CSF-FA in human obesity
Description
Hypothalamic inflammation (HI) is a key factor in the onset of obesity. Magnetic resonance imaging (MRI) studies have demonstrated the presence of hypothalamic alterations that might indicate HI in subjects with obesity. However, the contribution of dietary factors, such as fatty acids, to these alterations has not been explored in humans. We aimed to compare the cerebrospinal fluid fatty acid (CSF-FA) profile between individuals with obesity before and 1-year after bariatric surgery (BS) and normal-weight volunteers and, to assess the association between those CSF-FA differently expressed between groups with MRI markers of HI and the weight-loss trajectory post-BS. Herein, we present the results obtained through ultra-high performance liquid chromatography–mass spectrometry (UHPLC-MS).
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Metabolite extraction was accomplished by fractionating the CSF samples into pools of species with similar physicochemical properties, using appropriate combinations of organic solvents. The following method was used according to the target chemical classes: proteins were precipitated from the defrosted samples by adding 4 volumes of methanol in 1.5 mL microtubes on ice. The methanol used for extraction was spiked with metabolites not detected in unspiked human CSF extracts. After brief vortex mixing the samples were incubated for 1 hour at 4 ˚C. Supernatants were collected after centrifugation at 18,000 x g for 5 minutes, dried, and reconstituted in methanol before being transferred to vials for UHPLC-MS analysis. Additionally, different types of quality control (QC) samples were used to assess the data quality: • QC Calibration sample: It is a reference pool of all the analysed samples, used to correct the different response factors between and within batches. • QC Validation sample: It is a reference serum sample, used to assess how well the data pre-processing procedure improved the data quality. These samples were extracted and analysed at the same time as the individual samples. • QC blank sample: It is a blank sample in which extraction is performed as for biological samples. • QC system suitability blank: It is a blank sample of the solvents in which biological samples are reconstituted. Randomized sample injections were performed, with each of the QC calibration and validation extracts uniformly interspersed throughout the entire batch run. For all variables, the internal standard-corrected response in each batch was divided by its corresponding intra-batch drift trend, thereby normalizing the abundance values of the study samples relative to the batch-averaged QC calibration CSF samples (set arbitrarily to 1).