Development of an optimized method for processing peripheral blood mononuclear cells for 1H-nuclear magnetic resonance-based metabolomic profiling

Abstract

Human peripheral blood mononuclear cells (PBMCs) are part of the innate and adaptive immune system, and form a critical interface between both systems. Studying the metabolic profile of PBMC could provide valuable information about the response to pathogens, toxins or cancer, the detection of drug toxicity, in drug discovery and cell replacement therapy. The primary purpose of this study was to develop an improved processing method for PBMCs metabolomic profiling with nuclear magnetic resonance (NMR) spectroscopy. To this end, an experimental design was applied to develop an alternative method to process PBMCs at low concentrations. The design included the isolation of PBMCs from the whole blood of four different volunteers, of whom 27 cell samples were processed by two different techniques for quenching and extraction of metabolites: a traditional one using organic solvents and an alternative one employing a high-intensity ultrasound probe, the latter with a variation that includes the use of deproteinizing filters. Finally, all the samples were characterized by 1H-NMR and the metabolomic profiles were compared by the method. As a result, two new methods for PBMCs processing, called Ultrasound Method (UM) and Ultrasound and Ultrafiltration Method (UUM), are described and compared to the Folch Method (FM), which is the standard protocol for extracting metabolites from cell samples. We found that UM and UUM were superior to FM in terms of sensitivity, processing time, spectrum quality, amount of identifiable, quantifiable metabolites and reproducibility.

NMR-Based Mitochondria Metabolomic Profiling: A New Approach To Reveal Cancer-Associated Alterations

“…Moreover, sonication alone or combined with final sample ultrafiltration provides better-quality spectra than the Folch method. 38 Given these data, we chose to resuspend mitochondrial extracts in an NMR buffer (Buffer 2), quench them in liquid nitrogen, and then carry out centrifugal ultrafiltration to extract the metabolites for the present study. We also introduced 3 kDa centrifugal filters to eliminate broad protein signals during centrifugal ultrafiltration.…”

“…The use of NMR buffer (Buffer 2) in place of organic solvents has been used in combination with sonication or quenching in liquid nitrogen to avoid metabolite loss during solvent evaporation. Moreover, sonication alone or combined with final sample ultrafiltration provides better-quality spectra than the Folch method . Given these data, we chose to resuspend mitochondrial extracts in an NMR buffer (Buffer 2), quench them in liquid nitrogen, and then carry out centrifugal ultrafiltration to extract the metabolites for the present study.…”

Systematic Review of NMR-Based Metabolomics Practices in Human Disease Research

“…Whole blood from patients is often altered upon collection by the tubes and processing steps that come after. Serum and plasma are the most commonly analysed blood extracts but analysis can be extended to whole blood [ 181 ], platelets [ 103 ], red blood cells [ 56 ], peripheral blood mononuclear cells [ 182 ] and dried blood spots [ 183 ].…”

Methodological Developments for Metabolic NMR Spectroscopy from Cultured Cells to Tissue Extracts: Achievements, Progress and Pitfalls

“…Although reports on these applications are much sparser than publications on oncological or neurological applications, they will be briefly presented in Supplementary Materials (Section SI 1.4). These investigations concern studies of blood vessel tissue, skin, ascites cells, spermatozoa, serum lipids and milk lipids (mammalian origin) [84][85][86][87][88][89][90][91]; furthermore, measurements of mussels, corals, and plants (non-mammalian origin), as well as microorganisms (bacteria, yeast and other fungi, parasites) [92][93][94][95][96][97][98][99][100][101][102][103][104][105][106][107].…”