Parallel labeling experiments and metabolic flux analysis: Past, present and future methodologies

Abstract

Radioactive and stable isotopes have been applied for decades to elucidate metabolic pathways and quantify carbon flow in cellular systems using mass and isotope balancing approaches. Isotope-labeling experiments can be conducted as a single tracer experiment, or as parallel labeling experiments. In the latter case, several experiments are performed under identical conditions except for the choice of substrate labeling. In this review, we highlight robust approaches for probing metabolism and addressing metabolically related questions though parallel labeling experiments. In the first part, we provide a brief historical perspective on parallel labeling experiments, from the early metabolic studies when radioisotopes were predominant to present-day applications based on stable-isotopes. We also elaborate on important technical and theoretical advances that have facilitated the transition from radioisotopes to stable-isotopes. In the second part of the review, we focus on parallel labeling experiments for (13)C-metabolic flux analysis ((13)C-MFA). Parallel experiments offer several advantages that include: tailoring experiments to resolve specific fluxes with high precision; reducing the length of labeling experiments by introducing multiple entry-points of isotopes; validating biochemical network models; and improving the performance of (13)C-MFA in systems where the number of measurements is limited. We conclude by discussing some challenges facing the use of parallel labeling experiments for (13)C-MFA and highlight the need to address issues related to biological variability, data integration, and rational tracer selection.

Figure 1.

Experimental designs for single tracer experiments and parallel labeling experiments. Major advantages and disadvantages of both approaches are highlighted. [1]Gluc denotes [1-¹³C]glucose. [U]Gln denotes [U-¹³C]glutamine. A semicolon “;” denotes parallel experiments. A plus-sign “+” indicates multiple tracers used in a single experiment.

Figure 2.

Tracer experiment designs using parallel experiments, multiple labeled substrates, and multiple isotope types. A semicolon “;” denotes parallel experiments and a plus-sign “+” indicates multiple tracers used in a single experiment.

Figure 3.

Timeline of important technical and computational advances that facilitated the transition from radioisotopes to stable-isotopes and further development of ¹³C-MFA.