Isotopic labeling

Isotopic labeling is a technique used to track the passage of an isotope through chemical reaction, metabolic pathway, or a biological cell. The reactant is 'labeled' by replacing one or more specific atoms with their isotopes. The reactant is then allowed to undergo the reaction. The position of the isotopes in the products is measured to determine what sequence the isotopic atom followed in the reaction or the cell's metabolic pathway. The nuclides used in isotopic labeling may be stable nuclides or radionuclides. In the latter case, the labeling is called radiolabeling.
In isotopic labeling, there are multiple ways to detect the presence of labeling isotopes; through their mass, nuclear spin, vibrational mode, or radioactive decay. Mass spectrometry detects the difference in an isotope's mass, while vibrational spectroscopy detects the difference in the isotope's vibrational frequencies. Nuclear magnetic resonance detects nuclei with non-integer spin. The radioactive decay can be detected through an ionization chamber or autoradiographs of gels.
An example of the use of isotopic labeling is the study of phenol in water by replacing common hydrogen with deuterium. Upon adding phenol to deuterated water, a hydrogen-deuterium exchange is observed to affect phenol's hydroxyl group, indicating that phenol readily undergoes hydrogen-exchange reactions with water. Mainly the hydroxyl group is affected—without a catalyst, the other five hydrogen atoms are much slower to undergo exchange—reflecting the difference in chemical environments between the hydroxyl hydrogen and the aryl hydrogens.

Isotopic tracer

An isotopic tracer,, is used in chemistry and biochemistry to help understand chemical reactions and interactions. In this technique, one or more of the atoms of the molecule of interest is substituted for an atom of the same chemical element, but of a different isotope. Because the labeled atom has the same number of protons, it will behave in almost exactly the same way as its unlabeled counterpart and, with few exceptions, will not interfere with the reaction under investigation. The difference in the number of neutrons, however, means that it can be detected separately from the other atoms of the same element.
Nuclear magnetic resonance and mass spectrometry are used to investigate the mechanisms of chemical reactions. NMR and MS detects isotopic differences, which allows information about the position of the labeled atoms in the products' structure to be determined. With information on the positioning of the isotopic atoms in the products, the reaction pathway the initial metabolites utilize to convert into the products can be determined. Radioactive isotopes can be tested using the autoradiographs of gels in gel electrophoresis. The radiation emitted by compounds containing the radioactive isotopes darkens a piece of photographic film, recording the position of the labeled compounds relative to one another in the gel.
Isotope tracers are commonly used in the form of isotope ratios. By studying the ratio between two isotopes of the same element, we avoid effects involving the overall abundance of the element, which usually swamp the much smaller variations in isotopic abundances. Isotopic tracers are some of the most important tools in geology because they can be used to understand complex mixing processes in earth systems. Further discussion of the application of isotopic tracers in geology is covered under the heading of isotope geochemistry.
Isotopic tracers are usually subdivided into two categories: stable isotope tracers and radiogenic isotope tracers. Stable isotope tracers involve only non-radiogenic isotopes and usually are mass-dependent. In theory, any element with two stable isotopes can be used as an isotopic tracer. However, the most commonly used stable isotope tracers involve relatively light isotopes, which readily undergo fractionation in natural systems. See also isotopic signature. A radiogenic isotope tracer involves an isotope produced by radioactive decay, which is usually in a ratio with a non-radiogenic isotope.

Stable isotope labeling

Stable isotope labeling involves the use of non-radioactive isotopes that can act as tracers used to model several chemical and biochemical systems. The chosen isotope can act as a label on that compound that can be identified through nuclear magnetic resonance and mass spectrometry. Some of the most common stable isotopes are ²H, ¹³C, ¹⁵N, and ¹⁸O, which can further be produced into NMR solvents, amino acids, nucleic acids, lipids, common metabolites and cell growth media. The compounds produced using stable isotopes are either specified by the percentage of labeled isotopes or by the specifically labeled carbon positions on the compound.
A network of reactions adopted from the glycolysis pathway and the pentose phosphate pathway is shown in which the labeled carbon isotope rearranges to different carbon positions throughout the network of reactions. The network starts with fructose 6-phosphate, which has six carbon atoms with a label ¹³C at carbon positions 1 and 2. 1,2-¹³C F6P becomes two glyceraldehyde 3-phosphate, one 2,3-¹³C T3P and one unlabeled T3P. The 2,3-¹³C T3P can now be reacted with sedoheptulose 7-phosphate to form an unlabeled erythrose 4-phosphate and a 5,6-¹³C F6P. The unlabeled T3P will react with the S7P to synthesize unlabeled products. The figure demonstrates the use of stable isotope labeling to discover the carbon atom rearrangement through reactions using position specific labeled compounds.

Metabolic flux analysis using stable isotope labeling

using stable isotope labeling is an important tool for explaining the flux of certain elements through the metabolic pathways and reactions within a cell. An isotopic label is fed to the cell, then the cell is allowed to grow utilizing the labeled feed. For stationary metabolic flux analysis the cell must reach a steady state or a quasi-steady state. The isotope pattern of the output metabolite is determined. The output isotope pattern provides valuable information, which can be used to find the magnitude of flux, rate of conversion from reactants to products, through each reaction.
The figure demonstrates the ability to use different labels to determine the flux through a certain reaction. Assume the original metabolite, a three carbon compound, has the ability to either split into a two carbon metabolite and one carbon metabolite in one reaction then recombine or remain a three carbon metabolite. If the reaction is provided with two isotopes of the metabolite in equal proportion, one completely labeled, commonly known as uniformly labeled, and one completely unlabeled. The pathway down the left side of the diagram does not display any change in the metabolites, while the right side shows the split and recombination. As shown, if the metabolite only takes the pathway down the left side, it remains in a 50–50 ratio of uniformly labeled to unlabeled metabolite. If the metabolite only takes the right side new labeling patterns can occur, all in equal proportion. Other proportions can occur depending on how much of the original metabolite follows the left side of the pathway versus the right side of the pathway. Here the proportions are shown for a situation in which half of the metabolites take the left side and half the right, but other proportions can occur. These patterns of labeled atoms and unlabeled atoms in one compound represent isotopomers. By measuring the isotopomer distribution of the differently labeled metabolites, the flux through each reaction can be determined.
MFA combines the data harvested from isotope labeling with the stoichiometry of each reaction, constraints, and an optimization procedure resolve a flux map. The irreversible reactions provide the thermodynamic constraints needed to find the fluxes. A matrix is constructed that contains the stoichiometry of the reactions. The intracellular fluxes are estimated by using an iterative method in which simulated fluxes are plugged into the stoichiometric model. The simulated fluxes are displayed in a flux map, which shows the rate of reactants being converted to products for each reaction. In most flux maps, the thicker the arrow, the larger the flux value of the reaction.

Isotope labeling measuring techniques

Any technique in measuring the difference between isotopomers can be used. The two primary methods, nuclear magnetic resonance and mass spectrometry, have been developed for measuring mass isotopomers in stable isotope labeling.
Proton NMR was the first technique used for ¹³C-labeling experiments. Using this method, each single protonated carbon position inside a particular metabolite pool can be observed separately from the other positions. This allows the percentage of isotopomers labeled at that specific position to be known. The limit to proton NMR is that if there are n carbon atoms in a metabolite, there can only be at most n different positional enrichment values, which is only a small fraction of the total isotopomer information. Although the use of proton NMR labeling is limiting, pure proton NMR experiments are much easier to evaluate than experiments with more isotopomer information.
In addition to Proton NMR, using ¹³C NMR techniques will allow a more detailed view of the distribution of the isotopomers. A labeled carbon atom will produce different hyperfine splitting signals depending on the labeling state of its direct neighbors in the molecule. A singlet peak emerges if the neighboring carbon atoms are not labeled. A doublet peak emerges if only one neighboring carbon atom is labeled. The size of the doublet split depends on the functional group of the neighboring carbon atom. If two neighboring carbon atoms are labeled, a doublet of doublets may degenerate into a triplet if the doublet splittings are equal.
The drawbacks to using NMR techniques for metabolic flux analysis purposes is that it is different from other NMR applications because it is a rather specialized discipline. An NMR spectrometer may not be directly available for all research teams. The optimization of NMR measurement parameters and proper analysis of peak structures requires a skilled NMR specialist. Certain metabolites also may require specialized measurement procedures to obtain additional isotopomer data. In addition, specially adapted software tools are needed to determine the precise quantity of peak areas as well as identifying the decomposition of entangled singlet, doublet, and triplet peaks.
As opposed to nuclear magnetic resonance, mass spectrometry is another method that is more applicable and sensitive to metabolic flux analysis experiments. MS instruments are available in different variants. Different from two-dimensional nuclear magnetic resonance, the MS instruments work directly with hydrolysate.
In gas chromatography-mass spectrometry, the MS is coupled to a gas chromatograph to separate the compounds of the hydrolysate. The compounds eluting from the GC column are then ionized and simultaneously fragmented. The benefit in using GC-MS is that not only are the mass isotopomers of the molecular ion measured but also the mass isotopomer spectrum of several fragments, which significantly increases the measured information.
In liquid chromatography-mass spectrometry, the GC is replaced with a liquid chromatograph. The main difference is that chemical derivatization is not necessary. Applications of LC-MS to MFA, however, are rare.
In each case, MS instruments divide a particular isotopomer distribution by its molecular weight. All isotopomers of a particular metabolite that contain the same number of labeled carbon atoms are collected in one peak signal. Because every isotopomer contributes to exactly one peak in the MS spectrum, the percentage value can then be calculated for each peak, yielding the mass isotopomer fraction. For a metabolite with n carbon atoms, n+1 measurements are produced. After normalization, exactly n informative mass isotopomer quantities remain.
The drawback to using MS techniques is that for gas chromatography, the sample must be prepared by chemical derivatization in order to obtain molecules with charge. There are numerous compounds used to derivatize samples. N,N-Dimethylformamide dimethyl acetal and N--N-methyltrifluoroacetamide are two examples of compounds that have been used to derivatize amino acids.
In addition, strong isotope effects observed affect the retention time of differently labeled isotopomers in the GC column. Overloading of the GC column also must be prevented.
Lastly, the natural abundance of other atoms than carbon also leads to a disturbance in the mass isotopomer spectrum. For example, each oxygen atom in the molecule might also be present as a ¹⁷O isotope and as a ¹⁸O isotope. A more significant impact of the natural abundance of isotopes is the effect of silicon with a natural abundance of the isotopes ²⁹Si and ³⁰Si. Si is used in derivatizing agents for MS techniques.