# Metabolomics/Analytical Methods/NMR/H NMR

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Modern Application
Opium poppy is recognized as an ideal model for alkaloid plant metabolism study, because of its diverse array of bioactive benzylisoquinoline alkaloids. Cultivation of the plant has produced such commercial narcotic analgesics as morphine and codeine. Environmental stimuli are capable of impacting secondary plant metabolism, which is in turn capable of regulating other plant metabolic pathways. Researchers have employed proton nuclear magnetic resonance ($^1$H NMR) metabolomics in order to investigate the interrelationship between primary and secondary metabolisms in opium poppy cells that had been treated with a fungal elicitor. Insight from this research is expected to reveal direct benefits of available opium alkaloids on the human metabolome.
Metabolite fingerprinting and compound-specific profiling revealed the extensive reprogramming of primary metabolic pathways that had been associated with alkaloid biosynthesis induction, in response to the elicitor treatment. The content of 42 diverse metabolites, in control and elicitor-treated opium poppy cell cultures, were monitored over timed 100 hour trials, with the use of a Chenomx NMR Suite v. 4.6 NMR spectrometer. Within five hours of the elicitor treatment, dynamic changes in elicitor-treated cell metabolomes were most significantly observed in the cellular pools of carbohydrates, organic acids and non-protein amino acids. Control cultures exhibited abundant fluctuations approximately 80 hours into the trials, and were most evident in amino acid and phospholipid pathway intermediate content. Overall, modulations were specifically detected in primary metabolic processes that produce secondary metabolic precursors. These modulations necessitated metabolite fluctuation in glycolysis, the tricarboxylic acid cycle, nitrogen assimilation, phospholipid/fatty acid synthesis and the shikimate pathway.
The comprehensive reprogramming of primary and secondary metabolism, along with associated cofactor biosynthetic pathways was mandatory for the analysis of cell culture responses to elicitor treatment. Ultimately, collected data produced a high-resolution map of this metabolic reprogramming, under specified conditions.
The Chenomx NMR Suite v. 4.6 spectrometer used for the experiment was “capable of identifying and quantifying individual compounds based on their respective signature spectra.” For the conducted experiments, samples were analyzed in D2O by $^1$H NMR. Employing NMR spectroscopy instead of mass spectroscopy (MS) for metabolomic analysis allowed for easier sample preparation, an analytical method that did not destroy sample analytes, and a more efficient and comprehensive compound identification method that provided structural descriptions of unknown compounds. The application of $^1$H NMR had also exposed a myriad of cellular metabolites that had not yet been observed, in previous research involving MS analysis.
Prospects
Opium poppy NMR Spectra:
http://www.biomedcentral.com/1471-2229/8/5/figure/F1
References:
http://www.biomedcentral.com/1471-2229/8/5
Overview: Nuclear Magnetic Resonance (NMR) is a powerful spectroscopic technique that gives information about both the structural and chemical properties of certain molecules. NMR is unique as it is one of the few non-destructive methods for analyzing structure and molecular dynamics. Proton NMR works by exploiting the behavior of H1 atoms when they are placed in a very strong magnetic field.
Basically, when a sample is placed in the magnet the nuclei of the proton atoms align with the magnetic field in a way analogous to when the needle of a compass aligns itself in the earth's magnetic field. The magnets used in NMR spectroscopy are ten thousand times stronger than the earth’s magnetic field. The NMR experiment consists of the application of radio frequency pulses of energy to the sample. These pulses stimulate the nuclei to rotate away from their equilibrium position and they start to rotate around the axis of the magnetic field. The exact frequency at which the nuclei rotate is related to both the chemical and physical environment of the atom in the molecule. So therefore, by using different combinations of pulses and delays it is possible to determine how each atom in the molecule interacts with other atoms in the molecule. Finally, using a large set of these interactions it is possible to calculate the three-dimensional structures of molecules. Nuclear Magnetic Resonance can also be used to look at dynamic processes. Some of which are internal motions within regions of larger molecules such as loops in a protein or the base pairs in DNA or RNA. NMR can also be used to monitor chemical reactions.
Image- http://www.pharmacy.arizona.edu/faculty/yanglab/images/NMRFacility/NMR.JPG
Links to any related materials in Wikipedia http://en.wikipedia.org/wiki/Proton_NMR
http://en.wikipedia.org/wiki/NMR
http://en.wikipedia.org/wiki/NMR_spectroscopy
Online source #1: http://riodb01.ibase.aist.go.jp/sdbs/cgi-bin/direct_frame_top.cgi
Spectrum database for compounds
This website is a database with the spectroscopy images for numerous organic compounds. It organizes the compounds into a integrated spectral data base system for organic compounds that allows anyone to use it as a references. The website holds not only proton NMR images but a wide range of other visualization techniques too. Its main purpose is to organize all the existing information on the spectra of organic compounds into a manageable system. By knowing a the molecular weight, molecular formula, compound name, or even the atoms present in the molecule, one can get a list of organic compounds whose spectra match that criteria.
Terms-
Organic compounds- Chemical compounds based on carbon chains or rings and also containing hydrogen, with or without oxygen, nitrogen, and other elements
Raman spectrum- a spectroscopic technique used in condensed matter physics and chemistry to study vibrational, rotational, and other low-frequency modes in a system
Electron spin resonance spectrum- technique for studying chemical species that have one or more unpaired electrons, such as organic and inorganic free radicals or inorganic complexes possessing a transition metal ion.
SDBS- (Spectral Data Base System) an integrated Spectral Data Base System for organic compounds.
Spectrometers- An optical instrument used to measure properties of light over a specific portion of the electromagnetic spectrum.
Spectral width- the wavelength interval over which the magnitude of all spectral components is larger than that of the component in question.
Connection-
This website relates to our biochemistry class because almost all the molecules that we were are studying are in this database. The molecules in our book and from the discussions in class were discovered or analyzed through proton NMR techniques. The spectra of these molecules are organized into one large database on this website.
Image- http://freebsd.kde.org/img/screenshots/en_jon-d_screen4.png
Online source #2: http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmr1.htm
Description of how proton NMR works
This web page gives an in depth analysis of how Nuclear Magnetic Resonance works and the theory behind it. The site goes on to explain how nuclear spin and the splitting of energy levels in a magnetic field affect the picture that is produced by NMR. It clarifies that the magnetic field, produced by the NMR apparatus, changes the spin and chemical shift of the nuclei of proton atoms resulting in different signals picked up by the machine. These signals, each of which are unique to a certain bond to hydrogen and the molecule in question, are presented to the user in the form of graph. The user can then interpret this graph to determine specific bonds and structure of molecule.
Terms-
Nuclear spin- an intrinsic property of certain nuclei that gives them an associated characteristic angular momentum and magnetic moment
Quantum mechanics- The laws of physics that apply on very small scales, eg to elementary particles and atoms.
Nucleus- the positively charged dense center of an atom
Spin relaxation- describes the interaction between neighboring nuclei with identical precessional frequencies but differing magnetic quantum states
Spin-spin coupling- Interactions between the nuclear spins of nearby nuclei, that give rise to characteristic splittings of resonance lines in NMR spectra.
Connections-
This website relates to the topics in class by demonstrating the science behind a tool that is used greatly in understanding biochemistry. Biochemists are constantly trying to identify new proteins and define biological processes such as the ones in our book. To understand them they need to be able to visualize the atoms of the molecules and reactions. To visualize them they will use either electron microscopes, NMR, or other techniques. Proton NMR is, by far, one of the easiest and most efficient ways to gain a quick understanding of the molecules in question.
Pictures-
http://pages.unibas.ch/mdpi/ecsoc-4/c0030/Image528.gif
http://www2.ups.edu/faculty/hanson/Spectroscopy/NMR/splittingtree.gif
Online Source #3:http://www.stolaf.edu/depts/chemistry/courses/toolkits/380/js/nmr1/
Database for proton chemical shifts
This website is a great database to view the chemical shifts of different aromatic compounds. The site allows you to manipulate different the frequency, the compound, and them functional group shown on the graph. This allows one to gain a general understanding of what to look for in chemical shifts of certain molecules and provide a standard for certain molecules and functional groups. This database can act as a reference for people who are trying to identify the metabolite or molecule in question.
Terms:
Functional group- a specific structure of one or more atoms that is responsible for the chemical behavior of a substance
Chemical shift- The difference between the resonance frequencies of a given nucleus and a standard reference nucleus
Peaks- The parts of the graph that give rise to the identification of splitting and are indicators of chemical structure.
Aromaticity- is a chemical property in which a conjugated ring of unsaturated bonds, lone pairs, or empty orbitals exhibit a stabilization stronger than would be expected by the stabilization of conjugation alone.
NMR resonant frequency- A variable on the apparatus that for a particular substance is directly proportional to the strength of the applied magnetic field.
Connections-
This website is related to the biochemistry class as it is a good visual of how biochemists determine the structure of compound they are trying to identify. As this web page focuses on aromatic compounds, it becomes pertinent to biochemists as both Phenylalanine, Tyrosine and Tryptophan, all have benzene rings. So if a biochemist were trying to identify metabolite in a metabolic pathway for the metabolomic database, he could refer to this site. If the proton NMR of his metabolite matches that of an aromatic amino acid, he would be able to narrow down his choices.
Images-
http://www.le.ac.uk/by/teach/biochemweb/images/aromatic.gif
http://hyperphysics.phy-astr.gsu.edu/hbase/organic/imgorg/aromatic2.gif
Article #1:http://www.ncbi.nlm.nih.gov/pubmed/18277383?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum
Applications of proton NMR
This article is a study of how proton NMR is now being used to measure subtle metabolic changes that precede and accompany chronic vascular complications. These complications are the primary cause of premature death due to diabetes. Scientists are using proton NMR, in their study, to obtain a multimetabolite characterization of high-risk diabetes patients. To do this they measured the serum of numerous patients with type I diabetes. With the use of the NMR they were able to develop new metabonomic frameworks to visualize and interpret the data and to link the metabolic profiles to the underlying diagnostic and biochemical variables.
Terms-
Macrovascular- Pertaining to the macrovasculature, the portion of the vasculature of the body comprising the larger vessels, those with an internal diameter of more than 100 microns
Serum- The noncellular liquid phase resulting from the clotting of a sample whole Blood or plasma
Metabonomics – The study of metabolic responses to drugs, environmental changes and diseases
Biomarkers - Substances in the blood whose levels can indicate the presence or extent of disease
Metabolic profile- An examination of a sample of blood to determine its chemical, physical, or serologic characteristics
Connections-
This article relates to the discussions in class by showing the applications of metabolomics and how it can be applied to diagnose and possibly treat disease. This study focused on diabetes in particular and used the close cousin of metabolomics, metabonomics, to determine how each different metabolite affects a metabolic pathway. Proton NMR helps by allowing scientists to view metabolites in diabetic patients’ blood both in vivo and in vitro easier. This ability to view the metabolites allows researchers to understand the effects of metabolites on pathways and helps to complete the metabolomic study of patients with diabetes.
Pictures
http://www.wired.com/ly/wired/news/images/full/ulderleg1_f.jpg
http://www.greenfacts.org/images/glossary/serum.jpg
Article #2:http://www3.interscience.wiley.com/cgi-bin/abstract/112149537/ABSTRACT?CRETRY=1&SRETRY=0
Use of Proton NMR to determine metabolic pathways
This article focuses on a new technique that harnesses water-suppressed localized proton NMR spectroscopy and stimulated echoes to detect metabolites in the human brain in vivo. This technique is able to apply proton NMR to an area of 64 square milliliters in order to view the molecules in the occipital area. The metabolites that they were able to view were acetate, N-acetyl aspartate, -amino butyrate, glutamine, glutamate, aspartate, creatine and phosphocreatine, choline-containing compounds, taurine, and inositols. Using this technology they were able to compare different metabolomic pathways to each other in vivo.
Terms-
Spectroscopy- Is the analysis of the lines of light emitted from excited atoms as the electrons drop back through their orbitals.
In vivo- In a living organism, as opposed to in vitro (in the laboratory).
Occipital area of the brain- the visual processing center of the mammalian brain, containing most of the anatomical region of the visual cortex.
MRI- a noninvasive, non-x-ray diagnostic technique based on the magnetic fields of hydrogen atoms in the body.
Spectra- The plural of spectrum. A range of wavelengths.
Connections-
This article ties in very well with past class discussions about metabolomics. It is at the root of the new science and reveals a how an already pre-existing tool can be used to aid in the human metabolomic project. By being able to view the intermediates and end products of the system pathways in vivo it becomes much easier to determine the effects of different metabolites on pathways. It does this by if one were to add a certain metabolite to a location in the body, proton NMR would allow us to see the how it effects the end product of the pathway without removing tissue.
Pictures-
http://cis.jhu.edu/data.sets/cortical_segmentation_validation/photos/occipital75.jpg
http://www.nature.com/npp/journal/v32/n6/images/1301248f1.jpg
Article #3:http://www.ncbi.nlm.nih.gov/pubmed/16648611?ordinalpos=10&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum
Applications of proton NMR
This article is a study a comprehensive compilation of a study done on mice, which utilized proton NMR as its primary visualization tool. The main objective of this study was to characterize the adipose tissue deposits and other visceral organs and to establish a model of in vivo phenotyping. The experiment used proton NMR to do a morphological analysis and create a 1H-NMR spectra focalized on specific lipid deposits. The results of the experiment showed that proton NMR could be used to great accuracy to measure the amount of the different types of lipid deposits in the body. Along with determining the different types of adipose (subcutaneous and visceral) they determined proton NMR allowed them to determine the amount of saturation and unsaturation of the triglycerides stored.
Terms-
Paradigm- an example serving as a model
Phenotype- The observable physical or biochemical traits of an organism, as determined by genetics and the environment.
Subcutaneous- beneath the skin
Visceral- Pertaining to the membranous covering of the internal organs
ob/ob Mice- a well-known animal model for obesity and diabetes.
Connections-
This article relates to what we have studied in class in many ways. The most obvious is lipid synthesis and metabolism, in which the study showed ways to view the gross break down and formation triglyceride deposits. If one used proton NMR before and after applying different stimulants and environments to the subject, one would be able to determine the amount of lipid metabolism occurring. Also by fixing the diet of the subject, we would be able to find the percent of lipid metabolism occurring in contrast to glycolysis.
Pictures-
http://www.hsph.harvard.edu/GSH-LAB/images/obese_mice.jpg

## Articles and Web Pages for Review and Inclusion
## Peer-Reviewed Article #1:

Metabolomics as an Extension of Proteomic Analysis: Study of Acute Kidney Injury
Seminars in Nephrology, Volume 27, Issue 6, Pages 609-620 November 2007

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       Identify the main focus of the resource. Possible answers include specific organisms, database design, intergration of information, but there are many more possibilities as well.

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#### Relevance to a Traditional Metabolism Course

       Enter a 100-150 word description of how the material in this article connects to a traditional metabolism course. Does the article relate to particular pathways (e.g., glycolysis, the citric acid cycle, steroid synthesis, etc.) or to regulatory mechanisms, energetics, location, integration of pathways? Does it talk about new analytical approaches or ideas? Does the article show connections to the human genome project (or other genome projects)?

## Peer-Reviewed Article #2:

Metabolic Discrimination of Catharanthus roseus Leaves Infected by Phytoplasma Using 1H-NMR Spectroscopy and Multivariate Data Analysis
Plant Physiology 135:2398-2410 2004

#### Main Focus

       Identify the main focus of the resource. Possible answers include specific organisms, database design, intergration of information, but there are many more possibilities as well.

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#### Summary

       Enter your article summary here. Please note that the punctuation is critical at the start (and sometimes at the end) of each entry. It should be 300-500 words. What are the main points of the article? What questions were they trying to answer? Did they find a clear answer? If so, what was it? If not, what did they find or what ideas are in tension in their findings?

#### Relevance to a Traditional Metabolism Course

       Enter a 100-150 word description of how the material in this article connects to a traditional metabolism course. Does the article relate to particular pathways (e.g., glycolysis, the citric acid cycle, steroid synthesis, etc.) or to regulatory mechanisms, energetics, location, integration of pathways? Does it talk about new analytical approaches or ideas? Does the article show connections to the human genome project (or other genome projects)?