Hyperpolarized δ‐[1‐¹³C]gluconolactone as a probe of the pentose phosphate pathway

Abstract

The pentose phosphate pathway (PPP) is thought to be upregulated in trauma (to produce excess NADPH) and in cancer (to provide ribose for nucleotide biosynthesis) but simple methods for detecting changes in flux through this pathway are not available. Magnetic resonance imaging of hyperpolarized 13C-enriched metabolites offers considerable potential as a rapid, non-invasive tool for detecting changes in metabolic fluxes. In this study, hyperpolarized δ-[1-13C]gluconolactone was used as a probe to detect flux through the oxidative portion of the pentose phosphate pathway (PPPox) in isolated perfused mouse livers. The appearance of hyperpolarized H13CO3− within seconds after exposure of livers to HP-δ-[1-13C]gluconolactone demonstrates that this probe rapidly enters hepatocytes, becomes phosphorylated, and enters the PPPox pathway to produce HP-H13CO3− after three enzyme catalyzed steps (6P-gluconolactonase, 6PGDH, and carbonic anhydrase). Livers perfused with octanoate as their sole energy source show no change in production of H13CO3− after exposure to low levels of H2O2, while livers perfused with glucose and insulin showed a 2-fold increase in H13CO3− after exposure to peroxide. This indicates that flux through the PPPox is stimulated by H2O2 in glucose perfused livers but not in livers perfused with octanoate alone. Subsequent perfusion of livers with non-polarized [1,2-13C]glucose followed by 1H NMR analysis of lactate in the perfusate verified that flux through the PPPox is indeed low in healthy livers and modestly higher in peroxide damaged livers. We conclude that hyperpolarized δ-[1-13C]gluconolactone has the potential to serve as a metabolic imaging probe of this important biological pathway.

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).…”

“…However, this approach requires active gluconeogenesis. Hyperpolarized [1– 13 C 1 ]gluconolactone detects the PPP by production of hyperpolarized 13 C bicarbonate released in the 6‐phosphogluconate dehydrogenase reaction . Although this method may eventually be applicable for imaging the PPP, the technology is not widely available.…”

“…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...…”