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
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ABSTRACT |
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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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INTRODUCTION |
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THE OXIDATIVE AND nonoxidative branch of the pentose phosphate pathway (PPP) is commonly estimated by the analysis of the distribution of 14C or 13C in products of glucose 6-phosphate (as in glycogen glucose) or triose phosphate (as in lactate or fatty acids; see Refs. 4-6, 8, 9, 12, 13, 19). Among 14C tracer methods, glucose labeled with 14C in the two position (C-2) is the single tracer method most often used for in vivo and in vitro studies of the PPP (12, 13, 19). Because glycogen and ribose have to be isolated from the tissue for analysis, repeat analysis on the same tissue sample is not possible. The determinations of the location and amount of 14C in glucose and glycerol are labor intensive. An alternative approach for the determination of PPP activity is to measure the isotope yield in glycerol or lactate from parallel incubations of cells or tissues with [1-14C]- and [6-14C]glucose. However, the requirement of parallel incubation makes the method suitable only for in vitro studies in which substrate conditions can be strictly controlled. Recently, Ross and colleagues (1, 20) developed a single-tracer approach using [1,6-13C2,6,6-2H2]glucose. The oxidative branch of PPP was determined with a single incubation with [1,6-13C2,6,6-2H2]glucose and gas chromatography (GC)-mass spectrometry (MS) analysis of the derived lactate. Because carbon-1 (C-1) and carbon-6 (C-6) of glucose are labeled with different heavy isotopes, the recovery of C-1 in lactate is reflected by the lactate isotopomer with one 13C substitution (m + 1 or m1) and that of C-6 by the isotopomer with two deuterium and one 13C (m + 3 or m3). Because deuterium can be lost during the formation of lactate, PPP activity was calculated using the C-1-to-C-6 ratio in lactate after correction for the loss of deuterium.
We present here a novel single-tracer method for the study of both oxidative and nonoxidative branches of the PPP using [1,2-13C2]glucose. In this study, we measured the synthesis of pentose phosphate and the production of lactate from [1,2-13C2]glucose in human hepatoma cells (Hep G2). The pentose mass isotopomers [1-13C]ribose from the glucose-6-phosphate dehydrogenase (G-6-PDH) reaction and [1,2-13C2]- or [4,5-13C2]ribose from the reactions of the transketolase (TK) and transaldolase (TA) enzymes are extensively recycled to glucose phosphate and converted to triose phosphate. Because the distribution of isotopomers with either one or two 13C substitutions in ribose- and triose phosphate-derived products can be quantitated using GC-MS, the isotopomer data can be used for the calculation of parameters of oxidative and nonoxidative branches of the PPP using the model of Katz and Rognstad (9). We also showed that the pentose cycle (PC) activity determined from incubation with [1,2-13C2]glucose and isotopomer yield in lactate was identical to that determined using incubation with [1-13C]- and [6-13C]glucose in parallel studies.
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MATERIALS AND METHODS |
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Tissue Culture
The human hepatoma cell line Hep G2 obtained from the American Type Culture Collection (ATCC, Rockville, MD) was grown in Dulbecco's modified Eagle's media augmented with 10% (vol/vol) fetal bovine serum (15). When the cells were ~50% confluent (~2.5 × 106 cells/plate), the medium was removed, the cells were washed with phosphate-buffered saline, and the appropriate [13C]glucose-containing media were added as described below to begin the experiment, which lasted 3 days. Incubation medium from each culture plate was replaced with fresh media daily. The medium removed was saved for lactate analysis. Exactly 3 days after the start of the experiment, the media were saved and the cells were harvested after trypsin treatment. The harvested cell pellet was kept frozen for RNA and lipid extraction. During the experiment, Hep G2 cells doubled to ~0.5-1 × 107 cells per plate and remained 80-90% viable after harvest.Isotopes
D-[1-13C]-, [6-13C]-, and [1,2-13C2]glucose were purchased from Cambridge Isotopes Laboratories (Woburn, MA). The isotopic enrichments for the specified positions were 99%. [1-13C]-, [6-13C]-, and [1,2-13C2]glucose were added separately to the serum-free media to a final assayed enrichment of 30%. This glucose enrichment was chosen so that the probability of recombination of 13C through TK and TA reactions would be negligible. The final glucose concentration in the Dulbecco's modified Eagle's media with fetal bovine serum was adjusted to 100 mg/dl. Experiments for each condition were performed in duplicates unless specified otherwise.Extraction and Derivatization of Substrates
A 1-ml aliquot of medium was first acidified with hydrochloric acid. Lactate was extracted from the acidified medium with ethyl acetate. For GC-MS analysis, lactate was converted to its n-propylamide-heptafluorobutyric ester according to the method of Tserng et al. (22).RNA was extracted from the cell pellet after treatment with ice-cold 0.5% Tryzol (vol/vol). The purified RNA pellet was hydrolyzed in 2 ml 2 N HCl for 2 h at 100°C and neutralized with a solution of equivalent strength of sodium bicarbonate (2, 3). Ribose was separated from the nucleotide residues using a tandem set of cation/anion exchange columns (Dowex50/Dowex1) and eluting with water. Ribose was then derivatized to its aldonitrile acetate form for analysis on the GC-MS spectrometer (21).
Fatty acid was extracted according to the method described by Lowenstein et al. (18). One-half of a cell pellet was saponified with 1 ml 30% KOH-ethanol (1:1, vol/vol) at 70°C overnight. Neutral lipids were first removed with petroleum ether extraction. The solution containing the saponified fatty acids was then acidifed, and palmitate and other fatty acids were recovered with another petroleum ether extraction. Palmitate was methylated with 0.5 N HCl in methanol (Supelco, Bellfonte, PA) for GC-MS analysis (14).
GC-MS Analysis
A Hewlett-Packard (model 5840) gas chromatograph connected to a 5985 mass spectrometer was used. The GC-MS methods for lactate and palmitate analysis were previously described (14, 22). For ribose analysis, a glass column (1.2 m × 2 mm ID) was packed with either 3% SP-2340 or 10% SP-2250 (Supelco). The GC conditions were as follows: injector temperature 250°C; oven temperature programming 208-220°C at 10°C/min. Helium was used as the carrier gas for electron impact ionization at flow rates of 20-25 ml/min. Selected ion cluster monitoring was used to follow specific ions. The ions monitored by electron ionization were clustered as follows: mass-to-charge ratio (m/z) 241-244 (C-1 to C-4) and m/z 216-220 (C-3 to C-5) of ribose. The ions monitored by chemical ionization using methane as the carrier gas were m/z 255-261 (C-1 to C-5) of ribose, which correspond to the molecular ion of aldonitrile tetraacetate with the loss of an acetyl unit.Mass isotopomer distribution in lactate, ribose, and palmitate was calculated from spectral intensities after correcting for the contribution of the derivatization agent and natural abundance of 13C using established methods (16). The mass isotopomer thus calculated represents the isotopomer (in molar fraction) resulting from the introduction of 13C from labeled glucose above its natural abundance. In the results reported, the coefficient of variation of the corrected mass isotopomer is <5% for molar enrichment of >1%. The mass and position isotopomers of ribose were determined from the mass isotopomer distribution of C-1 to C-5 and C-3 to C-5 fragments. Under conditions in which the recombination of 13C through TK and TA reactions is negligible, there are two main m2 isotopomers of ribose ([1,2-13C2]- and [4,5-13C2]ribose) and three m1 isotopomers ([1-13C]-, [3-13C]-, and [5-13C]ribose) from [1,2-13C2]glucose. These different species can be quantitated from the isotopomer distribution of the C-1 to C-4 and C-3 to C-5 fragments.
PC Model and Calculations
The distribution of isotope in the various carbon positions of the PC intermediates can be expressed as functions of PC parameters according to model of Katz and Rognstad (9). Basically, these are equations of mass balance across the oxidative and nonoxidative branches of the PC. The Katz and Rognstad (9) model and the accompanying mass balance equations for mass isotopomers from [1-13C]-, [6-13C]-, and [1,2-13C2]glucose are presented in the APPENDIX.Estimation of the PC. The isotope
yields in triose phosphate products are traditionally used for the
calculation of the PC. When the fluxes across TK and TA reactions are
unidirectional toward formation of glucose, a practical equation
(Eq. 1), can be derived for the
calculation of PC flux. Commonly, the ratio of isotope yield in lactate
from [1-13C]- and
[6-13C]glucose ()
is determined, and the value is used with Eq. 2 to calculate PC
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(1) |
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(2) |
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(3) |
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(4) |
Fatty acid is another product of triose phosphate. Isotope yield, specific activity, in fatty acids after incubation in parallel experiments with [1-14C]- and [6-14C]glucose has been shown to give PC activity (8, 9, 15) equivalent to that derived from isotope yield in lactate. The application of a stable isotope differs from that of a radioisotope in several respects. In experiments with [1-13C]- and [6-13C]glucose, isotope yield can be determined as the total enrichment in the fatty acids such as used in the present study or as the precursor (acetyl-CoA) enrichment using isotopomer distribution analysis as in a previous report (15). In experiments with [1,2-13C2]glucose, the incorporation of labeled acetyl-CoA from m1 and m2 pyruvate results in fatty acids containing an even or odd number of 13C substitutions in fatty acids. The ratio of the singly labeled (m1) over the doubly labeled (m2) isotope in palmitate can be used as an approximation of the the m1-to-m2 ratio in lactate. PC can be calculated using Eq. 4.
Estimation of TK and TA activity. Because of the large number of variables in the system and the limited number of observations on isotope ratios, solution of the equations for TK and TA activities can be obtained by an iterative method. The procedure for the estimation of PC, TK, and TA is based on isotopomer ratios in ribose and lactate following an algorithm similar to the one employed by Katz and Rognstad (9): step 1, PC was approximated by the isotopomer ratio (yield from C-1 glucose/yield from C-6 glucose) in lactate; step 2, the value of TK (assuming TK1 = TK2) is varied and substituted into the 15 model equations to calculate values of P1 and P2 (APPENDIX). These values are compared with the observed ratio of [1-13C]ribose to [1,2-13C2]ribose. The procedure is repeated until an optimum fit is achieved; step 3, finally, the value of TA is varied to optimize the fit of the ratio of P1 and P5 to the ratio of [1,2-13C2]ribose to [4,5-13C2]ribose. The optimization steps 2 and 3 are repeated until the fit cannot be improved further. The resultant values for TK and TA from this algorithm represent optimum values that would give rise to the observed isotopomer distribution in ribose.
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RESULTS |
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Isotope Yield in Lactate and PC
The values of isotope yield,
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13C Label in Palmitate and PC
The distributions of mass isotopomers in palmitate from these studies are shown in Table 2. Isotope yields in fatty acid from stable isotope studies are determined either as molar enrichment in the case of C-1- and C-6-labeled glucose, which corresponds to specific activity in radioisotope studies, or in the case of [1,2-13C2]glucose as precursor enrichment using the mass isotopomer distribution analysis method (14, 15, 17). The isotope yield from C-1- and C-6-labeled glucose gave a ratio of 0.874 and 0.840. Both values and the calculated PC agreed with the isotope yield ratio in lactate. In experiments with [1,2-13C2]glucose, m1 is a small fraction of m2 in lactate. Thus the predominant isotopomers in palmitate are those with two or four 13C substitutions (multiples of m2). The ratio of m2 to m1 in acetyl units is approximated by m2 to m1 in palmitate from [1,2-13C2]glucose. The m1/m2 in acetyl units was 0.148 with calculated PC of 6.89%. These results confirm the usefulness of isotope yield ratio in fatty acids for the determination of PC. This can be either accomplished with parallel incubation with [1-13C]- and [6-13C]glucose or with a single incubation with [1,2-13C2]glucose.
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Mass Isotopomers in Ribose and Estimation of TK and TA
The observed mass isotopomer distributions in three mass fragments from ribose are shown in Table 3. Labeling of ribose in the C-1 position from [1-13C]glucose is possible only by the action of TK and TA. Labeling of C-5 of ribose arises from both isomerization of triose phosphate and TA action. Only C-5 of ribose is labeled from [6-13C]glucose. In the experiment with [1,2-13C2]glucose, m1 and m2 ribose are observed. These isotopomers are present both in the C-1 to C-4 or C-3 to C-5 fragments. The positional isotopomers from these experiments are shown pictorially in the top of Table 4. Each column represents an isotopomer species, and the enrichment information is provided directly beneath the column. We detected two labeled isotopomers ([1-13C]- and [5-13C]ribose) in ribose after incubation with [1-13C]glucose. Their enrichments are given by the enrichment in the C-1 to C-4 and C-3 to C-5 fragments (Table 3). The enrichments of isotopomers [1,2-13C2]- and [4,5-13C2]ribose from the incubation with [1,2-13C2]glucose are given by m2 in the C-1 to C-4 and C-3 to C-5 fragments (Table 3). Enrichments of [1-13C]- and [5-13C]ribose are deduced from m1 in the C-1 to C-4 and C-3 to C-5 fragments. The ratios of these mass and position isotopomers were then used to provide an estimate of the action of TK and TA using the iterative algorithm of Katz and Rognstad (9). The values of PC, TK, and TA that give isotopomer ratios that best match the observed values are PC 0.055, TK 0.032, and TA 0.85. From these values of TK, TA, and PC, the isotopomer distribution in ribose can be predicted. The correctness of these estimates can be seen in the close match between the observed and predicted isotopomer ratios. In the present method, PC, TK, and TA are determined based on the following three isotopomer ratios: m1/m2 ratio in lactate, ratio of [1-13C]- to [1,2-13C2]ribose, and the ratio of [1,2-13C2]- to [5,4-13C2]ribose. The propagation of error of these ratios results in a coefficient of variation of 20% in the parameter estimate. It is clear that calculations of TK and TA depend on a number of assumptions, such as isotope equilibrium among the pentose phosphates and the lack of formation of nontriose phosphate products (R). These assumptions cannot be independently validated until the isotope distribution in other PPP intermediates is determined.
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The numerical relationship between parameters PC, TK, and TA and the rates of these reactions is illustrated by an example using values estimated for Hep G2 cells in culture. The common denominator for these rates is the glucose flux. Let us assume glucose utilization to be 100 µmol/h. For a PC of 0.055 or 5.5%, it means that 5.5% of the glucose carbon is metabolized to CO2 via oxidation of glucose 6-phosphate. Thus the rate of G-6-PDH is 3PC or 3 × 0.055 × 100 = 16.5 µmol/h. The total rate of transformation of xylulose 5-phosphate by reactions II (Fig. 2C) and IV (Fig. 2D) is 2 × (PC + TK) or (2 × 0.055 + 2 × 0.032) × 100 = 17.4 µmol/h. The rate of conversion of sedoheptulose 7-phosphate by TA is TA + PC or (0.055 + 0.85) × 100 = 90.5 µmol/h. In the reverse direction, i.e., from hexose phosphate to xylulose 5-phosphate and sedoheptulose phosphate, the two TK reactions are 6.4 µmol/h and the TA 85 µmol/h. Because of the equilibrium reactions of TK and TA, the sum of flux via the Embden Meyerhof pathway, PC, TK, and TA, which is the total flux of glucose 6-phosphate, is 202.4 µmol/h, which is substantially greater than that of net glucose flux.2
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DISCUSSION |
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We have demonstrated theoretically and experimentally that isotopomer yield in triose phosphate products, such as lactate and fatty acid, after an incubation with [1,2-13C2]glucose can be used to provide results of PC identical to those obtained from parallel incubation with [1-13C]- and [6-13C]glucose. Unlike the use of [2-14C]glucose, the use of [1,2-13C2]glucose and application of positional and mass isotopomer analysis in the intermediates or triose phosphate products by GC-MS obviate the need for laborious chemical degradation. The single incubation with [1,2-13C2]glucose is an advantage over separate parallel incubations with [1-13C]- and [6-13C]glucose, making it suitable for dynamic studies of changes in the PPP by the measurement of the isotopomer ratio in lactate (1). However, unlike the single-isotope method of Ross et al. (1, 20) with deuterium-labeled glucose, the [13C]glucose method allows isotope yield in other triose phosphate products, such as fatty acid, to be determined. Therefore, [1,2-13C2]glucose is potentially suitable for more long-term studies of changes in the PPP by examining the isotope ratio in fatty acids or glycogen from tissue extracts (6, 8).
The movement of label in C-2 of [2-14C]glucose to C-1 of ribose by G-6-PDH and its subsequent relocation to C-1 and C-3 of glucose by TK and TA create many key isotopomers of ribose. Under metabolic conditions in which the nontriose phosphate products, predominately pentose in nucleic acids and glycogen, are relatively insignificant, the isotope distribution data can be used to solve for individual metabolic flux (PC, TK, or TA) according to the equations of Katz and Rognstad (9), assuming tracer equilibrium. We have shown that the same set of equations can be applied to the analysis of mass isotopomer data in ribose and lactate from [1,2-13C2]glucose for the determination of TK and TA, parameters of the nonoxidative pathways of the PC. Because [1,6-13C2,6,6-2H2]glucose does not produce labeled ribose isotopomers for such an analysis, [1,6-13C2,6,6-2H2]glucose cannot be used to study the nonoxidative pathways of the PC. Because of the limited information provided by the ribose isotopomer data, a number of assumptions are necessary for the calculations in the present method. Because GC-MS analysis of carbohydrate is relatively well established, such a method can be extended to quantitation of other intermediates, such as sedoheptulose, erythrose, and glycerol, providing an array of data necessary for the solution of a more complex pentose phosphate system, including the synthesis of pentose and glycogen. The isotopomer distribution in these intermediates may provide the necessary data for a more complete analysis of intermediary metabolism of the PPP in future studies.
The operation of the PPP leads to a number of products as follows: ribose for nucleic acid synthesis, glycerol phosphate for triglyceride synthesis, and reducing equivalents for fatty acid synthesis. Pentose phosphate, the precursor of ribose and deoxyribose of nucleic acids, can be synthesized from the oxidative decarboxylation of glucose 6-phosphate or from the nonoxidative interconversion with hexose phosphate in human cells (1). Under most metabolic conditions, the pentose phosphate from the oxidative path is recycled to fructose 6-phosphate by the action of TK and TA through a set of equilibrium exchanges (4, 6, 8, 12, 13, 19). It is generally accepted that the PC is mainly driven by the oxidative pathway and functions as a complete cycle to provide reducing equivalents for biosynthesis. For this reason, previous studies of the PC have mainly focused on the measurement of the oxidative pathway corresponding to the action of G-6-PDH. The role of TK and TA was considered to be of interest only in determining the redistribution of tracer label in pentose and hexose phosphate. The study of Katz and Rognstad (9) and our present study revealed that the substrate flux through these reactions is both rapid and large. Because of this rapid exchange, hexose phosphate and pentose phosphate behave essentially as one homogeneous pool. Thus a small change in TK and TA may potentially have a large effect on the partitioning between hexose phosphate and pentose phosphate as well as the relative amounts of reducing equivalents or pentose or hexose phosphate produced.3 In this manner, the action of TK and TA serves an important role to regulate the irreversible "oxidative" and the reversible "nonoxidative" pathways.
In summary, the use of [1,2-13C2]glucose provides a single tracer approach to the study of PPP similar to the application of [2-14C]glucose. When isotope yield as measured by m1/m2 is determined in lactate, such method can potentially be used to study metabolic processes over time in nondestructive studies. Because there is excellent agreement in isotopomer ratios in lactate and in fatty acid, in vivo and in vitro studies can also be designed such that isotope distribution in other intermediates or products of the PPP are determined from tissue extracts. Thus the application of [1,2-13C2]glucose in association with the use of modeling potentially yields more complete understanding on the PPP in terms of the contribution of oxidative and nonoxidative paths to glucose metabolism.
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APPENDIX |
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PC Model
Except for the determination of glucose oxidation from the release of labeled CO2 from C-1- and C-6-labeled glucose, isotope methods for the estimation of PPP are usually based on an assumed metabolic model consisting of three separate substrate pools, hexose phosphate, triose phosphate, and pentose phosphate, connected by either reversible or irreversible reactions (Ref. 9 and Fig. 1). The three irreversible reactions are hexose phosphate conversion to pentose phosphate, uridine diphosphoglucose, and fructose diphosphate. From these reactions, glucose carbons are removed from the pentose phosphate pool by the synthesis of nucleic acid and from the triose phosphate pool by the synthesis of glycerol, lactate, and fatty acids. Reversible exchange reactions, which are responsible for the interconversion of hexose phosphate and pentose phosphate, are catalyzed by phosphohexose isomerase, triosephosphate isomerase, pentose phosphate isomerase, epimerase, aldolase, TA, and TK. It is further assumed that 1) glucose is metabolized mostly by either the Embden Meyerhof or the pentose pathway; 2) the pentose phosphate produced is recycled to hexose phosphate, and the net loss of pentose phosphate to nucleic acid synthesis is relatively small; and 3) there is rapid tracer equilibrium of substrates in these pools. In tissues other than the liver or the mammary glands, it has been shown that these assumptions are often valid under many experimental conditions (8, 10, 13).
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In the case of labeling of pentose phosphate from [1,2-13C2]glucose, the reversible and irreversible reactions of the Embden Meyerhof pathway and of the PC result in the formation of positional and mass isotopomers, as illustrated in Fig. 2. In Fig. 2, isotopomers from four key reactions of glucose metabolism via the PC are shown. Reaction I (Fig. 2A) is one of the irreversible reactions showing the decarboxylation of glucose 6-phosphate to ribulose 5-phosphate, leading to the formation of [1-13C]ribose. Other irreversible reactions not shown are the loss of hexose phosphate via uridine diphosphoglucose to the synthesis of glycogen and other nontriose phosphate products, the loss of pentose phosphate via phosphoribosyl pyrophosphate to ribose in ribonucleic acid synthesis, and the loss of hexose phosphate to the formation of triose phosphate products pyruvate and lactate. Reactions II to IV (Fig. 2, B-D) are three reversible reactions catalyzed by TK (TK1 and TK2) and TA, which produce m1 and m2 isotopomers of pentose phosphate ([1-13C]- and [5-13C]ribose and [1,2-13C2]- and [4,5-13C2]ribose) and permit the recycling of pentose carbon to glucose, forming the corresponding isotopomers in hexose phosphate and its products.
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The five parameters of the model are PC, the fraction of hexose phosphate that passes through the PC; TK1 and TK2, the two TK reactions; TA, the TA reaction; and ribonucleic acid synthesis, the outflow of pentose phosphate in the synthesis of phosphoribosyl pyrophosphate. At isotopic and metabolic steady state, the distribution of isotope in the various carbon positions of pentose phosphate and its intermediates is a function of these parameters. If we designate each position of hexose by H1, H2, and H3, etc., each position in pentose by P1, P2, and P3, etc., and similarly, positions in sedoheptulose and erythrose by S1, S2, E1, and E4, etc., using the same convention as previously published (9), a set of equations can be written for the mass balance of every carbon position in these intermediates in terms of these parameters (see Ref. 9). The equations for the calculation of isotope distribution from [1-13C]glucose are shown in Table 5.
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Algorithm for the Estimation of TK and TA
We have rearranged the coefficients of the model equations (Eqs. 1A-15A in Table 5) in a matrix format, as shown in Table 6, for easy computation. The constant terms are either
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To determine TK by iteration, we first set PC to the
observed value of 0.055. Then we substitute an arbitrary value (between 0 and 1) for TK1 and
TK2. Because the relationship
between [1-13C]- to
[1,2-13C2]ribose
is equivalent to that of P1 to
P2 in experiments with [2-13C]glucose or
H2, we next calculate the relative
distribution of P1 to
P2 by setting the constant term in
Eq. 2A to 1.
Constant terms for other equations are zero. The ratio of
P1 to
P2 is then compared with the ratio
of the observed
[1-13C]- to
[1,2-13C2]ribose
isotopomers. If these ratios are different by >10%, another value is
chosen for TK, and the calculation is repeated until these ratios are
in good agreement within a preset range.
After an optimal value of TK is obtained, we determine TA by iteration
using the determined values of PC and TK. Because
m2 isotopomers in
ribose are the products of TK and TA only similar to the labeling of
ribose from
[1-13C]glucose or
H1, we calculate
P1 to
P5 by setting the constant term of
Eq. 1A in Table 5 to 1.
Constant terms for other equations are zero. The calculated ratio of
P1 to
P5 will be compared with the ratio
of the observed
[1,2-13C2]-
to
[3,4-13C2]ribose
isotopomers. If these ratios are different by >10%, another value is chosen for TA, and the calculation is repeated until
these ratios are in good agreement within a preset range. The processes of determining TK and TA are repeated until optimal values for both values are attained. Using this process, we determined TK to be 0.032 and TA to be 0.85. The isotopomers of ribose calculated from the model using these values are provided in Table 4.
In systems such as in tumor tissues or in lactating mammary glands in which nucleic acid synthesis and/or the nontriose phosphate use of hexose phosphate is increased, using Eqs. 1-15 of Ref. 9 to estimate PC, TK, and TA may result in unacceptably large errors. The above model should be modified using Eqs. 1'-15' of Ref. 9. In such application, measurement of nontriose phosphate and ribonucleic acid synthesis may be necessary. Substitution of these values and the isotopomer distribution in PPP intermediates permit more accurate estimation of PC, TK, and TA.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health (NIH) Grant R01-DK-46353. The gas chromatography-mass spectrometry facility was supported by NIH Grants M01-RR-00425 to the General Clinical Research Center and P01-CA-42710 to the University of California Los Angeles Clinical Nutrition Research Unit, Stable Isotope Core.
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FOOTNOTES |
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1 In theory, m1 of lactate can also be generated from the metabolism of labeled lactate through the tricarboxylic acid cycle. This is possible only under two conditions: 1) there is a high degree of pyruvate recycling, and 2) the anaplerotic flux is low, i.e., there is much tracer dilution due to exchange with unlabeled acetate. Neither of these conditions is likely in tissue culture.
2 The inflow into glucose 6-phosphate is the sum of inflow from glucose (100 µmol/h), recycled pentose (3PC, or 16.5 µmol/h), and exchange via TK and TA (6.4 ± 85 µmol/h). The outflow from glucose 6-phosphate is the sum of the Embden Myerhof pathway (94.5 µmol/h), G-6-PDH (3PC, 16.5 µmol/h), and exchange via TK and TA (91.4 µmol/h). Therefore, glucose 6-phosphate flux is 202.4 µmol/h, which is greater than net glucose flux.
3 It is not difficult to see that the ratio of the products, pentose phosphate to reducing equivalent, can be affected by the rate of recycling via the TK and TA pathways. The ratio is one when there is no recycling and zero when recycling is complete.
Address for reprint requests: W.-N. P. Lee, Dept. of Pediatrics, Harbor-UCLA Medical Center, 1124 W. Carson St., Torrance, CA 90502.
Received 12 September 1997; accepted in final form 20 January 1998.
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REFERENCES |
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1.
Ben-Yoseph, O.,
D. M. Camp,
T. E. Robinson,
and
B. D. Ross.
Dynamic measurement of cerebral pentose phosphate pathway activity in vivo using [1,6-13C2, 6,6-2H2-]glucose and microdialysis.
J. Neurochem.
64:
1336-1342,
1995[Medline].
2.
Chomczynski, P.
A reagent for the single step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples.
Biotechniques
15:
532-536,
1993[Medline].
3.
Chomczynski, P.,
and
N. Sacchi.
Single step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
164:
156-160,
1987[Medline].
4.
Green, M.,
and
B. R. Landau.
Contribution of the pentose cycle to glucose metabolism in muscle.
Arch. Biochem. Biophys.
111:
569-575,
1965[Medline].
5.
Horecker, B. L.,
G. Domagk,
and
H. H. Hiatt.
A comparison of C14-labeling patterns in deoxyribose and ribose in mammalian cells.
Arch. Biochem. Biophys.
78:
510-517,
1958.
6.
Hostetler, K. Y.,
and
B. R. Landau.
Estimation of the pentose cycle contribution to glucose metabolism in tissue in vivo.
Biochemistry
6:
2961-2964,
1967[Medline].
7.
Katz, J.,
and
N. Grunnet.
Estimation of metabolic pathways in steady state in vitro. Rates of tricarboxylic acid and pentose cycles.
In: Techniques in Metabolic Research, edited by H. L. Kornberg. Amsterdam: Elsevier/North Holland, 1979, p. 1-18.
8.
Katz, J.,
B. R. Landau,
and
G. E. Bartsch.
The pentose cycle, triose phosphate isomerization and lipogenesis in rat adipose tissue.
J. Biol. Chem.
241:
727-740,
1966
9.
Katz, J.,
and
R. Rognstad.
The labeling of pentose phosphate from glucose-14C and estimation of the rates of transketolase and transaldolase, the contribution of the pentose cycle and ribose phosphate synthesis.
Biochemistry
6:
2227-2247,
1967[Medline].
10.
Katz, J.,
and
P. A. Wal.
Pentose cycle and reducing equivalents in rat mammary-gland slices.
Biochem. J.
128:
879-899,
1972[Medline].
11.
Katz, J.,
and
H. G. Wood.
The use of glucose 14C for the evaluation of pathway of glucose metabolism.
J. Biol. Chem.
235:
2165-2177,
1960[Medline].
12.
Landau, B. R.,
and
G. E. Bartsch.
Estimation of pathway contributions to glucose metabolism and the transaldolase reactions.
J. Biol. Chem.
241:
741-749,
1966
13.
Landau, B. R.,
G. E. Bartsch,
J. Katz,
and
H. G. Wood.
Estimation of pathway contributions to glucose metabolism and of rate of isomerization of hexose phosphate.
J. Biol. Chem.
239:
686-696,
1964
14.
Lee, W. P.,
S. Bassilian,
Z. K. Guo,
D. A. Schoeller,
J. Edmond,
E. A. Bergner,
and
L. O. Byerley.
Measurement of fractional synthesis of fatty acids and cholesterol using deuterated water and mass isotopomer analysis.
Am. J. Physiol.
266 (Endocrinol. Metab. 29):
E372-E383,
1994
15.
Lee, W.-N. P.,
L. O. Byerley,
S. Bassilian,
H. O. Ajie,
I. Clark,
J. Edmond,
and
E. A. Bergner.
Isotopomer study of lipogenesis in human hepatoma cells in culture: contribution of carbon and reducing hydrogen from glucose.
Anal. Biochem.
226:
100-112,
1995[Medline].
16.
Lee, W.-N. P.,
J. Edmond,
L. O. Byerley,
and
E. A. Bergner.
Mass isotopomer analysis, theoretical and practical considerations.
Biol. Mass. Spectrom.
20:
451-458,
1990.
17.
Lee, W.-N. P.,
Z. K. Guo,
and
E. A. Bergner.
Mass isotopomer pattern and precursor-product relationship.
Biol. Mass Spectrom.
21:
114-122,
1992[Medline].
18.
Lowenstein, J. M.,
H. Brunengraber,
and
M. Wadke.
Measurement of rates of lipogenesis with deuterated and tritiated water.
Methods Enzymol.
34:
279-287,
1975.
19.
Magnusson, I.,
V. Chandramouli,
W. C. Schumann,
K. Kumaran,
J. Wahren,
and
B. R. Landau.
Pentose pathway in human liver.
Proc. Natl. Acad. Sci. USA
85:
4682-4685,
1988[Abstract].
20.
Ross, B. D.,
P. B. Kingsley,
and
O. Ben-Yoseph.
Measurement of pentose phosphate pathway activity in a single incubation with [1,6-13C 2, 6,6-2H2-]glucose.
Biochem. J.
302:
31-38,
1994[Medline].
21.
Szafranek, J.,
D. C. Pfaffenberger,
and
E. C. Horning.
The mass spectra of some per-O-acetylaldonitriles.
Carbohydr. Res.
38:
97-105,
1974[Medline].
22.
Tserng, K. Y.,
C. A. Gilfillan,
and
S. C. Kalhan.
Determination of carbon-13 labeled lactate in blood by gas chromatography/mass spectrometry.
Anal. Chem.
56:
517-523,
1984[Medline].