Quantitation of absolute 2H enrichment of plasma glucose by 2H NMR analysis of its monoacetone derivative.

A simple (2)H NMR method for quantifying absolute (2)H-enrichments in all seven aliphatic positions of glucose following its derivatization to monoacetone glucose is presented. The method is based on the addition of a small quantity of (2)H-enriched formate to the NMR sample. When the method was applied to [2-(2)H]monoacetone glucose samples prepared from [2-(2)H]glucose standards of known enrichments in the range of 0.2-2.5%, enrichment estimates derived by the NMR method were in good agreement with the real enrichment values of the [2-(2)H]glucose precursors. The measurement was also applied to monoacetone glucose derived from human plasma glucose samples following administration of (2)H(2)O and attainment of isotopic steady state, where glucose H2 and body water enrichment are equivalent. In these studies, the absolute H2 enrichment of plasma glucose estimated by the formate method was in good agreement with the (2)H-enrichment of body water measured by an independent method.     

Simple measurement of gluconeogenesis by direct 2H NMR analysis of menthol glucuronide enrichment from 2H2O.

The contribution of gluconeogenesis to fasting glucose production was determined by a simple measurement of urinary menthol glucuronide (MG) 2H enrichment from 2H2O. Following ingestion of 2H2O (0.5% body water) during an overnight fast and a pharmacological dose (400 mg) of a commercial peppermint oil preparation the next morning, 364 micromol MG was quantitatively recovered from a 2-h urine collection by ether extraction and a 125 micromol portion was directly analyzed by 2H NMR. The glucuronide 2H-signals were fully resolved and their relative intensities matched those of the monoacetone glucose derivative. The pharmacokinetics and yields of urinary MG after ingestion of 400 mg peppermint oil as either gelatin or enteric-coated capsules 1 h before breakfast were quantified in five healthy subjects. Gelatin capsules yielded 197 +/- 81 micromol of MG from the initial 2-h urine collection while enteric-coated capsules gave 238 +/- 84 micromol MG from the 2- to 4-h urine collection.     

Quantifying endogenous glucose production and contributing source fluxes from a single 2H NMR spectrum

Endogenous glucose production (EGP), gluconeogenic and glycogenolytic fluxes by analysis of a single ²H‐NMR spectrum is demonstrated with 6‐hr and 24‐hr fasted rats. Animals were administered [1‐²H, 1‐¹³C]glucose, a novel tracer of glucose turnover, and ²H₂O. Plasma glucose enrichment from both tracers was quantified by ²H‐NMR analysis of monoacetone glucose. The 6‐hr fasted group (n = 7) had EGP rates of 95.6 ± 13.3 μmol/kg/min, where 56.2 ± 7.9 μmol/kg/min were derived from PEP; 12.1 ± 2.1 μmol/kg/min from glycerol, and 32.1 ± 4.9 μmol/kg/min from glycogen. The 24‐hr fasted group (n = 7) had significantly lower EGP rates (52.8 ± 7.2 μmol/kg/min, P = 0.004 vs. 6 hr) mediated by a significantly reduced contribution from glycogen (4.7 ± 5.9 μmol/kg/min, P = 0.02 vs. 6 hr) while PEP and glycerol contributions were not significantly different (39.5 ± 3.9 and 8.5 ± 1.2 μmol/kg/min, respectively). These estimates agree with previous assays of EGP fluxes in fasted rats obtained by multinuclear NMR analyses of plasma glucose enrichment from ²H₂O and ¹³C‐glucose tracers. Magn Reson Med, 2009. © 2009 Wiley‐Liss, Inc.     

Sources of plasma glucose by automated Bayesian analysis of 2H NMR spectra.

Sources of blood glucose can be determined after oral ingestion of (2)H(2)O followed by isolation of plasma glucose and measurement of the relative (2)H enrichments in select positions within the glucose molecule. Typically, (2)H enrichments are obtained by mass spectrometry but (2)H NMR offers an alternative. Here it is demonstrated that the entire analysis may be automated by Bayesian analysis of a (2)H free induction decay signal of monoacetone glucose to obtain a direct readout of the relative contributions of glycogenolysis, glycerol, and phosphoenol pyruvate to plasma glucose production. Furthermore, Markov Chain Monte Carlo (MCMC) simulations of the posterior probability density provide uncertainties in all metabolic parameters from a single patient, thereby allowing comparisons in glucose metabolism from one individual to another. The combined MCMC Bayesian methodology is operationally simple and requires little intervention from the operator.     

Sources of hepatic glucose production by 2H2O ingestion and Bayesian analysis of 2H glucuronide enrichment

The contribution of gluconeogenesis to hepatic glucose production (GP) was quantified after ²H₂O ingestion by Bayesian analysis of the position 2 and 5 ²H‐NMR signals (H2 and H5) of monoacetone glucose (MAG) derived from urinary acetaminophen glucuronide. Six controls and 10 kidney transplant (KTx) patients with cyclosporine A (CsA) immunosuppressant therapy were studied. Seven KTx patients were lean and euglycemic (BMI = 24.3 ± 1.0 kg/m²; fasting glucose = 4.7 ± 0.1 mM) while three were obese and hyperglycemic (BMI = 30.5 ± 0.7 kg/m²; fasting glucose = 7.1 ± 0.5 mM). For the 16 spectra analyzed, the mean coefficient of variation for the gluconeogenesis contribution was 10% ± 5%. This uncertainty was associated with a mean signal‐to‐noise ratio (SNR) of 79:1 and 45:1 for the MAG H2 and H5 signals, respectively. For control subjects, gluconeogenesis contributed 54% ± 7% of GP as determined by the mean and standard deviation (SD) of individual Bayesian analyses. For the lean/normoglycemic KTx subjects, the gluconeogenic contribution to GP was 62% ± 7% (P = 0.06 vs. controls), while hyperglycemic/obese KTx patients had a gluconeogenic contribution of 68% ± 3% (P < 0.005 vs. controls). These data suggest that in KTx patients, an increased gluconeogenic contribution to GP is strongly associated with obesity and hyperglycemia. Magn Reson Med 60:517–523, 2008. © 2008 Wiley‐Liss, Inc.     

Unambiguous assignment of the H3S and H3R deuterations of cerebral (2‐13C) glutamate by 13C NMR at 18.8 tesla

We used high‐field ¹³C NMR (18.8 T) to assign unambiguously the isotopic shifts induced by the deuterium substitutions of the H3_proR and H3_proS hydrogens of (2‐¹³C) glutamate in extracts of the brain from deuterated animals. Monodeuterated H3R or H3S glutamate diastereoisomers were produced stereospecifically either by chemical synthesis or by coupling the reactions of isocitrate dehydrogenase and aspartate aminotransferase in deuterated medium, respectively. We show that the (3S‐²H) or (3R‐²H) deuterations induce characteristic small (Δ₂ = ?0.058 parts per million (ppm)) or large (Δ₂ = ?0.071 ppm) vicinal isotopic shifts upfield of the perprotonated (2‐¹³C) glutamate resonance (at 55.5 ppm). Isotopically shifted (2‐¹³C, 3S‐²H) or (2‐¹³C, 3R‐²H) glutamate singlets are conveniently observed by high‐field ¹³C NMR in brain extracts from deuterated rats. Since the (3S‐²H) or (3R‐²H) glutamate diastereoisomers are produced stereospecifically by the cytosolic or mitochondrial isoforms of aconitase and isocitrate dehydrogenase, our results will facilitate the ¹³C NMR investigation of these enzymatic activities and their role in subcellular glutamate trafficking. Magn Reson Med 63:1088–1091, 2010. © 2010 Wiley‐Liss, Inc.     

13C NMR study of the generation of C2- and C3,-deuterated lactic acid by tumoral pancreatic islet cells exposed to D-[1-13C]-, D-[2-13C]- and D-[6-13C]-Glucose in 2H2O

Tumoral pancreatic islet cells of the RIN5mF line were incubated for 120 min in media prepared in ²H₂O and containing D -[1-¹³C]glucose, and D -[2-¹³C]glucose, and D -[6-¹³C]glucose. The generation of C₂- and C₃- deuterated lactic acid was assessed by ¹³C NMR. The interpretation of experimental results suggests that a) the efficiency of deuteration on the C₁ of D-fructose 6-phosphate does not exceed about 47% and 4% in the phosphoglucoisomerase and phosphomannoisomerase reactions, respectively; b) approximately 38% of the molecules of D -glyceraldehyde 3-phosphate generated from D -glucose escape deuteration in the sequence of reactions catalyzed by triose phosphate isomerase and aldolase; and c) about 41% of the molecules of pyruvate generated by glycolysis are immediately converted to lactate, the remaining 59% of pyruvate molecules undergoing first a single or double back-and-forth interconversion with L -alanine. It is proposed that this methodological approach, based on high resolution ¹³C NMR spectroscopy, may provide novel information on the regulation of back-and-forth interconversion of glycolytic intermediates in intact cells as modulated, for instance, by enzyme-to-enzyme tunneling.     

In vivo monitoring of hepatic glutathione in anesthetized rats by 13C NMR.

A method for in vivo (13)C NMR monitoring of hepatic glutathione (GSH) in intact, anesthetized rats has been developed. Studies were conducted using a triple-tuned, surgically implanted surface coil designed for this animal model. The coil permitted complete decoupling and sufficient resolution in the (13)C NMR spectrum to monitor the time course of hepatic (13)C-metabolites of intravenously administered 2-(13)C-glycine, particularly GSH at 44.2 ppm and serine signals at 61.1 and 57.2 ppm, respectively. It further allowed concomitant monitoring of high-energy phosphagens and intracellular pH by (31)P NMR. To confirm in vivo NMR peak assignments, we compared high-resolution 2D (1)H[(13)C] heteronuclear multiple quantum coherence and 1D (13)C spectra of hepatic perchloric acid extracts to those of authentic standards. The fractional isotopic enrichment of hepatic (13)C-glycine increased exponentially at a rate of 1.68 h(-1) and reached its plateau level of 81% in 2 h. The (13)C fractional isotopic enrichment of GSH increased exponentially at a rate of 0.316 h(-1) and reached 55% after 4 h of 2-(13)C-glycine infusion, but without achieving a plateau. To confirm that the resonance at 44.2 ppm resulted from GSH, a rat was given an intravenous dose of 2-oxothiazolidine-4-carboxylic acid (OTC), a cysteine precursor that increases intracellular GSH. As expected, with OTC administration the hepatic (13)C GSH-to-glycine peak area increased more than sevenfold.     

Hepatic UDP-glucose 13C isotopomers from [U-13C]glucose: a simple analysis by 13C NMR of urinary menthol glucuronide.

Menthol glucuronide was isolated from the urine of a healthy 70-kg female subject following ingestion of 400 mg of peppermint oil and 6 g of 99% [U-(13)C]glucose. Glucuronide (13)C-excess enrichment levels were 4-6% and thus provided high signal-to-noise ratios (SNRs) for confident assignment of (13)C-(13)C spin-coupled multiplet components within each (13)C resonance by (13)C NMR. The [U-(13)C]glucuronide isotopomer derived via direct pathway conversion of [U-(13)C]glucose to [U-(13)C]UDP-glucose was resolved from [1,2,3-(13)C(3)]- and [1,2-(13)C(2)]glucuronide isotopomers derived via Cori cycle or indirect pathway metabolism of [U-(13)C]glucose. In a second study, a group of four overnight-fasted patients (63 +/- 10 kg) with severe heart failure were given peppermint oil and infused with [U-(13)C]glucose for 4 hr (14 mg/kg prime, 0.12 mg/kg/min constant infusion) resulting in a steady-state plasma [U-(13)C]glucose enrichment of 4.6% +/- 0.6%. Menthol glucuronide was harvested and glucuronide (13)C-isotopomers were analyzed by (13)C NMR. [U-(13)C]glucuronide enrichment was 0.6% +/- 0.1%, and the sum of [1,2,3-(13)C(3)] and [1,2-(13)C(2)]glucuronide enrichments was 0.9% +/- 0.2%. From these data, flux of plasma glucose to hepatic UDPG was estimated to be 15% +/- 4% that of endogenous glucose production (EGP), and the Cori cycle accounted for at least 32% +/- 10% of GP.     

Quantitation of erythrocyte pentose pathway flux with [2-13C]glucose and 1H NMR analysis of the lactate methyl signal.

A simple and sensitive NMR method for quantifying excess (13)C-enrichment in positions 2 and 3 of lactate by (1)H NMR spectroscopy of the lactate methyl signal is described. The measurement requires neither signal calibrations nor the addition of a standard and accounts for natural abundance (13)C-contributions. As a demonstration, the measurement was applied to approximately 3 micromol of lactate generated by erythrocyte preparations incubated with [2-(13)C]glucose to determine the fraction of glucose metabolized by the pentose phosphate pathway (PP). PP fluxes were estimated from the ratio of excess (13)C-enrichment in lactate carbon 3 relative to carbon 2 in accordance with established metabolic models. Under baseline conditions, PP flux accounted for 7 +/- 2% of glucose consumption while in the presence of methylene blue, a classical activator of PP activity, its contribution increased to 27 +/- 10% of total glucose consumption (P < 0.01).     

2H double quantum filtered (DQF) NMR spectroscopy of the nucleus pulposus tissues of the intervertebral disc.

Deuterium (2H) double-quantum filtered (DQF) NMR spectroscopy of nucleus pulposus (NP) tissues from human intervertebral discs is reported. The DQF spectral intensities, DQ build-up rates, and DQF-detected rotating-frame spin-lattice relaxation times are sensitive to the degree of hydration of the NP tissue, and display a monotonous correlation with age between 15 and 80 years. The implications of this work are that the changes in water dynamics as detected via DQF NMR spectroscopy may be used as a probe of tissue degeneration in NP, particularly in the early stages of degeneration to which most standard NMR methods are not sensitive.     

Investigating brain metabolism at high fields using localized 13C NMR spectroscopy without 1H decoupling.

Most in vivo 13C NMR spectroscopy studies in the brain have been performed using 1H decoupling during acquisition. Decoupling imposes significant constraints on the experimental setup (particularly for human studies at high magnetic field) in order to stay within safety limits for power deposition. We show here that incorporation of the 13C label from 13C-labeled glucose into brain amino acids can be monitored accurately using localized 13C NMR spectroscopy without the application of 1H decoupling. Using LCModel quantification with prior knowledge of one-bond and multiple-bond J(CH) coupling constants, the uncertainty on metabolites concentrations was only 35% to 91% higher (depending on the carbon resonance of interest) in undecoupled spectra compared to decoupled spectra in the rat brain at 9.4 Tesla. Although less sensitive, 13C NMR without decoupling dramatically reduces experimental constraints on coil setup and pulse sequence design required to keep power deposition within safety guidelines. This opens the prospect of safely measuring 13C NMR spectra in humans at varied brain locations (not only the occipital lobe) and at very high magnetic fields above 4 Tesla.     

13C and 1H NMR study of the metabolic degradation of 4-pentenoate in different dog nephron segments.

The metabolism of 4-pentenoate in isolated kidney tubules has been investigated by 1H and 13C NMR. The 4-pentenoate metabolite, 3-keto-4-pentenoyl-CoA, accumulated in proximal tubules only and its formation could be competitively inhibited by octanoate. 4-Pentenoate was metabolized in thick ascending limbs but not in papillary collecting ducts.     

Allergen-induced histamine secretion associated with lactate production in mast cells detected by 1H NMR

Allergen-induced histamine secretion in mast cells, obtained from rats infected with Nippostrongylus brasiliensis, was detected by a ¹H NMR semi-selective excitation technique. Lactate production was also detected in association with histamine secretion as observed in mast cells stimulated with other nonimmunological agents, such as compound 48/80, poly-myxin B, and melittin. The assignment of the lactate resonance was confirmed using lactate oxidase. Thus, ¹H NMR spectroscopy is a useful method to study the activation of mast cells in allergic reaction.     

Glucose metabolism in RIF-1 tumors after reduction in blood flow: An in Vivo 13C and 31P NMR study

Low pH appears to enhance the effectiveness of therapeutic hyperthermia. ¹³C and ³¹P NMR spectroscopy have been employed to examine the possibility that elevating glucose in a solid tumor while simultaneously reducing tumor blood flow would induce a more profound acidosis than either treatment alone. When blood flow in RIF-1 tumors was acutely reduced by administration of hydralazine and additional glucose was delivered locally by intratumoral injection, tumor acidosis (as determined by ³¹P NMR spectroscopy) during the period of reduced blood flow was not enhanced, relative to administration of hydralarine alone. Tumor NTP/P₁ ratios decreased significantly within 20 min of hydralazine administration, whether or not glucose was injected, although NTP/P₁ ratios were slightly higher in tumors that received extra glucose. Tumor lactate concentrations were not significantly different in glucose-supplemented tumors, despite glucose concentratlons that were 4 to 5 times higher. When the added glucose was labeled with ¹³C, no correlation was detected between the pH in an individual tumor and the intensity of the 3-[¹³C]-lactate resonance in the same tumor.     

Synthesis of [guanidino-13C]creatine and measurement of the creatine phosphokinase reaction in vitro by 13C NMR spectroscopy

[guanidino-13C]Creatine has been synthesized by reaction involving synthesis of [13C]cyanamide followed by its condensation with sarcosine. The creatine guanidino carbon 13C NMR frequency shifts upon the formation of phosphocreatine by 0.83 ppm with a splitting (2JCP = 3.3 Hz) due to two-bond phosphorus-carbon coupling. The high 13C enrichment (99%) at the guanidino carbon of creatine gave rise to an adequate signal-to-noise ratio for both creatine and phosphocreatine so that a study of the time course of the creatine phosphokinase reaction could be performed and provided the means for a determination of the Km (15.5 mM) of the reaction.     

Glycogen detection by in vivo 13C NMR: a comparison of proton decoupling and polarization transfer.

The performance of gated proton decoupling and polarization transfer with respect to glycogen detection by 13C NMR was investigated. Experiments were performed on a 1.5-T whole-body scanner using a 13C surface coil in combination with a proton head coil. Spectra were acquired from a glycogen phantom and from the lower leg of a healthy volunteer using proton decoupling and the polarization transfer method SINEPT. The signal strength of the C1 resonance of glycogen was determined and compared to a reference spectrum acquired without any form of sensitivity enhancement. In the phantom experiment both decoupling and SINEPT produced a signal gain of 3.5. Under in vivo conditions, the signal gain was approximately 2.5 for both techniques. We conclude that decoupling and polarization transfer are equivalently useful techniques for glycogen detection.     

1H NMR detection of vitamin C in human brain in vivo.

Vitamin C (ascorbate) is well established as an essential nutrient that functions as an antioxidant. Since it is present in the human brain at detectable concentrations, this study was designed to detect and quantify ascorbate in the human brain in vivo using 1H NMR spectroscopy (MRS). Ascorbate was consistently detected in all five study subjects, and was measured using MEGA-PRESS difference editing. The in vivo resonance pattern was consistent with that of ascorbate based on position, line width, peak pattern, and relative intensity. Metabolites with a potential for coediting were assessed using phantom solutions. The putative resonances of myo-inositol, lactate, glycerophosphocholine, phosphocholine, and phosphoethanolamine were detected at positions distinct from those of ascorbate. This study represents the first in vivo detection of vitamin C in the human brain using 1H MRS. A concentration of 1.3 +/- 0.3 micromol/g (mean +/- SD, N = 4) was estimated.