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Article
Expanding the Limits of Human Blood Metabolite
Quantitation using NMR Spectroscopy
G. A. Nagana Gowda, Yashas N. Gowda, and Daniel Raftery
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 26 Nov 2014
Downloaded from http://pubs.acs.org on November 28, 2014

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Page 1 of 25 Analytical Chemistry

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2 Expanding the Limits of Human Blood Metabolite Quantitation using NMR
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Spectroscopy
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G. A. Nagana Gowda,1* Yashas N. Gowda1 and Daniel Raftery1,2,3*
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14 Northwest Metabolomics Research Center, Anesthesiology and Pain Medicine, and
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Department of Chemistry, University of Washington, Seattle, WA 98109,
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19 Fred Hutchinson Cancer Research Center, Seattle, WA 98109
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28 *Address correspondence to
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G. A. Nagana Gowda (ngowda@uw.edu) or Daniel Raftery (draftery@uw.edu)
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2 ABSTRACT
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4 A current challenge in metabolomics is the reliable quantitation of many metabolites.
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6 Limited resolution and sensitivity combined with the challenges associated with unknown
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9 metabolite identification have restricted both the number and the quantitative accuracy of blood
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11 metabolites. Focused on alleviating this bottleneck in NMR-based metabolomics, investigations
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of pooled human serum combining an array of 1D/2D NMR experiments at 800 MHz, database
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16 searches, and spiking with authentic compounds enabled the identification of 67 blood
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18 metabolites. Many of these (~1/3rd) are new compared to those reported previously as a part of
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the Human Serum Metabolome Database. In addition, considering both the high reproducibility
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23 and quantitative nature of NMR, as well as the sensitivity of NMR chemical shifts to altered
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25 sample conditions, experimental protocols and comprehensive peak annotations are provided
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28 here as a guide for identification and quantitation of the new pool of blood metabolites for
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30 routine applications. Further, investigations focused on the evaluation of quantitation using
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32 organic solvents revealed a surprisingly poor performance for protein precipitation using
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35 acetonitrile. One third of the detected metabolites were attenuated by 10-67% compared to
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37 methanol precipitation at the same solvent to serum ratio of 2:1 (v/v). Nearly 2/3rd of the
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metabolites were further attenuated by up to 65% upon increasing the acetonitrile to serum ratio
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42 to 4:1 (v/v). These results, combined with the newly established identity for many unknown
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44 metabolites in the NMR spectrum, offer new avenues for human serum/plasma based
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metabolomics. Further, the ability to quantitatively evaluate nearly 70 blood metabolites that
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49 represent numerous classes including amino acids, organic acids, carbohydrates and heterocyclic
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51 compounds using a simple and highly reproducible analytical method such as NMR may
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54 potentially guide the evaluation of samples for analysis using mass spectrometry.
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56 Keywords: Quantitative NMR, qNMR, blood serum/plasma, metabolomics, NMR spectroscopy,
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58 protein precipitation, absolute quantitation
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2 INTRODUCTION
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4 Metabolite profiling of human serum/plasma is of major interest for the investigations of
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6 virtually all human diseases.1-3 This interest stems from the clinical relevance of blood arising
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9 from its close association (directly or indirectly) with essentially every living cell in the human
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11 body combined with its relatively easy access for investigations. Nuclear magnetic resonance
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(NMR) spectroscopy is one of the two most widely used analytical techniques to analyze blood
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16 metabolites, the other being mass spectrometry (MS). Metabolite profiling of serum/plasma is
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18 generally met with two major challenges: first, the need to alleviate interference from the
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massive amount of serum/plasma proteins (6-8 g/dL); and, second, the need to unravel the
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23 inherent complexity of the mixture of compounds in the biofluid to reliably detect, identify and
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25 quantitate metabolites, individually. To attempt to alleviate these challenges, MS employs
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28 protein precipitation and liquid- or gas-chromatography prior to detection. However, neither
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30 protein precipitation nor chromatography is generally deployed in NMR analysis; traditionally,
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32 intact serum/plasma is used in the analysis using 1D NMR, where protein signals are suppressed
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35 using the CPMG sequence.1 While analysis of intact serum/plasma is attractive, numerous
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37 limitations increasingly make this approach less suitable for metabolomics studies. In particular,
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(1) the number of metabolites detected using intact serum/plasma is restricted to about 30 or less,
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42 which is far fewer compared to the actual number of blood metabolites;2 (2) concentrations of
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44 many detected metabolites are grossly underestimated due to attenuation caused by metabolite
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binding to serum/plasma proteins;4-7 (3) residual macromolecule signals in the CPMG spectra
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49 often cause distorted spectral baseline, which deleteriously affects metabolite quantitation; and
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51 (4) the copious proteins present in serum/plasma cause reduced transverse relaxation (T2) times
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54 for metabolite signals and facilitate exchange between protein-bound and free metabolites, which
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56 together result in significantly broadened NMR peaks and poor quantitative accuracy.
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58 Efforts focused on alleviating these bottlenecks have included: physically removing
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2 serum/plasma proteins using ultra-filtration; solid phase extraction; or protein precipitation using
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4 an organic solvent such as methanol, acetonitrile, acetone, perchloric acid or trichloroacetic
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6 acid.8-11 These approaches have been shown to achieve significant improvements in the number
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9 of metabolites identified in blood. Notably, a comprehensive analysis of ultra-filtered serum, as a
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11 part of investigations of the human serum metabolome, resulted in identification of 49
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metabolites, the number believed to be an upper limit for human serum especially using 1H 1D
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16 NMR.2, 12 More recently, another exhaustive study of ultra-filtered human plasma focused on the
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18 global characterization of a NIST SRM (National Institute of Standards and Technology
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Standard Reference Material) identified a total of 39 metabolites.13 Incidentally, two studies
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23 from the same group involving applications to colorectal cancer14 and pancreatic cancer,15
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25 presumably using ultra-filtered serum, report identification of 55 and 58 metabolites,
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28 respectively. However, the lack of pertinent experimental and NMR spectral analysis details
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30 makes these studies difficult for translation to more routine applications. Further, both studies
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32 utilized Chenomx software,16 which often provides numerous hits in the identification of
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35 unknown metabolites and hence can lead to incorrect metabolite identification, especially for low
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37 concentration (~1 µM) metabolites. Chenomx also is a poor choice for analysis if there is not a
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40 resolved resonance.
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42 To make further progress in NMR-based metabolomics, there is a strong need to enhance
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44 the number of identified metabolites, provide experimental details to allow easy reproduction of
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47 spectra, and establish spectral assignment/analysis protocols for unambiguous metabolite
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49 identification. Establishment of a robust workflow will facilitate translation of the approach for
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routine use, particularly given the highly reproducible and quantitative nature of NMR. The
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54 current study is focused on enhancing the pool of quantifiable metabolites in blood using a
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56 protein precipitation approach which we recently showed has superior performance over
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ultrafiltration for blood metabolite quantitation.7 In the current work, comprehensive analyses of
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2 a pooled human serum were made combining an array of 1D and 2D NMR experiments,
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4 database searches, and spiking experiments using authentic compounds to achieve this goal
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6 resulting in 67 quantified metabolites (including two beta sugars). Importantly, apart from
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9 identifying a new pool of blood metabolites, detailed experimental protocols and exhaustive peak
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11 labeling especially for characteristic metabolite peaks are provided to enable easy reproduction
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of serum spectra, reliable metabolite identification and quantitation. Further, investigations based
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16 on the absolute concentrations of identified metabolites reveal a surprisingly poor performance
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18 for protein precipitation using acetonitrile compared to methanol, an important outcome that
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contradicts commonly held opinion that acetonitrile performs better as a serum protein
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23 precipitation solvent in metabolomics studies.
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26 MATERIALS AND METHODS
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Methanol, acetonitrile, sodium phosphate, monobasic (NaH2PO4), sodium phosphate,
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31 dibasic (Na2HPO4), and 3-(trimethylsilyl)propionic acid-2,2,3,3-d4 sodium salt (TSP) were
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33 obtained from Sigma-Aldrich (St. Louis, MO). Sixty-five standard compounds used for spiking
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36 to confirm peak assignments were all obtained from Sigma-Aldrich, except for 3-
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38 hydroxyisovaleric acid, which was obtained from Fisher Scientific (Waltham, MA)
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40 (Supplementary Table S1). Deuterium oxide (D2O) was obtained from Cambridge Isotope
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43 laboratories, Inc. (Andover, MA). Pooled human serum was obtained from Innovative Research,
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45 Inc. (Novi, MI). Deionized (DI) water was purified using an in-house Synergy Ultrapure Water
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System from Millipore (Billerica, MA). All chemicals were used with no further purification.
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52 Preparation of phosphate buffer
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Buffer solution was prepared by dissolving 928.6 mg anhydrous NaH2PO4 and 320.9 mg
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57 anhydrous Na2HPO4 in 100 g D2O and used without further pH correction.
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2 Solutions of authentic compounds for spiking experiments: One mL stock solutions (1 mM)
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4 for all 65 compounds (see Supplementary Table S1) were prepared, separately, in D2O by
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6 diluting their 50 mM stock solutions, which were first prepared by weighing the compounds and
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9 dissolving in D2O solvent.
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11 Serum protein precipitation using methanol: Sixteen 300 µL serum samples were mixed with
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14 methanol in a 1:1, 1:2, 1:3 or 1:4 ratio (v/v) (see Supplementary Table S2), vortexed and
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16 incubated at -20 °C for 20 min. The mixtures were centrifuged at 13,400 rcf for 30 min to pellet
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18 proteins. Supernatants were decanted to fresh vials and dried. The dried samples were mixed
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21 with 100 µL phosphate buffer in D2O containing 66.17 µM TSP, made up to 600 µL with
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23 phosphate buffer in D2O and transferred to 5 mm NMR tubes.
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26 Serum protein precipitation using acetonitrile: Sixteen 300 µL serum samples were mixed
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28 with acetonitrile in a 1:1, 1:2, 1:3 or 1:4 ratio (v/v) (see Supplementary Table S2), vortexed and
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incubated at -20 °C for 20 min. The mixtures were centrifuged at 13,400 rcf for 30 min to pellet
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33 proteins. Supernatants were decanted to fresh vials and dried. The dried samples were mixed
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35 with 100 µL solution of phosphate buffer in D2O containing 66.17 µM TSP, made up to 600 µL
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38 with phosphate buffer in D2O and transferred to 5 mm NMR tubes.
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40 Ultrafiltration: Centrifugal filters (3kDa cutoff; Amicon Microcon, YM-3; Sigma-Aldrich)
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were washed with water and centrifuged thrice with 300 µL water at 13,400 rcf for 20 min, each
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45 time. Two 300 µL serum samples were then transferred to filter tubes and centrifuged for 20 min
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at 13,400 rcf. The filtrates were mixed separately with a 100 µL solution of phosphate buffer in
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50 D2O containing 66.17 µM TSP. The solutions were made up to 600 µL with the phosphate buffer
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in D2O and transferred to 5 mm NMR tubes.
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57 NMR Spectroscopy
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2 All NMR experiments were performed at 298 K on a Bruker Avance III 800 MHz spectrometer
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4 equipped with a cryogenically cooled probe and Z-gradients suitable for inverse detection. A few
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6 spiking experiments were performed on a Bruker 700 MHz spectrometer equipped with a room
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9 temperature probe and Z-gradients suitable for inverse detection. The one-pulse or NOESY pulse
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11 sequence along with the CPMG (Carr-Purcell-Meiboom-Gill) pulse sequence, all with water
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suppression using presaturation, were used for 1H 1D NMR experiments. To confirm unknown
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16 metabolite identification, spectra were obtained after each addition of 5-10 µL stock solution (1
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18 mM) of the authentic compounds to the methanol precipitated (2:1 v/v) serum samples (see
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21 Supplementary Table S1); in the case of volatile compounds, such as acetone, ethanol, 2-butanol,
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23 dimethylamine and urea, spiking experiments were performed using ultra-filtered serum samples.
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25 To enable comparison of metabolite concentrations from various protein precipitation methods
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28 (Table S2), the CPMG experiments were performed with 128 transients and a sufficiently long
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30 recycle delay (D1=15 s). To aid unknown metabolite identification, homonuclear two-
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dimensional (2D) experiments, such as 1H-1H double quantum filtered correlation spectroscopy
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35 (DQF-COSY) and 1H-1H total correlation spectroscopy (TOCSY) experiments, were performed
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37 on serum samples after protein precipitation using methanol (2:1 v/v). The 2D experiments were
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40 performed with suppression of the residual water signal by presaturation during the relaxation
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42 delay. For DQF-COSY and TOCSY experiments, sweep widths of 9600 Hz were used in both
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44 dimensions; 512 or 400 FIDs were obtained with t1 increments for DQF-COSY or TOCSY,
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47 respectively, each with 2048 complex data points. The number of transients used was 16 and the
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49 relaxation delay was 2.0 s for DQF-COSY and 1.5 s for TOCSY. The resulting 2D data were
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zero-filled to 1024 points in the t1 dimension. A 90o shifted squared sine-bell window function
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54 was applied to both dimensions before Fourier transformation. Chemical shifts were referenced
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56 to the internal TSP signal for 1H 1D or 2D spectra. Bruker Topspin versions 3.0 or 3.1 software
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packages were used for NMR data acquisition, processing and analyses.
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2 Peak assignment, unknown metabolite identification and metabolite quantitation: Initial
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4 peak assignments relied on established literature values, specifically, the human metabolome
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6 database (HMDB),17 the biological magnetic resonance data bank (BMRB),18 and publications
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9 from our laboratory on the serum metabolome.7,19 Unknown metabolite identification involved a
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11 combination of literature/database searches,17 chemical shift, peak multiplicity and J couplings
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measurements, and comprehensive 2D DQF-COSY and TOCSY spectral analyses. The putative
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16 new compounds were finally confirmed by spiking with authentic compounds (see
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18 Supplementary Table S1). Chenomx NMR Suite Professional Software package (version 5.1;
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Chenomx Inc., Edmonton, Alberta, Canada) was used to quantitate the metabolites. This
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23 software allows fitting spectral lines using the standard metabolite library for 800 MHz 1H NMR
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25 spectra, and in particular the determination of concentrations in complicated, overlapped spectral
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28 regions. One complication that arises is that the proximity of chemical shift values for multiple
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30 metabolite signals often result in the software providing multiple library hits for the same
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32 metabolite peak; the correct metabolite identification therefore relied on the newly established
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35 metabolite identification as annotated for a typical 1H NMR spectrum (vide infra). Peak fitting
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37 with reference to the internal TSP signal enabled the determination of absolute concentrations for
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identified metabolites in protein precipitated serum except for 2-oxoisovaleric, which was absent
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42 in the Chenomx library and was therefore quantitated by manual integration using the Bruker
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44 Topspin versions 3.0 or 3.1 software package.
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49 RESULTS AND DISCUSSION
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51 Both protein precipitated and ultrafiltered serum provided highly resolved 1H NMR spectra for
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54 quantitative analysis of metabolites. A total of 67 blood metabolites were identified based on the
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56 comprehensive analyses of the NMR spectra. Importantly, more than 1/3rd of the identified
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58 metabolites are new compared to the previous comprehensive investigation, made as a part of an
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2 analysis of human serum metabolome.2 Table 1 lists all 67 identified metabolites with the newly
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4 identified NMR-detectable metabolites highlighted in bold. Figures 1 and 2 show typical spectra
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6 for protein precipitated serum and ultrafiltered serum along with the annotations for
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9 characteristic peaks for all 67 metabolites. Both anomers (α and β) for glucose as well as
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11 mannose were distinctly identified. Six of the metabolites, namely acetone, ethanol, methanol,
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14 dimethylamine, 2-propanol, and urea (See supplementary Table S3) were not observed in protein
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16 precipitated serum, as they are lost due to sample drying after protein precipitation. Further, 1,2-
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18 propanediol, despite being a liquid at room temperature with boiling point of 187 oC, is detected
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21 in protein precipitated serum; however its NMR peak intensity is attenuated due to sample drying
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23 (see Figures 1 and 2) and hence, under such circumstances, its peak intensity underestimates its
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25 concentration in blood. In principle, deuterated methanol could be used without drying, but this
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28 approach would add considerable expense, and the chemical shifts would of course be different.
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30 Despite the potential of NMR-based metabolomics of blood for the study of human
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diseases, advances that rely on intact serum/plasma analysis using CPMG sequence are met with
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35 numerous challenges including the limited resolution, unknown metabolite identification and the
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37 deleterious effects of copious proteins.20 In particular, the small number of quantifiable
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40 metabolites (~30) combined with the grossly attenuated metabolite concentrations7 has limited
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42 the utility of this popular NMR analysis method and yielded more limited data compared to that
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44 obtained from mass spectrometry. Several efforts that involve removal of serum/plasma proteins
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47 by ultrafiltration or precipitation have been made, which show significant improvements in the
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49 number of quantifiable metabolites.2,9-11 However, these approaches lack comprehensive
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evaluation for routine quantitation of blood metabolites. Thus far, many NMR studies have used
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54 ultrafiltration because of its efficient protein removal under the assumption that metabolites are
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56 recovered optimally.2,13-15 Only recently, based on the comprehensive analysis of 44 blood
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metabolites utilizing a variety of protein removal methods, we showed that protein precipitation
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2 exhibits superior performance over ultrafiltration for quantitating blood metabolites.7 In the
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4 current study, we have used the protein precipitation approach and identified an additional set of
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6 quantifiable blood metabolites.
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9 Two important aspects of the analysis of blood metabolites by NMR are unknown
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11 metabolite identification and unambiguous peak assignment for routine applications. The large
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chemical shift databases currently available help immensely to narrow peak assignments to a
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16 relatively small list of metabolites. However, what remains challenging is the unambiguous peak
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18 assignment to specific metabolites, especially for low concentration metabolites, most of whose
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peaks are often buried underneath abundant metabolite signals. Thus, although a rich metabolite
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23 library consisting of nearly 300 compounds is available in the Chenomx library, for example, and
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25 is helpful for unknown metabolite identification,16 the numerous hits that it gives often lead to
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28 ambiguous or incorrect identification. The remedy for this bottleneck is a one-time establishment
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30 of the peak identities for human blood serum/plasma NMR spectra. An advantage of using serum
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32 or plasma as the sample matrix results in stable chemical shift values that are very similar to
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35 those of other serum/plasma samples. Since, under the given experimental conditions virtually
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37 identical spectra for blood serum/plasma can be obtained, and due to the highly reproducible
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nature of NMR, a one-time establishment of peak assignments enables translation for routine
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42 applications. Therefore, in addition to the information provided in Table 1, Figures 1 and 2, with
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44 annotations of the characteristic peaks useful for integration for all 67 identified metabolites,
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serve as important visual guides to help to identify blood metabolites, routinely. Once the
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49 unambiguous identification of blood metabolites is achieved, they can be reliably quantitated
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51 using any quantitation software package such as Chenomx, Mnova (http://mestrelab.com) or the
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54 one available with any NMR instrument vendor.
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2 Utilizing the new and enhanced pool of blood metabolites, a comprehensive assessment
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4 of the performance of two commonly used protein precipitation solvents, methanol and
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6 acetonitrile, was made. The results reveal a surprisingly poor performance for protein
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9 precipitation using acetonitrile: nearly 1/3rd of metabolites were attenuated by 10 to 67%
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11 compared to methanol precipitation at the same solvent to serum ratio of 2:1. In addition, nearly
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2/3rd of metabolites were attenuated further by up to 65% upon increasing the acetonitrile to
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16 serum ratio to 4:1 (Figure 3, and Supplementary Tables S4 and S5). For a 3:1 acetonitrile to
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18 serum ratio nearly 50% of the metabolites were attenuated up to 53%. In contrast, only formic
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acid was attenuated by 10% upon increasing the methanol to serum ratio from 2:1 to 3:1,
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23 although 26% of the metabolites were attenuated by up to 33% upon increasing the methanol to
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25 serum ratio from 2:1 to 4:1. As an example, Supplementary Figures S1 illustrates the reduction
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28 in the NMR peak intensities for two typical metabolites, glutamic acid (50%) and threonine
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30 (39%), upon increasing the proportion of acetonitrile; under identical conditions, no appreciable
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32 changes in peak intensities were observed for methanol precipitation. These results indicate that
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35 the extraction of metabolites by methanol precipitation, apart from being more quantitative, is
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37 less susceptible to variation caused by different amounts of methanol. Acetonitrile, on the other
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hand, deleteriously affects metabolite quantities and hence is likely unsuitable for quantitative
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42 analysis of blood metabolites without employing recovery or internal standards. This is
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44 particularly important since, due to the lack of a comprehensive evaluation of blood metabolite
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quantitation, acetonitrile is still incorrectly (in our opinion) considered to be a better solvent for
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49 serum protein precipitation in metabolomics.7 It may be noted that protein precipitation using a
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51 1:1 solvent to serum ratio retained a high level of residual proteins compared to the 2:1 ratio for
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54 both methanol and acetonitrile, and hence the 1:1 ratio was not considered for further
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56 quantitative evaluation (See Supplementary Figures S2 and S3).
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2 The vast differences in metabolite recovery between methanol and acetonitrile solvents
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4 observed in the present study may be understood based on the differences in solubility of
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6 metabolites dissolved in these two solvents. Virtually all identified and quantified metabolites in
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9 this study are hydrophilic in nature and hence are all soluble in water. However, these
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11 metabolites have more limited solubility in organic solvents such as acetonitrile and to a much
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lesser extent, methanol. In the aqueous mixture of serum, methanol is quite hydrophilic and
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16 recovers all water soluble metabolites, although at higher methanol concentrations one can see
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18 some reduced recovery. Hydrophilic metabolites are less soluble in acetonitrile, and therefore at
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the higher proportions of this organic solvent, the metabolite recovery decreases substantially.
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23 Studies have shown that solubility of polar metabolites decrease significantly as the solvent is
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25 switched from water to alcohol. For example, it has been shown that the relative solubility of
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28 amino acids such as glycine, alanine, isoleucine, phenylalanine and asparagine decreases as
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30 much as 3 orders of magnitude from water to pure methanol.21 The solubility for highly polar
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32 metabolites such as histidine, glutamic acid, aspartic acid and asparagine could not even be
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35 measured in acetonitrile at a proportion greater than ~ 1:2 (w/v).22 In accordance with these
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37 results, the recovery of a number of highly polar metabolites including citric acid, succinic acid,
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lysine, aspartic acid, asparagine, glutamine, glutamic acid, threonine and histidine is reduced
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42 drastically as the proportion of acetonitrile increases (Figure 3 and Supplementary Figure S1).
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44 MS plays an important role in the metabolomics analysis of blood owing to its inherently
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high sensitivity and complementarity to NMR. In MS analysis, prior protein removal from blood
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49 serum/plasma is an important prerequisite. Considering the challenges involved in alleviating the
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51 interference of residual proteins in MS analysis, sample processing has been the subject of
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54 numerous investigations.20, 23-29
The performance of sample processing in such MS focused
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56 investigations is typically evaluated using the total number of ions detected and not on the
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58 comprehensive quantitative analysis of metabolites. Such an evaluation limits the quantitative
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2 accuracy, as identities for a majority of the ions thus detected remains unknown. In the current
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4 study, we have shown that absolute concentrations for numerous classes of metabolites, which
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6 represent amino acids, organic acids, carbohydrates and heterocyclic compounds, can be
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9 obtained from a simple NMR method. Beyond metabolomics studies and applications, this
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11 ability of NMR to quantitatively evaluate a relatively large number of metabolites representing
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many important classes may also offer new avenues to assess sample processing methods for
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16 analysis using MS.
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18 In conclusion, we describe the NMR identification of numerous unknown metabolites in
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blood and provide a simple optimized NMR approach to quantitate the enhanced pool of blood
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23 metabolites on a routine basis. A one-time establishment of the identity for 67 metabolites, a
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25 significant portion of which is newly established in this study, is made by a comprehensive
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28 analysis of pooled human blood serum using an array of 1D and 2D NMR techniques, database
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30 searches and spiking experiments using authentic compounds. An important aspect of this study
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32 is that the characteristic peaks for all identified metabolites are marked for easy identification
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35 and quantitation of metabolites in the NMR spectrum. This, we believe, is critical for widespread
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37 use of enhanced and NMR-based blood metabolite profiling, since the chemical shift databases
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alone are often insufficient for unambiguous assignment. This issue is particularly acute for low
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42 concentration metabolites, given the peak overlap and chemical shift sensitivity to sample
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44 conditions such as pH, salt and temperature. The high reproducibility of NMR combined with
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sample processing and analysis protocols provided here enable obtaining identical spectra for
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49 blood serum/plasma on a routine basis. Hence, even non-expert NMR users can easily identify
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51 and quantify blood metabolites following the described approach, which is important for
52
53
54 effective use of this tool. Additionally, based on the comprehensive analysis of the pool of
55
56 metabolites established in this study, the performance of serum protein precipitation using
57
58 different proportions of methanol or acetonitrile was evaluated. Methanol performs most
59
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2 optimally over a wide range of concentration, while acetonitrile shows a surprisingly poor
3
4 performance, especially at higher solvent to serum ratios. Finally, the ability to quantitatively
5
6 detect a large number of metabolites, which represent many classes, including amino acids,
7
8
9 organic acids, carbohydrates and heterocyclic compounds, using a simple NMR method
10
11 described in this study potentially offers new avenues to assess sample processing for analysis
12
13
using MS. The raw and annotated data from this study will be made available to the
14
15
16 metabolomics community through major metabolomics resources including the Metabolomics
17
18 Workbench (http://www.metabolomicsworkbench.org/), MetaboLights
19
20
21
(http://www.ebi.ac.uk/metabolights/) and the Northwest Metabolomics Research Center
22
23 (http://depts.washington.edu/nwmrc/).
24
25
26
27
28 Acknowledgments
29
30 We acknowledge financial support from the NIH (National Institute of General Medical Sciences
31
32 2R01GM085291).
33
34
35
36 Notes
37
38 The authors declare the following financial interest: Daniel Raftery reports holding equity and an
39
40
41 executive position at Matrix Bio, Inc.
42
43
44
45 Supporting Information Available
46
47
48 This information is available free of charge via the Internet at http://pubs.acs.org/.
49
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2 References
3
4 1. Beckonert, O.; Keun, H. C.; Ebbels, T. M.; Bundy, J.; Holmes, E.; Lindon, J. C.;
5 Nicholson, J. K. Nat Protoc. 2007, 2(11), 2692-703.
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10 Huang, P.; Hollander, Z.; Pedersen, T. L.; Smith, S. R.; Bamforth, F.; Greiner, R.;
11 McManus, B.; Newman, J. W.; Goodfriend, T.; Wishart, D. S. PLoS One 2011, 6 (2),
12 e16957.
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15 3. Nagana Gowda, G. A.; Raftery, D. Curr. Metabolomics 2013, 1 (3), 227-240.
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18 4. Nicholson, J. K.; Gartland, K. P. NMR Biomed. 1989, 2 (2), 77-82.
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20 5. Chatham, J. C.; Forder, J. R. Biochim. Biophys. Acta 1999, 1426 (1), 177-184.
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36 L. Anal. Biochem. 2008, 377 (1), 16-23.
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38 11. Fan, T.W. In The Handbook of Metabolomics, Methods in Pharmacology and
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40 pp7-27.
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43 12. Bouatra, S.; Aziat, F.; Mandal, R.; Guo, A. C.; Wilson, M. R.; Knox, C.; Bjorndahl, T.
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49 13. Simón-Manso Y, Lowenthal MS, Kilpatrick LE, Sampson ML, Telu KH, Rudnick PA,
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2 15. Bathe, O. F.; Shaykhutdinov, R.; Kopciuk, K.; Weljie, A. M.; McKay, A.; Sutherland, F.
3 R.; Dixon, E.; Dunse, N.; Sotiropoulos, D.; Vogel, H. J. Cancer Epidemiol Biomarkers
4 Prev. 2011, 20(1), 140-7.
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6 16. Weljie, A. M.; Newton, J.; Mercier, P.; Carlson, E.; Slupsky, C. M. Anal Chem. 2006, 78,
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10 17. Wishart, D.S.; Jewison, T.; Guo, A. C.; Wilson, M.; Knox, C.; Liu, Y.; Djoumbou, Y.;
11 Mandal, R.; Aziat, F.; Dong, E.; Bouatra, S.; Sinelnikov, I.; Arndt, D.; Xia, J.; Liu, P.;
12 Yallou, F.; Bjorndahl, T.; Perez-Pineiro, R.; Eisner, R.; Allen, F.; Neveu, V.; Greiner, R.;
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Scalbert, A. Nucleic Acids Res. 2013, 41(Database issue), D801-7.
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16 18. Ulrich, E. L.; Akutsu, H.; Doreleijers, J. F.; Harano, Y.; Ioannidis, Y. E.; Lin, J.; Livny,
17 M.; Mading, S.; Maziuk, D.; Miller, Z.; Nakatani, E.; Schulte, C. F.; Tolmie, D. E.; Kent
18 Wenger, R.; Yao, H.; Markley, J. L. Nucleic Acids Res. 2008, 36 (Database issue),
19 D402−8.
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22 19. Nagana Gowda GA, Tayyari F, Ye T, Suryani Y, Wei S, Shanaiah N, Raftery D. Anal
23 Chem. 2010, 82(21), 8983-90.
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25 20. Vuckovic, D. Anal. Bioanal. Chem. 2012, 403 (6), 1523-1548.
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28 21. Orella, C.J.; Kirwan, D.J. Ind. Eng. Chem. Res. 1991, 30, 1040-1045.
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30 22. Gekko, K.; Ohmae, E.; Kameyama, K.; Takagi, T. Biochim. Biophys. Acta 1998, 1387(1-
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23. Dunn, W. B.; Broadhurst, D.; Begley, P.; Zelena, E.; Francis-McIntyre, S.; Anderson, N.;
35 Brown, M.; Knowles, J. D.; Halsall, A.; Haselden, J. N.; Nicholls, A. W.; Wilson, I. D.;
36 Kell, D. B.; Goodacre, R.; Human Serum Metabolome (HUSERMET) Consortium. Nat
37 Protoc. 2011, 6(7), 1060-83.
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41 24. Ivanisevic, J.; Zhu, Z. J.; Plate, L.; Tautenhahn, R.; Chen, S.; O'Brien, P. J.; Johnson, C.
42 H.; Marletta, M. A.; Patti, G. J.; Siuzdak, G. Anal. Chem. 2013, 85 (14), 6876-6884.
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45 25. Bruce, S. J.; Tavazzi, I.; Parisod, V.; Rezzi, S.; Kochhar, S.; Guy, P. A. Anal. Chem.
46 2009, 81 (9), 3285-3296.
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48 26. Jiye, A.;, Trygg, J.; Gullberg, J.; Johansson, A. I.; Jonsson, P.; Antti, H.; Marklund, S. L.;
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Moritz, T. Anal. Chem. 2005, 77 (24), 8086-8094.
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52 27. Zelena, E.; Dunn, W. B.; Broadhurst, D.; Francis-McIntyre, S.; Carroll, K. M.; Begley,
53 P.; O'Hagan, S.; Knowles, J. D.; Halsall, A. Anal. Chem. 2009, 81 (4), 1357-1364.
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55 28. Tulipani, S.; Llorach, R.; Urpi-Sarda, M.; Andres-Lacueva, C. Anal. Chem. 2013, 85 (1),
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58 C.; Trauger, S. A.; Siuzdak, G. Anal. Chem. 2006, 78 (3), 743-752.
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2 29. Want, E. J.; O'Maille, G.; Smith, C. A.; Brandon, T. R.; Uritboonthai, W.; Qin, C.;
3 Trauger, S. A.; Siuzdak, G. Anal. Chem. 2006, 78 (3), 743-752.
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5 30. Cui, Q.; Lewis, I. A.; Hegeman, A. D.; Anderson, M. E.; Li, J.; Schulte, C. F.; Westler,
6 W. M.; Eghbalnia, H. R.; Sussman, M. R.; Markley, J. L. Nat Biotechnol. 2008, 26(2),
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162–164.
9 31. Prestegard JH, Miner VW, Tyrell PM. Proc Natl Acad Sci U S A. 1983, 80(23), 7192-6.
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3 Table 1: Chemical shifts (in ppm), J couplings (in Hz) and multiplicities for the pool of 67 metabolites identified in human serum by NMR.
4 Chemical shifts for characteristic peaks of metabolites that provide unambiguous information for identification and quantitation using 1D 1H
5 NMR are shown in bold. Chemical shifts for authentic compounds are also shown separately for comparison. Newly identified metabolites
6 are shown in bold.
7
8 Metabolite Human serum Authentic compounds2,30,31
9
1-Methylhistidine 7.116 (s), 7.908 (s) 3.07 (dd), 3.16 (dd), 3.68 (s), 3.96 (dd), 7.0 (s),
10 7.67 (s)
11 1,2-Propanediol 1.146 (d; J=6.461), 3.457 (dd), 3.545(dd), 3.894(m) 1.130 (d), 3.434 (dd), 3.537 (dd), 3.870 (m)
12
13 2-Hydroxybutyric acid 0.903 (t; J=7.459), 4.001 (m)
0.886 (t), 1.641 (m), 1.734 (m), 3.990 (dd)
14 0.82 (d), 0.95 (d), 2.01 (m), 3.84 (d)
15 2-Hydroxyisovaleric acid 0.839 (d; J=6.868)
0.982 (t), 1.906 (m), 3.718 (dd)
16 2-Aminobutyric acid 0.984 (t)
17 1.162 (d), 4.012 (m)
2-Propanol 1.177 (d
18
19 2-Oxoisocaproic acid 0.941 (d; J=6.633), 2.097 (m), 2.616 (d; J=7.021) 0.93 (d), 2.09 (m), 2.65 (d)
20
21 1.11 (d), 3.01 (m)
2-Oxoisovaleric acid 1.128 (d; J=7.058)
22 3-Hydroxybutyric acid 1.204 (d; J=6.233Hz), 2.311 (m), 2.414 (m), 1.204 (d), 2.314 (m), 2.414 (m), 4.160 (m)
23 4.156 (m)
24 1.26 (s), 2.35 (s)
3-Hydroxyisovaleric acid 1.274 (s)
25 3.24 (m), 3.70 (s), 3.93 (dd), 7.05 (s), 7.92 (s)
26 3-Methylhistidine 8.391 (s)
27 3-Methyl-2-oxovaleric 0.90 (t), 1.10 (d), 1.46 (m), 1.70 (m), 2.93 (m)
acid 0.899 (t; J=7.518), 1.104 (d; J=6.736)
28 1.91 (s)
29 Acetic acid 1.924 (s)
30 2.22 (s)
Acetone 2.236 (s)
31 2.13 (s), 2.48 (dd), 2.61 (dd), 3.18 (s) 3.61 (d), 3.82
32 Acetylcarnitine 3.201 (s) (dd). 5.57 (q)
33 2.05 (s), 3.76 (d), 8.0 (br.s)
N-Acetylglycine 3.751 (s)
34 1.46 (d), 3.76 (q)
Alanine 1.485 (d; J=7.296), 3.805 (q)
35 Arginine 1.664 (m), 1.731 (m), 1.918 (m), 3.255 (t), 3.775 (t) 1.68 (m), 1.90 (m), 3.23 (t), 3.76 (t)
36
37 2.861 (d; J=7.687), 2.883 (d; J=7.687), 2.84 (m), 2.94 (m), 4.00 (dd)
38 Asparagine 2.949 (d; J=4.294), 2.970 (d; J=4.294), 4.015 (dd)
39 Aspartic acid 2.675 (d; J=8.823) 2.697 (d; J=8.823), 2.66 (dd), 2.80 (dd), 3.89 (dd)
40 2.807 (d; J=3.713), 2.829 (d; J=3.713), 3.912 (dd)
41 7.473 (dd), 7.544 (t), 7.864 (d)
Benzoic acid 7.487 (t), 7.559 (t), 7.875 (d; J=8.041)
42 Betaine 3.272 (s)
3.25 (s), 3.89 (s)
43 2.425 (m), 3.215 (s), 3.419 (m), 4.555 (m)
44 Carnitine 3.233 (s)
3.189 (s), 3.507 (m), 4.058 (m)
45 Choline 3.209 (s), 3.526 (m), 4.070 (m)
46 2.53 (d), 2.65 (d)
Citric acid 2.553 (d; J=15.124), 2.676 (d; J=15.124 )
47 3.02 (s), 3.92 (s)
Creatine 3.040 (s), 3.935 (s)
48 3.03 (s), 4.05 (s)
49 Creatinine 3.051 (s), 4.066 (s)
50 2.50 (s)
Dimethylamine 2.726 (s)
51 Dimethylglycine 2.934 (s)
2.91 (s), 3.71 (s)
52 1.17 (t), 3.65 (q)
53 Ethanol 1.188 (t), 3.6635 (q)
8.44 (s)
54 Formic acid 8.459 (s)
55 6.51 (s)
Fumaric acid 6.524 (s)
56 α-Glucose 3.416 (m), 3.539 (m), 3.715 (m), 3.771 (m), 3.842 3.391, 3.524, 3.701, 3.762, 3.821, 3.831, 5.223 (d)
57 (m), 5.238 (d; J=3.798)
58
59 3.251 (dd), 3.414 (m); 3.490 (m), 3.729 (m), 3.902 3.232, 3.391; 3.461, 3.478; 3.707, 3.883, 4.634 (d),
60 β-Glucose (m), 4.652 (d; J=7.989), 18
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1 2.040 (m), 2.119 (m), 2.341 (m), 3.748 (dd)

2 Glutamic acid 2.064 (m), 2.130 (m), 2.357 (m), 3.766 (dd)
3 2.125 (m), 2.446 (m), 3.766 (t)
Glutamine 2.145 (m), 2.459 (m), 3.787 (t)
4 Glycerol 3.555 (d; J=6.636), 3.570 (d; J=6.636), 3.551 (m), 3.644 (m), 3.775 (tt)
5 3.647 (d; J=4.341), 3.662 (d; J=4.341)
6 3.54 (s)
Glycine 3.564 (s)
7 Hippuric acid 7.641 (t), 7.837 (d)
3.96 (d), 7.54 (m), 7.62 (tt), 7.82 (dd)
8 3.16 (dd), 3.23 (dd), 3.98 (dd), 7.09 (d), 7.90 (d)
9 Histidine 7.205 (s), 8.183 (s)
8.17 (s), 8.20 (s)
10 Hypoxanthine 8.199 (s), 8.221 (s)
11 1.21 (d), 2.59 (m)
Isobutyric acid 1.067 (d; J=6.823)
12 Isoleucine 0.944 (t; J=7.485), 1.015 (d; J=6.994), 0.926 (t), 0.997 (d), 1.248 (m), 1.457 (m), 1.968
13 3.678 (d; J=4.173) (m), 3.661 (d)
14
15 0.90 (d), 1.94 (dq), 2.05 (d)
Isovaleric acid 0.915 (d; J=6.645)
16 1.32 (d), 4.10 (q)
Lactic acid 1.332 (d; J=6.976), 4.115 (q; J=6.976)
17 0.961 (d; J=6.227), 0.972 (d; J=6.050), 1.718 (m), 0.948 (t), 1.700 (m), 3.722 (m)
18 Leucine 3.740 (m)
19 Lysine 1.452 (m), 1.512 (m), 1.733 (m), 1.913 (m), 1.46 (m), 1.71 (m), 1.89 (m), 3.02 (t), 3.74 (t)
20 3.030 (t; J=7.627)
21 3.66, 3.75, 3.80, 3.83, 3.85, 3.91, 5.17
22 α-Mannose 5.186 (d; J=1.708)
23
3.37, 3.56, 3.63, 3.72, 3.88, 3.92, 4.89
24 β-Mannose 4.907 (d; J=0.949)
25 3.341 (s)
Methanol 3.362 (s)
26 2.157 (m), 2.631 (t), 3.851 (dd)
Methionine 2.140 (s), 2.648 (t), 3.872 (m)
27
3.268 (t), 3.524 (dd), 3.613 (t), 4.053 (t)
28 Myoinositol 3.625 (t)
29 1.727 (m), 1.826 (m), 1.933 (m), 3.046 (t), 3.774 (t)
Ornithine 3.060 (t; J=7.489)
30 Phenylalanine 7.333 (d), 7.381 (t; J=7.356), 7.432 (t) 3.19 (m), 3.98 (dd), 7.32 (d), 7.36 (m), 7.42 (m)
31
32 Proline 4.138 (dd; J=6.372; J=8.716) 1.99 (m), 2.06 (m), 2.34 (m), 3.33 (dt), 3.41 (dt),
33 4.12 (dd)
2.02 (m), 2.39 (m), 2.50 (m), 4.17 (dd)
34 Pyroglutamic acid 2.407 (m), 2.507 (m), 4.182 (dd; J=5.904; J=9.081)
35 2.73 (s), 3.60 (s)
Sarcosine 2.742 (s)
36 Serine 3.832 (dd), 3.958 (m)
37 3.9450 (d), 3.960 (d), 3.986 (d), 4.000 (d)
38 2.393 (s)
Succinic acid 2.415 (s)
39 Sucrose 5.419 (d) 3.46 (t), 3.55 (dd), 3.57 (s), 3.75 (t), 3.82 (m), 3.87-
40 3.89 (m), 4.04 (t), 4.21 (d), 5.40 (d)
41 1.337 (d), 3.596 (d; J=4.931), 1.316 (d), 3.575 (d), 4.244 (m)
42 Threonine 4.261 (dq; J=4.918; J=6.536)
43 Tryptophan 7.208 (t), 7.289 (t), 7.329 (s), 7.546 (d), 3.292 (dd), 3.472 (dd), 4.046 (dd), 7.194 (m), 7.274
44 7.741 (d; J=8.017) (m), 7.310 (s), 7.531 (d), 7.723 (d)
45 Tyrosine 6.906 (d; J=8.503), 7.199 (d; J=8.503) 3.024 (dd), 3.170 (dd), 3.921 (dd), 6.877 (m), 7.170
46 (m)
47 5.78 (br. s)
Urea 5.787 (br.s)
48
4.250 (m), 4.3663 (m), 5.906 (d; J=8.010), 3.801 (dd), 3.907 (dd), 4.121 (m), 4.220 (dd), 4.344
49 Uridine 5.923 (d; J=4.532), 7.886 (d; J=8.010) (dd), 5.882 (d), 5.902 (d), 7.864 (d)
50
51 Valine 0.996 (d; J=7.061), 1.047 (d; J=7.061), 2.281 (m), 0.976 (d), 1.029 (d), 2.261 (m), 3.601 (d),
52 3.617 (d; J=4.408)
53 7.892 (s)
Xanthine 7.977 (s)
54 s, singlet; br.s, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; dt, doublet of triplets; q, quartet; dq, doublet of quartets; m,
55 multiplet.
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Lactic acid
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β -Glucose

Acetic acid
14

Lactic acid
15
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17 α-Glucose
18

Alanine
19
20
21
22

TSP
23
24
25 (a)
26
27
8 7 6 5 4 3 2 1 ppm
28
29
Histidine
Phenylalanine

30

Tyrosine
31
32
Tyrosine

33
34
Hippuric acid

Tryptophan

35
Benzoic acid

Tryptophan

36
37
3-Methylhistidine

1-Methylhistidine
Histidine
Hypoxanthine

38
1-Methylhistidine
Formic acid

39
40 Fumaric acid
Xanthine

Uridine

41
42
43
44
45
(b)
46
47
48 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 ppm
49
50
51
52
53 Figure 1: (a) A typical 800 MHz (cryo-probe) 1D CPMG 1H NMR spectrum of a pooled human
54 serum after protein precipitation using methanol with expanded regions (b-h) and annotations for
55 all identified metabolites.
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α-Glucose

α-Mannose
3
4
5

β -Mannose
6
7

Uridine
8

Sucrose
9
10
11
12
13 (c)
14
15 5.9 5.8 5.7 5.6 5.5 5.4 5.3 5.2 5.1 5.0 ppm
3-Hydroxybutyric acid

16

Valine
Pyroglutamic acid

Glycerol

Threonine
17 Lactic acid Glycine

N-Acetylglycine

Isoleucine
18

Alanine
Serine
19
Creatinine
Proline

Creatine
20
21

Myoinositol
Choline

22
23
24 Threonine
25
26 (d)
27
28
29
30 4.25 4.20 4.15 4.10 4.05 4.00 3.95 3.90 3.85 3.80 3.75 3.70 3.65 3.60 ppm
31
Glucose

32
33
34
35
36
37
Choline

38
39
40
Betaine

41
Creatinine

42
Carnitine

43
44
45
Creatine
Acetylcarnitine

46
47
Lysine
Ornithine

48
49
50
51
52
53 (e)
54 Figure 1 continued
55
56 3.25 3.20 3.15 3.10 3.05 ppm
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3-Hydroxyisovaleric acid
Pyroglutamic acid

1.25
2.5
2.95
Succinic acid

Figure 1 continued
Glutamine

2.4
Pyroglutamic acid

1.20
2.90
Glutamic acid

3-Hydroxybutyric acid
3-Hydroxybutyric acid

2.3
1,2-Propanediol

1.15
Asparagine Dimethylglycine

Valine 2.85
2-Oxoisovaleric acid

2.2
3-Methyl-2-oxovaleric acid Methionine

1.10
Glutamine
2.80

22
Isobutyric acid
Glutamic acid

2.1

1.05
2.75

Proline
Analytical Chemistry

Valine
Isoleucine
Aspartic acid Sarcosine

2.0

ACS Paragon Plus Environment

Isoleucine

1.00
Methionine

2.70

2-Aminobutyric acid
Leucine
Leucine

1.9
Arginine
2-Oxoisocaproic acid Lysine

0.95
2.65

Acetic acid

Isoleucine
1.8
Isovaleric acid
2-Hydroxybutyric acid Lysine

0.90
2.60

2-Oxoisocaproic acid
3-Methyl-2-oxovaleric acid Arginine
1.7
Citric acid

Arginine
2-Hydroxyisovaleric acid
ppm

ppm
ppm

(f)
(g)
(h)
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Page 23 of 25 Analytical Chemistry

1
2
3
4 Methanol
5 Urea
6
7
8
9
3.55 3.50 3.45 3.40 3.35 3.30 ppm
10
11 5.9 5.8 ppm
12
13
14 Dimethylamine
Acetone
15
16
17
18
19
20
21 2.75 2.70 2.65 2.60 2.55 2.50 2.45 2.40 2.35 2.30 2.25 2.20 ppm
22
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Ethanol
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27
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29
30 2-Propanol
31
32
33 1,2-Propanediol
34
35
36
37
38 1.20 1.15 1.10 1.05 1.00 0.95 0.90 0.85 ppm
39
40
41
42
Figure 2: Parts of the 700 MHz 1D 1H NMR spectrum of a pooled human serum sample
43 obtained after ultrafiltration using a 3kDa filter with expanded regions highlighting volatile
44 metabolites that were not detected in protein precipitated serum because of sample drying (see
45 Figure 1).
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 Analytical Chemistry Page 24 of 25
22 22
2:1 MeOH:Serum 2:1 ACN:Serum
20 20
18
3:1 MeOH:Serum 18 3:1 ACN:Serum
1 4:1 MeOH:Serum 16 4:1 ACN:Serum
16
2 14
14
3 12 (a) 12 (e)
4 10 10
5 8 8
6 6 6
7 4 4
8 2 2
9 0 0
10
11
12
13
14
15
16 100 100
17 90 90
18 80 (b) 80 (f)
70 70
19
60 60
20 50 50
21 40 40
22 30 30
23 20 20
24 10 10
25 0 0
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31 450 450
32
400 400
33
34 350 (c) 350 (g)
35 300 300
36 250 250
37 200 200
38
150 150
39
40 100 100
41 50 50
42 0 0
43
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45
46
47
48
49 (d) (h)
4500
50 4500
4000 4000 Figure 3: Comparison of absolute concentrations (in µM) of
51 3500
52 3500 metabolites detected in pooled human blood serum and
3000 3000
53 2500 quantitated using 800 MHz NMR spectroscopy after protein
2500
54 2000 2000 precipitation using methanol (MeOH) (a, b, c and d) or
55 1500 1500 acetonitrile (ACN) (e, f, g and h) at a solvent to serum ratios of
56 1000 1000 2:1, 3:1 and 4:1. Methanol performs most optimally over a wide
57 500 500
58 0 0 range and methanol to serum ratio of 2:1 provides the best
59 performance.
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Page 25 of 25

3-Hydroxyisovaleric acid Formic acid

3-Methylhistidine
Figure for TOC

Hypoxanthine
Histidine

Hypoxanthine

3-Hydroxbutyric acid
1-Methylhistidine
Serum/Plasma

1,2-Propanediol
Uridine
2-Oxoisovaleric acid
Benzoic acid
2-Hydroxyisovaleric acid
Hippuric acid

Tryptophan
Isobutyric acid
Phenylalanine
Methanol

Valine
Isoleucine
Acetonitrile

Tryptophan

2-Aminobutyric acid
Tyrosine Histidine
Leucine

25
Leucine 1-Methylhistidine
Isoleucine
2-Oxoisocaproic acid
Isovaleric acid
qNMR

Tyrosine
2-Hydroxbutyric acid
Analytical Chemistry

3-Methyl-2-oxovaleric acid

2-Hydroxyisovaleric acid
~70 Metabolites

Fumaric acid

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