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(Work in progress section for Radioactive tracer page)
Interpretation in biological experiments[edit]
Biological radioisotope tracing experiments, for example to establish if a candidate precursor metabolite is actually a precursor & becomes incorporated via metabolism into a target metabolite, is now infrequently done, likely due to the strict regulation of radioisotope use in research, and the necessity of specialized experimentation like HPLC-scintillation counters. But, radioisotope tracing experiment do have some advantages vs analogous stable isotope tracing techniques. For example, radioisotope detection is highly sensitive vs techniques like NMR, LC-UV, and even LC-MS. But overall this sensitivity necessitates careful purification to avoid misinterpretation. A classical best practice was purification of the target metabolite by at least two times recrystallization, as samples with even trace radiolabeled impurities could lead to misinterpretation. Later, HPLC-scintillation coupled instruments allowed for workable purification of a target metabolite, without laborious recrystallization.
more difficult to interpret vs stable isotope tracing experiments.
Since the absolute quantity of input radiolabeled tracer is known, and because radioisotope detection methods such as scintillation give the same responsivity per unit of incorporated radiolabel regardless of the molecular structure of its host molecule, allows final absolute quantification of the radiolabel into a target metabolite. This specific incorporation, or percentage of initial radiolabel which is incorporated into a target-metabolite, has been used as a rough scale of the reliability of interpretation. A scale which is often attributed to Sir Derek Barton, states that >= 1% specific incorporation is excellent support for a precursor-metabolite to target metabolite link, 0.1% is good, 0.01% is positive, 0.001% or less is dubious, and probably negative.[1] In contrast, due to the infrequency of performing absolutely quantified NMR or LC-MS experiments, stable isotope tracing experiments are interpreted typically in relative incorporation terms. For example, incorporation of 13C carbons into glucose, would be quantified relative to the native 13C isotopic abundance, perhaps 10% greater than normal. This can be misleading, as the relative incorporation depends on the pool (quantity within an organism) of the target metabolite. A metabolite like glucose may have a large pool, whereas a specialized metabolite may have a very small pool, meaning that specific incorporation cannot be calculated from relative incorporation measures. An example of a misleading relative incorporation tracing experiment, would be when a low quantity specialized metabolite is traced with a very high level of input tracer. Observed relative incorporation, may seem quite solid at >150% relative incorporation, but with a
- ^ Morgan, E. David (2004). Biosynthesis in insects (1st ed.). Cambridge: Royal Society of Chemistry. p. 70-71. ISBN 978-0-85404-691-1. OCLC 232637789.