Kinetic isotope effects significantly influence intracellular metabolite (13) C labeling patterns and flux determination

Abstract

Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from (13) C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of (13) C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the (13) C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the (13) C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in (13) C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC-MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in (13) C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions.

Keywords: Isotope Effects; Isotopomer Modeling; Metabolic Engineering; Metabolic Flux Analysis; Modeling errors.

Figure 1

The pyruvate node model system. We assume that pyruvate is produced through pyruvate kinase (PK) and consumed through one of two reactions. In the pyruvate dehydrogenase (PDH) reaction, the bond between C₁ and C₂ of pyruvate is broken (represented by the dashed line), yielding one molecule of CO₂ and one acetyl-CoA two-carbon unit. This reaction is subject to isotope effects, as measured by Melzer and Schmidt (see text). In the other reaction, pyruvate is converted to a metabolite X. In this reaction all carbon–carbon bonds in pyruvate remain intact, so we assume the isotope effects on this reaction are small relative to those on the PDH reaction. The ratio of the flux through PDH to the flux through PK is equal to f, while the ratio of the flux to metabolite X to the flux through PK is equal to 1 – f. Circles represent the carbon atoms of each metabolite.

Figure 2

Typical results for errors associated with neglecting isotope effects as a function of f. The predicted modeling errors associated with neglecting isotope effects on the E. coli PDH enzyme (assuming additivity of isotope effects for multiply labeled isotopomers of pyruvate) with (A) natural abundance glucose and (B) 20% U-¹³C₆-glucose substrates are plotted as a function of the ratio of flux through PDH to flux through PK, f. The errors for each of the three mass isotopomer mole fractions of the acetyl-CoA two-carbon unit MID are shown.

Figure 3

Expected errors associated with isotope effects at low values of f. The predicted modeling errors associated with neglecting isotope effects at a fixed value of f = 0.01 for different combinations of organism, tracer, and assumption on the additivity of isotope effects for multiply labeled isotopomers of pyruvate. (A) Predicted errors for each glucose substrate are plotted for the cases of E. coli assuming additive isotope effects, (B) S. cerevisiae assuming additive isotope effects, (C) E. coli assuming non-additive isotope effects, and (D) S. cerevisiae assuming non-additive isotope effects. NA, natural abundance glucose; 1 = 1-¹³C-glucose; 1,2 = 1,2-¹³C -glucose; 3,4 = 3,4-¹³C₂-glucose; 20%U = 20% U-¹³C₆-glucose; 75%1 + 25%U = 75% 1-¹³C-glucose + 25% U-¹³C₆-glucose.