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Metabolites 2018, 8(4), 93; https://doi.org/10.3390/metabo8040093

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

1
NMR Metabolomics Laboratory, Department of Clinical Pharmacy, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA
2
Department of Emergency Medicine, University of Minnesota, Minneapolis, MN 55455, USA
3
Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI 48109, USA
4
Akadeum Life Sciences, Ann Arbor, MI 48103, USA
5
Department of Emergency Medicine, University of Mississippi, Jackson, MS 39216, USA
*
Author to whom correspondence should be addressed.
Received: 17 October 2018 / Revised: 12 November 2018 / Accepted: 10 December 2018 / Published: 14 December 2018
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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. View Full-Text
Keywords: pharmacometabolomics; extraction; ultrafiltration; 1d-1H NMR; quantitative analysis; preanalytical processing pharmacometabolomics; extraction; ultrafiltration; 1d-1H NMR; quantitative analysis; preanalytical processing
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McHugh, C.E.; Flott, T.L.; Schooff, C.R.; Smiley, Z.; Puskarich, M.A.; Myers, D.D.; Younger, J.G.; Jones, A.E.; Stringer, K.A. Rapid, Reproducible, Quantifiable NMR Metabolomics: Methanol and Methanol: Chloroform Precipitation for Removal of Macromolecules in Serum and Whole Blood. Metabolites 2018, 8, 93.

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