A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes |
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Authors: | Michael C Schrader Clifford J Eskey Virgil Simplaceanu |
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Institution: | 1. Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA;2. Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA |
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Abstract: | We have used 2-13C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has 13C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with 1-13C]-, 2-13C]-, and 3-13C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway. |
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Keywords: | Erythrocyte Glucose metabolism Pentose phosphate pathway (Human) NMR nuclear magnetic resonance ppm parts per million FID free induction decay 3-PG 3-phosphoglycerate 1 3-DPG 1 3-diphosphoglycerate 2 3 -DPG 2 3-diphosphoglycerate F6P fructose 6-phosphate G3P glyceraldehyde 3-phosphate G6P glucose 6-phosphate GAPD glyceraldehyde 3-phosphate dehydrogenase H6P hexose 6-phosphate Hb hemoglobin oxy-Hb oxyhemoglobin met-Hb methemoglobin PCA perchloric acid Hepes N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid NOE nuclear Overhauser effect spin-lattice relaxation time P5P pentose 5-phosphate PC pentose cycle PFK phosphofructokinase PGK phosphoglycerate kinase PPP pentose phosphate pathway TA transladolase TK transkelolase X5P xylulose 5-phosphate R5P ribose 5-phosphate S7P sedoheptulose 7-phosphate and E4P erythrose 4-phosphate RBC red blood cell |
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