# Metabolomics/Analytical Methods/NMR/C NMR

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## Introduction

Researchers strive to identify and map enzymatic pathways that are critical to the functionality of endocrine cells, with the intention of applying this knowledge to therapeutic development for a myriad of disorders. $^{13}$C NMR spectroscopy and isotopomer analysis have proven to be very useful in quantitative analysis of cell metabolic processes via intermediate labeling, and estimate carbon entry into the TCA cycle. Furthermore, researchers have discussed and reported the details of NMR application to analyzing cell tissue extracts. The experiments conducted on neuroendocrine tissue samples have revealed specific cautionary measures that must be taken, ways in which to apply $^{13}$C NMR, and how to infer $^{13}$C NMR results.
Utilization of NMR spectroscopy entails manipulating the nuclei of atoms that contain a nuclear spin, with a magnetic field, ultimately generating a signal. This signal may then be interpreted to provide biochemical, structural, and anatomical information of an analyte. $^{13}$C NMR involves observing the non-radioactive $^{13}$C carbon isotope. Unlike the $^{12}$C carbon isotope, the $^{13}$C isotope has a nuclear spin, making it readily observable by NMR. Tracking of $^{13}$C through cell metabolism involved feeding cells a metabolizable $^{13}$C label (on glucose, for example), and using $^{13}$C NMR to monitor its progress through metabolic processes including the TCA cycle, citric acid cycle and glycolysis. Carbon flux through metabolic pathways was observed via $^{13}$C labeling of glutamate, an amino acid, and a 5-carbon TCA cycle byproduct, before employing NMR spectroscopy. This analytical method, however, was prone to a number of pitfalls. Reducing the probability of these errors mandates appropriate preventative measures, which include identifying any impact of media on cellular or carbon label behavior, as well as proper application and selection of metabolic models when interpreting $^{13}$C NMR data.
Experimental results indicated NMR application significance when researching pituitary adenomas. Specific results included the determination of the capacities of host metabolic modulators. Applications also might allow for observing the influences of a cell function modulator allowing for the observation of any existing interrelationships between metabolism and cell function. NMR application is expected to enable researchers to identify the fate of labeled carbons through metabolic pathways that are pertinent to hormone release. However, researchers must take care and employ the most physiologically relevant media and modern metabolic models lest their experimental results be invalid or inapplicable to previous results. In essence, eliciting proper application of $^{13}$C NMR spectroscopy is anticipated to provide information that will lead to a better understanding of cell energetics.
References:
http://www.actabiomedica.it/data/2007/supp_1_2007/simpson.pdf
13C Nuclear Magnetic Resonance Spectroscopy
Overview: 13C-NMR is an analytical technique that is used to identify the number and type of carbons in an organic molecule and the number of non-equivalent carbon atoms. It is also used to determine structure of compounds. Since 13-carbon has a spinning nucleus that has magnetic properties, NMR can be performed. The compound containing the 13-carbon atoms is placed in a very strong magnetic field and irradiated with electromagnetic energy. The nuclei absorb energy called magnetic resonance. This energy absorption is quantized and produces a spectrum for the particular compound. Spectrometers are instruments used to measure the absorption of energy by the 13C nuclei. They are very powerful magnets and irradiate the nuclei with electromagnetic radiation in the radio frequency region. The spectrum is produced as a graph with various peaks. Each unique carbon atom in a molecule produces only one peak. Where the peak is located on the graph depends on chemical shift. Chemical shift is determined by the surrounding electron density of each carbon in a molecule. Based on where the peak is and its chemical shift, the structure of organic molecules can be determined, as well as what functional groups or other atoms are attached to each carbon atom.
Online Source #1-
Wikipedia Carbon- 13 NMR
http://en.wikipedia.org/wiki/Carbon-13_NMR
Focus: This website gives a basic overview of what carbon-13 NMR is used for and how it works. It focuses mainly on the complications that this type of nuclear magnetic resonance has in comparison to proton NMR.
Terms: a) spin quantum number- the intrinsic angular momentum of an electron (or the spin of a electron) b) chemical shift- frequency of the resonance expressed with reference to a standard compound defined at 0 ppm; position on the NMR scale where the peak occurs (measured in ppm) c) ppm- parts per million; used to measure the chemical shift d) J-coupling- the coupling between two nuclear spins due to the influence of bonding electrons in the magnetic field running between the two nuclei (between carbon and hydrogen in 13C-NMR causes reduced sensitivity and makes the spectra harder to read) e) 1H-NMR (proton magnetic resonance imaging)- used to identify hydrogen atoms in an organic molecule
Connection: When talking about the glycolysis pathway, we traced the carbon atoms through the pathway to see where they ended up in pyruvate. 13C-NMR is what the carbons are marked with so that they can be followed throughout the pathway.
Online Source #2-
Carbon-13 NMR Tutorial
http://www.wfu.edu/~ylwong/nmr/c13/
Focus: This website gives a really good overview of 13C-NMR. It describes what 13C-NMR is used for and how it works. It also describes chemical shift and splitting of carbon from hydrogen. There are exercises that are interactive to help you to further understand how it works and how to interpret the 13C-NMR spectrum for different compounds.
Terms: a) resonance- when the energy required to flip the proton from its lower energy state to its higher energy state in the magnetic field is proportional to the strength of the magnetic field b) aliphatic- class of saturated or unsaturated carbon compounds, in which the carbon atoms are joined in open chains c) TMS (tetramethylsilane)- reference compound; signals a zero point on the spectrum d) aromatic compound- cyclic conjugated unsaturated molecule that is stabilized by electron delocalization
Connection: Most of the molecules formed in the metabolic pathways that we studied in class were probably identified using 13C-NMR. We can also find out where certain carbons end up in metabolic pathway products and how much of the product is produced by tracing them using 13C-NMR.
Online Source #3-
NMR Spectroscopy
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/nmr/nmr1.htm
Focus: This website provides a good overview of proton and 13C-NMR spectroscopy. There is a section on 13C-NMR spectroscopy which includes problems associated with it, improvements, what it is used for and it really goes into the physical principles behind it. They also explain the two spectrometers that are used, continuous wave and Fourier Transform. On another page they provide a link to, they include additional information on 13C-NMR such as off-resonance decoupling and insensitive nuclear enhancement by polarization transfer which are techniques for classifying carbon signals.
Terms: a) heteronuclear decoupling – an electronic technique that allows decoupling of spin-spin interactions between 13C nuclei and 1H nuclei b) pulse technology- irradiating the molecule with short pulses of electromagnetic radiation instead of slowly sweeping the magnetic field with electromagnetic radiation; another type of NMR spectroscopy that uses Fourier transform spectrometers c) isomer- one of two or more compounds that have the same chemical formula but different arrangements of the atoms within the molecules d) homotopic- giving superposable products upon substitution, and therefore spectroscopically and chemically identical e) continuous wave method- irradiation of a molecule with electromagnetic energy of a constant frequency while the magnetic field is varied or swept; type of NMR spectrometer
Connection: 13C-NMR would be a great tool to learn and use if we were to really go in depth about structures of metabolites or any other type of molecule in class. Like mentioned before, we did introduce 13C-NMR when we went over glycolysis by tracing the carbons through the pathway.
Peer Reviewed Article #1 –
Localized in vivo 13C NMR spectroscopy of the brain
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=14679498
Focus: This is a really good article that discusses the uses of 13C-NMR to learn about carbohydrate metabolism in the brain. The article first discusses the difficulties that are faced when using 13C-NMR and successful implementation of localized 13C-NMR spectroscopy. Then the author discusses the achievements of in vivo localized 13C-NMR spectroscopy of the brain which include the measurement of glutamine synthesis and brain glycogen metabolism in hypoglycemic rats and humans, to name a few.
Terms: a) in vivo- within a living organism b) autoradiography- technique that uses X-ray film to visualize radioactively labeled molecules or fragments of molecules c) tomography- series of detailed pictures of areas inside the body; the pictures are created by a computer linked to an x-ray machine d) RF (radio frequency)- the region of the electromagnetic spectrum that the radiation is in e) hypoglycemia- state of low blood sugar f) shimming- correction of inhomogeneity of the magnetic field produced by the main magnet of an NMR system due to imperfections in the magnet
Connection: In relation to what we did in class, we talked about glutamine synthesis and how glutamine carries Ammonia which is toxic to our body. 13C-NMR helps us study glutamine synthesis. If we were to alter gene expression or introduce certain chemicals into the rat, we could see if that affects the rate of glutamine synthesis. Ammonia accumulation is especially toxic to the brain and can cause death. By finding out what causes the low level of glutamine synthesis and over-accumulation of ammonia, we may be able to find a medical treatment. Also we discussed hypoglycemia in class.
Peer Reviewed Article #2 –
Metabolic Imaging by Hyperpolarized 13C Magnetic Resonance Imaging for In vivo Tumor Diagnosis
http://cancerres.aacrjournals.org/cgi/content/full/66/22/10855
Focus: This article discusses the use of 13C-NMR in cancer research, particularly in the diagnosis of tumors. Most tumors exhibit a high rate of aerobic glycolysis called the Warburg effect. They are using a method that involves magnetic resonance spectroscopy and a hyperpolarization technique that improves the signal strength for detectable compounds on a very short time scale. The glycolytic pathway compound that they are choosing to trace is pyruvate with 13C-NMR. They are watching how it is transformed to lactate and alanine and then they are able to trace all three substrates throughout cells. The results are interesting. They demonstrate that a high concentration of lactate was present in the tumor cells which indicate that tumor cells prefer the anaerobic pathways even in the presence of oxygen. Also the amount of pyruvate taken up by the tumor cell indicates that they require a lot of energy.
Terms: a) noninvasive- a procedure that does not cut or penetrate the skin b) hyperpolarized- an increase in nuclear spin polarization beyond thermal equilibrium conditions c) phenotype- the observable traits or characteristics of an organism (in this case, biochemical traits) d) 18FDG- radiopharmaceutical used in positron emission tomography e) necrosis- death of cells or tissue f) PET (Positron Emission Tomography)- special type of X-ray using a radioactive dye which shows areas of the brain that have a higher or lower metabolism than normal
Connection: In relation to biochemistry, this article is all about the glycolytic pathway in tumor cells. They were able to trace carbons in pyruvate through glycolysis and then through lactic acid fermentation. 13C-NMR is being used to detect tumors metabolically. This has many medical applications particularly in cancer research.
Peer Reviewed Article #3 –
Methyl-beta-D-glucopyranoside in higher plants: accumulation and intracellular localization in Geum montanum L. leaves and in model systems studied by 13C nuclear magnetic resonance.
http://www.ncbi.nlm.nih.gov/pubmed/15361539?ordinalpos=41&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum
Focus: This article discusses the use of 13C-NMR to discover a major component in leaves of an herb plant that is a product of this plant’s metabolism which aides in decreasing the accumulation of methanol in the plant cell’s cytoplasm. MeG is the compound that is found in the leaf cells and is synthesized from glucose and methanol.
Terms: a) senescence- process of aging, decline and death b) vacuole- large organelle in plant cells that contains water and waste products c) dicot- plants that produce two seed leaves d) cytosol- interior fluid of the cell e) overwinter- to remain alive in the winter
Connection: In class, we discussed how plants synthesize carbohydrates. MeG is a carbohydrate that is synthesized from glucose and methanol. MeG is beneficial to certain plants because it aides in ridding the plant of a toxic molecule. We did talk about how humans get rid of waste products but this could lead us into discussion of how plants get rid of their waste products.

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

Altered regulation of metabolic pathways in human lung cancer discerned by 13C stable isotope-resolved metabolomics (SIRM)
Molecular Cancer 2009, 8:41

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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:

Systematic NMR analysis of stable isotope labeled metabolite mixtures in plant and animal systems: coarse grained views of metabolic pathways
PLoS One. 2008;3(11):e3805. Epub 2008 Nov 25.

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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)?