Isotopic labeling (or isotopic labelling) is a technique used to track the passage of an
isotope
Isotopes are two or more types of atoms that have the same atomic number (number of protons in their nuclei) and position in the periodic table (and hence belong to the same chemical element), and that differ in nucleon numbers (mass numbers) ...
(an
atom
Every atom is composed of a nucleus and one or more electrons bound to the nucleus. The nucleus is made of one or more protons and a number of neutrons. Only the most common variety of hydrogen has no neutrons.
Every solid, liquid, gas, and ...
with a detectable variation in neutron count) through a
reaction
Reaction may refer to a process or to a response to an action, event, or exposure:
Physics and chemistry
*Chemical reaction
*Nuclear reaction
*Reaction (physics), as defined by Newton's third law
*Chain reaction (disambiguation).
Biology and me ...
,
metabolic pathway
In biochemistry, a metabolic pathway is a linked series of chemical reactions occurring within a cell. The reactants, products, and intermediates of an enzymatic reaction are known as metabolites, which are modified by a sequence of chemical reac ...
, or
cell
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...
. The
reactant
In chemistry, a reagent ( ) or analytical reagent is a substance or compound added to a system to cause a chemical reaction, or test if one occurs. The terms ''reactant'' and ''reagent'' are often used interchangeably, but reactant specifies a ...
is 'labeled' by replacing specific atoms by their isotope. The reactant is then allowed to undergo the reaction. The position of the isotopes in the
products
Product may refer to:
Business
* Product (business), an item that serves as a solution to a specific consumer problem.
* Product (project management), a deliverable or set of deliverables that contribute to a business solution
Mathematics
* Produ ...
is measured to determine the sequence the isotopic atom followed in the reaction or the cell's metabolic pathway. The
nuclide
A nuclide (or nucleide, from nucleus, also known as nuclear species) is a class of atoms characterized by their number of protons, ''Z'', their number of neutrons, ''N'', and their nuclear energy state.
The word ''nuclide'' was coined by Truman ...
s used in isotopic labeling may be
stable nuclide
Stable nuclides are nuclides that are not radioactive and so (unlike radionuclides) do not spontaneously undergo radioactive decay. When such nuclides are referred to in relation to specific elements, they are usually termed stable isotopes.
Th ...
s or
radionuclide
A radionuclide (radioactive nuclide, radioisotope or radioactive isotope) is a nuclide that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transfer ...
s. In the latter case, the labeling is called
radiolabeling
A radioactive tracer, radiotracer, or radioactive label is a chemical compound in which one or more atoms have been replaced by a radionuclide so by virtue of its radioactive decay it can be used to explore the mechanism of chemical reactions by ...
.
In isotopic labeling, there are multiple ways to detect the presence of labeling isotopes; through their
mass
Mass is an intrinsic property of a body. It was traditionally believed to be related to the quantity of matter in a physical body, until the discovery of the atom and particle physics. It was found that different atoms and different elementar ...
,
vibrational mode
A normal mode of a dynamical system is a pattern of motion in which all parts of the system move sinusoidally with the same frequency and with a fixed phase relation. The free motion described by the normal modes takes place at fixed frequencies. ...
, or
radioactive decay
Radioactive decay (also known as nuclear decay, radioactivity, radioactive disintegration, or nuclear disintegration) is the process by which an unstable atomic nucleus loses energy by radiation. A material containing unstable nuclei is consid ...
.
Mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
detects the difference in an isotope's mass, while
infrared spectroscopy
Infrared spectroscopy (IR spectroscopy or vibrational spectroscopy) is the measurement of the interaction of infrared radiation with matter by absorption, emission, or reflection. It is used to study and identify chemical substances or function ...
detects the difference in the isotope's vibrational modes.
Nuclear magnetic resonance
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a ...
detects atoms with different gyromagnetic ratios. The radioactive decay can be detected through an
ionization chamber
The ionization chamber is the simplest type of gas-filled radiation detector, and is widely used for the detection and measurement of certain types of ionizing radiation, including X-rays, gamma rays, and beta particles. Conventionally, the term ...
or
autoradiographs of gels.
An example of the use of isotopic labeling is the study of
phenol
Phenol (also called carbolic acid) is an aromatic organic compound with the molecular formula . It is a white crystalline solid that is volatile. The molecule consists of a phenyl group () bonded to a hydroxy group (). Mildly acidic, it req ...
(C
6H
5OH) in water by replacing common
hydrogen
Hydrogen is the chemical element with the symbol H and atomic number 1. Hydrogen is the lightest element. At standard conditions hydrogen is a gas of diatomic molecules having the formula . It is colorless, odorless, tasteless, non-toxic, an ...
(
protium) with
deuterium
Deuterium (or hydrogen-2, symbol or deuterium, also known as heavy hydrogen) is one of two Stable isotope ratio, stable isotopes of hydrogen (the other being Hydrogen atom, protium, or hydrogen-1). The atomic nucleus, nucleus of a deuterium ato ...
(deuterium labeling). Upon adding phenol to
deuterated water (water containing D
2O in addition to the usual H
2O), the substitution of deuterium for the hydrogen is observed in phenol's
hydroxyl group
In chemistry, a hydroxy or hydroxyl group is a functional group with the chemical formula and composed of one oxygen atom covalently bonded to one hydrogen atom. In organic chemistry, alcohols and carboxylic acids contain one or more hydroxy g ...
(resulting in C
6H
5OD), indicating that phenol readily undergoes hydrogen-exchange reactions with water. Only the hydroxyl group is affected, indicating that the other 5 hydrogen atoms do not participate in the exchange reactions.
Isotopic tracer
![Benzyneconversioncolouredlabels](https://upload.wikimedia.org/wikipedia/commons/1/1b/Benzyneconversioncolouredlabels.png)
An ''isotopic tracer'', (also "isotopic marker" or "isotopic label"), is used in
chemistry
Chemistry is the science, scientific study of the properties and behavior of matter. It is a natural science that covers the Chemical element, elements that make up matter to the chemical compound, compounds made of atoms, molecules and ions ...
and
biochemistry
Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...
to help understand chemical
reactions
Reaction may refer to a process or to a response to an action, event, or exposure:
Physics and chemistry
*Chemical reaction
*Nuclear reaction
*Reaction (physics), as defined by Newton's third law
*Chain reaction (disambiguation).
Biology and me ...
and interactions. In this technique, one or more of the
atom
Every atom is composed of a nucleus and one or more electrons bound to the nucleus. The nucleus is made of one or more protons and a number of neutrons. Only the most common variety of hydrogen has no neutrons.
Every solid, liquid, gas, and ...
s of the
molecule
A molecule is a group of two or more atoms held together by attractive forces known as chemical bonds; depending on context, the term may or may not include ions which satisfy this criterion. In quantum physics, organic chemistry, and bioch ...
of interest is substituted for an atom of the same
chemical element
A chemical element is a species of atoms that have a given number of protons in their nuclei, including the pure substance consisting only of that species. Unlike chemical compounds, chemical elements cannot be broken down into simpler sub ...
, but of a different
isotope
Isotopes are two or more types of atoms that have the same atomic number (number of protons in their nuclei) and position in the periodic table (and hence belong to the same chemical element), and that differ in nucleon numbers (mass numbers) ...
(like a
radioactive
Radioactive decay (also known as nuclear decay, radioactivity, radioactive disintegration, or nuclear disintegration) is the process by which an unstable atomic nucleus loses energy by radiation. A material containing unstable nuclei is consid ...
isotope used in
radioactive tracing). 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
neutron
The neutron is a subatomic particle, symbol or , which has a neutral (not positive or negative) charge, and a mass slightly greater than that of a proton. Protons and neutrons constitute the nuclei of atoms. Since protons and neutrons beh ...
s, however, means that it can be detected separately from the other atoms of the same element.
Nuclear magnetic resonance
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a ...
(NMR) and
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
(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
autoradiograph
An autoradiograph is an image on an X-ray film or nuclear emulsion produced by the pattern of decay emissions (e.g., beta particles or gamma rays) from a distribution of a radioactive substance. Alternatively, the autoradiograph is also available ...
s of gels in
gel electrophoresis
Gel electrophoresis is a method for separation and analysis of biomacromolecules ( DNA, RNA, proteins, etc.) and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size (IEF ...
. The radiation emitted by compounds containing the radioactive isotopes darkens a piece of
photographic film
Photographic film is a strip or sheet of transparent film base coated on one side with a gelatin photographic emulsion, emulsion containing microscopically small light-sensitive silver halide crystals. The sizes and other characteristics of th ...
, 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
Geology () is a branch of natural science concerned with Earth and other astronomical objects, the features or rocks of which it is composed, and the processes by which they change over time. Modern geology significantly overlaps all other Ear ...
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
Isotope geochemistry is an aspect of geology based upon the study of natural variations in the relative abundances of isotopes of various elements. Variations in isotopic abundance are measured by isotope ratio mass spectrometry, and can reveal ...
.
Isotopic tracers are usually subdivided into two categories:
stable isotope
The term stable isotope has a meaning similar to stable nuclide, but is preferably used when speaking of nuclides of a specific element. Hence, the plural form stable isotopes usually refers to isotopes of the same element. The relative abundanc ...
tracers and
radiogenic
A radiogenic nuclide is a nuclide that is produced by a process of radioactive decay. It may itself be radioactive (a radionuclide) or stable (a stable nuclide).
Radiogenic nuclides (more commonly referred to as radiogenic isotopes) form some ...
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
An isotopic signature (also isotopic fingerprint) is a ratio of non-radiogenic ' stable isotopes', stable radiogenic isotopes, or unstable radioactive isotopes of particular elements in an investigated material. The ratios of isotopes in a sample ...
. A radiogenic isotope tracer involves an isotope produced by
radioactive decay
Radioactive decay (also known as nuclear decay, radioactivity, radioactive disintegration, or nuclear disintegration) is the process by which an unstable atomic nucleus loses energy by radiation. A material containing unstable nuclei is consid ...
, which is usually in a ratio with a non-radiogenic isotope (whose abundance in the earth does not vary due to radioactive decay).
Stable isotope labeling
![StableIsotopeLabeling2](https://upload.wikimedia.org/wikipedia/commons/7/72/StableIsotopeLabeling2.png)
Stable isotope labeling involves the use of non-radioactive
isotopes
Isotopes are two or more types of atoms that have the same atomic number (number of protons in their nuclei) and position in the periodic table (and hence belong to the same chemical element), and that differ in nucleon numbers (mass numbers) ...
that can act as a 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
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a ...
(NMR) and
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
(MS). Some of the most common stable isotopes are
2H,
13C, and
15N, which can further be produced into
NMR solvents,
amino acids
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
,
nucleic acids
Nucleic acids are biopolymers, macromolecules, essential to all known forms of life. They are composed of nucleotides, which are the monomers made of three components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main clas ...
,
lipids
Lipids are a broad group of naturally-occurring molecules which includes fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. The functions of lipids include ...
, common
metabolites
In biochemistry, a metabolite is an intermediate or end product of metabolism.
The term is usually used for small molecules. Metabolites have various functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, c ...
and cell growth
media
Media may refer to:
Communication
* Media (communication), tools used to deliver information or data
** Advertising media, various media, content, buying and placement for advertising
** Broadcast media, communications delivered over mass el ...
. The compounds produced using stable isotopes are either specified by the percentage of labeled isotopes (i.e. 30% uniformly labeled
13C glucose contains a mixture that is 30% labeled with
13 carbon isotope and 70% naturally labeled carbon) or by the specifically labeled carbon positions on the compound (i.e. 1-
13C glucose which is labeled at the first carbon position of glucose).
A network of reactions adopted from the
glycolysis pathway and the
pentose phosphate pathway
The pentose phosphate pathway (also called the phosphogluconate pathway and the hexose monophosphate shunt and the HMP Shunt) is a metabolic pathway parallel to glycolysis. It generates NADPH and pentoses (5-carbon sugars) as well as ribose 5-pho ...
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
Fructose 6-phosphate (sometimes called the Neuberg ester) is a derivative of fructose, which has been phosphorylated at the 6-hydroxy group. It is one of several possible fructosephosphates. The β-D-form of this compound is very common in cells. ...
(F6P), which has 6 carbon atoms with a label
13C at carbon position 1 and 2. 1,2-
13C F6P becomes two
glyceraldehyde 3-phosphate
Glyceraldehyde 3-phosphate, also known as triose phosphate or 3-phosphoglyceraldehyde and abbreviated as G3P, GA3P, GADP, GAP, TP, GALP or PGAL, is a metabolite that occurs as an intermediate in several central pathways of all organisms.Nelson, D ...
(G3P), one 2,3-
13C T3P and one unlabeled T3P. The 2,3-
13C T3P can now be reacted with
sedoheptulose 7-phosphate
Sedoheptulose 7-phosphate is an intermediate in the pentose phosphate pathway.
It is formed by transketolase and acted upon by transaldolase.
Sedoheptulokinase is an enzyme that uses sedoheptulose and ATP to produce ADP and sedoheptulose 7-phosp ...
(S7P) to form an unlabeled
erythrose 4-phosphate
Erythrose 4-phosphate is a phosphate of the simple sugar erythrose. It is an intermediate in the pentose phosphate pathway and the Calvin cycle.
In addition, it serves as a precursor in the biosynthesis of the aromatic amino acids tyrosine, pheny ...
(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.
Metabolic flux analysis using stable isotope labeling
Metabolic flux analysis Metabolic flux analysis (MFA) is an experimental fluxomics technique used to examine production and consumption rates of metabolites in a biological system. At an intracellular level, it allows for the quantification of metabolic fluxes, thereby e ...
(MFA) using stable
isotope
Isotopes are two or more types of atoms that have the same atomic number (number of protons in their nuclei) and position in the periodic table (and hence belong to the same chemical element), and that differ in nucleon numbers (mass numbers) ...
labeling is an important tool for explaining the flux of certain elements through the
metabolic pathway
In biochemistry, a metabolic pathway is a linked series of chemical reactions occurring within a cell. The reactants, products, and intermediates of an enzymatic reaction are known as metabolites, which are modified by a sequence of chemical reac ...
s and
reactions
Reaction may refer to a process or to a response to an action, event, or exposure:
Physics and chemistry
*Chemical reaction
*Nuclear reaction
*Reaction (physics), as defined by Newton's third law
*Chain reaction (disambiguation).
Biology and me ...
within a
cell
Cell most often refers to:
* Cell (biology), the functional basic unit of life
Cell may also refer to:
Locations
* Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...
. 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
In systems theory, a system or a Process theory, process is in a steady state if the variables (called state variables) which define the behavior of the system or the process are unchanging in time. In continuous time, this means that for those p ...
(the isotopes entering and leaving the cell remain constant with time) or a quasi-steady state (steady state is reached for a given period of time). The isotope pattern of the output
metabolite
In biochemistry, a metabolite is an intermediate or end product of metabolism.
The term is usually used for small molecules. Metabolites have various functions, including fuel, structure, signaling, stimulatory and inhibitory effects on enzymes, c ...
is determined. The output isotope pattern provides valuable information, which can be used to find the magnitude of
flux
Flux describes any effect that appears to pass or travel (whether it actually moves or not) through a surface or substance. Flux is a concept in applied mathematics and vector calculus which has many applications to physics. For transport ph ...
, rate of conversion from
reactant
In chemistry, a reagent ( ) or analytical reagent is a substance or compound added to a system to cause a chemical reaction, or test if one occurs. The terms ''reactant'' and ''reagent'' are often used interchangeably, but reactant specifies a ...
s to
products
Product may refer to:
Business
* Product (business), an item that serves as a solution to a specific consumer problem.
* Product (project management), a deliverable or set of deliverables that contribute to a business solution
Mathematics
* Produ ...
, 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 (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. These patterns of labeled atoms and unlabeled atoms in one compound represent
isotopomer
Isotopomers or isotopic isomers are isomers with isotopic atoms, having the same number of each isotope of each element but differing in their positions. The result is that the molecules are either constitutional isomers or stereoisomers solely ...
s. 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
Stoichiometry refers to the relationship between the quantities of reactants and products before, during, and following chemical reactions.
Stoichiometry is founded on the law of conservation of mass where the total mass of the reactants equal ...
of each reaction,
constraints, and an optimization procedure resolve a flux map. The ir
reversible reaction
A reversible reaction is a reaction in which the conversion of reactants to products and the conversion of products to reactants occur simultaneously.
: \mathit aA + \mathit bB \mathit cC + \mathit dD
A and B can react to form C and D or, in the ...
s provide the thermodynamic constraints needed to find the fluxes. A
matrix
Matrix most commonly refers to:
* ''The Matrix'' (franchise), an American media franchise
** ''The Matrix'', a 1999 science-fiction action film
** "The Matrix", a fictional setting, a virtual reality environment, within ''The Matrix'' (franchis ...
is constructed that contains the stoichiometry of the reactions. The
intracellular
This glossary of biology terms is a list of definitions of fundamental terms and concepts used in biology, the study of life and of living organisms. It is intended as introductory material for novices; for more specific and technical definitions ...
fluxes are estimated by using an
iterative method
In computational mathematics, an iterative method is a Algorithm, mathematical procedure that uses an initial value to generate a sequence of improving approximate solutions for a class of problems, in which the ''n''-th approximation is derived fr ...
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
Isotopomers or isotopic isomers are isomers with isotopic atoms, having the same number of each isotope of each element but differing in their positions. The result is that the molecules are either constitutional isomers or stereoisomers solely ...
can be used. The two primary methods,
nuclear magnetic resonance
Nuclear magnetic resonance (NMR) is a physical phenomenon in which nuclei in a strong constant magnetic field are perturbed by a weak oscillating magnetic field (in the near field) and respond by producing an electromagnetic signal with a ...
(NMR) and
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
(MS), have been developed for measuring mass isotopomers in stable isotope labeling.
Proton NMR
Proton nuclear magnetic resonance (proton NMR, hydrogen-1 NMR, or 1H NMR) is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the struc ...
was the first technique used for
13C-labeling experiments. Using this method, each single protonated carbon position inside a particular
metabolite pool
Metabolite pool is a collective term for all of the substances involved in the metabolic process in a biological system.
Metabolic pools are within cells (or organelles such as chloroplasts) and refer to the reservoir of molecules upon which enzyme ...
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
Proton nuclear magnetic resonance (proton NMR, hydrogen-1 NMR, or 1H NMR) is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the struc ...
, 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 Metabolic flux analysis (MFA) is an experimental fluxomics technique used to examine production and consumption rates of metabolites in a biological system. At an intracellular level, it allows for the quantification of metabolic fluxes, thereby e ...
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 Hydrolysate refers to any product of hydrolysis. Protein hydrolysate has special application in sports medicine because its consumption allows amino acids to be absorbed by the body more rapidly than intact proteins, thus maximizing nutrient delive ...
.
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. 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.
Applications in human mineral nutrition research
The use of stable isotope tracers to study
mineral
In geology and mineralogy, a mineral or mineral species is, broadly speaking, a solid chemical compound with a fairly well-defined chemical composition and a specific crystal structure that occurs naturally in pure form.John P. Rafferty, ed. ( ...
nutrition and metabolism in humans was first reported in the 1960s. While radioisotopes had been used in human nutrition research for several decades prior, stable isotopes presented a safer option, especially in subjects for which there is elevated concern about radiation exposure, e.g. pregnant and lactating women and children. Other advantages offered by stable isotopes include the ability to study elements having no suitable radioisotopes and to study long-term tracer behavior.
Thus the use of stable isotopes became commonplace with the increasing availability of isotopically enriched materials and inorganic mass spectrometers. The use of stable isotopes instead of radioisotopes does have several drawbacks: larger quantities of tracer are required, having the potential of perturbing the naturally existing mineral; analytical sample preparation is more complex and
mass spectrometry
Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a ''mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is use ...
instrumentation more costly; the presence of tracer in whole bodies or particular tissues cannot be measured externally.
Nonetheless, the advantages have prevailed making stable isotopes the standard in human studies.
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
Iron () is a chemical element with symbol Fe (from la, ferrum) and atomic number 26. It is a metal that belongs to the first transition series and group 8 of the periodic table. It is, by mass, the most common element on Earth, right in f ...
,
zinc
Zinc is a chemical element with the symbol Zn and atomic number 30. Zinc is a slightly brittle metal at room temperature and has a shiny-greyish appearance when oxidation is removed. It is the first element in group 12 (IIB) of the periodi ...
,
calcium
Calcium is a chemical element with the symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar to ...
,
copper
Copper is a chemical element with the symbol Cu (from la, cuprum) and atomic number 29. It is a soft, malleable, and ductile metal with very high thermal and electrical conductivity. A freshly exposed surface of pure copper has a pinkis ...
,
magnesium
Magnesium is a chemical element with the symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 of the periodic ta ...
,
selenium
Selenium is a chemical element with the symbol Se and atomic number 34. It is a nonmetal (more rarely considered a metalloid) with properties that are intermediate between the elements above and below in the periodic table, sulfur and tellurium, ...
and
molybdenum
Molybdenum is a chemical element with the symbol Mo and atomic number 42 which is located in period 5 and group 6. The name is from Neo-Latin ''molybdaenum'', which is based on Ancient Greek ', meaning lead, since its ores were confused with lea ...
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
The gastrointestinal tract (GI tract, digestive tract, alimentary canal) is the tract or passageway of the digestive system that leads from the mouth to the anus. The GI tract contains all the major organ (biology), organs of the digestive syste ...
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. 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.
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
In pharmacology, bioavailability is a subcategory of absorption and is the fraction (%) of an administered drug that reaches the systemic circulation.
By definition, when a medication is administered intravenously, its bioavailability is 100%. H ...
, 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.
phytate
Phytic acid is a six-fold dihydrogenphosphate ester of inositol (specifically, of the ''myo'' isomer), also called inositol hexakisphosphate (IP6) or inositol polyphosphate. At physiological pH, the phosphates are partially ionized, resulting in ...
), disease and metabolic disorders (e.g.
environmental enteric dysfunction), the reproductive cycle, quantity of mineral in diet, chronic
mineral deficiency
Mineral deficiency is a lack of the dietary minerals, the micronutrients that are needed for an organism's proper health. The cause may be a poor diet, impaired uptake of the minerals that are consumed, or a dysfunction in the organism's use of the ...
, 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.
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. 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
The International Atomic Energy Agency (IAEA) is an intergovernmental organization that seeks to promote the peaceful use of nuclear energy and to inhibit its use for any military purpose, including nuclear weapons. It was established in 1957 ...
has recently published detailed and comprehensive descriptions of stable isotope methods to facilitate the dissemination of this knowledge to researchers beyond western academic centers.
Radioisotopic labeling
Radioisotopic labeling is a technique for tracking the passage of a sample of substance through a system. The substance is "labeled" by including
radionuclide
A radionuclide (radioactive nuclide, radioisotope or radioactive isotope) is a nuclide that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transfer ...
s in its chemical composition. When these
decay
Decay may refer to:
Science and technology
* Bit decay, in computing
* Software decay, in computing
* Distance decay, in geography
* Decay time (fall time), in electronics
Biology
* Decomposition of organic matter
* Tooth decay (dental caries ...
, their presence can be determined by detecting the
radiation
In physics, radiation is the emission or transmission of energy in the form of waves or particles through space or through a material medium. This includes:
* ''electromagnetic radiation'', such as radio waves, microwaves, infrared, visi ...
emitted by them. Radioisotopic labeling is a special case of isotopic labeling.
For these purposes, a particularly useful type of radioactive decay is
positron emission
Positron emission, beta plus decay, or β+ decay is a subtype of radioactive decay called beta decay, in which a proton inside a radionuclide nucleus is converted into a neutron while releasing a positron and an electron neutrino (). Positron ...
. When a positron collides with an electron, it releases two high-energy
photon
A photon () is an elementary particle that is a quantum of the electromagnetic field, including electromagnetic radiation such as light and radio waves, and the force carrier for the electromagnetic force. Photons are massless, so they always ...
s traveling in diametrically opposite directions. If the positron is produced within a solid object, it is likely to do this before traveling more than a millimeter. 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
Radiometric dating, radioactive dating or radioisotope dating is a technique which is used to date materials such as rocks or carbon, in which trace radioactive impurities were selectively incorporated when they were formed. The method compares t ...
uses a closely related principle.
Applications in proteomics
In
proteomics
Proteomics is the large-scale study of proteins. Proteins are vital parts of living organisms, with many functions such as the formation of structural fibers of muscle tissue, enzymatic digestion of food, or synthesis and replication of DNA. In ...
, the study of the full set of
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
s expressed by a
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding ge ...
, identifying
diseases
A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that ar ...
biomarker
In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, ...
s can involve the usage of
stable isotope labeling by amino acids in cell culture Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling. It is a popular method for quantitat ...
(SILAC), that provides isotopic labeled forms of amino acid used to estimate protein levels. In protein recombinant, manipulated proteins are produced in large quantities and isotope labeling is a tool to test for relevant proteins. The method used to be about selectively enrich nuclei with
13C or
15N or deplete
1H from them. The recombinant would be expressed in ''
E.coli
''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Esche ...
'' with media containing
15N-
ammonium chloride as a source of nitrogen. The resulting
15N labeled proteins are then purified by immobilized metal affinity and their percentage estimated. In order to increase the yield of labeled proteins and cut down the cost of isotope labeled media, an alternative procedure primarily increases the cell mass using unlabeled media before introducing it in a minimal amount of labeled media. Another application of isotope labeling would be in measuring DNA synthesis, that is cell proliferation ''in vitro''. Uses H
3-thymidine labeling to compare pattern of synthesis (or sequence) in cells.
Applications for ecosystem process analysis
Isotopic tracers are used to examine processes in natural systems, especially terrestrial and aquatic environments. In soil science
15N tracers are used extensively to study nitrogen cycling, whereas
13C and
14C, stable and radioisotopes of carbon respectively, are used for studying turnover of organic compounds and fixation of by
autotroph
An autotroph or primary producer is an organism that produces complex organic compounds (such as carbohydrates, fats, and proteins) using carbon from simple substances such as carbon dioxide,Morris, J. et al. (2019). "Biology: How Life Works", ...
s. For example, Marsh et al. (2005) used dual labeled (
15N- and
14C)
urea
Urea, also known as carbamide, is an organic compound with chemical formula . This amide has two amino groups (–) joined by a carbonyl functional group (–C(=O)–). It is thus the simplest amide of carbamic acid.
Urea serves an important r ...
to demonstrate utilization of the compound by
ammonia
Ammonia is an inorganic compound of nitrogen and hydrogen with the formula . A stable binary hydride, and the simplest pnictogen hydride, ammonia is a colourless gas with a distinct pungent smell. Biologically, it is a common nitrogenous was ...
oxidizers as both an energy source (ammonia oxidation) and carbon source (chemoautotrophic carbon fixation). Deuterated water is also used for tracing the fate and ages of water in a tree or in an ecosystem.
Applications for oceanography
Tracers are also used extensively in
oceanography
Oceanography (), also known as oceanology and ocean science, is the scientific study of the oceans. It is an Earth science, which covers a wide range of topics, including ecosystem dynamics; ocean currents, waves, and geophysical fluid dynamic ...
to study a wide array of processes. The isotopes used are typically naturally occurring with well-established sources and rates of formation and decay. However, anthropogenic isotopes may also be used with great success. The researchers measure the isotopic ratios at different locations and times to infer information about the physical processes of the ocean.
Particle transport
The ocean is an extensive network of particle transport. Thorium isotopes can help researchers decipher the vertical and horizontal movement of matter.
234Th has a constant, well-defined production rate in the ocean and a half-life of 24 days. This naturally occurring isotope has been shown to vary linearly with depth. Therefore, any changes in this linear pattern can be attributed to the transport of
234Th on particles. For example, low isotopic ratios in surface water with very high values a few meters down would indicate a vertical flux in the downward direction. Furthermore, the thorium isotope may be traced within a specific depth to decipher the lateral transport of particles.
Circulation
Circulation within local systems, such as bays, estuaries, and groundwater, may be examined with radium isotopes.
223Ra has a half-life of 11 days and can occur naturally at specific locations in rivers and groundwater sources. The isotopic ratio of radium will then decrease as the water from the source river enters a bay or estuary. By measuring the amount of
223Ra at a number of different locations, a circulation pattern can be deciphered. This same exact process can also be used to study the movement and discharge of groundwater.
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.
Tectonic processes and climate change
Isotopes with extremely long half-lives and their decay products can be used to study multi-million year processes, such as tectonics and extreme climate change. For example, in
rubidium–strontium dating
The rubidium-strontium dating method is a radiometric dating technique, used by scientists to determine the age of rocks and minerals from their content of specific isotopes of rubidium (87Rb) and strontium (87Sr, 86Sr). One of the two naturally ...
, the isotopic ratio of strontium (
87Sr/
86Sr) can be analyzed within ice cores to examine changes over the earth's lifetime. Differences in this ratio within the ice core would indicate significant alterations in the earth's geochemistry.
Isotopes related to nuclear weapons
The aforementioned processes can be measured using naturally occurring isotopes. Nevertheless, anthropogenic isotopes are also extremely useful for oceanographic measurements. Nuclear weapons tests released a plethora of uncommon isotopes into the world's oceans.
3H,
129I, and
137Cs can be found dissolved in seawater, while
241Am and
238Pu are attached to particles. The isotopes dissolved in water are particularly useful in studying global circulation. For example, differences in lateral isotopic ratios within an ocean can indicate strong water fronts or gyres. Conversely, the isotopes attached to particles can be used to study mass transport within water columns. For instance, high levels of Am or Pu can indicate
downwelling
Downwelling is the process of accumulation and sinking of higher density material beneath lower density material, such as cold or saline water beneath warmer or fresher water or cold air beneath warm air. It is the ''sinking'' limb of a convecti ...
when observed at great depths, or
upwelling
Upwelling is an oceanographic phenomenon that involves wind-driven motion of dense, cooler, and usually nutrient-rich water from deep water towards the ocean surface. It replaces the warmer and usually nutrient-depleted surface water. The nutr ...
when observed at the surface.
Methods for isotopic labeling
*
Chemical synthesis
As a topic of chemistry, chemical synthesis (or combination) is the artificial execution of chemical reactions to obtain one or several products. This occurs by physical and chemical manipulations usually involving one or more reactions. In moder ...
* Enzyme-mediated exchange
* Recombinant
protein expression in isotopic labeled media.
See also
*
Uses of radionuclides
*
Radioactivity in biology
Radioactivity is generally used in life sciences for highly sensitive and direct measurements of biological phenomena, and for visualizing the location of biomolecules radiolabelled with a radioisotope.
All atoms exist as stable or unstable iso ...
*
Radioactive tracer
A radioactive tracer, radiotracer, or radioactive label is a chemical compound in which one or more atoms have been replaced by a radionuclide so by virtue of its radioactive decay it can be used to explore the mechanism of chemical reactions by ...
*
Isotopomer
Isotopomers or isotopic isomers are isomers with isotopic atoms, having the same number of each isotope of each element but differing in their positions. The result is that the molecules are either constitutional isomers or stereoisomers solely ...
*
Isotopologue
In chemistry, isotopologues are molecules that differ only in their isotopic composition. They have the same chemical formula and bonding arrangement of atoms, but at least one atom has a different number of neutrons than the parent.
An exampl ...
*
Isobaric labeling
Isobaric labeling is a mass spectrometry strategy used in quantitative proteomics. Peptides or proteins are labeled with chemical groups that have (at least nominally) identical mass (isobaric), but vary in terms of distribution of heavy isotopes ...
*
Isotope dilution
Isotope dilution analysis is a method of determining the quantity of chemical substances. In its most simple conception, the method of isotope dilution comprises the addition of known amounts of isotopically enriched substance to the analyzed samp ...
*
Infrared spectroscopy of metal carbonyls
Metal carbonyls are coordination complexes of transition metals with carbon monoxide ligands. Metal carbonyls are useful in organic synthesis and as catalysts or catalyst precursors in homogeneous catalysis, such as hydroformylation and Reppe ch ...
*
Localization of organelle proteins by isotope method tagging
References
External links
{{Library resources box
, onlinebooks=no
, by=no
Synthesis of Radiolabeled Compounds
Labeling
Labelling or using a label is describing someone or something in a word or short phrase. For example, the label "criminal" may be used to describe someone who has broken a law. Labelling theory is a theory in sociology which ascribes labelling ...
Laboratory techniques
Physical chemistry
Biochemistry methods
Mass spectrometry
Spectroscopy
Nuclear physics