- Written by Administrator
- Created: 26 January 2014
Isotopic labeling is a technique for tracking the passage of a sample of substance through a system. The substance is 'labeled' by including unusual isotopes in its chemical composition. 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.
Isotopic labeling method can be used in biochemistry to help understand biochemical reactions and interactions. Isotops used in labeling may be radioactive or stable.
The use of radioactive isotops in ion uptake assays allows the calculation of Km, Ki and Vmax and determines the initial change in the ion content of the cells.
Examples of using radionuclides:
• Phosphorus-32 and Phosphorus-33 is used to label nucleotides.
• Sodium-22 and Chloride-36 are commonly used to study ion transporters.
• Sulfur-35 is used to label proteins and nucleic acids.
Stable isotopically labeled products can provide accurate and non-radioactive in vivo studies of both nutrition and metabolism.
Examples of using stable isotopes:
• Oxygen-17 is the only stable isotope of oxygen possessing a nuclear spin. The nuclear spin allows through NMR static and dynamic non-invasive studies of metabolic pathways of compounds incorporating oxygen.
• Carbon-13 is a natural, stable isotope of carbon . 13C-enriched compounds are used in the research of metabolic processes. 13C is also used to quantitate proteins and in Stable isotope labeling with amino acids in cell culture ( SILAC)
• Doubly-labeled water is water in which both the hydrogen and the oxygen has been partly or completely replaced with an uncommon isotope of these elements. In practice almost all applications of the "doubly-labeled water" method use water labeled with the heavy, non-radioactive forms of the elements deuterium and oxygen-18. The doubly-labeled water method is particularly useful for measuring average metabolic rate over relatively long periods of time (a few days or weeks), in subjects. This is done by administering a dose of doubly-labeled water, and then measuring the elimination rates of deuterium and O-18 in the subject.
In isotopic labeling, there are several ways to detect the presence of labeling isotopes. Since isotopes have different masses, they can be separated using mass spectrometry. Another consequence of the difference in mass is that molecules containing isotopes have different vibrational modes and can be detected by infrared spectroscopy. In combination with isotopic labeling of metabolic compounds, the GC-MS is used for determining metabolic activity. Most applications are based on the use of 13C as the labeling and the measurement of 13C/12C ratios. Isotopic labeling ins some cases may be used together with nuclear magnetic resonance spectroscopy. NMR detects not only isotopic differences, but also gives an indication of the position of the atom.
|Wolfe R. R., Chinkes D. L. Isotope tracers in metabolic research. – 2nd ed. Hoboken, New Jersey: John Wiley&Sons, Inc, 2005. – 464 p.|
|Isotopic labeling / Wikipedia|
|Doubly-labelled water (DLW) – resource centre|