MASS ISOTOPOMER STUDY OF THE NONOXIDATIVE PATHWAYS OF THE PENTOSE CYCLE WITH [1,2-C-13(2)]GLUCOSE

We present a single-tracer method for the study of the pentose phospha te pathway (PPP) using [1,2-C-13(2)]glucose and mass isotopomer analys is. The metabolism of [1,2-C-13(2)]glucose by the glucose-6-phosphate dehydrogenase, transketolase (TK), and transaldolase (TA) reactions re sults in unique pentose and lactate isotopomers with either one or two C-13 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 po sition isotopomers of ribose, and lactate and palmitate (products from triose phosphate) from human hepatoma cells (Hep G2) incubated with 3 0% enriched [1,2-C-13(2)]glucose were determined using gas chromatogra phy mass spectrometry. After 24-72 h incubation, 1.9% of lactate molec ules in the medium contained one C-13 substitution (m(1)) and 10% cont ained two C-13 substitutions (m(2)). A similar m(1)-to-m(2) ratio was found in palmitate as expected. Pentose cycle (PC) activity determined from incubation with [1,2-C-13(2)]glucose was 5.73 +/- 0.52% of the g lucose flux, which was identical to the value of PC (5.55 +/- 0.73%) d etermined by separate incubations with [1-C-13] and [6-C-13]glucose. C -13 was found to be distributed in four ribose isotopomers ([1-C-13]-, [5-C-13]-, [1,2-C-13(2)]-, and [4,5-C-13(2)]ribose). The observed rib ose isotopomer distribution was best matched with that provided from s imulation by substituting 0.032 for TK and 0.85 for TA activity relati ve to glucose uptake into the model of Katz and Rognstad. The use of [ 1,2-C-13(2)]glucose not only permits the determination of PC but also allows estimation of relative rates through the TK and TA reactions.