Direct Infusion Mass-Spectrometry of Human Blood Plasma Metabolome
Description
Scientists currently use only a small portion of the information contained in the blood metabolome. The identification of metabolites is a huge challenge because only highly abundant and well-separated compounds can be easily identified in complex samples. However, new approaches that enhance the identification of compounds have emerged; among them, the identification of compounds based on their involvement in a particular biological context is a recent development. In this work, this approach was first applied to identify metabolites in complex samples and, together with metabolite set enrichment analysis, was used for the evaluation of blood plasma from obese patients. The proposed approach was found to provide a statistically sound overview of the biochemical pathways, thus presenting additional information on obesity. Obesity progression was demonstrated to be accompanied by marked alterations in steroidogenesis, androstenedione metabolism, and androgen and estrogen metabolism. The findings of this study suggest that the workflow used for blood analysis is sufficient to demonstrate obesity at the biochemical pathway level as well as to monitor the response to treatment. The current dataset contains mass spectra of venous blood collected at the Federal State Budgetary Institution “Nutrition and Biotechnology” (Moscow, Russia), from 100 volunteers (20 healthy subjects, 20 overweight subjects, and 60 subjects with stage 1, 2, or 3 obesity, according to the World Health Organization classification of obesity by BMI). The blood samples were analyzed by a hybrid quadrupole time-of-flight mass spectrometer (maXis Impact, Bruker Daltonics, Billerica, MA, USA) equipped with an electrospray ionization (ESI) source. The detailed Materials and Methods and experimental design are described at https://doi.org/10.3390/ijms21020568
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The venous blood was collected from 100 volunteers (20 healthy subjects, 20 overweight subjects, and 60 subjects with stage 1, 2, or 3 obesity, according to the World Health Organization classification of obesity by BMI) into EDTA Vacutainer plasma tubes at the Federal State Budgetary Institution “Nutrition and Biotechnology” (Moscow, Russia). To reduce the effect of food intake on the metabolic composition of the blood, blood was collected before breakfast. The subjects were not treated with medication at the time of the blood sampling. An anamnesis was collected, the overall condition of the body was evaluated by a doctor, a laboratory study of blood (14 parameters) and urine (11 parameters) was carried out, resting energy expenditures were determined, and the body composition was estimated by bioimpedance measurements. Based on the results obtained, the doctor categorized the volunteers into the appropriate group. The groups of cases included volunteers with obesity of varying stages with a diagnosis of E 66.0, according to the International Classification of Diseases (obesity of exchange-alimentary origin). The presence of hyperuremia, dyslipidemia, and steatosis (in stage 3 obesity) in the case groups was allowed. The resultant blood plasma was stored at −80 °C until analysis (no more than two months). The analyzed samples were subjected to one freeze/thaw cycle. Plasma (10 μL) was mixed with 10 μL of water and 80 μL of methanol. After incubation at room temperature for 10 min, the samples were centrifuged at 13,000× g for 15 min. The supernatant was then transferred to clean plastic Eppendorf tubes, and fifty volumes of methanol containing 0.1% formic acid were added to each tube. The resulting solutions were subjected to mass spectrometry analysis. Samples were analyzed by a hybrid quadrupole time-of-flight mass spectrometer (maXis Impact, Bruker Daltonics, Billerica, MA, USA) equipped with an electrospray ionization (ESI) source. The mass spectrometer was set up to prioritize the detection of ions with a mass-to-charge ratio (m/z) ranging from 45 to 900 Da, with a mass accuracy of 1–3 parts per million (ppm). The spectra were recorded in the positive ion charge detection mode. The samples were injected into the ESI source using a glass syringe (Hamilton Bonaduz AG, Bonaduz, Switzerland) connected to a syringe injection pump (KD Scientific, Holliston, MA, USA). The rate of sample flow to the ionization source was 180 µL/h. Technical replicates were not performed. The detailed Materials and Methods and experimental design are described at https://doi.org/10.3390/ijms21020568