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Isotopic labeling (or isotopic labelling) is a technique used to track the passage of an isotope (an atom with a detectable variation in neutron count) 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 used in isotopic labeling may be or . 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, vibrational mode, or radioactive decay. Mass spectrometry detects the difference in an isotope's mass, while infrared spectroscopy detects the difference in the isotope's vibrational modes. Nuclear magnetic resonance detects atoms with different gyromagnetic ratios. 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 (C6H5OH) in water by replacing common hydrogen (protium) with deuterium ( deuterium labeling). Upon adding phenol to heavy water (water containing D2O in addition to the usual H2O), a hydrogen-deuterium exchange is observed to affect phenol's hydroxyl group (resulting in C6H5OD), indicating that phenol readily undergoes hydrogen-exchange reactions with water. Mainly the hydroxyl group is affected—without a catalyst, the other 5 hydrogen atoms are much slower to undergo exchange—reflecting the difference in chemical environments between the hydroxyl hydrogen and the aryl hydrogens.
Nuclear magnetic resonance (NMR) and mass spectrometry (MS) 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 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 Dickin, A. P., 2005. Radiogenic Isotope Geology, Cambridge University Press. involves an isotope produced by radioactive decay, which is usually in a ratio with a non-radiogenic isotope (whose abundance in the earth does not vary due to radioactive decay).
A network of reactions adopted from the Glycolysis 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 (F6P), which has 6 carbon atoms with a label 13C at carbon positions 1 and 2. 1,2-13C F6P becomes two glyceraldehyde 3-phosphate (G3P), one 2,3-13C T3P and one unlabeled T3P. The 2,3-13C T3P can now be reacted with sedoheptulose 7-phosphate (S7P) to form an unlabeled erythrose 4-phosphate(E4P) 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.
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 (blue circles), commonly known as uniformly labeled, and one completely unlabeled (white circles). 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. (1998). 9780126662603, Academic Press. ISBN 9780126662603 These patterns of labeled atoms and unlabeled atoms in one compound represent . 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.
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 (MS) 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 (2D-NMR), the MS instruments work directly with hydrolysate.
In gas chromatography-mass spectrometry (GC-MS), 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 (LC-MS), the GC is replaced with a liquid chromatograph.de Graaf, A. A. (2000c). Use of 13C labeling and NMR spectroscopy in metabolic flux analysis. In NMR in Biotechnology: Theory and Applications (J.-N. Barbotin and J.-C. Portais, Eds.), Horizon Scientific Press. 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 (DMFDMA) and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) Dauner, M., and Sauer, U. (2000). GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnol. Prog. 16, 642-649. 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.
For these purposes, a particularly useful type of radioactive decay is positron emission. When a positron collides with an electron, it releases two high-energy traveling in diametrically opposite directions. Suppose the positron is produced within a solid object. In that case, it will travel as little as 0.5 millimeters, up to 6 mm. If both of these photons can be detected, the location of the decay event can be determined very precisely.
Strictly speaking, radioisotopic labeling includes only cases where radioactivity is artificially introduced by experimenters, but some natural phenomena allow similar analysis to be performed. In particular, radiometric dating uses a closely related principle.
Most of the minerals that are essential for human health and of particular interest to nutrition researchers have stable isotopes, some well-suited as biological tracers because of their low natural abundance. Iron, zinc, calcium, copper, magnesium, selenium and molybdenum are among the essential minerals having stable isotopes to which isotope tracer methods have been applied. Iron, zinc and calcium in particular have been extensively studied.
Aspects of mineral nutrition/metabolism that are studied include absorption (from the gastrointestinal tract into the body), distribution, storage, excretion and the kinetics of these processes. Isotope tracers are administered to subjects orally (with or without food, or with a mineral supplement) and/or intravenously. Isotope enrichment is then measured in blood plasma, erythrocytes, urine and/or feces. (1996). 9780124905405, Academic Press. ISBN 9780124905405 (2025). 9780849387302, CRC Press. ISBN 9780849387302 Enrichment has also been measured in breast milk and intestinal contents. Tracer experiment design sometimes differs between minerals due to differences in their metabolism. For example, iron absorption is usually determined from incorporation of tracer in erythrocytes whereas zinc or calcium absorption is measured from tracer appearance in plasma, urine or feces.Davidsson, L. (Lena), 1957- (2025). 9789201265104, International Atomic Energy Agency. ISBN 9789201265104 The administration of multiple isotope tracers in a single study is common, permitting the use of more reliable measurement methods and simultaneous investigations of multiple aspects of metabolism.
The measurement of mineral absorption from the diet, often conceived of as bioavailability, is the most common application of isotope tracer methods to nutrition research. Among the purposes of such studies are the investigations of how absorption is influenced by type of food (e.g. plant vs animal source, breast milk vs formula), other components of the diet (e.g. Phytic acid), disease and metabolic disorders (e.g. environmental enteric dysfunction), the reproductive cycle, quantity of mineral in diet, chronic mineral deficiency, subject age and homeostatic mechanisms. When results from such studies are available for a mineral, they may serve as a basis for estimations of the human physiological and dietary requirements of the mineral. (2025). 9780309511995, National Academy Press. ISBN 9780309511995
When tracer is administered with food for the purpose of observing mineral absorption and metabolism, it may be in the form of an intrinsic or extrinsic label. (1996). 9780124905405, Academic Press. ISBN 9780124905405 An intrinsic label is isotope that has been introduced into the food during its production, thus enriching the natural mineral content of the food, whereas extrinsic labeling refers to the addition of tracer isotope to the food during the study. Because it is a very time-consuming and expensive approach, intrinsic labeling is not routinely used. Studies comparing measurements of absorption using intrinsic and extrinsic labeling of various foods have generally demonstrated good agreement between the two labeling methods, supporting the hypothesis that extrinsic and natural minerals are handled similarly in the human gastrointestinal tract.
Enrichment is quantified from the measurement of isotope ratios, the ratio of the tracer isotope to a reference isotope, by mass spectrometry. Multiple definitions and calculations of enrichment have been adopted by different researchers. Calculations of enrichment become more complex when multiple tracers are used simultaneously. Because enriched isotope preparations are never isotopically pure, i.e. they contain all the element's isotopes in unnatural abundances, calculations of enrichment of multiple isotope tracers must account for the perturbation of each isotope ratio by the presence of the other tracers.
Due to the prevalence of mineral deficiencies and their critical impact on human health and well-being in resource-poor countries, the International Atomic Energy Agency has recently published detailed and comprehensive descriptions of stable isotope methods to facilitate the dissemination of this knowledge to researchers beyond western academic centers.
Various isotopes of lead can be used to study circulation on a global scale. Different oceans (i.e. the Atlantic, Pacific, Indian, etc.) have different isotopic signatures. This results from differences in isotopic ratios of sediments and rocks within the different oceans. Because the different isotopes of lead have half-lives of 50–200 years, there is not enough time for the isotopic ratios to be homogenized throughout the whole ocean. Therefore, precise analysis of Pb isotopic ratios can be used to study the circulation of the different oceans.
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