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Measurement of pentose phosphate-pathway activity in a single incubation with [1,6-13C2,6,6-2H2]glucose.

ABSTRACT: The isotopically substituted molecule D-[1,6-13C2,6,6-2H2]glucose is introduced for measuring the relative activities of the pentose phosphate pathway (PPP) and glycolysis in a single incubation. PPP activity in cultured cells was determined by gas chromatography/mass spectrometric analysis of lactate produced by cells incubated with [1,6-13C2,6,6-2H2]glucose. Two other isotopes, [1,5,6-13C3]glucose and [6-13C,1,6,6-2H3]glucose, were not satisfactory for measurements of this activity. This method has four advantages over the traditional one in which 14CO2 production from [1-14C]glucose and [6-14C]glucose is compared: (1) repeated measurements can be made on a single set of cells, (2) only a single incubation is required, (3) extensive CO2 production by Krebs-cycle activity does not interfere with the measurements and (4) it is not necessary to measure the amount of glucose consumed in order to calculate relative activities of the PPP and glycolysis. Preliminary observation indicates that rat brain PPP activity may be measured in vivo with [1,6-13C2,6,6-2H2]glucose when combined with microdialysis techniques.

SUBMITTER: Ross BD

PROVIDER: S-EPMC1137187 | biostudies-other | 1994 Aug

REPOSITORIES: biostudies-other

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Multiple independent fusions of glucose-6-phosphate dehydrogenase with enzymes in the pentose phosphate pathway.

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Glial glucose fuels the neuronal pentose phosphate pathway for long-term memory.

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The pentose phosphate pathway of glucose metabolism. Measurement of the non-oxidative reactions of the cycle.

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Project description:The pentose phosphate pathway (PPP) is essential for reductive biosynthesis, antioxidant processes and nucleotide production. Common tracers such as [1,2-13 C2 ]glucose rely on detection of 13 C in lactate and require assumptions to correct natural 13 C abundance. Here, we introduce a novel and specific tracer of the PPP, [2,3-13 C2 ]glucose. 13 C NMR analysis of the resulting isotopomers is informative because [1,2-13 C2 ]lactate arises from glycolysis and [2,3-13 C2 ]lactate arises exclusively through the PPP. A correction for natural abundance is unnecessary. In rats receiving [2,3-13 C2 ]glucose, the PPP was more active in the fed versus fasted state in the liver and the heart, consistent with increased expression of key enzymes in the PPP. Both the PPP and glycolysis were substantially increased in hepatoma compared with liver. In summary, [2,3-13 C2 ]glucose and 13 C NMR simplify assessment of the PPP.

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Elevated pentose phosphate pathway flux supports appendage regeneration.

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The Pentose Phosphate Pathway Regulates the Circadian Clock.

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Overcompensation in response to herbivory in Arabidopsis thaliana: the role of glucose-6-phosphate dehydrogenase and the oxidative pentose-phosphate pathway.

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