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.     

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.     

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.     

Glucose production pathways by 2H and 13C NMR in patients with HIV-associated lipoatrophy.

Patients with HIV taking protease inhibitors were selected for the presence (five subjects) or absence (five subjects) of lipoatrophy. Following an overnight fast, subjects were given oral (2)H(2)O in divided doses (5 mL/kg body water), [U-(13)C(3)] propionate (10 mg/kg), and acetaminophen (1000 mg). Glucose (from plasma) or acetaminophen glucuronide (from urine) were converted to monoacetone glucose for (2)H NMR and (13)C NMR analysis. The fraction of plasma glucose derived from gluconeogenesis was not significantly different between groups. However, flux from glycerol into gluconeogenesis relative to glucose production was increased from 0.20 +/- 0.13 among subjects without lipoatrophy to 0.42 +/- 0.12 (P < 0.05) among subjects with lipoatrophy, and the TCA cycle contribution was reduced. Lipoatrophy was associated with an abnormal profile of glucose production as assessed by (13)C and (2)H NMR of plasma and urine.     

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.     

Acetaminophen glucuronide and plasma glucose report identical estimates of gluconeogenesis and glycogenolysis for healthy and prediabetic subjects using the deuterated water method

Plasma glucose ²H‐enrichment in positions 5 (²H5) and 2 (²H2) from deuterated water (²H₂O) provides a measure of the gluconeogenic contribution to endogenous glucose production. Urinary glucuronide analysis can circumvent blood sampling but it is not known if glucuronide and glucose enrichments are equal. Thirteen subjects with impaired fasting glucose/impaired glucose tolerance and 11 subjects with normal fasting glucose and normal glucose tolerance ingested ²H₂O to ～0.5% body water and acetaminophen. Glucose and glucuronide ²H5 and ²H2 were measured by ²H NMR spectroscopy of monoacetone glucose. For normal fasting glucose/normal glucose tolerance, ²H5 was 0.23 ± 0.02% and 0.25 ± 0.02% for glucose and glucuronide, respectively, whereas ²H2 was 0.47 ± 0.01% and 0.49 ± 0.02%, respectively. For impaired fasting glucose/impaired glucose tolerance, ²H5 was 0.22 ± 0.01% and 0.26 ± 0.02% for glucose and glucuronide, respectively, whereas ²H2 was 0.46 ± 0.01% and 0.49 ± 0.02%, respectively. The gluconeogenic contribution to endogenous glucose production measured from glucose and glucuronide were identical for both normal fasting glucose/normal glucose tolerance (48 ± 4 vs. 51 ± 3%) and impaired fasting glucose/impaired glucose tolerance (48 ± 2 vs. 53 ± 3%). Magn Reson Med 70:315–319, 2013. © 2012 Wiley Periodicals, Inc.     

Quantifying tracer levels of (2)H(2)O enrichment from microliter amounts of plasma and urine by (2)H NMR.

A simple and sensitive (2)H NMR measurement of (2)H(2)O enrichment from a 10 microl volume of body fluid is presented. The method allows (2)H-enrichment levels of 0.1% or above to be rapidly determined from 10 microl of plasma or urine. The measurement is insensitive to the presence of plasma protein, allowing direct analysis of (2)H(2)O enrichment from native plasma samples. Magn Reson Med 45:156-158, 2001.     

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).     

2H2O incorporation into hepatic acetyl‐CoA and de novo lipogenesis as measured by Krebs cycle‐mediated 2H‐enrichment of glutamate and glutamine

Deuterated water is widely used for measuring de novo lipogenesis on the basis of quantifying lipid ²H‐enrichment relative to that of body water. However, incorporation of ²H‐enrichment from body water into newly synthesized lipid molecules is incomplete therefore the true lipid precursor enrichment differs from that of body water. We describe a novel measurement of de novo lipogenesis that is based on a true precursor‐product analysis of hepatic acetyl‐CoA and triglyceride methyl enrichments from deuterated water. After deuterated water administration to seven in situ and seven perfused livers, acetyl‐CoA methyl enrichment was inferred from ²H nuclear magnetic resonance analysis of hepatic glutamate/glutamine (Glx) enrichment and triglyceride methyl enrichment was directly determined by ²H nuclear magnetic resonance of triglycerides. Acetyl‐CoA ²H‐enrichment was 71% ± 1% that of body water for in situ livers and 53% ± 2% of perfusate water for perfused livers. From the ratio of triglyceride‐methyl/acetyl‐CoA enrichments, fractional de novo lipogenesis rates of 0.97% ± 0.09%/2 hr and 7.92% ± 1.47%/48 hr were obtained for perfused and in situ liver triglycerides, respectively. Our method reveals that acetyl‐CoA enrichment is significantly less than body water both for in situ and perfused livers. Furthermore, the difference between acetyl‐CoA and body water enrichments is sensitive to the experimental setting. Magn Reson Med, 2011. © 2011 Wiley Periodicals, Inc.     

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.     

Comparison of [3,4‐13C2]glucose to [6,6‐2H2]glucose as a tracer for glucose turnover by nuclear magnetic resonance

A recently introduced tracer, [3,4‐¹³C₂]glucose, was compared to the widely used tracer, [6,6‐²H₂]glucose, for measurement of whole‐body glucose turnover. The rate of glucose production (GP) was measured in rats after primed infusions of [3,4‐¹³C₂]glucose, [6,6‐²H₂]glucose, or both tracers simultaneously followed by a constant infusion of tracer(s) over 90 min. Blood glucose was purified and converted into monoacetone glucose for analysis by ¹³C NMR (for [3,4‐¹³C₂]glucose) or ¹H and ²H NMR (for [6,6‐²H₂]glucose). The values of GP measured during infusion of each single tracer were not significantly different. In rats infused with both tracers simultaneously, GP was identical as reported by each tracer, 42 ± 4 μmol/kg/min. Since ²H and ¹³C enrichment in glucose is typically much less than 2% for in vivo studies, [3,4‐¹³C₂]glucose does not interfere with measurements of ¹³C or ²H enrichment patterns and therefore is valuable when multiple metabolic pathways are being evaluated simultaneously. Magn Reson Med 53:1479–1483, 2005. © 2005 Wiley‐Liss, Inc.     

孕期血糖逆向增高与妊娠结局的关系研究

目的 探讨孕期75g葡萄糖耐量试验(OGTT)结果逆向增高与妊娠结局的关系.方法 采用回顾性分析的研究方法,收集2014年南方医科大学珠江医院常规产检及分娩的孕产妇资料,选取服糖后2h血糖高于1h血糖者226例为观察组,随机抽取同期分娩的无妊娠期糖尿病(GDM)及糖尿病者235例为对照组.结果 观察组OGTT结果空腹血糖、服糖后1h血糖均低于对照组,而服糖后2h血糖高于对照组,差异均有统计学意义(P<0.01).观察组羊水过多、羊水过少、胎儿生长受限(FGR)、早产、妊娠合并高血压疾病、胎膜早破、胎死宫内、非疤痕子宫剖宫产的发生率以及新生儿发育异常率、窒息率均高于对照组,但差异无统计学意义(P>0.05).对照组新生儿转科率低于观察组,差异有统计学意义(P<0.01).结论 孕期OGTT结果逆向增高者存在血糖代谢延迟,可能影响新生儿的远期预后,临床上应加强对该部分孕妇的孕期管理.     

Concurrent quantification of tissue metabolism and blood flow via 2H/31P NMR in vivo. I. Assessment of absolute metabolite quantification.

In a series of three papers, we demonstrate and validate an approach for concurrent absolute quantification in situ of blood flow and energy metabolism with a modification of the NMR method for absolute concentration determination put forth by Thulborn and Ackerman [J. Magn. Reson. 55, 357 (1983)] and later expanded upon by Tofts and Wray. In this first paper of the series, we briefly review the theoretical basis for the concentration measurement and present, for the first time, a successful paired validation of metabolite quantification via 31P surface-coil NMR through corroborative in vitro enzymatic assays. The paired radiolabeled microsphere validation of blood flow measurement via 2H surface-coil NMR employing D2O as a freely diffusible tracer and the concurrent determination of blood flow and energy metabolism in a septic rat model are presented in the accompanying second and third paper to complete the series. In this article a classical RF tank circuit is employed to describe the effect of conductive sample loading on the NMR receiver by considering its apparent series resistance. It is shown in an easily visualized generalizable manner that the effect of sample loading on the observed NMR signal intensity can be accounted for quantitatively by monitoring changes in 90 degrees pulse width at constant power at a fixed reference point, i.e., Ssample = Sphantom (PW90phantom/PW90sample). In a series of paired experiments the absolute concentrations of high energy phosphates obtained from resting rat leg muscle (n = 4) in situ (NMR) and in vitro (enzymatic) were determined as follows: [PCr]NMR = 17.2 +/- 0.8 SD, [PCr]enzymatic = 17.3 +/- 2 SD, [ATP]NMR = 5.1 +/- 0.8 SD, [ATP]enzymatic = 5.0 +/- 0.2 SD mmol/kg tissue wet wt. Results of these two independent methods of concentration determination were not statistically different (P = 0.94 and P = 0.74 respectively) and serve to rigorously validate the Thulborn approach for absolute quantification of phosphorous metabolites in situ via NMR. Furthermore, these results strongly suggest that ATP and PCr in resting rat leg muscle under normal physiologic conditions are 100% NMR visible. The free cytosolic [ADP]NMR was estimated from the creatine kinase reaction equilibrium expression to be 0.022 +/- 0.003 SD mmol/kg tissue wet wt.     

In vivo identification and monitoring of changes in rat brain glucose by two-dimensional shift-correlated 1H NMR spectroscopy.

Intracerebral glucose resonance was directly detected and resolved in vivo by two-dimensional shift-correlated (COSY) 1H NMR spectroscopy in anesthetized rats (n = 4). The relative changes in brain glucose concentration were measured by volume integration of the alpha-D-glucose cross peak in the 2D COSY spectra. This report demonstrates the possibility of monitoring the variations in cerebral glucose following iv injection of glucose.     

A lineshape fitting model for 1H NMR spectra of human blood plasma.

A lineshape fitting model was constructed for classifying the overlapping information in the 1H NMR spectrum of human blood plasma. A reliable assignment of the overlapping fatty acid (-CH2-)n and -CH3 resonances of the various lipoproteins (VLDL, very low density lipoprotein; LDL, low density lipoprotein; HDL high density lipoprotein) is introduced, and for the first time detailed characteristics (chemical shifts, half linewidths, and relative intensities) of the individual lipoprotein components were obtained directly from the whole plasma spectrum. This was achieved by combining the constructed lineshape fitting model and the proper 400 MHz proton NMR measurements from blood plasma of a healthy donor, from fractions of the different lipoproteins, and from plasma samples in which the lipoprotein fractions were separately added. The results suggest fair promise of future applications of the rapid and easy NMR analysis of lipoprotein distribution in various research and clinical situations.     

Lactate isotopomer analysis by 1H NMR spectroscopy: consideration of long-range nuclear spin-spin interactions.

Lactate is a key metabolite and its rates of cellular uptake and release, its production rates from glucose and glycogen, and its interconversion rate with pyruvate are important determinants of cellular energy production. If lactate precursors such as pyruvate and glucose are labeled appropriately with (13)C, (1)H NMR spectroscopy provides a means of quantifying lactate production from each source and allows measurement of all these rates within a single experiment. However, due to the multiplicities of the resonance lines (from nuclear spin-spin couplings) in lactate (13)C isotopomers, the (1)H NMR spectra were found to be more complex than expected, requiring determination of all spin-spin interactions in this anion. All such values were determined for lactate and its precursor pyruvate. The method was then applied to simultaneously measure the rates of exogenous lactate uptake and rates of release of glucose-, glycogen-, and pyruvate-derived lactate in the isolated perfused rat heart.     

Determination of absolute phosphate metabolite concentrations in RIF-1 tumors in vivo by 31P-1H-2H NMR spectroscopy using water as an internal intensity reference.

The absolute metabolite quantification method of Thulborn and Ackerman [J. Magn. Reson. 55, 357 (1983)] in which the tissue water proton signal is used as an internal intensity standard and its more recent variation in which NMR peak intensities are referenced to that of the natural abundance deuterium signal of water [Li et al., SMRM Abstr. 2, 825 (1988); Song et al., Magn. Reson. Med. 25, 45 (1992) have been implemented to obtain absolute phosphate metabolite concentrations in subcutaneous RIF-1 tumors during untreated growth and following treatment with 5-fluorouracil. The equivalence of these two hydrogen isotopes as intensity standards and the validity of their use in the determination of absolute metabolite concentrations in vivo by NMR has been demonstrated. On matched in vivo and extract tumor samples (n = 5), excellent agreement has been obtained between nucleoside triphosphate concentrations determined by NMR and those derived by HPLC analysis for the control tumors. Following 3 days of untreated growth, absolute concentrations of phosphate metabolites in RIF-1 tumors (n = 10) decreased significantly, except for the Pi concentration which did not vary. For the treated tumors (n = 10) there were no changes in metabolite concentrations except for a decrease in the PCr and, possibly, Pi concentrations. The PCr/Pi ratio in the latter tumors did not change. These observations suggest that changes in absolute metabolite concentrations may be more sensitive indices of response to therapy than changes in metabolite peak amplitude ratios, a parameter commonly used to express in vivo NMR data.     

Quantitation of simulated short echo time 1H human brain spectra by LCModel and AMARES.

LCModel and AMARES, two widely used quantitation tools for magnetic resonance spectroscopy (MRS) data, were employed to analyze simulated spectra similar to those typically obtained at short echo times (TEs) in the human brain at 1.5 T. The study focused mainly on the influence of signal-to-noise ratios (SNRs) and different linewidths on the accuracy and precision of the quantification results, and their effectiveness in accounting for the broad signal contribution of macromolecules and lipids (often called the baseline in in vivo MRS). When applied in their standard configuration (i.e., fitting a spline as a baseline for LCModel, and weighting the first data points for AMARES), both methods performed comparably but with their own characteristics. LCModel and AMARES quantitation benefited considerably from the incorporation of baseline information into the prior knowledge. However, the more accurate quantitation of the sum of glutamate and glutamine (Glx) favored the use of LCModel. Metabolite-to-creatine ratios estimated by LCModel with extended prior knowledge are more accurate than absolute concentrations, and are nearly independent of SNR and line broadening.     

尿苷二磷酸葡萄糖脱氢酶的基因克隆及在原核细胞中的表达

目的：尿苷二磷酸葡萄糖脱氢酶是催化黄原胶生成的酶类之一。设想通过增加酶量来达到提高黄原胶产量的最终目的。方法：提取基因组ＤＮＡ作为模板,通过ＰＣＲ扩地得到 酶基因,经ＤＮＡ体外重组构建表达质粒。结果：实验在大肠杆菌中的表达,并成功转化黄原胶菌株。转化过程提示黄原胶菌株可能具有某些特殊性,与文献报道比较有异同点。     

Oxygen-17 compounds as potential NMR T2 contrast agents: enrichment effects of H2(17)O on protein solutions and living tissues

The isotopic enrichment of solutions, living tissues, and organisms with oxygen-17 in the form of H2(17)O shortens their proton NMR transverse relaxation times (T2) and produces changes in NMR image intensity. The transverse relaxation rate (1/T2) was found to be linearly dependent on the H2(17)O concentration in biological solutions up to 5% enrichment. The longitudinal relaxation time (T1) is not affected by enrichment. Equal concentrations of H2(17)O do not produce the same magnitude of T2 change in all physiological environments. The reasons for these differences are discussed. The results suggest that certain oxygen-17 compounds should be explored as "contrast agents" in magnetic resonance imaging.