Rapid, Reproducible, Quantifiable NMR Metabolomics: Methanol and Methanol: Chloroform Precipitation for Removal of Macromolecules in Serum and Whole Blood

Abstract

Background: Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-1H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. Methods: A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. Results: In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at −80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. Conclusions: In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.

A Multilevel Bayesian Approach to Improve Effect Size Estimation in Regression Modeling of Metabolomics Data Utilizing Imputation with Uncertainty

“…The multilevel Bayesian model and standard logistic regression models with or without corrections for multiple testing were fit on the original, unchanged NMR metabolomics data from patients with septic shock [19,20]. In our Bayesian model, 17/27 (63%) of the metabolites were significantly related to sepsis mortality.…”

“…We then applied the standard regression approach and our Bayesian model to the real NMR dataset of septic shock with 228 samples and 27 metabolites after filtering [19,20] a GC-MS dataset from patients with and without ARDS with 85 samples and 79 metabolites [22]. For these datasets, we compared the number of significant metabolites predicted by each model and we compared the estimated effect size of metabolites.…”

A Multivariate Metabolomics Method for Estimating Platelet Mitochondrial Oxygen Consumption Rates in Patients with Sepsis

“…The aqueous supernatant was removed, lyophilized, and resuspended in 50 mM phosphate buffer in deuterium oxide in preparation for NMR. WB samples were prepared for NMR by methanol:chloroform precipitation as previously described [39].…”

“…At the time of assay, samples were thawed on ice and prepared for NMR analysis, as previously described [39]. Details of NMR acquisition can be found in the Supplementary Materials (S4.7.1).…”

Serum Metabonomic Research of the Anti-Hypertensive Effects of Ogaja on Spontaneously Hypertensive Rats

“…Metabolism by internal or external factors can be understood from changes of comprehensive metabolic profiles. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for metabonomics study, as it is highly automatable, reproducible, reliable, and fast [ 2 ]. Indeed, proton NMR collection times for a single spectrum are often as short as a few minutes.…”