Metabolomics is the study of intracellular and extracellular metabolite profiles in biological systems, from which a systematic understanding of molecular physiology in cell, tissue, or organisms can putatively be achieved. As such, metabolomics plays important roles in biomarker discovery for disease diagnosis/drug responses and metabolic pathway analysis in response to perturbations. The commonly used analytical platforms for metabolomics, such as mass spectrometry (MS) and nuclear magnetic resonance (NMR) can detect the total amount of metabolites after complex metabolism in the body at a specific moment, but cannot accurately identify the specific metabolic pathway of metabolites. At present, to overcome such problems and further study of metabolomics, stable isotope-labeled compounds as tracers are introduced to delineate metabolic pathway and track the element fates in cell metabolites. The technology is also known as stable isotope-assisted metabolomics (SIAM).


Isotopic labeling approaches

The detection of metabolites and the quantification of intracellular metabolic fluxes in diverse organisms in metabolomics can be achieved by isotopic labeling organisms with stable isotope-labeled compounds. The isotopic labeling approaches include isotopic dilution (or enrichment), isotopic tracing and 13C-fingerprinting. The details are as follows and graphically illustrated in Fig. 1.

Isotopic dilution (or enrichment): The method is described briefly as follows. Grow cells are labeled with multiple carbon sources (some of them are labeled) and then the labeling situation of the metabolic products are measured. The method is used for studying cell nutrient utilizations.

Isotopic tracing: The method is described briefly as follows. Expose cell culture to a labeled compound and then measure change of labeling in downstream metabolites over time. The method allows isotopic non-stationary metabolic flux analysis to quantify cell fluxomes.

13C-fingerprinting: The method is described briefly as follows. Use specified labeled 13C-substrates to create steady state and position specific labeling patterns in metabolites, which delineate functional pathways. The method allows 13C-metabolic flux analysis to quantify cell fluxomes.

MetabolomicsFig. 1 Isotopic labeling approaches for metabolism analysis.


The practical applications of isotope-labeled compounds in metabolomics have a broad-scope. The details are as follows.

Analysis of tracking nutrient utilizations: A rich medium contains bottleneck nutrients to promote cell growth and product synthesis. When a 13C-labeled compounds is supplied in rich mediums, the metabolites synthesized de novo from the substrate will be labeled, while metabolites derived from the undefined nutrients in the medium are not. Therefore, 13C-labeling in biogenic and exogenous metabolites can identify the essential nutrients that are not effectively synthesized from primary substrates. It has particular value in medical research for disease diagnoses and pathological analysis.

Metabolite identifications: Since labeled isotopes are distinguishable from natural isotopes by m/z ratios, the biological samples labeling by 13C and 15N labeled compounds of often assist metabolite analysis.

Metabolite quantification analysis: The use of stable isotope-labeled metabolites as internal standards can be used to determine metabolite concentrations even under the noise of sample degradation and instrumental variations.

Pathway investigations: Stable isotope-labeled compounds are widely used to confirm or discover functional pathways. For example, a threonine-independent route for isoleucine synthesis was found in a cyanobacterium by using 2-13C glycerol to fingerprint its metabolites. Stable isotope-labeled compounds have also been used for elucidation of network-wide metabolic pathways. For example, U-13C glutamine or amine-labeled α-15N glutamine were supplied to cell culture with unlabeled glucose to fingerprint downstream metabolites to analyze the glutamine metabolism in A549 human lung carcinoma cells.

Metabolic analysis of microbial communities: Species in microbial communities has complex nutrient interactions. In ecology, stable isotope-labeled compounds are widely used to determine microbial community populations and carbon assimilation patterns. For example, after feeding 13C-toluene to a microbial consortium, toluene-degrading microorganisms were identified by analyzing 13C-fatty acids and sequencing 13C-labeled 16S rRNA.

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  • You, L. et al. Application of Stable isotope-assisted metabolomics for cell metabolism studies[J]. Metabolites, 2014, 4:142-165.
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