332
Y. Bai et al.
to define whether these differences are the cause or effect of the pathophysiology
of heart failure [32]. Newgard’s research group has applied target analysis of up to
250 metabolites to study heart-related diseases including myocardial ischemia and
planned myocardial infarction [33].
5.2 Nuclear Magnetic Resonance Spectroscopy
The sensitivity of NMR spectroscopy is not comparable to that of mass spectrometry, but it offers advantages for compounds that are difficult to be ionized or else
require derivatization for MS. Using stable isotope labeling, NMR spectroscopy
offers several advantages in elucidating the dynamics and mechanisms of metabolite
transformations and for exploring the compartmentalization of metabolic pathways
by its inherent capability to monitor isotope enrichment. Nuclear magnetic resonance
spectroscopy can be used to measure a wide variety of spin 1/2 nuclei, including the
biologically relevant
1 H,
13 C,
31 P, and
15 N as well as the pharmaceutically relevant
19 F. Among these,
1 H,
19 F, and
31 P are at high abundance, while the lower natural
abundance of
13 C and
15 N can be useful in measuring enrichment due to catabolism
of labeled precursors as a function of specific biological processes or pathways. The
positional enrichment of a molecule in both
13 C and
15 N NMR spectroscopic methods
may be used to establish which pathways are being activated or inactivated.
Samples for NMR metabolomic profiling are collected in a uniform way to minimize variability and are analyzed in terms of their NMR profiles to collect data on all
metabolites potentially present in the sample. Using pattern recognition (e.g., principal component analysis and partial least squares discriminant analysis), the potential biomarkers usually can be identified using visualization tools. The identified
markers eventually will be placed in a metabolic pathway to provide insight into the
biochemical phenomena are observed. Two-dimensional (2D) NMR methods offer
an improved resolution for unambiguous identification of metabolites in a mixture,
including COSY (correlated spectroscopy, TOCSY (total correlation spectroscopy),
and HSQC (heteronuclear single-quantum correlation). Automation of metabolite
identification can be achieved using software with combined TOCSY and HSQC
data. By setting tolerance levels for the matching of
1 H and
13 C NMR signals, one
can maximize compound identification efforts while minimizing false positives [35].
The use of
1 H NMR spectroscopy is well documented, and its application has
been used in a wide range of fields and sample matrices, such as biofluids, plants,
and natural product mixtures.
Biofluids NMR metabolomics offers the potential for a thorough understanding
of disease pathogenesis and the identification of disease biomarkers. For a metabolic
biomarker to be useful clinically, its level must clearly associate with the disease
risk or its progression and should be insensitive to variables (e.g., ethnicity, diet, and
location). Blood plasma or urine
1 H NMR spectra usually are measured on samples
obtained from both healthy and unhealthy volunteers. In a recent investigation to
catalog the metabolic components of urine, a comprehensive study of the human
Y. Bai et al.
to define whether these differences are the cause or effect of the pathophysiology
of heart failure [32]. Newgard’s research group has applied target analysis of up to
250 metabolites to study heart-related diseases including myocardial ischemia and
planned myocardial infarction [33].
5.2 Nuclear Magnetic Resonance Spectroscopy
The sensitivity of NMR spectroscopy is not comparable to that of mass spectrometry, but it offers advantages for compounds that are difficult to be ionized or else
require derivatization for MS. Using stable isotope labeling, NMR spectroscopy
offers several advantages in elucidating the dynamics and mechanisms of metabolite
transformations and for exploring the compartmentalization of metabolic pathways
by its inherent capability to monitor isotope enrichment. Nuclear magnetic resonance
spectroscopy can be used to measure a wide variety of spin 1/2 nuclei, including the
biologically relevant
1 H,
13 C,
31 P, and
15 N as well as the pharmaceutically relevant
19 F. Among these,
1 H,
19 F, and
31 P are at high abundance, while the lower natural
abundance of
13 C and
15 N can be useful in measuring enrichment due to catabolism
of labeled precursors as a function of specific biological processes or pathways. The
positional enrichment of a molecule in both
13 C and
15 N NMR spectroscopic methods
may be used to establish which pathways are being activated or inactivated.
Samples for NMR metabolomic profiling are collected in a uniform way to minimize variability and are analyzed in terms of their NMR profiles to collect data on all
metabolites potentially present in the sample. Using pattern recognition (e.g., principal component analysis and partial least squares discriminant analysis), the potential biomarkers usually can be identified using visualization tools. The identified
markers eventually will be placed in a metabolic pathway to provide insight into the
biochemical phenomena are observed. Two-dimensional (2D) NMR methods offer
an improved resolution for unambiguous identification of metabolites in a mixture,
including COSY (correlated spectroscopy, TOCSY (total correlation spectroscopy),
and HSQC (heteronuclear single-quantum correlation). Automation of metabolite
identification can be achieved using software with combined TOCSY and HSQC
data. By setting tolerance levels for the matching of
1 H and
13 C NMR signals, one
can maximize compound identification efforts while minimizing false positives [35].
The use of
1 H NMR spectroscopy is well documented, and its application has
been used in a wide range of fields and sample matrices, such as biofluids, plants,
and natural product mixtures.
Biofluids NMR metabolomics offers the potential for a thorough understanding
of disease pathogenesis and the identification of disease biomarkers. For a metabolic
biomarker to be useful clinically, its level must clearly associate with the disease
risk or its progression and should be insensitive to variables (e.g., ethnicity, diet, and
location). Blood plasma or urine
1 H NMR spectra usually are measured on samples
obtained from both healthy and unhealthy volunteers. In a recent investigation to
catalog the metabolic components of urine, a comprehensive study of the human