A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance

Luca Goldoni,Tiziana Beringhelli,Walter Rocchia,Natalia Realini,Daniele Piomelli

doi:10.1016/j.ab.2016.02.009

Abstract

Absolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. Here we report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] = 5−25 μM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. The method does not require the use of databases and uses PULCON (pulse length-based concentration determination) quantitative NMR to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (Carr–Purcell–Meiboom–Gill) sequence or post-processing filtering approaches. Three practical applications of the method highlight its flexibility under different cell culture conditions. We identified and quantified (i) metabolic differences between genetically engineered human cell lines, (ii) alterations in cellular metabolism induced by differentiation of mouse myoblasts into myotubes, and (iii) metabolic changes caused by activation of neurotransmitter receptors in mouse myoblasts. Thus, the new protocol offers an easily implementable, efficient, and versatile tool for the investigation of cellular metabolism and signal transduction.

Highlights

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance
The method does not require the use of databases and uses pulse length-based concentration determination (PULCON) quantitative nuclear magnetic resonance (NMR) to obtain results that are significantly more accurate and reproducible than those obtained by CPMG (CarrePurcell eMeiboomeGill) sequence or post-processing filtering approaches
We describe a new and efficient protocol based on PULCON quantitative nuclear magnetic resonance (q-NMR) that, after pH-controlled removal of serum proteins, allows for the sensitive quantification (LOD 1⁄4 5À25 Mean measured concentration (mM)) of the principal hydrophilic nutrients and metabolites found in the extracellular medium of mammalian cells in cultures without the use of commercial databases [18]

Summary

Introduction

A simple and accurate protocol for absolute polar metabolite quantification in cell cultures using quantitative nuclear magnetic resonance. Absolute analyte quantification by nuclear magnetic resonance (NMR) spectroscopy is rarely pursued in metabolomics, even though this would allow researchers to compare results obtained using different techniques. We report on a new protocol that permits, after pH-controlled serum protein removal, the sensitive quantification (limit of detection [LOD] 1⁄4 5À25 mM) of hydrophilic nutrients and metabolites in the extracellular medium of cells in cultures. Compared with other analytical techniques, q-NMR does not require chromatographic separation, generates signals that are directly proportional to the number of NMR-active nuclei in the targeted analyte [4,5], and offers a high degree of assay reproducibility [6] along with reduced uncertainty [7,8]. Advances in hardware developmentdsuch as the introduction of high-field magnets and cryogenic probesdhave progressively lowered the limit of detection (LOD) for analytes, thereby improving the overall sensitivity of the technique [4]

Methods

Results

Conclusion