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 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.     

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.     

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 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.     

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.     

Stability of ethyl glucuronide in urine, post-mortem tissue and blood samples

The stability of ethyl glucuronide (EtG) under conditions of degradation was examined in urine samples of nine volunteers and in post-mortem tissue (liver, skeletal muscle) and blood taken from seven corpses at autopsies. Analysis was performed via LC-MS/MS. EtG concentrations in urine samples ranged from 2.5 to 296.5 mg/l. When stored at 4°C in airtight test tubes, EtG concentrations remained relatively constant; when stored at room temperature (RT) for 5 weeks in ventilated vials, variations of EtG concentrations ranged from a 30% decrease to an 80% increase, with an average of 37.5% increase. Liver and skeletal muscle tissue of three corpses with positive blood alcohol concentrations (BAC; ranging from 0.106 to 0.183 g%) were stored for 4 weeks and analysed periodically. EtG concentrations decreased 27.7% on average in 4 weeks storage at RT but EtG was still detectable in all samples with initial EtG concentrations higher than 1 μg/g. Blood and liver samples of four corpses with negative BACs were stored at RT after addition of 0.1 g% ethanol, and no new formation of EtG was observed.     

Conjunction analysis and propositional logic in fMRI data analysis using Bayesian statistics

Purpose

To evaluate logical expressions over different effects in data analyses using the general linear model (GLM) and to evaluate logical expressions over different posterior probability maps (PPMs).

Materials and Methods

In functional magnetic resonance imaging (fMRI) data analysis, the GLM was applied to estimate unknown regression parameters. Based on the GLM, Bayesian statistics can be used to determine the probability of conjunction, disjunction, implication, or any other arbitrary logical expression over different effects or contrast. For second‐level inferences, PPMs from individual sessions or subjects are utilized. These PPMs can be combined to a logical expression and its probability can be computed. The methods proposed in this article are applied to data from a STROOP experiment and the methods are compared to conjunction analysis approaches for test‐statistics.

Results

The combination of Bayesian statistics with propositional logic provides a new approach for data analyses in fMRI. Two different methods are introduced for propositional logic: the first for analyses using the GLM and the second for common inferences about different probability maps.

Conclusion

The methods introduced extend the idea of conjunction analysis to a full propositional logic and adapt it from test‐statistics to Bayesian statistics. The new approaches allow inferences that are not possible with known standard methods in fMRI. J. Magn. Reson. Imaging 2008;28:1533–1539. © 2008 Wiley‐Liss, Inc.     

A fatal clomipramine intoxication case of a chronic alcoholic patient: application of postmortem hair analysis method of clomipramine and ethyl glucuronide using LC/APCI/MS

Toxicological investigations of postmortem specimens of a 26-year-old man were performed with the use of LC/APCI/MS. They revealed in the blood of the deceased clomipramine (9.49 microg/g) and its main metabolite norclomipramine (1.10 microg/g) at concentrations explaining the fatal outcome. The presence of these xenobiotics in a 12-cm-long strand of hair (clomipramine, 7.60 ng/mg in I segment; 4.19 ng/mg in II segment; 1.86 ng/mg in III segment; norclomipramine, 5.71 ng/mg in I segment; 9.71 ng/mg in II segment; 4.13 ng/mg in III segment) confirmed the fact obtained from the medical history that the deceased had been receiving clomipramine as an antidepressant for 1 year prior to his death. The analysis demonstrated ethanol in autopsy blood (2.5mg/ml) and urine (3.2mg/ml); ethyl glucuronide as a marker of chronic alcohol abuse was detected in the deceased's hair (0.44 ng/mg in I segment; 0.07 ng/mg in II segment; n.d. in III segment). These findings may suggest the contribution of alcohol in the mechanism of drug-ethanol interaction, which in consequence might have affected the biotransformation of clomipramine in the final period of his life and evoked the ultimate toxic effect.     

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.     

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.     

Improved intravoxel incoherent motion analysis of diffusion weighted imaging by data driven Bayesian modeling

In addition to the diffusion coefficient, fitting the intravoxel incoherent motion model to multiple b‐value diffusion‐weighted MR data gives pseudo‐diffusion measures associated with rapid signal attenuation at low b‐values that are of use in the assessment of a number of pathologies. When summary measures are required, such as the average parameter for a region of interest, least‐squares based methods give adequate estimation accuracy. However, using least‐squares methods for pixel‐wise fitting typically gives noisy estimates, especially for the pseudo‐diffusion parameters, which limits the applicability of the approach for assessing spatial features and heterogeneity. In this article, a Bayesian approach using a shrinkage prior model is proposed and is shown to substantially reduce estimation uncertainty so that spatial features in the parameters maps are more clearly apparent. The Bayesian approach has no user‐defined parameters, so measures of parameter variation (heterogeneity) over regions of interest are determined by the data alone, whereas it is shown that for the least‐squares estimates, measures of variation are essentially determined by user‐defined constraints on the parameters. Use of a Bayesian shrinkage prior approach is, therefore, recommended for intravoxel incoherent motion modeling. Magn Reson Med 71:411–420, 2014. © 2013 Wiley Periodicals, Inc.     

Spectral analysis of99mTc-HMPAO for estimating cerebral blood flow: A comparison with H2 15O PET

Cerebral blood flow (CBF) can be quantified non-invasively using the brain perfusion index (BPI), which is determined using radionuclide angiographic data obtained through the use of technetium-99m hexamethylpropylene amine oxime (99mTc-HMPAO). The BPI is generally calculated using graphical analysis (GA). In this study, BPI was measured using spectral analysis (SA), and the usefulness of SA was compared with that of GA. Thirteen patients with various brain diseases and four healthy male volunteers were examined using radionuclide angiography with 99mTc-HMPAO. The BPI was measured for each subject using both SA and GA. In the four healthy volunteers, the BPI was examined at rest and after the intravenous administration of 1 g of acetazolamide (ACZ). An H2(15)O PET examination was also performed in the 13 patients; the BPIS and BPIG values were compared with the CBF measurements obtained using H2(15)O PET (CBFPET). The BPI values obtained by SA (BPIS) (x) and by GA (BPIG) (y) were correlated (y = 0.568x + 0.055, r = 0.901) in the 13 patients and four healthy volunteers at rest, although the BPIG values were underestimated by 36.1 +/- 7.5% (mean +/- SD) compared with the BPIS values. The degree of underestimation tended to increase with increasing BPIS values. The increase in the BPIS was 32.1 +/- 8.0% after the intravenous administration of ACZ, while the increase in BPIG was only 8.1 +/- 2.8%. This discrepancy was considered to be the result of the BPIG values being affected by the first-pass extraction fraction of the tracer. Although both BPIS and BPIG values were significantly correlated with the CBFPET values, the correlation coefficient for BPIS was higher than that for BPIG (BPIS: r = 0.881; BPIG: r = 0.832). These results suggest that SA produces a more reliable BPI for quantifying CBF using 99mTc-HMPAO than the conventional method using GA. The SA method should be especially useful for activation studies involving pharmacological intervention and/or clinical cases with an increased CBF.     

实验性2型糖尿病大鼠模型的建立和评价(Ⅰ)——体重、血糖和肝糖原的变化

目的：用高脂饲料加小剂量STZ腹腔注射建立实验性2型糖尿病大鼠模型。方法：采用Wistar大鼠,分为正常对照组、高脂饲料组、小剂量STZ组（30 mg/kg）和高脂饲料加小剂量STZ（30 mg/kg）组,每10 d测定大鼠体重、空腹血糖和肝糖原等指标,连续50 d,对各组大鼠上述指标进行比较和评价。结果：①体重：高脂饲料组大鼠体重呈连续上升趋势,小剂量STZ组和高脂饲料加小剂量STZ组大鼠体重呈先降低再恢复的趋势;②空腹血糖：高脂饲料组大鼠空腹血糖轻微升高,小剂量STZ组大鼠空腹血糖呈上升的趋势,高脂饲料加小剂量STZ组大鼠空腹血糖呈明显上升趋势;③肝糖原：高脂饲料组大鼠肝糖原明显升高,小剂量STZ组、高脂饲料加小剂量STZ组大鼠肝糖原未见明显改变;结论：高脂饲料加小剂量STZ腹腔注射能成功诱导2型糖尿病大鼠模型,这种造模方法具有成模后血糖维持在较高水平、自身缓解较少、症状较轻和动物状态良好等优点。     

A series target system for the rapid,sequential production of [15O]H2O and O2, and [18F]F2

A two chambered, series target was developed for use in the rapid, sequential production of [¹⁵O]H₂O and O₂, and [¹⁸F]F₂ using an FN-6 tandem van de Graaff accelerator. The ¹⁵O labeled tracer gases are produced on a continuous flow basis in an aluminum target chamber using the ¹⁴N(d,n)¹⁵O reaction on 5% H₂ in N₂ and 2% O₂ in N₂, respectively. Fluorine-18 labeled F₂ is produced on a batch basis using the ²⁰Ne(d,α)¹⁸F reaction on 0.1% F₂ in Ne in a high pressure, polished nickel chamber. The two target chambers are separated by an aluminum-nickel foil sandwich, and the ¹⁵O target is evacuated during ¹⁸F production. Switching between ¹⁵O target gases as well as choice of ¹⁸F or ¹⁵O production is completely remote. System performance is comparable to that obtained previously using individually mounted targets.     

A system for continuous production and infusion of [15O]H2O for PET activation studies.

A system for continuous production and infusion of [15O]H2O has been designed for Positron Emission Tomography brain activation studies. The infusion system consists of two Horizon Nxt infusion pumps, a four-port-two-position valve and a sterile 50 ml vial. The line and the back check valve between the furnace and the reservoir were heated in order to reduce vapor condensation in the line. The variation of the production of [15O]H2O was < 1%. The activity delivered as measured by scanner counts varied < 2% during the steady state period. The system has been demonstrated to be capable of delivering activity over a wide range of conditions.