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

Abstract

a b s t r a c tAbsolute 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] ¼ 5À25 mM) 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 (CarrePurcell eMeiboomeGill) 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.© 2016 Elsevier Inc. All rights reserved.Interest in quantitative nuclear magnetic resonance (q-NMR) analysis has steadily increased since the seminal works of Jungnickel and Forbes [1] and Hollis [2], owing to the unique ability of this methodological approach to quantify, at the same time, a wide range of structurally diverse biological substances with high throughput and automation [3]. 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]. Moreover, 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].The PULCON (pulse length-based concentration determination) procedure [9], one of the most promising methods for q-NMR [4], finds its mathematical and physical foundation in the principle of reciprocity [10e12]. This principle states that the strength of a signal is inversely proportional to the 90 pulse length in the active volume of the NMR tube. The PULCON method can be implemented in all types of commercial NMR spectrometers and requires only one Abbreviations used: q-NMR, quantitative nuclear magnetic resonance; LOD, limit of detection; PULCON, pulse length-based concentration determination; CPMG,

Recent Advances in NMR-Based Metabolomics

“…73 This method uses the procedure PULCON (pulse length-based concentration determination), which was first applied for protein concentration measurements, requires one reference spectrum for quantitation of metabolites in all samples.…”

HDAC inhibition in glioblastoma monitored by hyperpolarized 13C MRSI

“…Proton water pre‐saturation was achieved using the ‘zgpr’ sequence. 10mM trimethylsilyl propionate dissolved in 600 μL of deuterium oxide was used as an external concentration reference, and the concentration of metabolites within a sample was determine using PULCON (pulse length‐based concentration determination) as described previously . Metabolites were then integrated using Topspin AU programs ‘quant_calibrate’ and ‘quant’.…”

“…32 Spectra were acquired using the following parameters: 90°pulse; 3 s relaxation delay; eight dummy scans; 16 k data points; rium oxide was used as an external concentration reference, and the concentration of metabolites within a sample was determine using PULCON (pulse length-based concentration determination) as described previously. 32,33 Metabolites were then integrated using Topspin AU programs 'quant_calibrate' and 'quant' . All detectable metabolites (threonine (Thr), lactate, alanine (Ala), acetate (Ace), UDP-N-acetylglucosamine, glutamate (Glu), glutamine (Gln), glutathione (GSH), aspartate (Asp), creatine (Cr), phosphocreatine (PCr), Cho, PC, GPC, glycine (Gly) and myo-inositol (m-Ins)) then quantified using Mnova (MestreLab Research), then peak integrals were corrected for saturation and normalized to cell number.…”

Quantitative NMR Studies of Multiple Compound Mixtures

“…Among various qNMR techniques, 1D 1 H qNMR is extensively used for quantitative metabolomics in complex biological samples (see Table 1), which is mainly classified as: (a) biofluids, such as plasma [71], serum [72][73][74][75][76][77], urine [71][72][73]76,[78][79][80], and cerebral spinal fluid [72]; (b) cell [83,86] and culture media [86,87]; (c) tissue extracts [30,73,88]; (d) plants [89,90]; and (e) microorganisms [73,91]. In spite of a good number of strengths for quantitatively detecting multiple components in complex mixture by 1 H 1D qNMR, its critical drawback is severe spectral overlap.…”