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1 Department of Pediatrics, Research and Education Institute, Harbor-University of California Los Angeles Medical Center, Torrance, California 90502; 2 The Ohio State University College of Medicine, General Surgery Research Laboratories, Department of Surgery, Columbus, Ohio 43210; and 3 Department of Biochemistry and Molecular Biology, University of Barcelona, 08028 Barcelona, Spain
We present a single-tracer method for the study of the pentose phosphate pathway (PPP) using [1,2-13C2]glucose and mass isotopomer analysis. The metabolism of [1,2-13C2]glucose by the glucose-6-phosphate dehydrogenase, transketolase (TK), and transaldolase (TA) reactions results in unique pentose and lactate isotopomers with either one or two 13C substitutions. The distribution of these isotopomers was used to estimate parameters of the PPP using the model of Katz and Rognstad (J. Katz and R. Rognstad. Biochemistry 6: 2227-2247, 1967). Mass and position isotopomers of ribose, and lactate and palmitate (products from triose phosphate) from human hepatoma cells (Hep G2) incubated with 30% enriched [1,2-13C2]glucose were determined using gas chromatography-mass spectrometry. After 24-72 h incubation, 1.9% of lactate molecules in the medium contained one 13C substitution (m1) and 10% contained two 13C substitutions (m2). A similar m1-to-m2 ratio was found in palmitate as expected. Pentose cycle (PC) activity determined from incubation with [1,2-13C2]glucose was 5.73 ± 0.52% of the glucose flux, which was identical to the value of PC (5.55 ± 0.73%) determined by separate incubations with [1-13C] and [6-13C]glucose. 13C was found to be distributed in four ribose isotopomers ([1-13C]-, [5-13C]-, [1,2-13C2]-, and [4,5-13C2]ribose). The observed ribose isotopomer distribution was best matched with that provided from simulation by substituting 0.032 for TK and 0.85 for TA activity relative to glucose uptake into the model of Katz and Rognstad. The use of [1,2-13C2]glucose not only permits the determination of PC but also allows estimation of relative rates through the TK and TA reactions.
oxidative and nonoxidative pentose phosphate pathways; ribose; gas chromatography-mass spectrometry; glucose-6-phosphate dehydrogenase
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