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All animal procedures in this study were approved by the Animal Experimental Ethics Committee of the University of Barcelona. Moreover animal handling and procedures were performed according the applicable regulations and guidelines of the Animal Experimental Ethics Committee of the University of Barcelona (Permit number: 2012/7684). The New Zealand pregnant rabbits were provided by a certified breeder and dams were housed for 1 week before surgery in separate cages on a reversed 12/12 hour light cycle, with free access to water and standard chow. IUGR was induced in two New Zealand pregnant rabbits at 25 days of gestation by uteroplacental vessels ligation as described in [24]. Briefly, after tocolysis and antibiotic prophylaxis administration, an abdominal midline laparotomy was performed under anaesthetic condition. Gestational sacs of both horns were identified and, in one uterine horn, 40–50% of the uteroplacental vessels of all gestational sacs were ligated, the contra lateral horn was used as control. After the procedure the abdomen was closed in two layers with a single suture of silk (3/0). Postoperative analgesia was administered and animals were again housed with free access to water and standard chow for 5 days until delivery and well-being was controlled each day. Cesarean section was performed at 30 days of gestation (term at 31 days) and nine living (five control and four IUGR) and seven stillborn (one control and six IUGR) fetuses were collected. After delivery, all living newborns were weighed and sacrificed by immediate decapitation. The fetal brains were removed, weighed and anterior hemisphere, posterior hemisphere, basal ganglia and brain stem regions were dissected on ice. The dissected brain tissues were snap frozen in liquid nitrogen and thereafter stored at −80°C for subsequent metabolomics analysis.
The frozen brain tissue regions were weighted and transferred to an Eppendorf tube. For metabolite extraction (25 µL/mg of tissue) ice cold high purity Methanol/Water mixture v/v 70/30 (Sigma Aldrich, Spain) was added. The brain tissues were disrupted (on ice) using an ultrasound sonicator (Qsonica, CT, USA) for 3–4 minutes until no more cellular debris was visible in the tissue homogenate. To control for potential differences in tissue quantities the total protein content of the homogenates was quantified using a protein assay (BioRad). A standard volume of 250 µL containing 10 mg of tissue was transferred to a new Eppendorf tube, stored for 1 hour at −20°C and centrifuged at 16,000 rcf, 4°C for 10 min. Supernatants were collected, transferred to a new eppendorf tube and evaporated to dryness at room temperature in a speedvac concentrator (Thermo Scientific). The dried samples were reconstituted in 60 µL of 60% MeOH with 0.1% FA and clarified by centrifugation at 14,000 rcf, 4°C for 5 minutes. The clarified samples were transferred to plastic HPLC vials for LC-QTOF-MS measurements.
Liquid Chromatography-quadrupole Time-of-flight Mass Spectrometry Measurements: The LC-QTOF-MS measurements were performed using an Agilent 1260 series LC binary pump and wellplate autosampler coupled to a 6520 accurate-mass Q-TOF LC-MS system equipped with a dual electrospray (ESI) ion source operated in negative-ion mode (Agilent Technologies, Santa Clara, CA). A Cogent Diamond Hydride ™ (MicroSol, Eatontown, NJ) aqueous normal phase (ANP) column (150×2.1 mm i.d., 4 µm particle size, 100 µm pore size) was used for separation of metabolites. The LC parameters were as follows: autosampler temperature, 4°C; injection volume, 5 µl; column temperature, 35°C; flow rate, 0.4 ml/min. The solvents and optimized gradient conditions for LC were: Solvent A, 50% methanol/50% water/0.05% formic acid; Solvent B, 90% acetonitrile with 5 mM ammonium acetate; elution gradient: 0 min–100% B; 20–25 min –40% B; post-run time for equilibration, 10 min in 100% B. A blank injection was run after every 3 samples. The optimized ESI Q-TOF parameters for MS experiments were: ion polarity, negative; gas temperature, 325°C; drying gas, 10 l/min; nebulizer pressure, 45 psig; capillary voltage, 4,000 V; fragmentor, 140 V; skimmer, 65 V; mass range, 70–1,100 m/z; acquisition rate, 1.5 spectra/s; instrument state, extended dynamic range (1,700 m/z, 2 GHz); Spectra were internally mass calibrated in real time by continuous infusion of a reference mass solution using an isocratic pump connected to a dual sprayer feeding into an electrospray ionization source. Data were acquired with MassHunter Acquisition software (Agilent Technologies, Santa Clara, CA).
Data Processing and Metabolite Identification: Following LC-QTOF-MS data acquisition, the acquired raw data files were processed with Agilent MassHunter Qualitative Analysis software (version 5.0). Reproducibility of chromatograms was first inspected by overlaying the Total Ion Chromatograms (TICs) of all samples. Data files that showed extraneous peaks were excluded for further processing. To normalize the samples for differences in tissue quantities the OD values of the protein assay were integrated into the ion intensity quantification procedure. The normalization procedure was confirmed by a comparison of the total ion intensity of peaks in MS profiles. Initial, putative metabolite identification was achieved by searching the accurate m/z values of the peaks against an in-house built database derived from HMDB, KEGG, METLIN and other public databases. At the same time, the Extracted Ion Chromatograms (EICs) for these matched putative metabolites were generated by performing Find by Formula function integrated into the software. The abundance of the EICs was calculated by summing the intensities of all compound-related peaks (e.g. isotopic peaks, adduct peaks, etc.). The pre-processed data files were imported into Agilent Mass Profiler Professional software (version 12.1) for further statistical analysis. MS/MS spectra and retention times acquired from reference metabolites were used for confirmation of the identification of statistically significant metabolites. More specifically, the exact m/z values and intensities of fragment ions from the acquired MS/MS spectra of putative metabolites must have a reasonable match with that of reference metabolites or the fragment ions from public databases (e.g. METLIN, MassBank), if available.
To identify statistical significant differences between the control and IUGR fetal brain tissue samples the processed data files were imported into Agilent Mass Profiler Professional software version 12.1 (Agilent Technologies, Santa Clara, CA) for statistical analysis. The intensities of the 78 identified metabolites in control and IUGR brain tissue samples were compared using a t-test (cut-off p<0.05) followed by a Benjamini-Hochberg Multiple Testing Correction. To visualize the variation within the dataset and most contributing variables (metabolites), the significant metabolites were used to perform a Principal Component Analysis (PCA) using the first three components for data classification. A hierarchical cluster analysis was used to demonstrate if control and IUGR fetal brain tissue samples could be distinguished based on the profile of the significantly altered metabolites. Correlations between fetal birth weight and metabolite intensities were performed in Graphpad Prism version 5 (GraphPad software, San Diego, CA).
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