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.     

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.     

Proton NMR imaging of cerebral blood flow using H2(17)O.

Cerebral blood flow was quantitatively mapped by monitoring the cerebral washout of H2(17)O using rapid, single-shot proton NMR imaging. H2(17)O acts as a freely diffusible contrast agent for proton imaging via its scalar-coupled term, enhancing T2 relaxation. Measured values for CBF ranged from 29 to 106 ml/min/100 g over a range of arterial pCO2 between 23 and 81 Torr.     

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.     

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 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.     

1H NMR studies of urine from premature and sick babies

High-frequency proton nuclear magnetic resonance (1H NMR) has been applied to a study of urine from premature and sick newborn babies with a variety of clinical disorders. These included severe birth asphyxia, necrotizing enterocolitis, ketosis, and drug treatment. Abnormal levels of metabolites such as lactate, ketone bodies, betaine, dicarboxylic acids, and 4-hydroxyphenolic acids were detected, as was the antibiotic metronidazole and one of its metabolites. The assignment of betaine was confirmed by two-dimensional 1H NMR studies of urine obtained from two adults with homocystinuria receiving treatment with oral betaine. High-resolution 1H NMR spectroscopy of urine is a potentially useful research technique for monitoring metabolism during early life.     

In vivo measurement of CBF using 17O NMR signal of metabolically produced H217O as a perfusion tracer

The cerebral metabolic rate of oxygen of small animals can be reliably imaged using the in vivo ¹⁷O magnetic resonance approach at high field. However, a separate measurement is required for imaging the cerebral blood flow in the same animal. In this study, we demonstrate that the ¹⁷O NMR signal of metabolically produced H₂¹⁷O in the rat brain following an ¹⁷O₂ inhalation can serve as a perfusion tracer and its decay rate can be used to determine the absolute values of cerebral blood flow across a wide range of animal conditions. This finding suggests that the in vivo ¹⁷O magnetic resonance approach is capable of imaging both cerebral metabolic rate of oxygen and cerebral blood flow simultaneously and noninvasively; and it provides new utilities for studying the cerebral oxygen metabolism and perfusion commonly associated with brain function and diseases. Magn Reson Med 70:309–314, 2013. © 2012 Wiley Periodicals, Inc.     

Single-shot diffusion trace (1)H NMR spectroscopy.

Ignoring diffusion anisotropy can severely hamper the quantitative determination of water and metabolite diffusion in complex tissues. The measurement of the trace of the diffusion tensor provides unambiguous and rotationally invariant ADC values, but usually requires three separate experiments. A single-shot technique developed earlier, originally designed for diffusion trace MR imaging (Mori and van Zijl, Magn Reson Med 1995;33:41-52), was improved and adapted for diffusion trace MR spectroscopy. A double spin-echo pulse sequence was incorporated with four pairs of bipolar gradients with specific predetermined relative signs in each of the three orthogonal directions. The combination of gradient directions leads to cancellation of all off-diagonal tensor elements while constructively adding the diagonal elements. Furthermore, the pulse scheme provides complete compensation for cross-terms between static magnetic field gradients and the applied diffusion gradients, while simultaneously avoiding cross-terms with localization gradients. The sequence was tested at 4.7 T in vivo on rat brain for MRI and on rat skeletal muscle and brain for MRS. It is shown that the average ADC as determined from the measurement of the ADCs in the three orthogonal directions is in close agreement with the ADC obtained along the trace of the diffusion tensor in a single acquisition, for both water and metabolite diffusion. The large differences in water and metabolite diffusion coefficients as measured in the individual orthogonal directions illustrate the need for diffusion trace measurements when accurate and rotationally invariant diffusion quantitation is required. The pulse scheme presented here may be applied for such purposes in MRS and MRI studies.     

Evaluation of radiation injury by 1H and 31P NMR of human urine

¹H and ³¹P NMR techniques were applied to study the changes in metabolite profiles in human urine resulting from radiation exposure following the Chemobyl reactor accident. In cases of acute leukemia and different accumulated doses of external radiation (from 0.20 to 4.00 Sv), the proton spectra were classified on the basis of the peaks due to N-trimethyl groups, creatinine, citrate, glycine, and hippurate. Unidentified resonances were observed between 15.9 and 21.4 ppm in six phosphorus spectra of patients with preleukemia and acute leukemia. Characteristic spectral changes were similar for external radiation and Incorporation-induced internal irradiation. The spectral patterns described may serve as a criterion of radiation injury.     

Measurement of regional cerebral blood flow in cat brain using intracarotid 2H2O and 2H NMR imaging

Cerebral blood flow (CBF) was measured in cat brain in vivo at 2.7 T using 2H NMR to monitor the washout of deuterated saline injected into both carotid arteries via the lingual arteries. In anesthetized cats, global CBF varied directly with PaCO2 over a range of 20-50 mm Hg, and the corresponding global CBF values ranged from 25 to 125 ml.100 g-1.min-1. Regional CBF was measured in a 1-cm axial section of cat brain using intracarotid deuterated saline and gradient-echo 2H NMR imaging. Blood flow images with a maximum pixel resolution of 0.3 x 0.3 x 1.0 cm were generated from the deuterium signal washout at each pixel. Image derived values for CBF agreed well with other determinations, and decreased significantly with hypocapnia.     

Clinical Applications of [(15)O] H(2)O PET Activation Studies

Localization of eloquent cortical regions is an important aspect of neurosurgical practice. When lesions lie close to or within areas such as somatosensory, motor or language cortex, the goal of surgical treatment is maximal resection without causing neurological deficit. Hence precise functional mapping combined with accurate delineation of the extent of pathology is important for deciding whether surgery is feasible at all and defining the safe limit of resection and the optimal operative approach. In addition, when the electroclinical pattern of intractable seizures suggests a specific localization in sensorimotor cortex (e.g., seizures originating in the face area), functional mapping may be of use in directing the surgical treatment at the seizure focus. Activation studies using positron emission tomography (PET) and the tracer [(15)O] labeled water ([(15)O]H(2)O) can be used in presurgical planning and in intraoperative surgical guidance in individual patients. The use of PET technology for these purposes is critically dependent on advances in registration or alignment of images acquired using different modalities; the development of neurosurgical guidance devices; and the development of methods of statistical analysis suitable for use in single subjects.     

Brain blood flow measured with intravenous H2(15)O. II. Implementation and validation

We have adapted the well-known tissue autoradiographic technique for the measurement of regional cerebral blood flow (CBF), originally proposed by Kety and his colleagues, for the measurement of CBF in human subjects using positron emission tomography (PET) and intravenously administered oxygen-15-labeled water. This report describes the steps necessary for the implementation of this PET/autoradiographic technique. In order to establish the accuracy of the method, we measured CBF with intravenously administered oxygen-15-labeled water and PET in anesthetized adult baboons and compared the results with blood flow measured by a standard tracer technique that uses residue detection of a bolus of oxygen-15-labeled water injected into the internal carotid artery. The correlation between CBF measured with PET and the true CBF for the same cerebral hemisphere was excellent. Over a blood-flow range of 10-63 ml/(min X 100 g), CBF (PET) = 0.90 CBF(true) + 0.40 (n = 23, r = 0.96, p less than 0.001). When blood flow exceeds 65 ml/(min X 100 g) CBF was progressively underestimated due to the known limitation of brain permeability to water.     

Mapping of local renal blood flow with PET and H(2)(15)O.

We developed a noninvasive method for the mapping of regional renal blood flow in humans using PET and H(2)(15)O. METHODS: Fifteen subjects participated in the study, 5 with normal renal function and 10 with renal disease. The protocol used a whole-body PET scanner, intravenous bolus injection of 1,110-1,850 MBq H(2)(15)O and sequential imaging at 3 s per frame. (131)I-Iodohippuran was used to independently assess effective renal plasma flow in each subject. Hippuran clearance and renal blood flow (RBF) were measured twice, before and after treatment with probenecid, to verify that RBF is not affected. Flow analysis was based on the Kety model, according to the operational equation: C(t) = F integral C(a)(u)du - k integral C(u)du, where F is the RBF, k is the tissue-to-blood clearance rate, C is the PET concentration, and C(a) is the tracer concentration in the abdominal aorta. F and k were estimated by linear least squares on a pixel-by-pixel basis to produce quantitative maps (parametric images) of RBF. The flow maps were analyzed by regions of interest (largely excluding the medulla and collecting system) for each kidney on each slice and pooled to yield mean RBF. RESULTS: In the 5 healthy subjects, mean RBF was 3.4 +/- 0.4 mL/min/g. There was no difference in flow between kidneys (t = -0.59; n = 11; P > 0.95). Before treatment with probenecid, RBF was linearly related to hippuran clearance (r(2) = 0.92). Probenecid treatment significantly reduced hippuran clearance (P < 0.003), but RBF was unchanged (P > 0.17). Compared with healthy control subjects, RBF was significantly decreased in patients with renal disease (P < 0.002). Flow maps were of good quality in all subjects, exhibiting characteristic patterns, with higher values in regions composed largely of renal cortex. CONCLUSION: Parametric mapping of RBF with PET and H(2)(15)O provides a straightforward, noninvasive method for quantitative mapping of RBF, which may prove useful in research applications and in the management of patients whose therapy alters renal tubular transport.     

回心草单体成分对H2O2诱导心肌细胞损伤的保护作用研究

目的 建立过氧化氢（H202）诱导心肌细胞损伤模型,并在此模型上研究胡椒碱、胡椒酸甲酯、咖啡酸甲酯、尿嘧啶苷对心肌细胞损伤的保护作用。方法 用H202诱导造成原代培养的心肌细胞损伤模型,将单体化合物分为3个剂量组,作用于正常和损伤的心肌细胞,利用全自动生化分析仪对细胞悬液中乳酸脱氢酶（LDH）和肌酸激酶同功酶（CK-MB）含量进行测定。结果 回心草各单体成分对正常心肌细胞无明显影响。胡椒碱和胡椒酸甲酯各剂量组作用的心肌细胞悬液中LDH和CK-MB值与模型组对照有非常显著性差异（P＜0．01）,咖啡酸甲酯对CK-MB有非常显著性差异（P＜0．01）,尿嘧啶苷无显著性差异（P＞0．05）。结论 对H202诱导的心肌细胞损伤,胡椒酸甲酯和胡椒碱具有明显的保护作用,咖啡酸甲酯具有部分保护作用,尿嘧啶苷无保护作用。     

Brain blood flow measured with intravenous H2(15)O. I. Theory and error analysis

The tissue autoradiographic method for the measurement of regional cerebral blood flow (rCBF) in animals was adapted for use with positron emission tomography (PET). Because of the limited spatial resolution of PET, a region of interest will contain a mix of gray and white matter, inhomogeneous in flow and in tracer partition coefficient (lambda). The resultant error in rCBF, however, is less than 4%. Although the tissue autoradiographic method requires a monotonically increasing input function to ensure a unique solution for flow, the PET adaptation does not, because of an additional integration in the operational equation. Simulation showed that the model is accurate in the presence of ischemia or hyperemia of the gray matter. Inaccuracy in timing of the arterial input function will result in large errors in rCBF measurement. Propagation of errors in measurement of tissue activity is largely independent of flow, reflecting the nearly linear flow compared with activity relationship.     

Anisotropic and restricted diffusion of water in the sciatic nerve: A (2)H double-quantum-filtered NMR study.

The signals of water in the different compartments of rat sciatic nerve are resolved in the (2)H double-quantum-filtered NMR spectrum, due to their different quadrupolar splittings and relaxation rates. This resolution allowed the independent measurement of the water diffusion coefficients in the different compartments. The water diffusion in all three compartments, the endoneurium, the epineurium and the axon was found to be anisotropic. Parallel to the nerve fiber the average intraxonal water diffusion coefficient was 1.11 x 10(-5) cm(2)/sec, while in the perpendicular direction the diffusion is heavily restricted. The average perpendicular diffusion coefficient ranged from 0.29 x 10(-5) cm(2)/sec to 0.05 x 10(-5) cm(2)/sec for diffusion times of 7 msec and 50 msec, respectively. Assuming restricted diffusion in nonpermeable cylinders, intra-axonal mean diameters of 6.0, 7.4 and 9.0 microm were obtained for nerves taken from three different rats. Magn Reson Med 42:461-466, 1999.     

High-resolution (1)H NMR and magic angle spinning NMR spectroscopic investigation of the biochemical effects of 2-bromoethanamine in intact renal and hepatic tissue.

The metabolic consequences of xenobiotic-induced toxicity were investigated using high-resolution magic angle spinning (MAS) NMR spectroscopy of intact tissue. Renal papillary necrosis (RPN) was induced in Sprague-Dawley rats (n = 12) via a single i.p. dose of 250 mg/kg 2-bromoethanamine (BEA) hydrobromide. At 2, 4, 6, and 24 h after treatment with BEA, three animals were killed and tissue samples were obtained from liver, renal cortex, and renal medulla. Tissue samples were also removed at 2 and 24 h from matched controls (n = 6). (1)H MAS NMR spectroscopic techniques were used to analyze samples of intact tissue ( approximately 10 mg). Decreased levels of nonperturbing renal osmolytes (glycerophosphocholine, betaine, and myo-inositol) were observed in the renal papilla of BEA-treated animals at 6 and 24 h postdose (p.d.), concomitant with a relative increase in the tissue concentration of creatine. Increased levels of glutaric acid were found in all tissues studied in BEA-treated animals at 4 and 6 h p.d., indicating the inhibition of mitochondrial fatty acyl CoA dehydrogenases and mitochondrial dysfunction. Increased levels of trimethylamine-N-oxide occurred in the renal cortex at 6 h p.d. Changes in the metabolite profile of liver included an increase in the relative concentrations of triglycerides, lysine, and leucine. The novel application of (1)H MAS NMR to the biochemical analysis of intact tissues following a toxic insult highlights the potential of this technique as a toxicological probe in providing a direct link between urinary biomarkers of toxicity and histopathological evaluation of toxicological lesions.