A roadmap for interpreting (13)C metabolite labeling patterns from cells

Abstract

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments.

Figure 1

Labeling basics. a) Time dependent metabolic changes: metabolism reaches a metabolic steady state when the parameters of interest (e.g. glucose uptake rate) are constant over time. b) Time dependent labeling changes: upon addition of an isotopically labeled carbon source, the isotopic enrichment will increase in the metabolites until the steady state enrichment is reached. c) Mass distribution vector (MDV) (also known as mass distribution (MID) vector): Labeling patterns are MDVs that consist of the fractional abundance of each isotopologue (mass isotopomer). M denotes mass of the unlabeled metabolite. d) Cellular compartmentalization: Most labeling pattern detection methods cannot resolve different cellular compartments, thus the whole cell average labeling pattern is measured.

Figure 2

Interpretation of labeling data. a) Steady state labeling data are independent from the metabolite levels. b) Fractional contribution quantifies the contribution of a labeled nutrient to the metabolite of interest. c) Exchange fluxes can lead to rapidly labeled metabolites although the net flux of the nutrient to the metabolites is small. d) Dynamic labeling patterns are metabolite level dependent: The flux from glutamine to glutamate is the same in condition A and B, but in condition A the glutamate levels are greater than in condition B. Consequently, the labeling dynamics of glutamate in condition A are slower than in condition B although the flux from glutamine to glutamate is the same in both conditions. e) Relative flux activity between two conditions can be evaluated without kinetic flux calculations if both the labeling dynamics and all metabolite levels of the pathway of interest are altered in the same direction.