HDO production from [2H7]glucose Quantitatively Identifies Warburg Metabolism

Abstract

Increased glucose uptake and aerobic glycolysis are striking features of many cancers. These features have led to many techniques for screening and diagnosis, but many are expensive, less feasible or have harmful side-effects. Here, we report a sensitive 1 H/ 2 H nMR method to measure the kinetics of lactate isotopomer and HDO production using a deuterated tracer. To test this hypothesis, HUH-7 hepatocellular carcinoma and AML12 normal hepatocytes were incubated with [ 2 H 7 ]glucose. 1 H/ 2 H nMR data were recorded for cell media as a function of incubation time. The efflux rate of lactate-CH 3 , lactate-cH 2 D and lactate-cHD 2 was calculated as 0.0033, 0.0071, and 0.0.012 µmol/10 6 cells/min respectively. Differential production of lactate isotopomers was due to deuterium loss during glycolysis. Glucose uptake and HDo production by HUH-7 cells showed a strong correlation, indicating that monitoring the HDO production could be a diagnostic feature in cancers. Deuterium mass balance of [ 2 H 7 ]glucose uptake to 2 H-lactate and HDO production is quantitatively matched, suggesting increasing HDO signal could be used to diagnose Warburg (cancer) metabolism. Measuring the kinetics of lactate isotopomer and HDO production by 1 H and 2 H MR respectively are highly sensitive. increased t 1 of 2 H-lactate isotopomers indicates inversion/ saturation recovery methods may be a simple means of generating metabolism-based contrast.

Deuterated water imaging of the rat brain following metabolism of [2H7]glucose

“…Previous work suggests that it could account for a maximum of 8% of the HDO generation when using [6,6‐ 2 H 2 ]glucose, 10 indicating that its contribution in this case should be very small in relative terms. In our recent work in Huh‐7 cancer cells incubated with [ 2 H 7 ]glucose, we observed that unlabeled lactate increased with time, suggesting significant deuterium loss during glycolysis 11 . The stoichiometry of the metabolism of the deuterated tracer is such that the number of HDO molecules produced by glycolysis versus glx and lactate 2 H enrichment varies depending upon whether the glycolytically derived pyruvate methyl group is produced from either the C1 or C6 position of glucose (Figure 5).…”

“…In our recent work in Huh-7 cancer cells incubated with [ 2 H 7 ]glucose, we observed that unlabeled lactate increased with time, suggesting significant deuterium loss during glycolysis. 11 The stoichiometry of the metabolism of the deuterated tracer is such that the number of HDO molecules produced by glycolysis versus glx and lactate 2 H enrichment varies depending upon whether the glycolytically derived pyruvate methyl group is produced from either the C1 or C6 position of glucose ( Figure 5). Multiple reactions, including equilibration at M6P, alanine, or glucose, HDO, glx, and lactate was calculated using the fitted areas for the 2 H-MRS data from rat brain.…”

“…These peaks are slowly enriched as a function of time, and are most easily imaged using the CSI technique, as they are smaller peaks relative to the deuterated water (HDO) signal that arises both from natural abundance contributions and from a limited set of exchange reactions that eliminate the 2 H label from the methyl position of lactate. We recently proposed [ 2 H 7 ]glucose as an alternative agent, and showed in cell culture that HDO evolution accurately reflected total glucose uptake, 11 in analogy to FDG‐PET, 12 but with detection of glucose downstream metabolism and without the use of ionizing radiation.…”

“…Deuterium ( 2 H) loss has been shown in the form of 2 HOH and NAD 2 H. Small and large red-filled circles represent one and two deuterium atoms, respectively, and black-filled and empty circles represent hydrogen atoms and quaternary carbons, respectively. Enzymes involved in glycolysis and TCA cycle are as follows: (1) hexokinase (HK), (2) glucokinase (GK), (3) glucose-6-phosphate isomerase (GPI), (4) phosphomannoseisomerase (PMI), (5) phosphofructokinase (PFK), (6) fructose bisphosphate aldolase (FBPA or aldolase), (7) triose phosphate isomerase (TPI), (8) glyceraldehyde phosphate dehydrogenase (GAPDH), (9) phosphoglycerate kinase (PGK),(10) phosphoglyceromutase (PGM),(11) enolase, (12) pyruvate kinase (PK), (13) lactate dehydrogenase (LDH),(14) alanine aminotransferase (ALT),…”

Deuterium MRSI characterizations of glucose metabolism in orthotopic pancreatic cancer mouse models

“…Figure 4 also hints at statistically significant differences between the rates of 2 H‐water generation in kidneys and in tumors; in fact, for both the PDAC models, 2 H‐water images were also found to highlight the tumors over the remaining organs in the abdomen. Water has been reported as an end product of glycolysis and of other metabolic processes including the pentose phosphate and the TCA pathways 39,40 ; some of these pathways may also be stimulated in cancer cells, 41 and the DMI data could be reflecting this. However, neither the experimental kinetic curves for water nor their statistical modeling led to the kind of clearer trends evidenced by the glucose and lactate clearance rates, and hence the detailed message conveyed by the 2 H‐water production in these experiments remains to be elucidated.…”