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Glucose Production, Gluconeogenesis, and Hepatic Tricarboxylic Acid Cycle Xuxes Measured by Nuclear Magnetic Resonance Analysis of a Single Glucose Derivative

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Glucose Production, Gluconeogenesis, and Hepatic Tricarboxylic Acid Cycle Xuxes Measured by Nuclear Magnetic Resonance Analysis of a Single Glucose Derivative View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Estudo Geral ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 327 (2004) 149–155 www.elsevier.com/locate/yabio Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle Xuxes measured by nuclear magnetic resonance analysis of a single glucose derivative Eunsook S. Jin,a John G. Jones,b Matthew Merritt,a Shawn C. Burgess,a Craig R. Malloy,a,c and A. Dean Sherrya,d,¤ a The Mary Nell and Ralph B. Rogers Magnetic Resonance Center, Department of Radiology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA b Center of Neurosciences and Department of Biochemistry, University of Coimbra, Coimbra, Portugal c VA North Texas Health Care System, Dallas, TX 75216, USA d Department of Chemistry, University of Texas at Dallas, Richardson, TX 75080, USA Received 9 May 2003 Abstract A triple-tracer method was developed to provide absolute Xuxes contributing to endogenous glucose production and hepatic tri- carboxylic acid (TCA) cycle Xuxes in 24-h-fasted rats by 2H and 13C nuclear magnetic resonance (NMR) analysis of a single glucose 13 derivative. A primed, intravenous [3,4- C2]glucose infusion was used to measure endogenous glucose production; intraperitoneal 2 H2O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal 13 X [U- C3] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle ux. Plasma glucose was converted to monoacetone glucose (MAG), and a single 2H and 13C NMR spectrum of MAG provided the following metabolic data (all in units of mol/kg/min; n D 6): endogenous glucose production (40.4 § 2.9), gluconeogenesis from glycerol (11.5 § 3.5), gluconeogenesis from the TCA cycle (67.3 § 5.6), glycogenolysis (1.0 § 0.8), pyruvate cycling (154.4 § 43.4), PEPCK Xux (221.7 § 47.6), and TCA cycle X V 2 13 ux (49.1 § 16.8). In a separate group of rats, glucose production was not di erent in the absence of H2O and [U- C]propionate, demonstrating that these tracers do not alter the measurement of glucose turnover. 2004 Elsevier Inc. All rights reserved. Keywords: Liver metabolism; Glucose turnover; Gluconeogenesis; NMR; Stable isotope tracers; Citric acid cycle Measurements of whole-body glucose production glucose production to the level of gluconeogenic TCA (GP)1 by dilution of intravenously infused labeled glu- cycle substrates. These sophisticated studies typically cose are simple, in principle. Simultaneous measures of combine measures of GP with contributions from glyco- related metabolic Xuxes involved in glucose production genolysis measured by in vivo magnetic resonance spec- are far more challenging due to the various precursors troscopy/imaging of hepatic glycogen [1], contributions available for glucose synthesis in vivo and their intimate from glycogen, glycerol, and the TCA cycle assessed by links to acetyl-CoA oxidation and energy production by distribution of 2H in plasma glucose [2–4], or distribu- the TCA cycle. Multiple tracer molecules and experi- tion of 13C in products of the TCA cycle [5–7]. ments are required to obtain a comprehensive picture of Recent studies have emphasized the use of stable iso- tope tracers for quantifying glucose turnover and sources of glucose production because of convenience, ¤ Corresponding author. Fax: 1-214-883-2925. safety, and ethical considerations for experiments with E-mail address: [email protected] (A.D. Sherry). V 1 Abbreviations used: GP, glucose production; TCA, tricarboxylic human subjects. Although mass spectrometry o ers acid; MAG, monoacetone glucose; PEP, phosphoenolpyruvic acid; superior sensitivity [8], NMR may be better at resolving OAA, oxaloacetate; PEPCK, phosphoenolpyruvate carboxykinase. complex enrichment patterns which may involve more 0003-2697/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2003.12.036 150 E.S. Jin et al. / Analytical Biochemistry 327 (2004) 149–155 than one tracer (e.g., 13C and 2H). 13C NMR also allows Protocol multiple 13C tracers to be used if distinctive 13C–13C cou- plings are exploited in data analysis. For example, the The study was approved by the Institutional Animal 13 13 combination of [1,6- C2]glucose, and [U- C]propionate Care and Use Committee. Male Sprague–Dawley rats tracers coupled with 13C NMR spectroscopy of plasma weighing 211 § 26 g were fasted for 24 h with free access glucose provides a comprehensive picture of the Xuxes at to water after which the jugular vein was cannulated the level of pyruvate and the TCA cycle that support under ketamine/xylazine anesthesia. Rats in group I 13 13 glucose production [9]. This mixture of C tracers can (nD6) received a bolus infusion of 3.5 mg [3,4- C2]glucose 2 13 be supplemented with an additional H tracer, such (99%) followed by continuous infusion of [3,4- C2] 2 as H2O, since, at low enrichment levels (04%), observa- glucose at 1.60 mol/min/kg for 90 min. Rats in group II tion of the 13C nucleus is not signiWcantly perturbed (n D 6) received an intraperitoneal injection of a solution by the presence of 2H or vice versa. Thus, glucose (20 L/g rat) containing 5 mg of [U-13C]propionate/mL 2 13 enrichment and isotopomer patterns from a combina- H2O followed by the same infusion of [3,4- C2]glucose 13 13 2 tion of [1,6- C2]glucose, [U- C]propionate, and H2O as the group I rats. At the end of each infusion protocol, tracers was resolved by 13C and 2H NMR spectroscopy. »8 mL of blood was drawn from the descending aorta 2 The H2O tracer resolves the contributions of glycogen, and the liver was freeze-clamped with liquid N2. glycerol, and gluconeogenic Krebs cycle outXow to glu- cose production, thereby complementing the informa- Sample processing tion from the 13C tracers and providing a more complete description of glucose synthesis [4]. This method had Blood was immediately centrifuged and plasma 13 one experimental annoyance in that [1,6- C2]glucose supernatant was deproteinized by treatment with cold (to measure glucose turnover) could be quantiWed only perchloric acid. After neutralization with KOH and cen- in the native glucose sample while enrichment from trifugation, the supernatant was lyophilized. To convert 2 13 the remaining H2O and [U- C]propionate tracers plasma glucose into MAG (Fig. 1), the dried residue required conversion to monoacetone glucose (MAG) was suspended in 3.0 mL of acetone containing 120 L for analysis.2 of concentrated sulfuric acid. The mixture was stirred Here, we introduce a simpler and faster method to for 4 h at room temperature to yield diacetone glucose. estimate whole-body glucose turnover using [3,4- 13 C2]glucose as a tracer that may also be used in combi- 2 13 nation with H2O and [U- C]propionate. In this case, all plasma glucose is converted to MAG and a complete metabolic analysis is provided by one 13C NMR spec- trum and one 2H NMR spectrum, thereby streamlining the analysis. Methods Materials 13 [3,4- C2]glucose (99%) was purchased from Omicron Biochemicals (South Bend, IN). [U-13C]propionate 2 (99%), H2O (99.9%), and deuterated acetonitrile (99.8%) were obtained from Cambridge Isotopes (Andover, MA). DSC-18 solid-phase extraction gel was obtained from Supelco (St. Louis, MO). Other common chemicals were purchased from Sigma (St. Louis, MO). 2 13 13 13 [1,6- C2]glucose is resolved from [1- C] and [6- C]glucose as a result of long-range 13C–13C coupling between carbons 1 and 6 of glucose. On conversion to MAG, this coupling is abolished and the 13 W 13 [1,6- C2]glucose tracer can no longer be resolved and quanti ed by C Fig. 1. T1 values of MAG carbons for a sample dissolved in deuterated NMR. Therefore, the method requires NMR analysis of glucose both acetonitrile. The two methyl carbons are arbitrarily labeled M1 and before and after derivatization to MAG. M2 without regard to spatial arrangement in the molecule. E.S. Jin et al. / Analytical Biochemistry 327 (2004) 149–155 151 W V W After ltering o any remaining precipitate and adding 80.5 ppm) was de ned as AC4D34, and the total area of 3 mL of water, the pH was adjusted 2.0 by dropwise the two methyl resonances (at 26.1 and 26.7 ppm) was W addition of a 1.5 M Na2CO3 solution. The mixture was de ned as Amethyl. The fraction of a glucose sample that 13 stirred for 24 h at room temperature to convert diace- was [3,4- C2]glucose was determined from the ratio tone glucose into MAG. The pH was then further (AC3D34 + AC4D34)/Amethyl of MAG obtained from stan- increased to 8.0 using Na2CO3 and the sample was dried. dard samples (Fig. 2). Samples of known standards 13 MAG was extracted (5£) with 3 mL of hot ethyl acetate. showed a linear correlation between [3,4- C2]glucose These solutions were combined and the ethyl acetate was enrichment and the ratio (AC3D34 + AC4D34)/Amethyl: removed by vacuum evaporation. The resulting MAG L D 1.4278x + 0.0626, where L is the percentage of total W 13 was further puri ed by passage through a 3-mL Discov- glucose that is [3,4- C2]glucose and x D (AC3D34 + 2 ery DSC-18 cartridge using 5% acetonitrile as eluant. AC4D34)/Amethyl (r D 0.998). The two carbons of methyl The eZuent was freeze-dried and stored dry prior to groups (labeled M1 and M2 in Fig. 1) of MAG origi- NMR analysis. nated from natural abundance acetone introduced Standard MAG samples (»22 mol) were prepared during the conversion of glucose to MAG.
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