Can stable isotope mass spectrometry replace ‎radiolabelled approaches in metabolic studies?

Abstract

Metabolic pathways and the key regulatory points thereof can be deduced using isotopically labelled substrates. One prerequisite is the accurate measurement of the labeling pattern of targeted metabolites. The subsequent estimation of metabolic fluxes following incubation in radiolabelled substrates has been extensively used. Radiolabelling is a sensitive approach and allows determination of total label uptake since the total radiolabel content is easy to detect. However, the incubation of cells, tissues or the whole plant in a stable isotope enriched environment and the use of either mass spectrometry or nuclear magnetic resonance techniques to determine label incorporation within specific metabolites offers the possibility to readily obtain metabolic information with higher resolution. It additionally also offers an important complement to other post-genomic strategies such as metabolite profiling providing insights into the regulation of the metabolic network and thus allowing a more thorough description of plant cellular function. Thus, although safety concerns mean that stable isotope feeding is generally preferred, the techniques are in truth highly complementary and application of both approaches in tandem currently probably provides the best route towards a comprehensive understanding of plant cellular metabolism.

Modulation of auxin signalling through DIAGETROPICA and ENTIRE differentially affects tomato plant growth via changes in photosynthetic and mitochondrial metabolism

“…We directly evaluated the rate of R L in the mutants lines ( dgt and entire ) by measuring the 14 CO 2 evolution following incubation of leaf discs with positionally labelled 14 C‐glucose ( 14 C‐Glc) to assess the relative rate of flux through the TCA cycle and the metabolic flux analysis using 14 C‐label (Figure and Table ). This information is of pivotal significance given that metabolic pathways, and the key regulatory points thereof can be deduced using isotopically labelled substrates (Batista Silva, Daloso, Fernie, Nunes‐Nesi, & Araújo, ). For this purpose, we recorded the evolution of 14 CO 2 following incubation of leaf discs in light (approximately 150 μmol m −2 s −1 ), supplied with [1‐ 14 C]‐Glc and [3,4‐ 14 C]‐Glc over a period of 6 hr.…”

“…Data were normalized with respect to mean response calculated from wild type. Values are presented as means ± SE (n = 6) Daloso, Fernie, Nunes-Nesi, & Araújo, 2016). For this purpose, we recorded the evolution of 14 CO 2 following incubation of leaf discs in light (approximately 150 μmol m −2 s −1 ), supplied with [1-14 C]-Glc and [3,4-14 C]-Glc over a period of 6 hr.…”

Analytical Considerations of Stable Isotope Labelling in Lipidomics

“…Historically, radiolabelled tracers have frequently been used in metabolism research, but they have now largely been replaced by stable isotope-labelled tracers. While radioactive tracers may have the advantage of higher analytical sensitivity, this is somewhat offset by strict legal restrictions on handling and waste disposal, limitations of use of radiolabelled tracers in humans and potential interference with physiological metabolism [8,15,16].…”

Advances in metabolic flux analysis toward genome-scale profiling of higher organisms

“…From an experimental viewpoint, there have been relatively few advances in recent years, with gradual shift away from using radioisotopes toward stable isotopes [ 16 ]. Given the fact that companies specializing in radiochemicals have dramatically reduced the number of labeled metabolites they offer, this shift is likely to rapidly gather pace.…”