An overview on NMR spectroscopy based metabolomics

Introduction: A fi eld of bioscience analysis that uses High throughput (HT) technologies to identify the products of metabolism formed in the cell metabolic processes (i.e. the metabolome). This process is termed metabolomics.The fi rst NMR based metabolic studies were carried out by Wilson and Burlingame in the year 1974. Nuclear magnetic spectroscopy is one technique using which one can identify and characterize the metabolites formed due to metabolism.


Metabolomics
Metabolomics is defi ned as the comprehensive analysis of metabolites i.e., identifi cation and quantifi cation in a biological system Figure 1 [1].
The metabolites known by metabolomics square measure intermediary metabolites used to form the macromolecular structures and some other molecules that help in necessary metabolic functions of the body. Also metabolites those are involved in signalling can also be identifi ed along with the secondary metabolites [2].
The study of molecules whose molecular size is less than or equal to 1500 Daltons is called as metabolomics. The metabolomics studies do not involve the quantifi cation of sugars, polymers and amino acids since their molecular weight will be more than 1500 Daltons [3].

Metabolite profi ling
The metabolite profi ling is a technique which enables is said to be one of the major tools that helps to identify large number of metabolites. But this process is done on a biased scale because of the limitations involved in the methodology and differences in analytical platforms. This is one limitation for metabolite profi ling. Despite the bias introduced, this method is still used widely for the analysis of metabolites that enables to give true and complete picture of the metabolome.

Metabolite fi ngerprinting
The high throughput technologies like NMR spectroscopy are used to identify and characterise the structure of metabolome. Just like every person has a unique fi ngerprint, similarly, every metabolite has unique characteristic comparable to the fi ngerprint of the human being. Comparison of these metabolites with change in time can be identifi ed using the metabolite fi ngerprinting. This technique is relatively simple and coarser compared to metabolite profi ling.

Metabonomics
Metabonomics is the study of biological and scientifi c processes that involve the metabolites. The Metabonomics mainly focuses on the metabolic response that the organism produces in response to the pathophysiological stimuli or due to the genetic modifi cations occurring in the body. Generally, the study of Metabonomics is restricted to analysis of metabolites in microbiological and non-botanical studies [12].
Although, these three different approaches generate the copious amounts of productive information about the metabolite, they do not provide the comprehensive analysis of the metabolome. But, they allow the scientist to get an idea of the metabolic state of an organism at a particular period of time i.e. it provides a snapshot of the metabolic state. Metabolomics can lead to better understanding of the organization and functioning of the organism when used along with other omic studies like genomics, proteomics, and other physiological assays [12].

Applications
The metabolomic studies have found to have great potential in plant biology. They are used to study various parameters like accumulation of biomass, study of production of secondary metabolites in plants, and also stress resistance [13].
Thanatometabolomics-study of metabolite modulations after death i.e., forensic studies [14]. Importance 1. Biomass accumulation and resistance to sure environmental stressors area unit vital as plants area unit sought-after as a possible supply of different energy production like biofuel . [12].

Precision medicine
3. Diagnosis of disease with analysis of metabolite responsible for the disease. 4. The presence of adulterants in food and medicines can also be identifi ed with the help of metabolomics.

To know the time of death of person and to help in
Tissue transplantation [13].

Measurement of the metabolome
The two approaches used in designing the metabolomics studies are:

Targeted approach
The targeted approach is used when one wants to measure a particular set of metabolites.
Ex: the effect of drug on the specifi c enzyme is one type of targeted approach where the therapeutic modifi cations and genetic modifi cations occurring because of the drug metabolism are studied.

Untargeted approach
Untargeted approach can be used when one wants to identify and quantify metabolites as many as possible present in the biological sample. Various types of metabolites can be identifi ed using the untargeted approach.
The two techniques that are used to study the metabolic state in an organism are:  Mass spectrometry (MS) based metabolomics.
The ideal technique for metabolomic studies is the coupling of Mass spectroscopy with NMR spectroscopy. Though this process is quite expensive, better results can be expected

Mass spectroscopy based metabolomics
The mass spectroscopy basically studies the effect of ionising energy on molecules. The principle involved in the technique of mass spectroscopy is as follows.
A mass spectrometer generated multiple ions form the sample that is under investigation. Then, electron ionisation occurs where the gas phase ion of the compund is produced. • When the external magnetic fi eld is removed, the atoms come back to their original state into the lower energy levels. During this process, they emit energy at a particular frequency.
• The NMR spectrum of that particular nucleus is obtained for these energy transfers and frequencies [15].
The NMR spectrum gives the following information about the metabolite: 1. Number of signals produced by protons.

2.
Position of the signals.

3.
Integration and intensity of the signals.

4.
Splitting of the signals produced.

NMR Spectroscopy
The nuclear magnetic resonance spectroscopy is a spectroscopic technique is used to determine the organic compounds. The molecular structure of the metabolite can be elucidated using the NMR spectroscopy.

Principle involved in NMR spectroscopy
• All the metabolites are made up of atoms that consists of nuclei. Every nucleus consists of positive charges that is responsible for the spin of the nuclei.
• Whenever an external magnetic fi eld is applied using any source, the atoms absorb the energy and excitation occurs. The electrons excite into the higher energy levels i.e. the energy transfer occurs at a particular wavelength.
• The wavelength at which energy transfer occurs corresponds to the frequency.

The workfl ow pattern of NMR based metabolomics
The workfl ow pattern can be categorized into 3 steps namely;

Sample preparation
The Silica nanoparticles can also be used to remove proteins in the plasma [16].

Tagging
Tagging of compound with NMR active labels to is one best approach for compounds that show overlapping of 1H signals or those that are present in very less concentrations. Tagging with an NMR active label are useful in identifi cation of the metabolite.
Nitrogen-15 with a hydrogen attached us the most widely used active label because of the advantage that it provided high sensitivity spectrum without the interference of other signals. 4. To fi nd out changes in the redox potentials when exposed to toxins form external source of from within the cell.

Accelerating the new drug discovery process by
identifying the lead compounds. The potency and toxicity of the lead compound that is known to elicit the pharmacological action are identifi ed.
6. Removal of compounds that are known to cause certain problems and adverse effects in the body. Figure 6 [3].

Future considerations
Though the method is fully developed, there is a need to concentrate more on developing the suitable databases for NMR data. Integrated and valid databases have to be developed in order to yield better results.
Suitable procedures of preparation of sample, NMR data collection, NMR data analysis should be suggested to enhance the accuracy of the results [2].

Advantages
1. NMR is ideal when the compounds are not possible to be ionized using the mass spectroscopy method.
2. Can also be used to identify those compounds that have identical masses.
3. For determining structures of unknown compounds.
4. Used to determine the mechanisms of the changes that the metabolite undergoes.

5.
In experiments of screening of activity of drug [3].

Conclusion
In the upcoming years, it is expected that more works will be reported depending upon the advantages and strengths that the NMR spectroscopy offers with regard to metabolomics.
The NMR spectroscopy technique offers high versatility, and specifi city with respect to spectra. This technique will gain high potential in the future because of its reproducibility and the ability to quantify the metabolites.