PROTOCOL

Metabolic profiling, metabolomic and metabonomic

procedures for NMR spectroscopy of urine, plasma,
serum and tissue extracts
Olaf Beckonert, Hector C Keun, Timothy M D Ebbels, Jacob Bundy, Elaine Holmes, John C Lindon &
Jeremy K Nicholson
Department of Biomolecular Medicine, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, UK. Correspondence
should be addressed to J.K.N. (j.nicholson@imperial.ac.uk).
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

Published online 25 October 2007; doi:10.1038/nprot.2007.376

Metabolic profiling, metabolomic and metabonomic studies mainly involve the multicomponent analysis of biological fluids, tissue
and cell extracts using NMR spectroscopy and/or mass spectrometry (MS). We summarize the main NMR spectroscopic applications
in modern metabolic research, and provide detailed protocols for biofluid (urine, serum/plasma) and tissue sample collection and
preparation, including the extraction of polar and lipophilic metabolites from tissues. 1H NMR spectroscopic techniques such as
standard 1D spectroscopy, relaxation-edited, diffusion-edited and 2D J-resolved pulse sequences are widely used at the analysis stage
to monitor different groups of metabolites and are described here. They are often followed by more detailed statistical analysis or
additional 2D NMR analysis for biomarker discovery. The standard acquisition time per sample is 4–5 min for a simple 1D spectrum,
and both preparation and analysis can be automated to allow application to high-throughput screening for clinical diagnostic and
toxicological studies, as well as molecular phenotyping and functional genomics.

INTRODUCTION
Metabolomics1 and metabonomics2 encompass the comprehensive
and simultaneous systematic profiling of multiple metabolite con-
centrations and their cellular and systemic fluctuations in response
to drugs, diet, lifestyle, environment, stimuli and genetic modula-
tions, in order to characterize the beneficial and adverse effects of
such interactions3–30.
Multiparametric metabolic profiling technologies are mainly
centered on NMR spectroscopy22–27,29,31,32 and mass spectrometry
(MS) (usually with a chromatographic separation step)1,29,32–34,
because both spectroscopic platforms can give extensive structural
and conformational information on multiple chemical classes in a
single analytical procedure. Multivariate spectroscopic data are
typically analyzed using chemometric and pattern recognition
techniques to extract latent metabolic information, and enable
sample classification and biomarker discovery2,28,35–40. NMR
spectroscopy has been used extensively for multivariate metabolic
profiling of cells, tissues and biological fluids since the 1970s22–27,31.
Many NMR-based applications of metabonomics have been pub-
lished, including the extensive study of physiological variation in
experimental animals, such as male/female differences, age-related
changes, dietary modulation, diurnal effects and phenotyping of
mutant and transgenic animals3–10,27,29,30. This has led to the
development of toxicological applications in which metabonomic
biofluid profiles were used to identify specific biomarkers of organ
toxicity, an important economic factor of attrition in the preclinical
pharmaceutical discovery process10,28,41. A recently completed
large-scale project in the COMET group (COnsortium on MEta-
bonomics in Toxicology), where the effects of 147 different model
toxins and treatments were studied, was successful in implementing
this technology for safety screening, initially for liver and kidney
toxicity, across five pharmaceutical companies (Bristol-Myers-
Squibb, Eli Lilly and Co., Hoffmann–La Roche, NovoNordisk
and Pfizer Inc.)41,42,43.
Metabonomics and metabolomics have also found application
not only in the study of many diseases12–14,44–46 but also of factors
such as nutrition and gut microflora47,48. Since the possibility of
predicting postdose drug effects from predose metabolic profiles
has been shown, this pharmaco-metabonomics approach has been
identified as a potential precursor for personalized medicine49.
These and other large-scale applications, for example, in
epidemiological studies50, show the necessity for standardized
protocols to ensure high reproducibility. In this protocol, we
describe the methodology that is required to perform NMR-
based metabonomic analysis of biofluids and tissue samples for
metabolite profiling.
Background
Analytical approaches in metabonomics and metabolomics.
The main analytical techniques that are employed for metabonomic
studies are based on NMR spectroscopy and MS, the latter requir-
ing a preseparation of the metabolic components using either gas
chromatography after chemical derivatization, or LC29.
Capillary electrophoresis coupled to MS has also shown some
promise. Other more specialized techniques such as Fourier trans-
form infrared (FTIR) spectroscopy and arrayed electrochemical
detection have been used, too. Both MS and NMR are suitable
techniques for metabonomic analysis but have different analytical
strengths and weaknesses and give complementary information,
and those have been comprehensively reviewed32. All metabolomic
and metabonomic studies result in complex multivariate data sets
that require visualization software and chemometric and bioinfor-
matic methods for interpretation. The aim of these procedures is to
produce biochemically based fingerprints that are of diagnostic or
other classification value. A second stage, crucial in such studies, is
to identify the substances causing the diagnosis or classification, as
these become the potentially complex set of biomarkers that define

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the biological or clinical context and help explain the mechanisms
related to tissue damage or disease.
NMR-based metabolite profiling. High-resolution NMR
spectroscopy is a quantitative nondestructive, noninvasive,
nonequilibrium perturbing technique that provides detailed infor-
mation on solution-state molecular structures, based on atom-
centered nuclear interactions and properties. NMR spectroscopic
methods can also be used to probe metabolite molecular dynamics
and mobility (such as ligand–protein binding) through the inter-
pretation of NMR spin relaxation and molecular diffusion proper-
ties51. NMR is a robust and reliable technique for metabonomic
applications in which high reproducibility is paramount50. It allows
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols
the detection of a wide range of structurally diverse metabolites
simultaneously, providing a metabolic ‘snapshot’ at a particular
time point. Metabolite concentrations down to the low micromole
per liter range are readily detected in B4–5 min acquisition time
using current-generation high-field spectrometers. Usually, the
whole spectral information is used for further chemometric ana-
lysis, but specific NMR pulse sequences can be employed to select
subsets of metabolites, if necessary. On the other hand, using no
selection or extraction of metabolites up-front to investigate all
possible variables is advantageous in metabonomics studies in
which no prior information about metabolites is known or in
which it still remains to be established whether several metabolites
are linked to factors such as toxicity, disease.
Given the diversity of applications of this technology across a
broad swathe of biomedicine, efforts have been underway to
standardize the reporting of metabonomic and metabolomic
data, and inter alia52,53 the Standard Metabolic Reporting Struc-
tures (SMRSs) group has recently provided a comprehensive report
(http://www.smrsgroup.org) and summary publication of the
major issues involved54. Following SMRS, the Metabolomics
Standards Initiative (MSI), which through a set of working groups
is building comprehensive descriptions of the reporting needs
for the technologies and various applications of the subject
(http://msi-workgroups.sourceforge.net), has been set up. This
process is designed not to be proscriptive and does not recommend
detailed protocols for data acquisition, for example. In fact,
although standardization of reporting experimental data is highly
desirable, it would be detrimental to the exploratory nature of the
subject to allow only ‘validated’ or ‘approved’ procedures to be used
in experimental metabolism studies.
Biological sample types. Metabonomic studies generally use
biofluids or cell or tissue extracts as primary sources of metabolic
fingerprint data. Biofluids are usually relatively easy to obtain,
particularly urine and serum or plasma, and this is important in
animal and human studies. A wide range of fluids have been studied
in addition to urine and plasma, including seminal fluids, amniotic
fluid, cerebrospinal fluid, synovial fluid, saliva and other digestive
fluids, blister and cyst fluids, lung aspirates and dialysis fluids30. In
addition, a number of NMR-based studies have used nondestruc-
tive analysis of tissue biopsy samples and their lipid and aqueous
extracts. Extensive evaluations of extraction methods for NMR-
based metabolic analyses have been performed55,56. Metabolic
profiling can also be used to characterize in vitro cell systems such
as yeast57, tumor cells58 and tissue spheroids15, which can be used as
model systems for liver or tumor investigations, for example.
PROTOCOL
Sample preparation approaches. Automatic sample preparation
is possible for NMR spectroscopy, and standard NMR spectra
typically take only a few minutes to acquire using robotic
flow-injection methods59. For large-scale studies, bar-coded vials
containing the biofluid can be used and the contents of these can be
transferred and prepared for analysis using robotic liquid handling
technology into 96-well plates (volume ¼ 1 ml) under Laboratory
Information Management System (LIMS) control. Larger density
plates are becoming available for high-throughput measurements,
too, especially when using capillary microprobes. Currently, using
such approaches, hundreds of samples per day can be measured on
one spectrometer, each taking a total data acquisition time of
typically 5 min with minimal sample handling or pretreatment,
although it is possible to acquire data faster than this. Alternatively,
for more precious samples or for those of limited volume, conven-
tional 5-mm diameter (or less) NMR tubes are usually used, either
individually or using a commercial sample tube changer and auto-
matic data acquisition. For more specialist microsample applications,
it is possible to use much smaller tubes, for example, 1–3-mm
diameter, and even microprobes with volumes as low as 10 ml.
NMR data. A typical 1H NMR spectrum of urine contains
thousands of sharp lines from predominantly low molecular weight
metabolites (Fig. 1). Blood plasma and serum contain both low and
high molecular weight components, and these give a wide range of
signal line widths (Fig. 2): Broad bands from protein and lipopro-
tein signals contribute strongly to the 1H NMR spectra, with sharp
peaks from small molecules superimposed on them. The large
interfering NMR signal arising from water in all biofluids is easily
eliminated by the use of appropriate standard NMR solvent
suppression methods. The reference compound used in aqueous
media is usually the sodium salt of 3-trimethylsilylpropionic acid
(TSP) with the methylene groups deuterated to avoid giving rise to
peaks in the 1H NMR spectrum. Other reference standards are DSS
Aromatic signals
TMAO
Citrate
Allantoin 2-Oxoglutarate
Hippurate
9 8 7 6 Creatinine
Urea
Hippurate Taurine Alanine
branched-chain
amino acids and
* organic acids
t-Aconitate
TSP
9 8 7 6 5 4 3 2 1
Sugars, polysols, amino acid CH
Figure 1 | 600 MHz 1H NMR spectrum of control rat urine, displaying
hundreds of resolved peaks. All peaks are referenced to the resonance of TSP
(sodium salt of 3-trimethylsilylpropionic acid) at 0 p.p.m. The spectrum
displays a wide range of metabolites such as aromatic, aliphatic compounds,
sugars, amino acids and other osmolytes. The asterisk denotes the suppressed
signal of water. TMAO, trimethylamine N-oxide.
NATURE PROTOCOLS | VOL.2 NO.11 | 2007 | 2693
 PROTOCOL

[2,2-dimethyl-2-silapentane-5-sulfonate sodium salt], or for

organic solvents, tetramethylsilane (TMS). The peak integrals relate
directly to the number of protons giving rise to the peak, and hence
to the relative concentrations of the substances in the sample.
Absolute concentrations can be obtained if the sample contains an Glucose, Lactate
added internal standard of known concentration, or if a standard amino acid CH Lipid CH2
(VLDL and LDL)
addition of the analyte of interest is added to the sample, or if the *
concentration of a substance is known by independent means Alanine
(e.g., glucose in plasma can be quantified by a conventional Glucose Lipid
biochemical assay). Recently, a synthetic electronic reference signal =CH-CH2
Lipid CH3
(ERETIC, Electronic REference To access In vivo Concentrations) (VLDL and LDL)
has been introduced for quantitation purposes, and this method Creatine
Lactate Valine
does not require any internal standards60.
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

Standard NMR pulse sequences, where the observed peak Lipid

Citrate
=CH
intensities are edited on the basis of molecular diffusion coefficients
Tyrosine
or on NMR relaxation times [such as the Carr–Purcell–Meiboom–
Gill (CPMG) spin-echo sequence], can be used to largely enhance Acetate
only the contributions from macromolecules, or to selectively
Lipid = CH-CH2CH=
highlight the signals from the small molecule metabolites,
8 7 6 5 4 3 2 1 0
respectively20 (Fig. 3).
Identification of biomarkers can involve the application of a Figure 2 | 600 MHz 1H NMR spectrum of blood serum sample. The spectrum
range of techniques, but 1H NMR spectra of urine and other shows signals of low molecular weight metabolites as well as of larger
biofluids, even though they are complex, allow many resonances to molecules such as lipoproteins. The large molecules give rise to much broader
be assigned directly16 based on their chemical shifts, signal resonances in both the aromatic and the aliphatic area due to their fast
multiplicities and by adding authentic material, and further infor- relaxation properties in the NMR experiment. The signal intensities of low
mation can be obtained by using spectral editing techniques molecular weight compounds from d 6–9.5 are low in this type of acquisition.
LDL, low density lipoprotein; VLDL, very low density lipoprotein.
or interrogation of spectral databases of authentic substances.

Structure elucidation using 2D NMR spectroscopy. 2D NMR

spectroscopy can be useful for increasing signal dispersion and for
elucidating the connectivities between signals, thereby helping to
identify biochemical substances. These include the 2D J-resolved
experiment61, which yields information on the multiplicity and
coupling patterns of resonances, aiding molecule identification.
Correlation spectroscopy (COSY)62 and total correlation spectro-
scopy (TOCSY) experiments63 provide spin–spin coupling con-
nectivities, giving information on which hydrogens in a molecule
are close in chemical bond terms. Use of other types of nuclei of
spin I ¼ 1/2, such as naturally abundant 13C, 15N or 31P, can be
important to help assign NMR peaks making use of heteronuclear
correlation NMR experiments. The lower sensitivity or less abun-
dant nucleus NMR spectrum (such as 13C) is detected indirectly
using the more sensitive/abundant nucleus (1H) by making use of
spin–spin interactions such as the one-bond 13C–1H spin–spin
coupling between the nuclei to effect the connection. Generally,
2D experiments require much longer acquisition times than the
standard 1D pulse sequences (in the range of hours while 1D
spectra can generally be performed within minutes). These experi-
ments, in particular, have benefited from the development of
cryogenic probes where the detector coil and preamplifier (but
not the samples) are cooled to B20 K. This has provided an
improvement in spectral signal-to-noise ratios of up to a factor of
4 by increasing the detection sensitivity of the radio frequency
(RF) coil and by reducing the thermal noise in the electronics of
the spectrometer. With optimized cryoprobe design, direct 13C
detection at natural abundance also can be made practical64.
Hyphenated technologies. For biomarker identification, it is also
possible to separate out the substances of interest on a larger scale
from a complex biofluid sample using techniques such as solid-
phase extraction or HPLC. For metabolite identification, directly
Unedited spectum T2 edited
(1D NOESY) (CPMG)

Figure 3 | Example of edited spectra of a plasma sample [all were acquired

with solvent (water) suppression]. 1D nuclear Overhauser enhancement
spectroscopy (NOESY) spectra generally give the best overview over all types
of molecules in biofluids. Diffusion properties can be used to select mainly J-resolved (2D)
Diffusion-edited
macromolecular signals (diffusion-edited), while Carr–Purcell–Meiboom–Gill (× 10 scale)
spectral
projection
(CPMG) spectra use the fast relaxation of protons in macromolecules (short T2)
to filter particularly those signals out and leave peaks from small molecules or
signals from molecules with significant segmental motion. Spectral projections
from J-resolved spectra show signals from small metabolites, too; but here
resolution of peaks is improved, since resonances do not show any multiplicity
and appear as individual singlets. 5 4 3 2 1 ppm 5 4 3 2 1 ppm

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coupled chromatography–NMR spectroscopy methods can also be
used. The most general of these ‘hyphenated’ approaches is
HPLC–NMR–MS in which the eluting HPLC fraction is split,
with parallel analysis by directly coupled NMR and MS techni-
ques65. This can be operated in on-flow, stopped-flow and loop-
storage modes, and thus can provide the full array of NMR- and
MS-based molecular identification tools. These include 2D NMR
spectroscopy as well as MS–MS for the identification of fragment
ions and FT-MS or time-of-flight MS (TOF-MS) for accurate
mass measurement and hence derivation of molecular empirical
formulae. The analytical strategies for metabolic profiling have
recently been reviewed32.
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols
High-resolution NMR of intact tissues. The development of
high-resolution 1H magic angle spinning (MAS) NMR spectro-
scopy has made feasible the acquisition of high-resolution NMR
data on small pieces of intact tissues with no pretreatment11–14.
Rapid spinning of the sample (typically at B4–6 kHz) at an angle
of 54.71 relative to the applied magnetic field serves to reduce the
loss of information caused by line broadening effects seen in
nonliquid samples such as tissues. MAS NMR spectroscopy has
straightforward, but manual, sample preparation. NMR spectro-
scopy on a tissue sample in an MAS experiment is the same as
solution-state NMR, and all common pulse techniques can be
employed in order to study metabolic changes and to perform
molecular structure elucidation and molecular dynamic studies.
Statistical spectroscopy. A new method for identifying multiple
NMR peaks from the same molecule in a complex mixture, based
on the concept of Statistical Total Correlation Spectroscopy
(STOCSY), has been demonstrated39,66. This takes advantage of
the multicolinearity of the intensity variables in a set of spectra to
display the correlation among the intensities of the various peaks
across the whole sample. This method is not limited to the usual
connectivities that are deducible from more standard 2D NMR
spectroscopic methods. Added information is available by examin-
ing lower correlation coefficients or even negative correlations,
since this leads to the connection between two or more molecular
species involved in the same biochemical process. In an extension of
the method, the combination of STOCSY with supervised chemo-
metrics methods offers a new framework for the analysis of
metabonomic data. In a first step, a supervised multivariate
discriminant analysis can be used to extract the parts of NMR
spectra related to the discrimination between two sample classes.
This information is then combined with the STOCSY results to
help identify the molecules responsible for metabolic variation.
The STOCSY approach has also been extended to include
heterogeneous forms of data. In this form, it is known as statistical
heterospectroscopy (SHY) and this has been applied to the coana-
lysis of both NMR and mass spectra in metabonomic toxicity
studies40. This allowed better assignment of biomarkers of the toxin
effect by using the correlated, but complementary, information
available from the NMR and mass spectra taken on a whole sample
cohort.
Recently, a new approach, statistical diffusion-ordered spectro-
scopy (S-DOSY), has been presented, which combines diffusion-
ordered (DO) NMR spectroscopy with STOCSY for the analysis of
complex biofluids to give enhanced information recovery using the
diffusion properties of biomolecules. Furthermore, a visualization
PROTOCOL
tool was developed in which the apparent diffusion coefficients
from DO spectra are projected onto a 1D NMR spectrum
(diffusion-ordered projection spectroscopy, DOPY)67.
Experimental design
Before performing the spectroscopic analysis, it is necessary to
consider aspects such as sample numbers per group and randomi-
zation. If this is a pilot study, smaller sample numbers per group are
often sufficient to identify trends between groups. In order to be
able to validate results, sufficient sample numbers should be
analyzed to achieve statistically significant results (consult and
involve a statistician as early as possible in the study planning
process). Generally, NMR-based metabonomics studies of biofluids
have shown a high reproducibility when using NMR21,50. There-
fore, in most cases it is sufficient to have one sample per time point.
Using flow automation, problems with the injection of samples are
rare (and often due to problems of sample volume), but can occur.
This implies that it is important to keep aliquots of samples at the
sample collection stage in order to be able to repeat acquisitions or
prepare fresh samples for additional 2D acquisitions. When per-
forming the work under Good Laboratory Practice (GLP) condi-
tions, it may be necessary to analyze multiples of every sample,
which should be randomized across the whole run.
In work with tissue samples, it is important to keep the time of
tissue in the defrosted stage as short as possible to avoid enzymatic
activity and degradation processes. Different collection and extrac-
tion protocols for tissue samples will lead to observation of
different fractions in the metabolite profile. If it is important for
a study to get as accurate a possible reflection of in vivo metabolism,
then tissue needs to be frozen as rapidly as possible to prevent any
enzymatic changes (e.g., labile phosphates and glycolytic inter-
mediates can change on a millisecond timescale) and loss of
volatiles. In most cases, it is sufficient to freeze samples directly
in liquid nitrogen; it may be decided to freeze larger samples by
freeze-clamping. It needs to be considered that these conditions are
not always easy to achieve, for example, in clinical surgery where
the care of the patient has priority, but it is important to discuss and
arrange these practical aspects with personnel to achieve optimum
results. Grind tissue samples in the frozen stage in a mortar and
pestle or a ball mill cooled with liquid nitrogen, or transfer them
directly into organic solvents (preferably ice-cold) for homogeniza-
tion while still frozen. The ground samples must not be allowed to
thaw before coming into contact with perchloric acid/solvents.
Do not use TSP as a reference standard in plasma, serum or any
other samples with a high protein content, since the compound
binds to proteins resulting in a much reduced signal with a very
broad line width. An alternative is the use of formate.
When preparing samples into well plates, substitute H2O for
urine or serum/plasma in the first and last sample in each plate to
produce blank samples. This provides a check for carry-over
between samples and prevents biofluid remaining in the NMR
probe at the end of the run. It is also advisable to run one to two
aliquots of a representative biofluid sample per well plate across the
whole run—this serves as a quality control (QC) measure.
Limits of applicability and practical considerations
NMR spectroscopy of biofluids and tissue extracts is most efficient
in the millimole per liter down to the micromole per liter range.
Strong pH variations between urine samples (which can have
NATURE PROTOCOLS | VOL.2 NO.11 | 2007 | 2695
 PROTOCOL
considerable buffering capacity) can lead to signal shifts, despite
additional buffering. In most studies this is not problematic,
but it should be considered in any study in which acidosis or
alkalosis is induced. Dilute samples (e.g., urine) can be difficult
to analyze, in particular in cryoprobes where a strong water signal
gives rise to radiation damping. Generally, water suppression
through presaturation has to be employed to reduce the water
signal of biofluids, and this helps targeting the dynamic range to
the metabolites of interest. Where simple presaturation is not
efficient, excitation sculpting has been found to be beneficial (see
EQUIPMENT SETUP). High salt concentrations and samples of
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols
MATERIALS
REAGENTS
. Na2HPO4, 99+% ACS, anhydrous (Sigma-Aldrich)
. NaH2PO4, 99%, anhydrous (Sigma-Aldrich)
. 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt, 98 atom %D
(Sigma-Aldrich)
. Sodium azide (NaN3; Sigma-Aldrich)
. Perchloric acid (PCA), ACS reagent, 70% wt/wt (Sigma-Aldrich)
. K2CO3, ACS reagent, Z99.0% (Sigma-Aldrich)
. D2O, DM-4LC (Goss Scientific Instruments)
. Water, HiPerSolv for HPLC, BDH (VWR International Ltd.)
. Chloroform, AnalaR, Z99%, BDH (VWR International Ltd.)
. Methanol, HiPerSolv, Z99.8%, BDH (VWR International Ltd.)
. Acetonitrile, NMR-Chromasolv, Z99.6%, Riedel-de-Ha¸n (Sigma-Aldrich)
. CDCl3, ‘100’, Z99.96 atom %D, contains 0.03 vol/vol TMS (Sigma-Aldrich)
. Methanol-d4, 99.8% (CD3OD; Goss Scientific)
EQUIPMENT
. 600 MHz Avance DRX NMR spectrometer (Bruker Biospin) or similar
(Varian, Jeol, etc.). 600 MHz is not the only frequency that can be used in
metabonomics applications, but it is a good compromise for cost-benefit
. Flow-injection probe FI TXI 600 SB 5 mm with Z gradient (Bruker Biospin)
or similar
. Tube probe TXI 600 MHz S3 5-mm XYZ gradient (Bruker Biospin) or
similar
. Gilson 215 flow-injection system with Icon-NMR or similar
. Autosampler BACS60 for tube NMR (Bruker Biospin) or similar
. Gilson 215 sample preparation robot with SampleTrack (Bruker Biospin)
or similar
. Tissue homogenizer T25 basic (IKA Labortechnik), Potter homogenizer
(B. Braun Biotech Co.) or similar
. Genevac EZ2 solvent evaporator (Genevac Ltd.) or similar
. Eppendorf tubes, 1.5 ml (VWR International Ltd.)
. 96 deep well-plate, Ritter Riplate, 1 ml, with sealing mats (Ritter)
REAGENT SETUP
Urine samples For animal urines or human urines possibly containing
bacterial contamination: collect into labeled tubes containing NaN3
(to result in a total concentration of azide of min 0.05% wt/vol) over ice or in
a refrigerator (
2 to +2 1C), and store frozen at –40 1C until analyzed. For
details about sample storage see ref. 69.
Prepare urine samples into 96-well plates for NMR spectroscopy using a
Bruker SampleTrack system and a Gilson 215 preparation robot or equivalent
technology. Alternatively, samples can be made up manually into standard
5 mm NMR tubes.
In order to provide some stabilization of the urinary pH, mix urine
aliquots with phosphate buffer (pH 7.4), see PROCEDURE.
Blood samples Collect B0.80 ml into lithium heparin tubes (e.g., BD
vacutainer, Li-heparin) to give plasma, or leave on ice to coagulate
(e.g., BD vacutainer, no additive) and, after centrifugation, to result in
serum. For these processes, use standard site-specific procedures.
Avoid EDTA, citrate and other added stabilizers since they give additional signals
in the NMR spectra. Also, collection tubes should be avoided, which use gel to
separate blood cells from plasma. The time before separation of blood cells
should ideally not exceed 30 min. Centrifuge samples at 1,600g for 15 min
at 4 1C. Store supernatant at –40 1C until analyzed. More details about sample
storage can be found in ref. 17.
2696 | VOL.2 NO.11 | 2007 | NATURE PROTOCOLS
high ionic strength (e.g., buffered urine samples) can affect the
tuning and matching of probes, which may make longer pulse
lengths necessary; smaller NMR tube diameters can be chosen
to reduce these effects, which are particularly prominent when
using cryoprobes. Very recently, autotune devices have become
available on the market, which may optimize tuning and matching
for each sample by option, therefore assuring optimum conditions.
Sample storage: Blood serum and plasma68 (
80 1C) and urine
(own results,
40 1C) samples can be stored for 9 months without
problems, and do not show any significant differences when
analyzed.
Prepare blood samples into 96-well plates for NMR spectroscopy using a
Bruker SampleTrack system and a Gilson 215 preparation robot or equivalent
technology. Alternatively, they can be made up manually into standard 5-mm
NMR tubes.
In order to provide diluted samples including deuterated lock solvent
for NMR spectroscopy, mix sample aliquots with saline solution, see
PROCEDURE.
Tissue samples Freeze the tissue sample rapidly in liquid nitrogen after
collection to immediately stop any enzymatic or chemical reactions
(see EXPERIMENTAL DESIGN). Store samples at
80 1C. The typical sample
size is 100 mg (wet mass) although as little as 20 mg can be used. Use a manual
grinding method with a mortar and pestle to disrupt the tissue or, alternatively,
use an electric homogenizer for homogenization in solvents. These approaches
are consistent with previous evaluation studies using wet and dry, ground or
homogenized, tissue samples, and in general it has been found that homogenizing
provides less inter-sample variability compared with grinding wet tissue56.
Phosphate buffer Prepare phosphate buffer (pH 7.4) by weighing 28.85 g
Na2HPO4, 5.25 g NaH2PO4, 1 mM TSP and 3 mM NaN3 into a 1 l volumetric
flask. Add 200 ml of D2O and fill up to 1 l with water. Shake thoroughly, and
leave in a sonicator at 40 1C, interspersed by shaking the flask, until the salts are
dissolved.
Saline solution Prepare a 0.9% NaCl (wt/vol) solution by weighing 9 g of NaCl
into a 1-l volumetric flask. Add 100 ml of D2O and fill up to 1 l with water, then
shake until salt is dissolved.
EQUIPMENT SETUP
General NMR setup Experiment setup for the individual samples:
Depending on the questions asked about the biochemical properties of the
compounds, the user can choose from a range of experiments, which are
exemplified here for Bruker Avance spectrometers: normal one-pulse sequence
(zg), 1D nuclear Overhauser enhancement spectroscopy (NOESY)-presat
(noesypr1d), CPMG-presat (cpmgpr), J-resolved and diffusion-edited experi-
ments. The most common are 1D NOESY-presat for urine, 1D NOESY-presat
and CPMG-presat for blood and a normal one-pulse sequence (zg) for tissue
extracts. The details of these sequences, which are used unmodified, can
be found in the Supplementary Note (NMR Pulse Sequences) and in
PROCEDURE. Other spectrometer manufacturers provide the same pulse
sequences, but they have different names and syntax, please discuss
with the supplier.
Generally, all NMR experiments should be acquired at a constant tempera-
ture; we perform experiments at 300 K. Ensure that temperature has been
calibrated using a standard methanol sample, for example.
All NMR experiments require that the correct 901 pulse length is deter-
mined on a representative sample—this is generally the first biofluid sample
in the run. The same sample is used to determine the offset of the water sig-
nal for the water suppression. Both parameters are then used for the whole
data set. Where an automatic pulse calibration per sample is possible, choose
this option to improve quality.
Water suppression. In order to observe the dynamic range of metabolite
concentrations efficiently, the water signal needs to be suppressed, ideally to
or below the highest metabolite peak. Generally, the normal water presatura-
tion is effective and the power level is optimized so that optimum suppression
is achieved without suppressing metabolite resonances next to it. Due to the
chemical exchange with water, the urea peak is influenced by water
presaturation—this requires the elimination of this peak in later chemometric

analysis. In very dilute samples, sufficient water suppression may not be
achieved using presaturation, and excitation sculpting has proven useful to
eliminate the water peak70,71.
The user may decide to run all experiments either with the same receiver
gain (urine, blood) or with an automatically adjusted receiver gain when the
sample concentration varies too much (urine or tissue extracts).
The following procedure describes recommendations for the number of scans
per experiment for the individual biological matrices. The user may decide to
increase the number of scans per experiment for the individual bio-
logical matrices if the signal-to-noise ratio is too low for the metabolites of
interest, but this will have an impact on the overall experiment time per sample.
Several aspects of the NMR acquisition can be automated in programs that
are linked with the pulse sequence (Bruker: ‘au’ programs). Among these are,
in consecutive order, a waiting delay for the sample to reach a constant
temperature (0.5–1 min on flow-injection probes with a heated transfer line,
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols
PROTOCOL
4–5 min on tube probes), automatic locking onto the reference signal of the
deuterated solvent, homogenization of the magnetic field (‘shimming’),
receiver gain adjustment, acquisition and automatic processing (apodization,
Fourier transformation, phasing and baseline correction).
Recent developments of NMR vendors now include, besides automated
pulse calibration, automatic tuning and matching on every sample in automa-
tion. This is especially of advantage in samples where salt concentration varies
between samples (e.g., urines). Choose this option, where possible.
General maintenance of the flow-injection NMR system The push solvent is
0.1% NaN3 in H2O (wt/vol; deionized). After each run, flush the system
using hydrogen peroxide (10%)(wt/vol), followed by HCl (0.1 M) and
finally H2O containing azide (0.1%). Once a week, flush the probe manually
with disinfectant and allow the solution to stand in the probe for 1 h.
Periodically, as required by continuous monitoring of performance, thoroughly
clean the probe by flushing for several hours after removal from the magnet.

PROCEDURE
Sample preparation
1| Prepare urine, blood and tissue samples using the guidelines described in options A (Biofluids) and B, C or D (extraction of
tissues with acetonitrile, perchloric acid or methanol/chloroform/water, respectively). Again, quantities apply to Bruker 5 mm
tube NMR and the above-described flow-NMR conditions, respectively. Adjust the quantities accordingly depending on different
vendor requirements and/or when varying tube size, for example.
(A) Biofluids (urine and blood plasma/serum)
(i) Rat urine, human urine: Mix 400 ml of urine with 200 ml of phosphate buffer (pH 7.4). Adjust these volumes according to
the NMR probe used. Prepare samples either into well plates (flow-injection) or Eppendorf tubes (tube NMR).
(ii) Mouse urine: Mix 200 ml of urine with 200 ml H2O and 200 ml of phosphate buffer (pH 7.4). Adjust these volumes
according to the NMR probe used. Prepare samples either into well plates (flow-injection) or Eppendorf tubes (tube NMR).
(iii) Plasma or serum: Add aliquots (200 ml) to 400 ml of 0.9% saline. Adjust these volumes according to the NMR probe used.
Prepare samples either into well plates (flow-injection) or Eppendorf tubes (tube NMR).
(iv) Well plates: Substitute H2O for urine or serum/plasma in the first and last sample in each plate to produce blank samples.
Prepare 1–2 QC biofluid aliquots into each well plate. Centrifuge the well plate at 1,800g for 5 min to remove insoluble
material, before positioning in the NMR flow-injection Gilson robot. Inject 500 ml of sample into the probe (Bruker
flow-injection probe, this volume may vary between different makes of probes); depending on the requirements and make
of equipment, it may be decided to use air gaps in between sample and push solvent or similar.
(v) NMR tubes: Centrifuge Eppendorf tubes at 12,000g for 5 min at 4 1C, and transfer 550 ml of sample into 5 mm NMR tubes.
(vi) Proceed to Step 3 (NMR DATA ACQUISITION AND PREPROCESSING).
(B) Extraction of polar metabolites from tissues using acetonitrile
(i) Prepare ice-cold solvents.
(ii) Weigh intact frozen tissue, then homogenize in 50% acetonitrile/50% H2O (vol/vol) (5 ml g
1 tissue). Centrifuge at
12,000g for 10 min at 4 1C.
(iii) Collect the supernatant and lyophilize.
’ PAUSE POINT Store samples at
80 1C until NMR acquisition18,19.
(iv) Proceed to Step 2.
(C) Extraction of polar metabolites from tissues using perchloric acid
(i) Weigh the frozen tissue and grind it in a mortar cooled with liquid nitrogen. Then add 5 ml g
1 of 6% ice-cold perchloric
acid and grind further to completely mix the acid and sample, and then allow it to thaw in the mortar. Alternatively weigh
the frozen tissue and homogenize in the ice-cold solution straight away.
(ii) Vortex the sample and then place on ice for 10 min.
(iii) Centrifuge for 10 min at 12,000g at 4 1C.
(iv) Neutralize the supernatant (e.g., 500 ml) to pH 7.4 with 2 M K2CO3 and leave for 30 min on ice to precipitate the
potassium perchlorate salts, taking care to add K2CO3 slowly to avoid loss of sample during effervescence.
(v) Check and adjust the pH if necessary, then centrifuge the sample and freeze–dry the supernatant.
’ PAUSE POINT Store samples at
80 1C (refs. 72, 73).
(vi) Proceed to Step 2.
(D) Combined extraction of polar and lipophilic metabolites from tissues using methanol/chloroform/water
(i) Prepare ice-cold solvents: methanol, chloroform, water.
(ii) Weigh intact frozen tissue, then transfer into a glass vial.
(iii) Homogenize after adding 4 ml of methanol per gram of tissue and 0.85 ml g
1 water to the sample. Vortex the sample,
then add 2 ml g
1 chloroform to the sample and vortex again.
(iv) Add 2 ml g
1 chloroform and 2 ml g
1 water to the sample and vortex again.

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 PROTOCOL

(v) Leave sample on ice or in the fridge for 15 min. Centrifuge at 1,000g for 15 min at 4 1C. The solutions should now separate
into an upper methanol/water phase (with polar metabolites) and a lower chloroform phase (with lipophilic compounds),
separated by protein and cellular debris. Centrifuge again if still no clear separation.
(vi) In turn, transfer the upper and lower layers of each sample into separate glass vials. Remove the solvents from the samples
using a speed vacuum concentrator or under a stream of nitrogen.
’ PAUSE POINT Store aqueous extract samples at
80 1C until required. Keep the lipophilic phase in deuterated organic
solvent (see Step 2(B); to reduce effects of oxidation) in the refrigerator until required, but preferably not longer than 2–
3 d before NMR acquisition56,74–76.
(vii) Proceed to Step 2.

Processing of tissue extracts for NMR

2| Process the tissue extracts for NMR acquisition using option A for aqueous extracts/water soluble metabolites and option B
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

for lipophilic extracts/lipid metabolites.

(A) Preparation of aqueous extracts/water-soluble metabolites for NMR spectroscopy
(i) Before NMR acquisition, resuspend the polar tissue extracts in either 580 ml NMR buffer (100 mM sodium phosphate buffer,
pH 7.4, in D2O, containing 0.1–0.5 mM TSP and optionally 0.2% NaN3) or in D2O containing TSP as a chemical shift
reference (d ¼ 0 p.p.m.).
(ii) Vortex samples and then centrifuge at 12,000g for 5 min.
(iii) Transfer 550 ml of the supernatant into an NMR tube and proceed to Step 3.
(B) Preparation of lipophilic extracts/lipid metabolites for NMR spectroscopy
(i) Resuspend the lipophilic tissue extracts in 580 ml deuterated NMR solvent (2:1 mixture of chloroform-d (CDCl3)
containing 0.03 vol/vol TMS, and CD3OD) and then vortex. This method works well when running NMR experiments
manually. For automated runs, we have found that resuspending the lipophilic tissue extracts in 580 ml deuterated CDCl3
containing 0.03 vol/vol TMS only is robust and reliable with regards to locking and shimming.
(ii) After centrifugation (1,000g, 5 min), transfer 550 ml of the supernatant into an NMR tube and proceed to Step 3.

NMR data acquisition and preprocessing

3| Set temperature to 300 K (one may decide to set the temperature higher for some plasma analysis).

4| Load sample into probe and leave sufficient time to equilibrate (see EQUIPMENT SETUP).

5| Using a representative sample (this could be the QC sample): (i) Tune and match the probe. (ii) Set the RF carrier frequency
offset value to the H2O resonance and determine the water saturation power. For excitation sculpting, adjust the power for the
shaped pulse. (iii) Determine the 901 pulse length at a given power level. (iv) Readjust the frequency offset for water signal
suppression, if necessary. (v) Transfer these settings to the experiments to be submitted.

6| Select suitable experiments for the samples: 1D NOESY-presat (option A) for urine, 1D NOESY-presat and CPMG-presat
(option B) for plasma and serum [optionally also J-resolved (option C) and diffusion-edited (option D)] and a single-pulse
sequence (option E) (or 1D NOESY-presat or CPMG-presat) for extracts. Processing parameters: If not mentioned otherwise,
the 1D spectra are generally processed by applying a line broadening of 0.3–1 Hz and zero-filling by a factor of 2 to give 64k
frequency domain data points.
(A) 1D NOESY-presat sequence
(i) Measure the 1H NMR spectra of biofluids and aqueous extracts using a specified water suppression pulse sequence
such as 1D NOESY-presat, which employs the first increment of a NOESY pulse sequence with water irradiation during the
relaxation delay and also during the mixing time (on a Bruker instrument, this is called noesypr1d). This has the
form –RD-901-t-901-tm-901-ACQ, where RD is the relaxation delay, t is a short delay typically of B3 ms, 901 represents a
901 RF pulse, tm is the mixing time and ACQ is the data acquisition period16. The mixing time is tm ¼ 100–150 ms (but
the presaturation quality is not very sensitive to this value), and other parameters are spectral width ¼ 20 p.p.m., number
of time domain data points ¼ 32,768, relaxation delay, RD ¼ 2.0 s, acquisition time ¼ 1.36 s, number of scans ¼ 64 for
urine and n ¼ 128 for plasma, serum and tissue extracts, and the receiver gain is set to fill the digitizer as closely as
possible. This results in a total acquisition time of B4–5 min per sample (64 scans). In addition to these routine settings,
there has been some suggestion that a similar sequence could be employed, which uses gradients in the sequence and
improves solvent suppression quality (e.g., noesygppr1d). Then, only a short mixing time of 10 ms can be used, which is
of benefit for suppressing the relaxation effects for quantification.
(ii) For processing, apply a line broadening of 0.3–1 Hz to the FID and zero-fill to double the number of Fourier domain
points to 64k. A target spectral resolution is required, typically a line width at half height on the TSP resonance of not

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 PROTOCOL

42.5 Hz (with a line broadening of 1 Hz) and sufficient resolution in the manufacturer’s ‘hump’ test (Bruker, compare to
similar procedures from other providers) to ensure that the 29Si satellite peaks are evident. For urine samples containing
high levels of protein or salt concentration, this will not be achieved. Failure to reach this resolution need not cause the
data acquisition to be halted, but it can be used as a cut-off for subsequent data analysis. A target signal-to-noise ratio
on the TSP signal is not necessary, although this might be useful for making comparisons of spectrometer performance
at different field strengths and NMR probe volumes. A target signal-to-noise ratio on the peaks from the endogenous
metabolites is not possible due to some toxins causing large changes in urinary volumes and hence dilution.
(B) CPMG relaxation-editing sequence with presaturation
(i) In addition for serum or plasma, acquire relaxation-edited spectra using the CPMG-presat sequence, J-resolved spectra
and diffusion-edited spectra. At times, it might also be appropriate to use the CPMG-presat sequence for water-soluble
metabolites to attenuate the NMR signals of any remaining proteins. The CPMG-presat pulse sequence77 has the form
–RD-901-(t-1801-t)n-ACQ, where the definitions are as above, plus 1801 is a 1801 RF pulse, t is the spin-echo delay and
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

n represents the number of loops. Typically, the number of scans is 8 n n, number of dummy scans ¼ 16, number of
scans ¼ 128, number of time domain points ¼ 32k, spectrum width ¼ 20 p.p.m., relaxation delay ¼ 2 s, acquisition
time ¼ 1.36 s, number of loops, n ¼ 80 or higher (depending on the intensity of macromolecular peaks), spin-echo delay,
t ¼ 400 ms, giving a total echo time ¼ 64 ms. Irradiation is at the water peak during RD.
(C) J-resolved
(i) Set up the J-resolved pulse sequence61 in the form –RD-901t1-1801-t1-ACQ, where t1 is an incremented time period,
number of scans ¼ 4 n n, number of dummy scans ¼ 16, number of scans per increment ¼ 16, number of time domain
points ¼ 32k, number of increments ¼32, spectral widths ¼ 20 p.p.m. (F2) and 40 Hz (F1), acquisition time ¼ 1.36 s,
relaxation delay ¼ 2 s.
(ii) After Fourier transformation (it might be necessary to use linear prediction to improve results), perform baseline
correction, then tilt by 451 to ensure that all the J-coupled multiplets (the F1 axis) are orthogonal to the chemical
shift axis (F2), baseline-correct again, then symmetrize the spectra about the J ¼ 0 Hz line. Although the 2D J-resolved
spectrum is obtained, pattern recognition is often carried out on the sum or skyline F2 projection.
(D) Diffusion-edited
(i) Acquire diffusion-edited spectra using a pulse sequence78 with bipolar gradients and the LED scheme that has the
form -RD-901-G1-1801-G1-901-G2-T-901-G1-1801-G1-901-G2-t-901-acquire FID, where RD is a relaxation delay, 901 is a 901
RF pulse, G1 is the pulsed-field gradient that is applied to allow editing, 1801 is a 1801 RF pulse, G2 is a spoil gradient
applied to remove unwanted magnetization components. The diffusion delay D is the time during which the molecules are
allowed to diffuse—this is the period (901-G1-1801-G1-901-G2-T-); and t is a delay to allow the longitudinal eddy currents
caused within the sample to decay. The settings are relaxation delay ¼ 2 s, number of scans ¼ 64, number of time domain
points ¼ 32k, spectral width ¼ 20 p.p.m., acquisition time ¼ 1.36 s, D ¼ 0.1 s, t ¼ 5 ms, the length of G1 is 1 ms
(recovery delay: 100 ms) and of G2 is 2 ms, the ratios of the gradients are given in Supplementary Notes.
(E) Normal one-pulse sequence
(i) The 1H NMR spectra of lipidic extracts do not require presaturation of the water resonance. Here, use a simple 901
pulse-acquire sequence, if measuring fully relaxed spectra. Alternatively, the Ernst angle can be used to acquire a higher
number of scans in shorter time and to obtain an optimum signal-to-noise ratio79,80. Typically, collect 64 transients into
32,768 data points, other parameters are relaxation delay ¼ 2 s, spectral width ¼ 20 p.p.m., acquisition time ¼ 1.36 s.
? TROUBLESHOOTING

 TIMING
Sample preparation
Sample preparation of biofluids can be automated and takes B1–1.5 h per 96-well plate. Tissue extract preparation is generally
performed manually and takes B4–6 h per sample batch; the process takes longer if drying down of organic solvents with
nitrogen gas is involved.

NMR acquisition
For the NMR experiments, a throughput of 120 urine samples a day, 48 serum/plasma samples (1D NOESY-presat & CPMG-presat)
or 72 extracts can be achieved.

? TROUBLESHOOTING
Main problems can be avoided by ensuring that water offset and 901 pulse length are adjusted on representative samples. Also
very important: ensure that sample volumes are constant to avoid problems with automatic locking or shimming.
Problems with baseline rolling (‘wiggles’) can occur when sample concentrations vary too much and the receiver may be
overloaded. In this case, select automatic receiver gain adjustment.

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 PROTOCOL

Another problem can be the water presaturation when 4

sample concentration is low. This can be specifically 3 Ctrl Low dose High dose
+168
+72 +96
problematic when the aqueous samples are run in a 2 +120
cryoprobe where radiation damping effect is much stronger. 1 +144
+48
In these cases, excitation sculpting has proven very useful +0
–16

t [2]
0 +48 +0
(a possible sequence in Bruker terminology is called zgesgp).
–1
+24 +24

ANTICIPATED RESULTS –2
+8 +8
Reproducibility –3

Over the past decade, NMR-based metabonomics has been –4

–12 –10 –8 –6 –4 –2 0 2 4 6
applied successfully to investigate many different areas of t [1]
physiological variation, toxicity, disease and, recently,
© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

Figure 4 | Mean score trajectories of principal component analysis of urinary

response prediction. All studies have in common that they
NMR spectral data for each dose group (control, low-dose, high-dose) showing
require thorough study planning and experimental execution. progression of metabolic effects of hydrazine treatment. Time of sampling is
Studies, which can be treated as pilot studies have smaller indicated. High-dose data also show standard deviations across replicates
sample numbers and are sufficient to spot trends or strong (n ¼ 8 up to 48 h; n ¼ 4 post 48 h). NMR data from one site only
responses in metabolism. Studies with larger sample numbers is shown21. Reprinted with permission from ref. 21. Copyright (2002)
American Chemical Society.
are necessary to evaluate findings using, for example, training
and test set classifications. For all these applications, it is
paramount that the analytical technology has a high reproducibility, implying that the metabolic effects of disease or
treatment as detected with metabonomic technology, as well as the intersubject variation, are much higher than the
analytical variability.
Two large-scale studies have been used to validate the reproducibility of the metabonomic platform, as well as for the
investigation of metabolic effects. One is a population study (INTERMAP50) analyzing phenotypic differences between 24-h
urine samples obtained from volunteers in Japan (n ¼ 259), USA (n ¼ 315) and China (n ¼ 278). The study was used to
investigate analytical reproducibility, urine sample storage procedures, inter-instrument variability and split-specimen detection.
It was found that the multivariate analytical reproducibility of the NMR screening platform was 498%, and most classification
errors were due to urine specimen handling variability. As a further result of this study, novel combinations of biomarkers were
identified, which separated the samples from different populations. These findings were attributed to differences in
genetic, dietary and gut microbiological factors.
Particularly in the analysis of toxicity, a comprehensive exploration was achieved by the COMET consortium formed between
five pharmaceutical companies and Imperial College London, UK, with the aim of developing methodologies for the acquisition
and evaluation of metabonomic data generated using 1H NMR spectroscopy of urine and blood serum from rats and mice for
preclinical toxicological screening of candidate drugs41. Numerous methodological publications on the use of metabonomics
in developing screening modules arose from the COMET project42. The COMET group showed that it is possible to construct
predictive and informative models of toxicity using NMR-based metabonomic data, delineating the whole time course of
toxicity43. The project goals of the generation of comprehensive metabonomic databases (B35,000 NMR spectra covering a
wide range of model toxins and treatments, 147 in total) and successful and robust multivariate statistical models (expert sys-
tems) for prediction of toxicity, initially for liver and kidney toxicity in the rat and mouse, were achieved42,43.
As part of this project, the analytical reproducibility of metabonomic protocols, sample preparation and NMR data acquisition
were investigated. These were performed at two sites (one using a 500 MHz and the other using a 600 MHz NMR system)
analyzing two identical (split) sets of urine samples from an 8-d acute study of hydrazine toxicity in the rat21. Figure 4
shows the response profile after treatment with a low and a
4
high dose of hydrazine in a principal component analysis
3
(PCA) plot. A position on the map symbolizes one metabo-
nomic fingerprint (an NMR spectrum) of a urine sample. 2

Figure 5 | Score (t) scatter plots for PC1 versus PC2 from a principal 0
t [2]

component analysis model using the data acquired at two different sites using
–1
the same protocols for the analysis of split aliquots in a study on hydrazine
toxicity (triangles and circles represent the two different datasets, –2
respectively). The ellipse denotes the 95% significance limit of the model. An –3
asterisk indicates a putative outlier. The plot clearly highlights that
–4
interaliquot differences are very low compared to the physiological
changes seen at different time points: the composition changes from
16 h –5
(right hand corner) to 72–96 h (left hand corner) and back again to 168 h *
(right-hand corner), following an L-shape21. Reprinted with permission from –10 –8 –6 –4 –2 0 2 4 6 8
ref. 21. Copyright (2002) American Chemical Society. t [1]

2700 | VOL.2 NO.11 | 2007 | NATURE PROTOCOLS

 PROTOCOL

If this position changes in the course of a study, it means that 8

at least the concentration of one of the metabolites in the 6

urine NMR spectrum changes (often, several metabolites
4
change at the same time). In this particular example, every
dot is the average of either four or eight spectra (four or eight 2

individuals) at a certain time point. It can be clearly seen that

PC2 score
0
the magnitude of response to the toxin is dose-dependent, but
–2
follows a similar trajectory (time profile).
Mercury chloride
The same metabonomics protocol for sample preparation and –4
Hexachlorobutadiene
NMR acquisition was performed at another site, preparing and Hydrazine (Study B)
–6
analyzing split aliquots of the same sample. Figure 5 Hydrazine (Study E)

illustrates that, despite analysis on different spectrometers, –8 Streptozotocin

© 2007 Nature Publishing Group http://www.nature.com/natureprotocols

the obtained results were very similar and gave near-identical –10
descriptions of the metabolic response (trajectory) to –10 –5 0 5 10 15 20 25 30
PC1 score
hydrazine treatment. The main consistent difference between
the datasets was related to the efficiency of water resonance Figure 6 | Metabonomics identifies different responses from the urine profiles
suppression in the spectra. of animals treated with different toxins. A high similarity between trajectories
In this model of both datasets combined, describing all sys- can be seen where toxins which affect the same organ were given
(hydrazine: liver; mercury chloride and hexachlorobutadiene: kidney;
tematic dose- and time-related variation, differences between
streptozotocin: pancreas). Each dot on the PCA plot symbolizes the
the two datasets (split-sample differences) accounted for only mean of ten urine samples up to 48 h, and five urine samples until 168 h
3% of the total modeled variance compared to B15% for (courtesy of H. Keun, IC).
normal physiological (predose) variation. Furthermore,
o3% of spectra displayed distinct inter-site differences, and these were clearly identified as outliers in their respective
dose group PCA models. It was noted that no samples produced clear outliers in both data sets, suggesting that the outliers
observed did not reflect an unusual sample composition, but rather sporadic differences in sample preparation leading to, for
example, very dilute samples.
Any variation in the comparison of split samples needs to be seen in relation to the intersample variation between control
animals, and, eventually, to the metabonomic effect of a treatment or disease. Both, interanimal variation between controls as
well as the metabonomic effect, were much higher, and this underlines the robustness and high reproducibility of the NMR-
based metabonomic technology.
Metabonomics is well suited for the detection of different toxic episodes and other metabolic alterations. Our example in
Figure 6 highlights that different toxins, for example, affecting kidney, liver and pancreas, give rise to different metabolic
trajectories with different magnitude, direction, onset and recovery. This metabolic information was used to establish an expert
system for the predicition of toxicity within COMET43.
The protocols described in this publication for the analysis of urine, blood samples and tissue extracts have been optimized
to provide high-quality data with a range of sample types; but experiments can always be tuned to give optimal performance for
particular samples. Hence, they are not to be regarded as being unchangeable. However, if large-scale statistical analysis is to
be performed on such data sets, then it is imperative to maintain a constancy of detailed experimental protocol; otherwise,
systematic analytical differences will impair data set comparison and may give rise to false biomarker reporting.

Note: Supplementary information is available via the HTML version of this article.
ACKNOWLEDGMENTS We thank our academic and industrial collaborators for
helpful discussions in the formulation of this paper, including those participating
in the COMET project.
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.com/
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