOCs) was carried out directly in the headspace of the cell culture medium, while some other volatile organic compounds such as volatile carbonyl compounds (VCCs) (aldehydes and ketones) were determined in the headspace of the cell culture medium after a previous derivatization step with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA). Fiber selection was performed using a univariate mode, whereas a central composite design (CCD) was used in the optimization of several other parameters that affect the extraction conditions. VOCs showed optimal extraction results using a DVB/CAR/PDMS fiber, by adding 0.43 g of NaCl to a sample volume of 2 mL and allowing the sample to equilibrate for 10 min at 45 °C with a subsequent extraction for 39 min at the same temperature. For VCCs, the best extraction response was achieved after in-solution (2 mL) derivatization with 0.94 g L-1 of PFBHA (final concentration), followed by an incubation period of 6 min and an extraction time of 37 min at 53 °C, using a PDMS/DVB fiber. The applicability of both optimized methods was then tested, through a untargeted study, on cell culture medium samples obtained from primary mouse hepatocytes (PMH) exposed to three low concentrations (LC01, LC10 and LC30) of the well-known hepatotoxic agent galactosamine (GalN). The results obtained by both methods showed that volatile compounds from GalN exposed cells are separated from controls in a concentration-dependent manner. Several volatile compounds, namely aldehydes, ketones and alcohols, suffered significant alterations, suggesting that GalN induces marked metabolic alterations in cells even at low, non-toxic concentrations. Although preliminary, this metabolomics approach proved its potential to be used in future studies to evaluate toxicity of different xenobiotics.
- Analysis of volatile human urinary metabolome by solid-phase microextraction in combination with gas chromatography–mass spectrometry for biomarker discovery: application in a pilot study to discriminate patients with renal cell carcinomaPublication . Monteiro, Márcia; Carvalho, Márcia; Henrique, Rui; Jerónimo, Carmen; Moreira, Nathalie; Bastos, Maria de Lourdes; Guedes de Pinho, PaulaA new and simple analytical approach consisting of headspace-solid phase microextraction (HS-SPME) sampling coupled with gas chromatography-ion trap/mass spectrometry (GC-IT/MS) was developed to study the volatile human urinary metabolome. A central composite design (CCD) was used in the optimisation of extraction conditions. Fibre selection and evaluation of pH influence were performed using an univariate mode and the influence of other parameters, such as the time and temperature of extraction, time of incubation and salt addition, that affect the efficiency of the SPME sampling, was carried out using a CCD. With a sample volume of 2 mL, the optimal conditions in terms of total response values and reproducibility were achieved by performing analyses with a divinylbenzene/polydimethylsiloxane (DVB/PDMS) fibre, in an acidic pH (pH 2) with the addition of 0.59 g of NaCl, allowing the sample to equilibrate for 9 min and extracting at 68 °C for 24 min. The applicability of the optimised method was then tested in a pilot non-target analysis of urine samples obtained from patients with renal cell carcinoma (RCC) and healthy individuals. Chemometric unsupervised analyses performed on the volatile pattern acquired for these samples clearly showed the potential of volatile urinary metabolome to discriminate between RCC and control patients.
- GC-MS metabolomics-based approach for the identification of a potential VOC-biomarker panel in the urine of renal cell carcinoma patientsPublication . Monteiro, Márcia; Moreira, Nathalie; Pinto, Joana; Pires-Luís, Ana S.; Henrique, Rui; Jerónimo, Carmen; Bastos, Maria de Lourdes; Gil, Ana M.; Carvalho, Márcia; Guedes de Pinho, PaulaThe analysis of volatile organic compounds (VOCs) emanating from biological samples appears as one of the most promising approaches in metabolomics for the study of diseases, namely cancer. In fact, it offers advantages, such as non-invasiveness and robustness for high-throughput applications. The purpose of this work was to study the urinary volatile metabolic profile of patients with renal cell carcinoma (RCC) (n = 30) and controls (n = 37) with the aim of identifying a potential specific urinary volatile pattern as a non-invasive strategy to detect RCC. Moreover, the effect of some confounding factors such as age, gender, smoking habits and body mass index was evaluated as well as the ability of urinary VOCs to discriminate RCC subtypes and stages. A headspace solid-phase microextraction/gas chromatography-mass spectrometry-based method was performed, followed by multivariate data analysis. A variable selection method was applied to reduce the impact of potential redundant and noisy chromatographic variables, and all models were validated by Monte Carlo cross-validation and permutation tests. Regarding the effect of RCC on the urine VOCs composition, a panel of 21 VOCs descriptive of RCC was defined, capable of discriminating RCC patients from controls in principal component analysis. Discriminant VOCs were further individually validated in two independent samples sets (nine RCC patients and 12 controls, seven RCC patients with diabetes mellitus type 2) by univariate statistical analysis. Two VOCs were found consistently and significantly altered between RCC and controls (2-oxopropanal and, according to identification using NIST14, 2,5,8-trimethyl-1,2,3,4-tetrahydronaphthalene-1-ol), strongly suggesting enhanced potential as RCC biomarkers. Gender, smoking habits and body mass index showed negligible and age-only minimal effects on the urinary VOCs, compared to the deviations resultant from the disease. Moreover, in this cohort, the urinary volatilome did not show ability to discriminate RCC stages and histological subtypes. The results validated the value of urinary volatilome for the detection of RCC and advanced with the identification of potential RCC urinary biomarkers.
- Optimisation and validation of a HS-SPME–GC–IT/MS method for analysis of carbonyl volatile compounds as biomarkers in human urine: application in a pilot study to discriminate individuals with smoking habitsPublication . Calejo, Isabel; Moreira, Nathalie; Araújo, Ana Margarida; Carvalho, Márcia; Bastos, Maria de Lourdes; Guedes de Pinho, PaulaA new and simple analytical approach consisting of an automated headspace solid-phase microextraction (HS-SPME) sampler coupled to gas chromatography-ion trap/mass spectrometry detection (GC-IT/MS) with a prior derivatization step with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride (PFBHA) was developed to detect volatile carbonyl metabolites with low molecular weights in human urine. A central composite design (CCD) was used to optimise the PFBHA concentration and extraction conditions that affect the efficiency of the SPME procedure. With a sample volume of 1 mL, optimal conditions were achieved by adding 300 mg/L of PFBHA and allowing the sample to equilibrate for 6 min at 62°C and then extracting the samples for 51 min at the same temperature, using a divinylbenzene/polydimethylsiloxane (DVB/PDMS) fibre. The method allowed the simultaneous identification and quantification of 44 carbonyl compounds consisting of aldehydes, dialdehydes, heterocyclic aldehydes and ketones. The method was validated with regards to the linearity, inter- and intra-day precision and accuracy. The detection limits ranged from 0.009 to 0.942 ng/mL, except for 4-hydroxy-2-nonenal (15 ng/mL), and the quantification limits varied from 0.029 to 1.66 ng/mL, except for butanal (2.78 ng/mL), 2-butanone (2.67 ng/mL), 4-heptanone (3.14 ng/mL) and 4-hydroxy-2-nonenal (50.0 ng/mL). The method accuracy was satisfactory, with recoveries ranging from 90 to 107%. The proof of applicability of the methodology was performed in a pilot target analysis of urine samples obtained from 18 healthy smokers and 18 healthy non-smokers (control group). Chemometric supervised analysis was performed using the volatile patterns acquired for these samples and clearly showed the potential of the volatile carbonyl profiles to discriminate urine from smoker and non-smoker subjects. 5-Methyl-2-furfural (p<0.0001), 2-methylpropanal, nonanal and 2-methylbutanal (p<0.05) were identified as potentially useful biomarkers to identify smoking habits.