The importance of accurately correcting for the natural abundance of stable isotopes

Abstract

The use of isotopically labeled tracer substrates is an experimental approach for measuring in vivo and in vitro intracellular metabolic dynamics. Stable isotopes that alter the mass but not the chemical behavior of a molecule are commonly used in isotope tracer studies. Because stable isotopes of some atoms naturally occur at non-negligible abundances, it is important to account for the natural abundance of these isotopes when analyzing data from isotope labeling experiments. Specifically, a distinction must be made between isotopes introduced experimentally via an isotopically labeled tracer and the isotopes naturally present at the start of an experiment. In this tutorial review, we explain the underlying theory of natural abundance correction of stable isotopes, a concept not always understood by metabolic researchers. We also provide a comparison of distinct methods for performing this correction and discuss natural abundance correction in the context of steady state 13C metabolic flux, a method increasingly used to infer intracellular metabolic flux from isotope experiments.

Assessing the pentose phosphate pathway using [2, 3‐13C2]glucose

“…It is important to be aware that [2,3-13 C 2 ]glucose produces [U-13 C 3 ]lactate through the PPP (in addition to [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]lactate) if the fractional enrichment in the glucose pool is high. Aside from brain under a fasted condition, [U-13 C 3 ]lactate was not detected in other organs or brain from fed animals in the current study.…”

“…This must be due to tracer dilution by endogenous glucose or stored glycogen. Because the possibility of [1,2,[3][4][5][6][7][8][9][10][11][12][13] C 3 ]F6P generation (and consequently [U-13 C 3 ]lactate) must be higher with a higher fraction of [2,3-13 C 2 ]glucose, we further examined 13 C enrichment of glucose using NMR analysis of β-glucose C3 assuming a singlet as natural abundance. Indeed, the brain from fasted animals had the highest enrichment (18%) by [2,3-13 C 2 ]glucose.…”

“…A typical MR image of hepatoma and 13 C NMR of lactate C2 in hepatoma extracts are shown in Figure 4A,B. The ratio of D23/D12 in hepatomas was not altered, but the levels of both [2,3-13 C 2 ]lactate (D23/DSS) and [1,[2][3][4][5][6][7][8][9][10][11][12][13] C 2 ]lactate (D12/DSS) were substantially higher compared with surrounding liver tissues; [2,3-13 C 2 ]lactate was increased 9-fold and [1,2-13 C 2 ]lactate was increased 8-fold. The concentration of lactate was also much higher in hepatomas ( Figure 4C).…”

“…With this labeling pattern, glycolysis produces [2,3‐ 13 C 2 ]lactate while flux in the PPP produces [3‐ 13 C 1 ]lactate. In principle this is an attractive method, but the informative metabolic product, [3‐ 13 C 1 ]lactate, needs to be distinguished from natural 13 C abundance . With additional measurements corrections are possible but require the assumption that the lactate pool is fully mixed.…”

“…Thus, the assay with [2,3-13 C 2 ]glucose eliminates corrections associated with natural abundance. Furthermore, the critical lactate isotopomer, [2,[3][4][5][6][7][8][9][10][11][12][13] C]lactate, cannot arise due to metabolism in the tricarboxylic acid (TCA) cycle and export of pyruvate. Metabolism of [1,[2][3][4][5][6][7][8][9][10][11][12][13] C 2 ]pyruvate via pyruvate carboxylation to [1,2-13 C 2 ]oxaloacetate or via pyruvate decarboxylation to [1-13 C 1 ]acetyl-CoA (or both pathways) cannot generate [2,[3][4][5][6][7][8][9][10][11][12][13] C 2 ]oxaloacetate that would be a competing source of [2,[3][4][5][6][7][8][9][10][1...…”