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Published on 04 January 2018 on http://pubs.rsc.org | doi:10.1039/9781782627937-FP001
NMR-based Metabolomics
View Online
New Developments in NMR
Editor-in-chief:
William S. Price, Western Sydney University, Australia
Series editors:
Sharon Ashbrook, University of St Andrews, UK
Bruce Balcom, University of New Brunswick, Canada
István Furó, Industrial NMR Centre at KTH, Sweden
Masatsune Kainosho, Tokyo Metropolitan University, Japan
Maili Liu, Chinese Academy of Sciences, Wuhan, China
Titles in the series:

1: Contemporary Computer-Assisted Approaches to Molecular Structure
Elucidation
2: New Applications of NMR in Drug Discovery and Development
3: Advances in Biological Solid-State NMR
4: Hyperpolarized Xenon-129 Magnetic Resonance: Concepts, Production,
Techniques and Applications
5: Mobile NMR and MRI: Developments and Applications
6: Gas Phase NMR
7: Magnetic Resonance Technology: Hardware and System Component
Design
8: Biophysics and Biochemistry of Cartilage by NMR and MRI
9: Diffusion NMR of Confined Systems: Fluid Transport in Porous Solids
.
and Heterogeneous Materials

10: NMR in Glycoscience and Glycotechnology
11: Fast NMR Data Acquisition: Beyond the Fourier Transform
12: Cross-relaxation and Cross-correlation Parameters in NMR: Molecular
Approaches
13: Contrast Agents for MRI: Experimental Methods
14: NMR-based Metabolomics
How to obtain future titles on publication:

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delivery of each new volume immediately on publication.
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Edited by
Hector C. Keun
Imperial College London, UK
Email: h.keun@imperial.ac.uk
.
Published on 04 January 2018 on http://pubs.rsc.org | doi:10.1039/9781782627937-FP001 View Online
New Developments in NMR No. 14
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Preface
NMR spectroscopy has long been a leading technique in the study of meta-
bolic biochemistry, with a rich literature predating the terms ‘metabolomics’
and ‘metabonomics’. However, with the advent of metabolic profiling as an
independent field of research the power and versatility of a technique that
can be readily applied to many biological matrices to provide highly compa-
rable, inherently quantitative, largely unselective compositional descriptions
has been revealed. This is why, despite the superior sensitivity and metab-
olome coverage of rival techniques such as mass spectrometry, NMR still
offers so much to metabolic profiling studies.
As a final year undergraduate I was first introduced to the concept of
taking NMR spectra of biological samples and using pattern recognition
analysis to discern the information about pathology and drug response. It
seemed at the time an impossible and incredible task and I was glad not to
.
be attempting it. The spectra generated and the biological problem appeared
too complex, with too many possible sources of uncertainty and variability.
After many years working in the field many of these sources of variation
are better rationalised, with a growing understanding of genetic and envi-
ronmental influences. Indeed, this complexity is increasingly embraced as
an advantage, for example, in the study of symbiotic microbes and health
where NMR offers a window into the metabolic cross-talk between multiple
genomes and a myriad of exposures. However, I personally have found that
the most rewarding aspect of using NMR spectroscopy in this context is its
ability to reveal the completely unexpected, sometimes a simple and obvious
but critical observation hidden just out of sight of the investigative team. I
can attest to many projects, laboratory and clinical, rescued by the unique
perspective offered by NMR, and even in today’s world of large-scale studies

Edited by Hector C. Keun
Published by the Royal Society of Chemistry, www.rsc.org
v
View Online
vi Preface
using established methodology, the data generated are continuing to teach
us new and unpredicted things about metabolism.
I salute all the pioneers of the field, cited throughout this volume, for
having the vision and perseverance to realise the potential of the approach,
which, in the context of biofluid NMR, is now well on the way to successful
translation into an important clinical tool. I hope that readers of this volume
will find it a useful introduction to the methodology and many of the appli-
cations of NMR-based metabolomics, and that it will help to germinate new
success stories for the field.
Hector C. Keun
.
Acknowledgements
I would like to thank all the colleagues who contributed to this volume for
their great patience and efforts. In particular I am very grateful to Toby
Athersuch for the unstinting enthusiasm, creativity and hard work he has
given to this project and many of our other joint endeavours over the years.
Finally I would like to thank my wife Ellie, for her tolerance and support for
all that I try to do, and who has heard infinitely more about this book than
she would ever have wished to.
.

vii
Contents
Chapter 1 Instrumental Platforms for NMR-based Metabolomics 1

Anthony C. Dona
1.1 H istory of NMR Hardware Development 1

1.2 Components of NMR Hardware 3
1.2.1 Magnet 3
1.2.2 Shim Coils 4
1.2.3 Sample Probe 4
1.2.4 Digital Filtering 8
1.2.5 Computational Support to Hardware 9
1.3 Automation of Metabolomic Profiling 10
1.3.1 Sample Preparation 11
1.3.2 Automated NMR 13
.
1.3.3 Automated Acquisition 16

1.3.4 Integrated Metabolic Profiling NMR Systems 19
References 20
Chapter 2 NMR Pulse Sequences for Metabolomics 22

Bénédicte Elena-Herrmann
2.1 I ntroduction 22
2.2 Standard 1D NMR Pulses Sequences for
High-throughput Metabolomics 23
2.2.1 One-dimensional 1H NOESY Experiment 23
2.2.2 The Carr–Purcell–Meiboom–Gill (CPMG)
Experiment 25
2.2.3 Diffusion-edited NMR Spectroscopy 25

ix
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x Contents
2.3 W ater and Solvent Suppression for NMR of
Biofluids 27
2.3.1 Water Pre-saturation 27
2.3.2 Advanced Water Suppression Schemes 28
2.4 Multidimensional NMR Techniques for Metabolite
Identification and Quantification 28

2.4.1 Proton Homonuclear Correlation
Spectroscopy 29
2.4.2 2D 1H–13C Heteronuclear Correlation
Experiments 31
2.4.3 Metabolite Quantification from
Two-dimensional NMR 32
2.5 Novel Strategies for Fast NMR Data Acquisition
in Metabolomics 33
2.5.1 Non-uniform Sampling 33
2.5.2 Fast Targeted Multidimensional NMR
Spectroscopy 33
2.5.3 Ultrafast 2D NMR for Metabolite
Quantification 36
2.6 Conclusion 37
References 37
Chapter 3 NMR Spectroscopy of Urine 39

Barry Slaff, Arjun Sengupta and Aalim Weljie
.
3.1.1 NMR Spectra of Urine and Conventional
Normalization to Creatinine 40
3.1.2 Early Applications: Urine NMR
Metabolomics in Toxicology 43
3.1.3 Analytical Reproducibility of Urine NMR
Spectra 46
3.2 NMR Spectroscopy of Urine in Metabolomics
Studies 48
3.2.1 Collection and Storage of Urine Samples 48
3.2.2 Preparation of Urine Samples for NMR 53
3.2.3 Recommendations Pertaining to Urine
Sample Collection, Storage and Treatment
with a Focus on Diagnostic Study 56
3.2.4 One-dimensional NMR Experiments and
Suppression Methods for Use with Urine 57
3.2.5 Two-dimensional NMR Experiments and
Suppression Methods for Use with Urine 59
3.2.6 Normalization of Urine NMR Spectral
Datasets 60
3.2.7 Multivariate Statistical Analysis of
NMR Urine Spectra 63
View Online
Contents xi
3.3 N MR Spectroscopy of Urine: Systems Biology
Applications 66
3.3.1 Metabolite Variation in Urine from Healthy
Subjects 66
3.3.2 Metabolite Variation in Urine Between
Healthy Population Groups 66

3.3.3 Unhealthy Pathophysiologies, Disease
Diagnosis, and Pharmacometabolomics 70
3.4 Conclusion 72
References 73
Chapter 4 NMR Spectroscopy of Serum and Plasma 85

Hector C. Keun
4.1.1 Sample Composition and Metabolome
Coverage 86
4.2 Methodology 89
4.2.1 Comparison of Sample Preparation Methods 90
4.2.2 Data Acquisition 94
4.3 Applications 105
4.3.1 Sample Collection and Pre-analytical Variation 105
4.3.2 Confounding and Normal Variation 107
4.3.3 Cancer 108
4.3.4 Cardiovascular Disease 110
.
4.3.5 Diabetes Risk 112

4.3.6 Genetic Influences on the Serum NMR
Metabolome 113
4.3.7 Toxicology 114
4.4 Future Perspectives 115
References 116
Chapter 5 High-resolution Magic-angle Spinning (HR-MAS)

NMR Spectroscopy 133
Alan Wong and Covadonga Lucas-Torres

5.2 HR-MAS Basic Concepts 134
5.2.1 Magnetic Susceptibility Broadening 134
5.2.2 Magic-angle Spinning 136
5.3 Hardware and Practical Considerations 138
5.3.1 Magnetic Susceptibility Components 138
5.3.2 B0 Field Correction 138
5.3.3 B0 Field Locking 140
5.3.4 Sample Temperature 140
5.3.5 Pulse-field Gradient 140
5.3.6 Pulse Experiments 141
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xii Contents
5.4 R ecent (HR)-MAS Developments Towards
NMR-based Metabolomics 142
5.4.1 In vivo Studies 142
5.4.2 Slow MAS Experiments 142
5.4.3 Magic-angle Field Spinning 144
5.4.4 Microscopic Quantity 145

5.5 Concluding Remarks 147
Acknowledgements 147
References 147
Chapter 6 Investigation of Tumor Metabolism by High-

resolution Magic-angle Spinning (HR-MAS)
Magnetic Resonance Spectroscopy (MRS) 151
May-Britt Tessem, Siver A. Moestue and Tone F. Bathen
6.1 H R-MAS MRS for Studies of Metabolic

Abnormalities in Cancer 151
6.2 HR-MAS MRS in Metabolic Profiling of
Intact Tumor Tissue—Clinical Studies 154
6.2.1 Breast Cancer 154
6.2.2 Prostate Cancer 154
6.2.3 Brain Tumors and Brain Metastases 155
6.2.4 Other Cancer Types 156
6.3 HR-MAS MRS in Metabolic Profiling of Intact
Tumor Tissue—Preclinical Disease Models 157
.
6.4 Technology Developments and Optimized

Protocols for HR-MAS MRS Analysis 158
6.4.1 Tissue Sampling and Harvesting 158
6.4.2 Protocols and Sequences 158
6.4.3 Quantification of Metabolites Using
HR-MAS MRS 159
6.4.4 Multivariate Analysis 160
6.4.5 Correlation with in vivo Spectroscopy 160
6.4.6 HR-MAS MRS and Gene Expression Analysis 161
6.5 HR-MAS MRS of X-Nuclei 161
6.5.1 13
C HR-MAS MRS 161
6.5.2 31
P HR-MAS MRS 162
6.6 Future Prospects of HR-MAS MRS in Cancer
Metabolomics 162
References 163
Chapter 7 NMR in Environmental and Nutritional Research 168

Toby J. Athersuch and Anisha Wijeyesekera

7.2 NMR-based Analytical Methods in
Environmental and Nutritional Research 170
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Contents xiii
7.2.1 S ample Types 170
7.2.2 Common NMR Spectroscopy Experiments 171
7.2.3 Practical Aspects of NMR Spectroscopic
Analysis in Nutritional and Environmental
Research 171
7.3 Applications of NMR-based Metabolomics in

Nutritional Research 173
7.3.1 Nutritional Exposure Characterisation—
Compositional Analysis of
Foodstuffs 173
7.3.2 NMR-based Biomarkers of Food
Consumption 174
7.3.3 Assessing the Influences of Diet on the
Metabolome 176
7.4 Application of NMR-based Metabolomics in
Environmental Health Research 177
7.4.1 Environmental Exposure Characterisation 177
7.4.2 Deriving Biomarkers of Environmental
Exposure 178
7.4.3 Assessing the Influences of the Environment
on the Metabolome 178
7.5 Summary 179
References 180
Chapter 8 NMR Foodomics 183

.
Francesco Savorani, Bekzod Khakimov, Nanna Viereck

and Søren Balling Engelsen
8.1 I ntroduction to NMR Analysis of Food 183

8.2 Sampling and Measuring Food with NMR 186
8.3 Data Analysis in NMR Foodomics Studies 193
8.3.1 Use PCA 195
8.3.2 Use Alignment 195
8.3.3 Use Intervals 197
8.3.4 Use Test Set and ROC 199
8.3.5 Use ASCA to Explore Designed Experiments 200
8.4 Selected NMR Foodomics Studies 202
8.4.1 Wine 202
8.4.2 Olive Oil 205
8.4.3 Tomato 207
8.4.4 Alginate 210
8.4.5 Milk 213
8.4.6 Cheese 219
8.4.7 Fish 221
8.4.8 Meat 225
8.5 Foodomics Outreach 226
References 228
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xiv Contents
Chapter 9 NMR-based Metabolomics: Understanding Plant
Chemistry and Identification of Biologically Active
Compounds 246
M. Jahangir, T. R. Nuringtyas, K. Ali, E. G. Wilson,
Y. H. Choi and R. Verpoorte

9.2 What Is the Metabolome? 248
9.2.1 Selective Extraction 248
9.2.2 The Metabolomics Process 248
9.3 Plant Under Stress 249
9.3.1 Host Plant Resistance 249
9.3.2 Biotic/Abiotic Stress 251
9.3.3 Species Characterization: Chemotaxonomy 251
9.4 Metabolomics to Evaluate Synergy 252
9.5 Bioactivity Screening 253
9.6 Food Metabolomics 255
9.7 Single-cell Metabolomics 256
9.7.1 Limitations of Single-cell Metabolomics 258
9.8 Conclusions 259
References 259
Chapter 10 1H NMR-based Metabolic Profiling in Infectious

Disease Research 264
Jasmina Saric, Sabrina D. Lamour and Jia V. Li
.

10.2 Human and in vivo Studies 267
10.2.1 Diagnostic and Prognostic Biomarkers 267
10.2.2 HIV/AIDS, Tuberculosis, and Malaria 268
10.2.3 Neglected Tropical Diseases 269
10.3 Systemic Characterisation of Parasite Effects in vivo 273
10.3.1 Global Metabolic Changes in the
Murine Host 273
10.3.2 Immune–Metabolic Co-development 273
10.4 In vitro Studies 274
10.5 Conclusions 276
References 277
Chapter 11 Imaging Metabolic Processes in Living Systems

with Hyperpolarised 13C Magnetic Resonance 280
Deborah K. Hill, Erika Mariotti and Thomas R. Eykyn
11.1 H
yperpolarisation 280
11.2 Dynamic Nuclear Polarisation 283
11.3 Magnetic Resonance Detection of Hyperpolarised
Metabolites 286
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Contents xv
11.4 Deriving Kinetic Parameters 291
11.5 Biomedical Applications of DNP 295
11.6 Hyperpolarised 13C Imaging in vivo 299
References 303
Chapter 12 Advances in Computational Analysis of

Metabolomic NMR Data 310
Timothy M. D. Ebbels, Andrea Rodriguez-Martinez,
Marc-Emmanuel Dumas and Hector C. Keun

12.2 Bayesian Methods in NMR Data Processing 311
12.3 Developments in Statistical Correlation Analysis 313
12.4 Statistical Association Networks 313
12.5 Genetic Mapping of Metabolic Phenotypes 317
12.5.1 mQTL Mapping Workflow 317
12.5.2 Lessons Learnt from mQTL Studies 318
12.6 The Role of Data Standards 319
References 320
Chapter 13 NMR Spectroscopy of Cell Culture, Tissues,

and Other Biofluids 324
Toby J. Athersuch, Chung-Ho Lau, Volker Behrends
and Hector C. Keun
.
13.1 G eneral Introduction 324

13.2 Sampling, Extraction and Analysis of Cellular
Material 325
13.2.1 Introduction 325
13.2.2 Sampling Cellular Material 327
13.2.3 Quenching Metabolism 329
13.2.4 Extraction of Intracellular Metabolites 330
13.2.5 NMR Spectroscopy of Cellular Materials 334
13.3 Cellular Material Profiling Applications 336
13.4 Other Biofluids 338
13.4.1 Introduction 338
13.4.2 Faeces 341
13.4.3 Cerebrospinal Fluid 343
13.4.4 Milk 345
13.4.5 Seminal Fluid 348
13.4.6 Bile 349
13.4.7 Less Commonly Reported Biofluids 352
13.5 Summary and Future Developments 352
Acknowledgements 353
References 353
Subject Index 360

Published on 04 January 2018 on http://pubs.rsc.org | doi:10.1039/9781782627937-00001
Chapter 1
Instrumental Platforms for

Anthony C. Dona†
MRC-NIHR National Phenome Centre, Department of Surgery & Cancer,

Imperial College of London, South Kensington Campus SW7 2AZ, United
Kingdom
*E-mail: anthony.dona@sydney.edu.au
1.1 History of NMR Hardware Development

The basic function of an NMR spectrometer is to measure the frequency of
.
the resonance of a given nucleus. After the first decade of discovery (1946–
1955), the basic NMR relationship was established (eqn (1.1)), which sug-
gests the resonance frequency of a nucleus (ω) to the magnetogyric ratio (γ,
specific to nucleus type) and the external magnetic field (B).

ω = γB (1.1)

At this stage it was thought that the frequency of a nucleus depended
entirely on the strength of the magnetic field in which it was placed. In 1949–
1950, however, observations from 19F and 31P showed variations in frequency
†
urrent address - Northern Medical School, Kolling Institute of Medical Research, The Univer-
C
sity of Sydney, Royal North Shore Hospital, St. Leonards, NSW 2065, Australia.

1
View Online
2 Chapter 1
between the two types of nucleus beyond the still rather large experimental
error. Furthermore, after developments in the stability and homogeneity of
the magnetic field, two separate resonances where observed from the hydro-
gen atoms in ethanol1 and separately for nitrogen atoms in different chemi-
cal environments. This phenomenon soon became known as ‘chemical shift’
as the frequency of a resonating nucleus is largely dependent on the local

chemical environment surrounding the nucleus itself. Further improve-
ments in the resolution allowed separate resonance lines to be observed
from a single chemical resonance, allowing the discovery of the concepts
of indirect spin–spin coupling or scalar coupling.2 In the late 1950s and the
into the 1960s the strength of the field increased to 100 MHz and commercial
instruments were developed by Varian that maintained a constant relation-
ship between the magnetic field and the radiofrequency (RF) applied so spec-
tra could be recorded at a known scan rate. In this time the first 13C spectrum
was recorded,3 made difficult at the time by the low natural abundance of 13C
atoms (1.1%). Carbon spectroscopy really became popular with the advent
of double resonance, where two RF fields are applied to a sample simulta-
neously in order to measure one spin system while the other is perturbed.
From double resonance, applications such as spin decoupling experiments
and nuclear Overhauser effect (nOe) were introduced to aid studies of molec-
ular conformation.4
In 1966, Ernst and Anderson published work5 showing that Fourier Induc-
tion Decay (FID) following a short RF pulse was all that was necessary to
produce a spectrum measuring a range of frequencies. Additionally, mini-
computers were being developed to interface directly with the spectrometer,
.
allowing Fourier Transform NMR (FT-NMR). These hardware advances rev-

olutionised NMR spectroscopy, supporting the enhancement of sensitivity
(which was NMR's main disadvantage compared with other spectroscopy
techniques) and enabling exploitation of time-dependent NMR phenome-
non, namely relaxation.
Since these major advances in NMR hardware, many pulse sequences and
applications have developed over the last 40 years, making NMR spectro
scopy an incredibly versatile tool in chemical and biochemical research
areas. Magnetic Resonance Imaging (MRI) was also made possible by impos-
ing magnetic field gradients across a sample in vivo. Two-dimensional and
eventually three-dimensional NMR imaging was made possible with wide
bore magnets (wide enough to fit animals or humans through) and the mea-
sure of a frequency across a spatial gradient.6 Techniques are also currently
being produced to integrate spectroscopy with imaging to obtain localised
spectra in living creatures.
These days, the hardware available for modern-day NMR measurements
allows for routine acquisitions with relative ease, in small (metabolite, organ-
ically synthesised) and large (protein) molecules, in either purified solutions
(for molecular structure elucidation) or complex mixtures (for solution com-
position elucidation).
View Online
Instrumental Platforms for NMR-based Metabolomics 3
1.2 Components of NMR Hardware

1.2.1 Magnet
The NMR magnet itself is generally considered the most important part of
the NMR spectrometer and is commonly the most expensive piece of equip-
ment for a standard NMR laboratory. Before the advent of superconducting

magnets, iron-core electromagnets had enabled field strengths of 2.35 T to be
reached. At this stage iron saturates, so a great effort was required to develop
a magnet that could be housed in a liquid helium Dewar to obtain the tem-
peratures required to cool a superconducting solenoid. Today, commercial
NMR magnets are generally superconducting and range in field strength
from 6 to 23.5 T. Other physical chemistry laboratories are developing larger
magnets, not intended for commercial distribution, with magnetic field
strengths of up to 45 T.7 With increasing strength, not only does the size of
the magnet need to increase but technologies need to be developed to break
ground in new magnet generation. As magnets increase in size (and so mag-
netic field strength) their resolution and sensitivity of frequencies improves,
but their cost also increases substantially, meaning larger magnets are much
less accessible.
Superconducting wires are generally made from Nb3Sn or (NbTaTi)3Sn,
which are wrapped hundreds of times into a coil making up a length of wire
up to 100 km long. Few higher temperature superconducting materials are
suitable for manufacturing in the quality and quantity required for NMR
magnets, so very low temperature superconductors (∼10 K) are usually con-
structed. The wire has a rectangular cross section allowing maximum cur-
.
rent density and therefore maximum magnetic field strength. This coil is
kept inside a large Dewar containing liquid helium, keeping the coil at super-
conducting temperatures, which is in turn surrounded by a liquid nitrogen
reservoir acting as a buffer between the room temperature air and the liquid
helium. A significant property of the wire is the maximum critical current
(Ic), which is a function of the operational temperature (T) and magnetic field
(B). If the critical value is reached there is a transition in the wire from a
superconductive to a resistive state, which in turn generates heat. The heat
propagates rapidly through the coil prompting the energy store to be con-
verted to heat, which induces the helium store to boil extremely rapidly. The
loss of the superconductive state is known as a ‘quench’ and magnetic coils
are developed to avoid quenches at all costs. Modern day magnets have an
additional coil outside the main coil, which is used to contain the strong
magnetic field by cancelling (shielding) the stray field, restricting it to a rela-
tively small area.
The cryostat, which is the vessel surrounding the magnetic coil, must also
be designed to be insensitive to variations in the environment and other dis-
turbances such as helium evaporation rate, ambient temperature and pres-
sure, and the cryogen levels inside the cryostat. New technologies in this area
are focused around either using the complete enthalpy stored in the helium
View Online
4 Chapter 1
gas to enable further cooling of the system or otherwise recycling helium
such that it is not lost to the atmosphere. These technologies are aimed at
having the lowest possible helium consumption as it becomes a rarer and
more expensive commodity.
NMR laboratories are often forced to compromise on magnetic field
strength owing to available funding and space. Modern day magnets between
400 and 600 MHz are commonplace in metabolomics laboratories as they are
now constructed to fit in a room without roof height modification. With mod-
ern day shielding, the footprint required for the magnetic field is not much
larger than the magnet itself and so magnets of this size can essentially be
lined up next to one another. Magnets of 600 MHz are produced much more
readily than those of larger size and so the production cost is far cheaper and
more reliable, making them the magnet size of choice for routine metabolo-
mics studies. On the other hand, magnet sizes smaller than 400 MHz do not
enable researchers to resolve important metabolite signatures in complex
biofluids and so these are generally overlooked when purchasing a magnet
for metabolomics purposes.
1.2.2 Shim Coils

Shim coils are a set of conducting coils used to adjust the homogeneity of a
magnetic field. In the past, shimming (the process of optimising the homo
geneity of the magnetic field) a magnet consisted of attaching thin metal
shims in various positions around the permanent magnet. Coincidently, the
term ‘shimming’ is used to describe the modern day process of homogenising
.
the magnetic field across which the samples nuclei frequencies are measured.
Modern high-resolution spectrometers alter the current in various conduct-
ing coils, which surround the external magnetic field, to alter it homogeneity.
When a spectrometer is installed, the local environment can disturb the
magnetic field. Iron constructs in the walls and floor of the surrounding
building disturb the homogeneity of the spectrometer's magnetic field. There-
fore, upon installation spectrometers need to be roughly shimmed, after the
initial activation of the magnetic field, with regards to the external environ-
ment. Once relative homogeneity is achieved, relatively minor changes in
the magnetic field as a result of variations in the sample, the tube thickness
and movement of ferromagnetic materials around the magnet are corrected
before sample acquisition. These minor changes in the magnetic field are
adjusted by changing the current in one or more (of up to 40) small shim
coils with various gradients along all three spatial axes. In high-resolution
spectrometers the magnetic field often demands homogeneity better than
1 part per billion in a tube, which is generally less than a millilitre in volume.
1.2.3 Sample Probe

The probe is the part of the spectrometer that does a lot of the physical exper-
imental work and laboratories can often gain significant improvements in
spectral quality by upgrading their spectrometer's probe. The probe contains
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the RF coils tuned to specific frequencies to excite particular nuclei and coils
to detect the NMR signal. Pulsed Field Gradient coils, which allow for the
application of field gradients, are also commonplace, allowing for the appli-
cation of field-gradient pulses. The probe must also consist of the necessary
hardware to measure and control the temperature of samples (a thermocou-
ple device).
An important aspect of probe design is the size of the bore, which can alter
to accommodate various sizes of tube. Small volume probes (3 mm) or nano-
tubes (1.7 mm) are able to give the greatest sensitivity, benefiting from the
dramatic decrease in the diameter of the NMR detection coil. Some modern
day probes are able to record good quality spectra from microlitre or even
picolitre sample volumes. Nevertheless, in many cases larger volume bores
(5 mm, 10 mm or wide bore) are necessary as the solubility or concentra-
tion of the sample is an issue. Larger volume bores are also recommended
when measuring samples of higher viscosity or samples with micro-scale
inhomogeneities (blood or emulsions), and are necessary when imaging
small animals.
1.2.3.1 Radiofrequency Coils
Modern day probes are constructed with two coils to record NMR signal
(known as observe coils). They are wrapped around one another such that
there is an inner coil and an outer coil, allowing the probe to respond to dif-
ferent frequencies during the one experiment. This design also allows mul-
tiple nuclei to be excited during one pulse sequence. There are two main
.
approaches to the design of these coils:

-- A ‘Broadband Observe Probe’ is constructed with the inner coil tuned to
a broadband nucleus, and so is optimised for maximum sensitivity for
nuclei at lower frequency (13C, 31P, etc.).
-- ‘Inverse Probes’ or ‘Indirect Detection Probes’ have the inner coil tuned
to measure the frequency of 1H atoms and so get maximum sensitivity
for proton experiments with much lower sensitivity when observing
lower frequency nuclei.
1.2.3.2 Cryoprobes
Sensitivity of detection has dramatically improved with the advent of cryo-
probes. These probes are significantly more expensive in design but ensure
2–4 times better sensitivity than standard probes. Cryoprobes have achieved
the single largest jump in sensitivity enhancement by probe development in
the last few decades by cryogenically cooling probe detector coils and pre-
amplifier coils. As long as these coils, along with the tuning and matching
circuits, are maintained at low temperatures the noise generated owing to
random thermal motion of electrons is kept at a minimum. The resistivity
in metals based in the preamplifier and filters is also cooled, improving
the level of noise generated owing to the probe's electronic components.
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6 Chapter 1
There are currently two types of cryoprobe available, the first is cooled a by
closed cycle helium cooler system, which yields a signal-to-noise enhance-
ment up to a factor of five. The second type is a liquid nitrogen-cooled sys-
tem, which has the advantage of being able to be cooled down and warmed
up relatively quickly, although is only able to provide a sensitivity enhance-
ment of up to a factor of three. Either one of these probes is generally applied

to compensate for the lack of sensitivity owing to magnet strength, or simply
to enhance a laboratory's run time capability.
1.2.3.3 Microprobes
The development of microprobes (or microcoil probes) was driven by the
pharmaceutical industry where detection of small sample amounts was
required with a relatively high throughput rate. The heart of the problem lied
in developing a system with increased sensitivity for small sample volumes.
The solution was simply to reduce the diameter of the RF coils; microprobes
are generally considered to have an RF coil of less than 1.6 mm inner diam-
eter. Therefore, there are few differences between standard NMR probes and
microcoil probes apart from the fundamental sample volume required. How-
ever, as the volume required is much less, the microprobes allow for many
more molecular biological applications.
While regular volume probes are based on Helmholtz saddle coils (coils
that lie parallel to the external magnetic field),8 microprobes of less than 45 µL
require a detection coil, which is typically a solenoid, that is placed orthog-
onal to the external magnetic field.9 The sample insertion into the magnetic
.
field therefore cannot be performed traditionally from the top of the magnet
using a conventional NMR tube. Sample insertion into a solenoidal coil is
generally controlled by a flow injection probe (Section 1.2.3.4). Solenoidal
probe interfaces are advantageous as they have a sensitivity level about 2–3
times larger than a traditional coil and only need an order of magnitude 1–2
times less than traditional NMR probes.
1.2.3.4 Flow Injection Probes

Flow injection probes were historically the first approach to conducting
high-throughput NMR. Physically a flow probe contains a glass chamber
within the RF coils with a total volume of between 1 µL and 120 µL. Flow
injection probes with volumes less than 10 µL are considered to be ‘micro-
coil’ flow probes (Section 1.2.3.3). The probe contains an inlet for solution to
enter the chamber and an outlet for the sample to be evacuated and rinsed
out. The probes can either be ‘stop-flow’, which means during the time of
acquisition the sample is motionless in the probe's chamber, or alternately
the probe can be ‘on-flow’ where the sample is flowing during the short ana-
lytical measurement of the sample by NMR. When using an on-flow probe,
though, the shim quality is compromised owing to the flowing solution mak-
ing it difficult to homogenise the magnetic field. Therefore, the results from
View Online

an on-flow probe are generally less resolved than a stop-flow probe, although
the throughput is higher. Furthermore, the concentration of the metabolites
of interest in the flowing sample needs to be considered before a measure-
ment and optimisation of the methodology for each sample type is almost
always necessary.
All flow techniques can be classified into one of two further separate
categories. The first simply uses flow injection as a means to transport an
untreated sample into the NMR analysis coil (almost always performed in a
stop-flow environment to better distinguish samples and improve the shim
quality), simplifying automation and speeding up throughput. The second
category subjects the sample to a separation technique such as chroma-
tography or electrophoresis before NMR analysis and can be performed in
either stop-flow (separating fractions) or on-flow (continuous analysis of the
probe's contents, which can be later processed separately). Flow injection
probes have had many applications over the last couple of decades and more
recently have been successfully applied to metabolic profiling of biofluids.10
Flow injection probes were invented at the turn of the century and have
since been applied in multiple applications relating to the pharmaceutical
industry and complex mixture analysis. Some examples of this are:

(1) NMR screening applications to discover ligands with a high affinity
to bind to proteins with various medical relevancies. Dubbed SAR-
by-NMR, organic molecules are screened to develop an understand-
ing of proximal subsites of proteins.11 The molecules are either used
themselves or as base molecules to develop high-affinity ligands.12 The
.
application is commonplace in the pharmaceutical industry and is a

great example of a successful flow probe application as sensitivity and
throughput are integral.
(2) A high-throughput method has also been developed using microcoil
probes for the production and analysis of large natural product librar-
ies for drug discovery.13 High-performance liquid chromatography
(HPLC) is coupled online with flow injection NMR to rapidly isolate and
characterise bioactive products from plant roots, stems, leaves, flowers
or fruits. Developing libraries of naturally occurring molecules through
the use of HPLC-NMR is considered a technique as valuable to develop-
ing drug leads as combinatorial chemistry.
(3) Identification of metabolite signals in complex mixtures is classically
considered more robust when multiple forms of spectral information
are acquired on a given sample or fraction. Alphanumeric metabolite
identification metrics have been proposed recently to rank identifica-
tion confidence based on the types of spectral data and comparisons
to authentic standards.14 As a result of metabolic sciences seeking
more robust metabolite identification, the direct coupling of HPLC
with NMR has been extended into a triple hyphenated online tech-
nique enabling UPLC-NMR-MS.15 The technique splits the eluent after
HPLC separation with conventional detection, with 95% of the eluent
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8 Chapter 1
flowing into an NMR flow injection probe and the other 5% directed
to the inlet of an ion-trap multipole mass spectrometer. Owing to the
technique's online approach, the development of automated routines
and high-throughput applications is feasible. The approach initially
allowed for the unequivocal identification of phenylacetylglutamine
in human urine, an endogenous metabolite not previously identified

by proton NMR owing to extensive overlapping with other resonances.
This research underlined the technology's scope for success in meta-
bolic profiling.
1.2.3.5 Magic-angle Spinning Probes

Magic-angle spinning (MAS) probes allow for broad resonances in solid
materials to be narrowed by spinning the sample at the magic angle (54.74°)
with respect to the magnetic field. This process averages internal interactions
such as chemical shift anisotropy, dipolar coupling and first order quadru-
pole coupling, enabling high-resolution NMR by reducing line broadening.
Spinning of the sample is generally achieved by using an air turbine system
to drive a rotor at a rate of anywhere between 1–100 kHz. The complex design
of these probes did mean that cryogenically cooling the coils wound around
the sample is very difficult without decreasing the sample temperature at
the same time. Recently, however, a cryogenically cooled MAS-NMR probe
was designed allowing the measurement of the proton resonance of solid
samples at room temperature. The desired increase in signal-to-noise was
confirmed using this approach.16
.
1.2.4 Digital Filtering

The digital filter, when applied to NMR data, has many advantages over the
pure application of analog filters, including improved baseline performance,
better dynamic range, and improved overall sensitivity.17 The availability of
fast digital signal processors (DSPs) has made the application of digital fil-
tering a reality in NMR spectrometers, with very similar improvements in
quality to other applications where source data from audio or visual sources
are handled.
The specific path of data through the digital filter of an NMR spectrome-
ter starts with unfiltered analog data entering an analog to digital converter
(ADC). Because of a faster sampling rate, the number of points increases,
which in turn needs to be reduced by extracting the spectral window of
interest. The sampled digital signal is filtered and reduced in real time with
a DSP. Finally, the filtered FID is sent to a workstation where Fourier trans-
formations provide us with the spectral data for analysis. The quality of data
has dramatically improved since the advent of digital filters as:

-- The transition region of analog filters is rather large compared to digital
filters and folding of signals and noise into the spectral window occurs.
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-- A digital filter has the ability to adapt and change the way in which a
signal is processed.
-- The characteristics of an analog filter are subject to drift and depend on
the environmental surrounds, including the temperature and electro-
magnetic radiation. Digital filters are particularly stable with both time
and temperature.
1.2.5 Computational Support to Hardware

Although computational support is not technically hardware, and so is not
in the current chapter's scope, as hardware technologies improve, so does
the computational support to achieve the full benefit of improvements in
spectrometers. Areas of computational support need to constantly evolve
with spectral instrumentation and data quality. Metabolomic data col-
lection is still evolving and so the metabolomics community has a large
volume of complex algorithms written in an array of languages to support
the high-throughput analysis of metabolomics sample cohorts. There are
already many user-orientated computational platforms specifically for pro-
cessing complex NMR spectral datasets.18 Some of the fastest evolving ele-
ments of computation support with regards to NMR data are mentioned in
this section.
1.2.5.1 Databases
.
NMR analysis has the advantage of being completely quantitative, accu-

rately measuring the amount of protons, under given conditions. Spectral
data can therefore be directly compared and spectral features of interest
elucidated. Databases of standards are generally easy to come upon and
there is a lot of information used to help assign NMR signals. Unfortu-
nately, NMR suffers from comparably poor resolution and issues relating
to molecules of similar structure producing similar magnetic resonance
signatures, making them difficult to confidently assign without further
work. Current research is aimed at producing reliable tools to deconvolve
NMR signals from complex biofluid spectra producing an accurate mea-
sure of a number of metabolites automatically in large profiling studies.
Issues of varying chemical shifts between samples, along with overlap-
ping signals of varying shape, means creating automated packages to
measure metabolites and their concentrations from spectral data is very
difficult and is a form of on-going research. Metabolomics databases of
NMR spectral data are readily available,19 including a urine-specific data-
base.20 Databases need to address a series of needs, including their avail-
ability, experimental validation, easily searched, readily interpreted and
comprehensibility. These needs are constantly adapting and so databases
are constantly in need of updating as spectral data improves as a result of
hardware improvements.
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10 Chapter 1
1.2.5.2 Laboratory Information Management Systems

Sometimes referred to as a Laboratory Information System (LIS) or a Labo-
ratory Management System (LMS), a Laboratory Information Management
System (LIMS) supports the operation of a modern laboratory, particularly
high-throughput laboratories where sample numbers are large and processes

require mediation. Aside from the key functions of sample management,
instrument application and data transfer, LIMS are also able to provide basic
laboratory functions like document management, calibration, maintenance
requirements, and data entry.
There are many open-source LIMS customised towards specific types of
biomaterials or research data. More generic or general LIMS have come
about as a necessity to catalogue large-scale biobanks of human specimens
and manage and track their progress through a cascade of complex analytical
procedures and experimental procedures.21
1.2.5.3 Metabolic Modelling
Metabolomics data sets, along with other phenomic data, generate increas-
ingly complex data tables, which are becoming more difficult to summarise
and visualise. For many years continuous spectral data, like that produced
by an NMR spectrometer, has been statistically correlated with disease
groups of other regions of the spectrum.22 Taking advantage of the multi-
collinearity of the intensities of signals in a set of spectra, spectral regions
can be correlated with one another, enabling the association of peaks from
the same molecule or similar biochemical pathways. Similarly, OPLS-DA
.
analysis can be visualised in the same fashion by highlighting spectral

regions associated with a discrete or continuous variable of physiological
interest. Displaying highly correlated areas of a complex spectrum can even
help identify the molecules responsible for the given signals or metabolic
variation.
1.3 Automation of Metabolomic Profiling

The process of acquiring NMR spectral data begins with a submission of
a request and finishes with data distribution and archiving. With the era
of ‘big data’ upon us and the demand to automate processes, many types
of hardware systems have recently become available to automate sample
preparation, sample insertion and sample acquisition. These hardware
accessories are linked with software packages often controlled by a LIMS
to enable the organisation of spectral data and the corresponding meta-
data. The specific automation hardware systems are generally accessories
to the components of NMR spectrometers (Section 1.2), although work in
closely timed conjunction with the spectrometer to produce accurate and
precise spectra without the need for constant supervision by a member of
the laboratory.
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1.3.1 Sample Preparation

In many NMR applications, as sample acquisition time is indeed the limiting
factor for throughput, sample preparation does not need to be automated. In
this case, the amount of a spectroscopist's time taken for sample preparation
is considered negligible. In most situations robotic preparation is considered

more precise and accurate; however, this is not always the case, and so there
is no direct advantage to developing an automated preparation routine when
sample preparation does not limit throughput. On the other hand, sample
acquisition can be routinely very quick and therefore the manual preparation
of samples can be tedious, tiring and unpractical. When hundreds, or even
thousands, of samples are being acquired a day the development of auto-
mated routines for sample preparation is required.
Automation of metabolomic sample preparation for NMR is made difficult
by the large variety of NMR applications and various types of biofluids. The
variety in applications leads to a large number of preparative protocols that
need to be catered for by robotic systems. Variations in total volume, solvent
viscosities, tube diameters, sample temperatures and sample types all need to
be accounted for when developing an automated routine to prepare a cohort
of samples. The sample preparation (much like the collection) is ideally per-
formed in such a way that as little variation as possible in the metabolic con-
tent of samples is encouraged during the process. In many cases, however,
preparative and analytical variation cannot be avoided. Inconsistencies in
quantification of metabolites can arise from many sources during sampling,
sample storage, sample extraction, derivatisation, analysis and/or detection.
During the sampling and preparation of samples, undesired changes in the
.
metabolic content of samples may occur owing to enzyme activity, high reac-
tivity of metabolites, or breakdown or degradation of metabolites. Owing to
the nature of metabolomics samples, a very inert analytical preparation is
required to minimize absorption and degradation of metabolites, particu-
larly relatively polar compounds. Furthermore, the degree of adsorption and
degradation can vary between different samples with different biomass con-
centrations and different sample matrices. Consequently, such matrix effects
should be evaluated.
Preparation of quality control samples (both internal and external) in the
project design helps understand the amount of preparative and analytical
variance introduced during a study. Three sample types are useful for these
measures, a composite quality control (QC), a stable quality control, and an
external quality control. A composite study reference (SR) is simply a refer-
ence solution made from equal amounts of each sample within the study.
A composite QC is made from the SR in the same way that each individual
sample is prepared. A stable QC is prepared on a much larger scale with the
reagents or buffers necessary and then transferred into multiple tubes or
vials for analysis. An external QC is simply produced with samples of the
biofluid of interest, obtained completely independently of the study, and pre-
pared similarly to each individual sample. Buffer blanks can also be prepared
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12 Chapter 1
by simply replacing a sample with pure water. These samples are important
to ensure the robot is performing as expected and that the prepared buffer or
samples are not being contaminated during the preparation process.
1.3.1.1 Robotics for Sample Preparation

There are many brands (more than 15), each with multiple makes, of com-
mercial liquid handling devices currently available to aid laboratory prepa-
ration of samples. A complete review of these devices is outside the scope of
this chapter; however, it is important to note that many devices do not cater
for NMR tube sample preparation. Robotic preparation into standard 5 mm
tubes requires that samples are prepared with a syringe and the robot is able
to clear the classical 7 inch length of the tube. The nature of how the tubes
are held in position within the robot while not confusing the individually
held tubes is also an issue. A simple solution to this came about with the pio-
neering of SampleJet Racks (compatible with SampleJet automation systems,
Section 1.3.2), which position 3 mm or 5 mm tubes into a 96 well plate for-
mat. The tubes are also only 4 inches in length and so are generally compati-
ble with robots that are capable of preparation using well plates (Figure 1.1).
Preparation of samples within a 96 tube rack generally occurs one at a
time, or alternately a robotic solution with a multi-syringe is required. Fur-
thermore, robotic preparation requires that each tube either has its cap off
during preparation (which entails the tedious process of taking each tube cap
off before preparation and replacing after preparation) or alternately a small
entry hole in the cap to allow the preparative syringe to enter through. The
.
most straightforward solution to this problem currently is to have a small
Figure 1.1 tube sample rack containing 5 mm sample tubes developed to enable
96
sophisticated automation of the NMR preparation and acquisition
process of biofluids.
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hole in each cap (Figure 1.1) and then following sample preparation sealing
each cap with a small ball (POM) to contain the biofluid sample inside.
Further complications are met as buffer solutions, urine, plasma/serum
and other biofluids manipulated regularly for routine metabolomics analysis
all have varying viscosities. Varying viscosities lead to variations in aspirated
and dispensed volumes when using robots that use either push solvents or
air pressure. Furthermore, during study design the amount of biofluid avail-
able is generally small and metabolomics assays can require relatively large
volumes compared with other available screening techniques. Modern bio-
banks spend a lot of time debating sample amounts that should be spared
for particular types of analysis. During the preparative procedure it is there-
fore also important that the volume of sample ‘wasted’ by preparative robots
is considered and preferably kept to a minimum, making metabolomics
approaches more reasonable for clinicians of population research groups.
All these complications lead to many forms of optimisation and understand-
ing of robotic preparation before routine high-throughput preparation can
occur.
Currently, the Gilson Automatic Liquid Handler is the most versatile robot
available for preparation into a SampleJet 96 tube rack, although not nec-
essarily the best for compatibility with tubes >7′ in length. The Gilson 215
robot has a single syringe format, so samples must be prepared one at a time,
while the Gilson 274 allows preparation of up to four samples at a time. Both
robots are well-suited to house refrigeration mats while preparation is occur-
ring, minimising the time biofluids are exposed to room temperature. A Liq-
uid Handler 215 takes 3 h to prepare a 96 tube rack, including the addition
.
of reagent to the sample, injection into the tube, and a mixing and cleaning
procedure for each sample.
1.3.2 Automated NMR

Given the large numbers of samples to be analysed and the need for excep-
tional levels of reproducibility, new analytical protocols have had to be devel-
oped for high-throughput metabolomics by NMR.23 This protocol describes
a significantly enhanced and augmented version of the NMR spectroscopy
protocol24 previously published for any large-scale phenotyping projects uti-
lizing the latest generation digital spectrometers. The previous version of the
protocol is still suitable for small scale studies that do not rely on multiple
batch analysis and where interstudy and interlaboratory comparisons are
less important. NMR spectroscopy is intrinsically both quantitative and, with
the current state of commercially available equipment, highly reproducible.
With modern digital electronics-based machines, exact parameter settings
can be set, but there are still other considerations and procedures required
to obtain reproducible sample chemistries. Before performing the spectro-
scopic analysis, it is necessary to consider aspects such as sample numbers
per group and randomization. In order to be able to validate results, suffi-
cient sample numbers should be analysed to achieve statistically significant
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14 Chapter 1
results (the numbers required can be estimated using multivariate power
calculations25).
Generally, NMR-based metabolic studies of biofluids have shown very high
reproducibility,26 but new protocols linked to new technology as described
herein have enabled even greater levels of reproducibility to be obtained
without compromising the level of throughput. Using current automation

provided by the SampleJet system, samples can be kept chilled while await-
ing analysis (Section 1.3.2.1) and problems with insertion of samples into
the magnet are rare. It is important to keep aliquots of samples at the sample
collection stage in order to be able to repeat acquisitions or for subsequent
2D NMR spectroscopy necessary for biomarker identification. Consideration
has to be given to quality control at all stages of the phenotyping process
(i.e., quality of the sample subject, quality of the study design, quality of the
sample collection, quality of sample aliquoting, quality of the sample stor-
age, quality of the preparation of samples for NMR, quality of the acquiring
of data, and quality of the upkeep of the parameters desired over time). Ulti-
mately, to achieve a spectral snapshot of the metabolic content of a biofluid
at the time of collection, care must be taken at each stage of sample handling
to ensure that a true indication of the biofluid content is observed.
1.3.2.1 Hardware for Automation

Many sample automation units are available for high-throughput analysis.
Historically, sample automation was performed by a carousel (generally
holding either 24 or 60 samples), which simply rotated as each sample was
.
analysed. The user would need to place each sample in a regular tube and
then the tube into a spinning shuttle. The tube already measured to a depth
in the shuttle was placed in a specific position in the carousel along with
the small cohort of samples and through the relevant software programmed
to guide the spectrometer to perform particular experiments on each sam-
ple. The carousel could be emptied of acquired samples and restocked with
further samples if necessary. There are four main types of technology cur-
rently available for installation onto an NMR magnet, SampleMail™, Sam-
pleCase™, SampleXpress™ and SampleJet™, each designed to completely
automate the acquisition process. The first three automation devices are
based on the classical carousel whereas the SampleJet provides a complete
makeover of high-throughput NMR and is the ideal device for automating
metabolomic profiling research.

-- The SampleMail is simply a device that allows a single sample to be
placed into and out of the magnet without the need for reaching over
the top of the magnet (avoiding the use of a ladder).
-- The SampleCase is a similar system to the SampleMail however allows
up to 24 samples to be held at user height in a carousel. The SampleCase,
like older carousels, still requires samples to be placed into individual
shuttles by the user, ordered in the carousel manually and programmed
via the software. The major advantage to metabolic research over
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classical automation methods is that samples can be cooled to a given
temperature (refrigerated) while they wait on the carousel to be analysed.
-- The SampleXpress is not a refrigerated storage unit reducing its poten-
tial for running large biofluid sample cohorts. It does however incor-
porate an automatic barcode reader, which automatically locates and
identifies samples that are being analysed, avoiding user error.

-- The SampleJet is the ideal solution for automated high-throughput
metabolomics research as it was designed specifically with biofluid
applications in mind (Figure 1.2). Samples are able to be prepared as
usual or in a 96 tube well plate format. When samples are prepared into
a 96 tube rack, the sample tubes do not need to be placed into individ-
ual shuttles but rather a single shuttle, which is controlled by the robot,
is placed in the magnet's shaft and samples are simply inserted into and
removed from the shuttle automatically. Samples can be refrigerated to
a given temperature in one of five rack positions while they are queued
for analysis. Tracking sample position can be performed by imaging
barcodes on individual tube caps or alternately samples can be tracked
by simply using their position in a given rack. Finally, SampleJet auto-
mation units are also compatible with classical individual 7 inch tubes;
however, unlike classical automation devices, NMR tubes do not need to
first be placed in a shuttle (Figure 1.2c).
.
Figure 1.2
Bruker SampleJet automation unit installed on a 600 MHz magnet (A);
access to temperature-controlled SampleJet unit (B); and interior of
SampleJet system displaying samples queued for analysis in both 96
well plate racks (at rear) and individual 7 inch tubes loaded into NMR
spinner collars (C).
Another random document with
no related content on Scribd:
To conveniently support a clothespin basket along the line on
which the clothes are being hung, a wire support can be provided,
bent to form a hook at both ends and the center shaped into a V-
bend. With the basket supported by the two ends, the wire can be
slid along the clothesline as required.—Contributed by N. R. Moore,
Cherokee, Iowa.
¶Varnishing should as a rule be done in a room having a

temperature of 80° F., and in some instances 15° higher is desirable.
Leather Tire Patch
A leather patch fixed over a tire puncture with shellac will be found
to give satisfaction and may be attached easily. Cut the patch
somewhat larger than the puncture and thin out its edges with a
knife. Melt flakes of shellac in a flame, fusing them, and rub the hot
mixture on the patch and tire, smoothing it down quickly. Such a
patch may be placed over a plug and will aid in holding it in place.—
Contributed by Robert C. Knox, Petersburg, Fla.
A Perpetual Whirligig
Camphor is the motive power which drives the device shown in the
illustration, and it will cause the whirligig to revolve for several days,
or until the camphor is consumed.
The whirligig is made of a piece of cork, ¹⁄₂ in. square, with a

needle stuck into each of its four sides. Smaller pieces of cork, to
which pieces of camphor have been fixed by means of sealing wax,
are attached to the ends of the needles. Care should be taken to
keep the needles and cork free from oil or grease, as this will retard
their movement. As soon as the device is placed in a dish of water it
will start whirling and continue to do so as long as motive power is
supplied. A small flag or other ornament may be attached to the
center cork.
Testing and Caring for Files
To test a file hold it so that the light will be reflected sharply from
the teeth and observe whether their edges are flattened and appear
as white lines. If so, the file is dull and should be recut if of
considerable size and value.
Files should not be thrown into drawers and mixed with other
tools, but should be carefully set in racks or drawers for the purpose.
A mechanic would not throw a straightedge into a drawer containing
other tools, and a file should be given similar consideration, as every
nick in the teeth impairs the efficiency of the file.
Files may be sharpened by dipping them into sulphuric acid, but
care must be taken not to permit the acid to come into contact with
one’s clothes or person. Water is used to wash off the acid.
Files should be provided with individual handles. This prevents
injury to the hand of the worker and aids in the proper use of the file.
Handles should be carefully fitted and be made of a size
proportionate to the file. In removing a handle from a file, strike the
handle at the end nearest the file, by sliding a piece of hard wood
along the surface of the file, as the blow is struck with it. Do not use
another file or metal object in thus removing a handle, as it will injure
the latter.
Catching Large Fish with a Teaspoon
Teaspoons may be made into alluring trolling spoon hooks, of a
size suitable for catching large fish, by the addition of hooks, as
shown in the sketch.
Drill ¹⁄₈-in. holes near the end of the spoon handle, the tip of the
bowl, and near the handle of the latter.
Procure three sets of triple hooks, a line swivel, and a strip of lead,
about 1 in. long. Rivet one end of the swivel and the loop of one of
the triple hooks into the hole of the handle. Wire the lower end of this
triple hook to the handle and with the same piece of copper wire
secure a second triple hook at the thin part of the handle. Drill a hole
through the lead strip and rivet it, together with a third triple hook,
into the upper hole of the bowl. Fix the lower end of this hook by
binding it with copper wire, through the hole near the tip of the bowl.
Once a Fish has Struck This Bait, It Is Seldom Able to Escape

This hook has been tested in the waters of Puget Sound and is a
deadly lure for rock cod, and other fish weighing up to 12 lb. The
famous barracuda and rock bass of the Catalina Islands have also
been caught with it. By permitting the lure to sink to the bottom and
bringing it up a yard or two with a quick jerk, it acts as a “jig” bait. It
may also be used in trolling. Once a fish has struck, it is seldom able
to escape.—Contributed by O. P. Avery, Los Angeles, Cal.
An Easily Made Counter
An Accurate Account can be Kept of Parts or Score for Any Game by Pulling
the Strips
From unruled paper cut a piece, as shown at A in the sketch, and

make slits parallel and evenly spaced with a sharp knife. Also cut six
strips, similar to the one shown at B, to fit the slits cut in A. The strips
are numbered as shown and inserted on the under side of A, and by
pulling the strips as shown, one can count up the number of parts or
keep tally on any game. By making more slits and using more strips
very large numbers can be recorded.
¶Be sure to keep the screw and nut in the jaws of a drill chuck clean
and well oiled, to prevent broken screws.
To Uncork a Bottle with a String
A convenient method of uncorking a bottle, from which liquid is to
be poured frequently, is to thread a strong string or cord through the
cork, tying it in a loop, which remains at the opening of the bottle.
The cork may be removed easily by drawing on the string. This is
more satisfactory than the use of a corkscrew, as the latter frequently
tears the cork.—Lee A. Collins, Louisville, Ky.
Wood Turning on an Emery Grinder
The Hand Emery Grinder of the Home Workshop Used as a Substitute for a
Lathe
The experimenter often requires small turned-wood pulleys,

circular bases for switches, etc. To produce these it is not necessary
to have recourse to a wood lathe, if a good emery grinder is at hand.
Simply clamp the grinder firmly to the workbench, remove the
grinding wheel, and fasten on a block to serve as a faceplate. This
may be held in place by the nut that holds the wheel and should be
trued up with a small chisel when in place. A tool rest may be
improvised by temporarily nailing one or more blocks of wood to the
bench. The article desired should be first roughed out with a saw and
then fastened to the faceplate with screws or brads, after which the
actual wood turning will require very little time.
Three Bathroom Kinks
The devices for the bathroom illustrated may be made easily and
contribute to the comfort, convenience, and, in the case of the fixed
window pole, to the safety of the room. A wall curtain, A, placed on
the towel rod, or hung on the wall beside the washbasin, is
especially convenient in keeping the walls unsoiled by children who
make use of the room and are likely to splash suds while washing.
Double roller shades on the window, as at B and C, give light and
privacy as well.
The Fixed Window Pole Is an Inducement to Ventilation; the Curtain Protects
the Wall, and the Lower Shade Gives Light with Privacy
Poor ventilation in bathrooms occasionally causes asphyxiation

and is often a menace. The permanent fixing of the window pole D
makes it convenient to open the window, which operation is often
neglected through fear of drafts from the lower sash and the lack of a
pole. Fig. 2 shows the top of the pole P, provided with a screw eye,
S, which is fastened to a metal strip, H.—D. L. Hough, Toledo, Ohio.
Prevents Soiling Goods after Oiling Sewing
Machine
To prevent a sewing machine that has been oiled from soiling the
material, the following is a good method: Tie a small piece of ribbon
or cotton string around the needle bar near the point at which it grips
the needle.
A Pigeon House
By Robert Baker
P igeon houses need not be eyesores, as is often the case, but may
be made to harmonize with the surroundings, adding beauty to a
dull spot, and even making the grounds of a home more attractive.
The house described will accommodate 20 pigeons, and additional
stories of the same type may be added to provide for more. Nearly
all of the wood necessary may be obtained from boxes, and the
other materials are also readily available at small cost. The
construction is such that a boy handy with ordinary carpentry tools
may undertake it successfully.
The house is constructed in general on principles used in
buildings, having a framed gable roof, rough-boarded and shingled.
The interior arrangement is original, being based on the Indian
swastika or good-luck sign. While the construction is simple, it must
be carried out systematically. The process outlined also follows in
general the typical methods in building construction.
The foundation need not be considered, since the house rests
upon a post, and the construction thus begins with the lower story.
The floor and the ceiling are similar in construction, as shown in Fig.
1. In framing them into the lower story, as may be observed in Fig. 8,
the cleats are placed below on the floor and above in the ceiling. The
construction is identical, however. The cleats are fastened to the
boards with screws, although nails, clinched carefully, may be used.
The 4-in. hole at the center should be made accurately, so as to fit
the shoulder portion at the top of the post, shown in Fig. 2. The latter
may be cut of a length to suit; about 9 ft. will be found convenient.
The notches in the top of the post are to fit the ridge pole and center
rafters of the roof frame, as shown in Fig. 10. They should not be
made until the house is ready for the roof boards.
The pieces for the compartments, as arranged on the floor in Fig.
3, are made next. Figs. 4 and 5 show the detailed sizes of these
pieces, of which four each must be made. The sizes shown must be
followed exactly, as they are designed to give the proper space for
entrances and to fit around the 4-in. square hole, through which the
post is to fit. The pieces marked A, B, and C, in Figs. 4 and 5,
correspond to those similarly marked in Fig. 3.
The pieces are nailed together to form the swastika in the
following manner:
Mark the pieces A, B, and C, as shown. Measure 4 in. from one
end of each piece marked A, and square a pencil line across, 4 in.
from the end. Arrange the pieces in pairs. Place one end of one
piece against the side of the other piece in the pair, so that the pencil
line is even with the end, permitting the 4-in. portion to project. Nail
both pairs in this position. Then fit the two parts together to form a 4-
in. square in the center, as shown in Fig. 3.
Fit the pieces C to the pieces B at an angle, as shown in Fig. 3,
trimming off the projecting corners where the pieces are joined. Nail
them together, and they are ready to be fixed to the end of the
pieces A, already nailed. By nailing the joined pieces B and C to the
end of the pieces A, as shown in Fig. 3, the swastika is completed.
Fix it into place, with the center hole exactly over the square hole in
the floor, by means of nails or screws driven through the floor.
Two small strips must now be nailed to the floor at each side of the
swastika. They should be exactly 4¹⁄₂ in. long, and are to hold the
slides, Fig. 9, which shut off the various compartments. The slides
are shown hanging by chains in the headpiece of this article, and are
shown in place in Fig. 8.
Fix the ceiling into place in the same manner, being careful that
the square holes fit together, and that the cleats are on the upper
side. Turn the construction over and fix into place the small strips for
the slides, as was done on the floor.
The fixed screens, Fig. 6, and the doors, Fig. 7, are constructed
similarly. They are built up of ¹⁄₂-in. wood, and vary in size to fit their
respective places in the framework. Observe that the fixed screens
are ¹⁄₄ in. higher than the doors, and that they are fastened between
the ceiling and floor, bracing them. The wire grating is ¹⁄₂-in. square
mesh, and is fixed between the pieces of the doors and the screens
when they are built up.
The doors are shown secured by combination strap hinges, bent
over the baseboard. Plain butts may be used and the lower portion
of the hinge covered by the baseboard, a recess being cut to receive
the part covered. In the latter instance the doors should be fixed into
place immediately after the screens are set. Catches and chains
may then be placed on the doors. Next nail the baseboards into
place. They are 2¹⁄₂ in. wide and may be mitered at the corners, or
fitted together in a square, or butt, joint. The latter joint may be nailed
more readily.
The slides, shown in Fig. 9, may now be made and fitted into their
grooves. The handles are made of strips of band iron, drilled for
screws and bent into the proper shape. It is important that the slides
be constructed of three pieces, as shown, so that they will not warp
or curve from exposure. The main piece is cut 7³⁄₄ in. long, and the
strips, ¹⁄₂ in. square, are nailed on the ends.
The construction of the framing for the roof should next be taken
up. This probably requires more careful work than any other part of
the pigeon house, yet it is simple, as shown in Fig. 10. Note that the
rafters are set upon a frame, or plate as it is called, built up of pieces
3 in. wide. It should be made ¹⁄₄ in. wider and longer on the inside
than the ceiling board, so as to fit snugly over it. The joints at the
corners are “halved” and nailed both ways. This gives a stronger
structure than butting them squarely and nailing them. The end
rafters should be fitted in before fixing the others. It is best to make a
diagram of the end of the roof framing on a sheet of paper, or a
board, and to fit the rafter joints in this way before cutting them. The
rafters are then nailed into place.
The “rough boards” to cover the rafters may now be nailed down.
They are spaced ¹⁄₂ in. apart so as to permit thorough drying, as is
done in larger buildings. They project 2 in. beyond the ends of the
plate frame, supporting the rafters. A ¹⁄₂-in. strip is nailed over the
ends to give a neat finish. The roof may be shingled, or covered with
tar paper, or any roofing material.
Nail a 1-in. strip under each end of the roof and nail the gable
ends into place. One gable end is provided with a door, as shown,
and the other has an opening fitted with a wire screen of the same
size as the door.
The gable story rests on the lower story, and the notches in the top
of the post should fit snugly to the ridge and center rafters, as shown
in Fig. 10. This will aid in supporting the house firmly. If additional
stories are added it would be well to place a post at each corner of
the house. The upper story may be removed for cleaning, or for
transporting the house.

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NMR based Metabolomics 1st Edition

LC-NMR: Expanding the Limits of Structure Elucidation

Metabolomics Paul L. Wood

Evidence-based Public Health Ross C. Brownson

Metabolomics Practical Guide to Design and Analysis 1st

High-Throughput Metabolomics: Methods and Protocols

The Fundamentals of C C Game Programming Using Target

Microbial Metabolomics Applications in Clinical

Applications of EPR and NMR spectroscopy in homogeneous

New Developments in NMR

Titles in the series:

and Heterogeneous Materials

How to obtain future titles on publication:

For further information please contact:

New Developments in NMR No. 14

Print ISBN: 978-1-84973-643-5

© The Royal Society of Chemistry 2018

All rights reserved

permission in writing of The Royal Society of Chemistry or the copyright owner, or in

For further information see our web site at www.rsc.org

New Developments in NMR No. 14

New Developments in NMR No. 14

Chapter 1 Instrumental Platforms for NMR-based Metabolomics  1

1.1 H istory of NMR Hardware Development  1

1.3.3 Automated Acquisition  16

Chapter 2 NMR Pulse Sequences for Metabolomics  22

New Developments in NMR No. 14

Identification and Quantification  28

Chapter 3 NMR Spectroscopy of Urine  39

Healthy Population Groups  66

Chapter 4 NMR Spectroscopy of Serum and Plasma  85

4.3.5 Diabetes Risk  112

Chapter 5 High-resolution Magic-angle Spinning (HR-MAS)

5.1 I ntroduction  133

5.4.4 Microscopic Quantity  145

Chapter 6 Investigation of Tumor Metabolism by High-

6.1 H R-MAS MRS for Studies of Metabolic

6.4 Technology Developments and Optimized

Chapter 7 NMR in Environmental and Nutritional Research  168

7.1 I ntroduction  168

7.3 Applications of NMR-based Metabolomics in

Chapter 8 NMR Foodomics  183

Francesco Savorani, Bekzod Khakimov, Nanna Viereck

8.1 I ntroduction to NMR Analysis of Food  183

9.1 I ntroduction  246

Chapter 10 1H NMR-based Metabolic Profiling in Infectious

10.1 I ntroduction  264

Chapter 11 Imaging Metabolic Processes in Living Systems

Chapter 12 Advances in Computational Analysis of

12.1 I ntroduction  310

Chapter 13 NMR Spectroscopy of Cell Culture, Tissues,

13.1 G  eneral Introduction  324

Subject Index  360

Instrumental Platforms for

MRC-NIHR National Phenome Centre, Department of Surgery & Cancer,

1.1 History of NMR Hardware Development

New Developments in NMR No. 14

as the frequency of a resonating nucleus is largely dependent on the local

allowing Fourier Transform NMR (FT-NMR). These hardware advances rev-

Instrumental Platforms for NMR-based Metabolomics 3

1.2 Components of NMR Hardware

ment for a standard NMR laboratory. Before the advent of superconducting

1.2.2 Shim Coils

Chapter 1 Instrumental Platforms for NMR-based Metabolomics 1

1.1 H istory of NMR Hardware Development 1

1.3.3 Automated Acquisition 16

Chapter 2 NMR Pulse Sequences for Metabolomics 22

Identification and Quantification 28

Chapter 3 NMR Spectroscopy of Urine 39

Healthy Population Groups 66

Chapter 4 NMR Spectroscopy of Serum and Plasma 85

4.3.5 Diabetes Risk 112

Chapter 5 High-resolution Magic-angle Spinning (HR-MAS)

5.1 I ntroduction 133

5.4.4 Microscopic Quantity 145

Chapter 6 Investigation of Tumor Metabolism by High-

6.1 H R-MAS MRS for Studies of Metabolic

6.4 Technology Developments and Optimized

Chapter 7 NMR in Environmental and Nutritional Research 168

7.1 I ntroduction 168

7.3 Applications of NMR-based Metabolomics in

Chapter 8 NMR Foodomics 183

8.1 I ntroduction to NMR Analysis of Food 183

9.1 I ntroduction 246

Chapter 10 1H NMR-based Metabolic Profiling in Infectious

10.1 I ntroduction 264

Chapter 11 Imaging Metabolic Processes in Living Systems

Chapter 12 Advances in Computational Analysis of

12.1 I ntroduction 310

Chapter 13 NMR Spectroscopy of Cell Culture, Tissues,

13.1 G eneral Introduction 324

Subject Index 360

1.1 History of NMR Hardware Development

1.2 Components of NMR Hardware

1.2.2 Shim Coils

1.2.3 Sample Probe

1.2.3.4 Flow Injection Probes

1.2.3.5 Magic-angle Spinning Probes

1.2.4 Digital Filtering

1.2.5 Computational Support to Hardware

1.2.5.2 Laboratory Information Management Systems

1.3 Automation of Metabolomic Profiling

1.3.1 Sample Preparation

1.3.1.1 Robotics for Sample Preparation

1.3.2 Automated NMR

1.3.2.1 Hardware for Automation