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PubMed2007

In vivo 2D mapping of impaired murine cardiac energetics in NO-induced heart failure.

(31)P MRS studies in humans have shown that an impairment of cardiac energetics is characteristic of heart failure. Although numerous transgenic mouse models with a heart-failure phenotype have been generated, current methods to analyze murine high-energy phosphates (HEPs) in vivo are hampered by limited spatial resolution. Using acquisition-weighted 2D (31)P chemical shift imaging (CSI) at 9.4 Tesla, we were able to acquire (31)P MR spectra over the entire thorax of the mouse with high spatial resolution in defined regions of the heart (the anterior, lateral, posterior, and septal walls) within a reasonable acquisition time of about 75 min. Analysis of a transgenic cardiomyopathy model (double mutant: cardiospecific inducible nitric oxide synthase (iNOS) overexpression and lack of myoglobin (tg-iNOS(+)/myo(-/-)) revealed that cardiac dysfunction in the mutant was associated with an impaired energy state (phosphocreatine (PCr)/adenosine triphosphate (ATP) 1.54 +/- 0.18) over the entire left ventricle (LV; wild-type (WT): PCr/ATP 2.06 +/- 0.22, N = 5, P < 0.05), indicating that in the absence of efficient cytosolic NO scavenging, iNOS-derived NO critically interferes with the respiratory chain. In vivo data were validated against (31)P MR spectra of perchloric acid extracts (PCr/ATP: 1.87 +/- 0.21 (WT), 1.39 +/- 0.17 (tg-iNOS(+)/myo(-/-), N = 5, P < 0.05). Future applications will substantially benefit studies on the cause-and-effect relationship between cardiac energetics and function in other genetically well-defined models of heart failure.

Animals↗
PubMed1989

A 13C NMR study on fluxes into the Krebs cycle of rabbit renal proximal tubular cells.

Suspensions of rabbit renal proximal tubular (PCT) cells were incubated with [2-13C] and [3-13C]pyruvate. The perchloric acid extracts of the cell pellets were examined by 13C NMR. All experiments showed that enriched lactate, alanine, glutamate, and glutamine were the main metabolic intermediates, and that enrichment to a minor extent was found in the glutamate residue of glutathione (GSH). From these experiments, it could be deduced that PCT cells show a highly glycolytic activity, whereas enrichment of glucose exhibits gluconeogenesis. The estimation by 13C NMR of the ratio of the flux into the Krebs cycle via pyruvate carboxylase to the flux via pyruvate dehydrogenase is discussed. From incubations with 10 mM 13C-labelled pyruvate, we calculated from the relative enrichments of the glutamate carbon atoms that the ratio of pyruvate carboxylase to pyruvate dehydrogenase is 1.44 +/- 0.04 in rabbit renal proximal tubules.

Animals↗
PubMed1991

Approaches to editing, assignment and interpretation of proton spectra.

Clinical 1H spectroscopy of the brain is complemented by parallel analyses of biopsy specimens and by studies of animal models of disease. 1H spectroscopy has been carried out on perchloric acid extracts of biopsy specimens from patients with intracranial tumours. The data suggest that clinical spectroscopy may be useful in the identification and grading of these tumours. In addition, the spectra from extracts derived from normal white matter add weight to the possibility that acetyl-containing compounds other than N-acetylaspartate may make a significant contribution to the signal at 2.0 ppm in vivo. Edited 1H spectra of brain metabolites in rats with acute liver failure demonstrate an elevation of glutamine and of lactate, suggesting a role for 1H spectroscopy in clinical investigations of metabolic encephalopathies. However, the observation and resolution of signals from glutamate and glutamine is more difficult at the lower fields that are available for clinical spectroscopy. Finally, some studies of patients with inborn errors of metabolism are described. It is shown that in a disorder of oxidative metabolism, brain lactate can be detected without the need for complex spectral editing techniques. Investigations of the metabolic abnormalities associated with Canavan's disease have shed further light on a possible role for N-acetylaspartate.

Animals↗
PubMed1991

Is cellular integrity responsible for the partial NMR invisibility of ATP in isolated ischemic rat liver?

The observability of nucleoside triphosphate (NTP) by 31P NMR spectroscopy was studied in the isolated rat liver during hypothermic perfusion and a subsequent 4-h cold ischemia. The influence of hypothermia (4 degrees C) was examined because of its delaying effects on cell injury induced by the ischemic conditions. The viability of the liver after hypothermic ischemia was assessed by measuring the recovery of the beta-NTP resonance after reperfusion. In 4-h cold ischemic liver, recovery was found to be in the range of 90-100% and consequently NTP visibility was studied under these conditions. Because the individual purine (or pyrimidine) NTPs are not distinguishable in the liver on the basis of their 31P NMR chemical shifts, the contributions of UTP and GTP were investigated by HPLC. The changes in liver NTP content measured either by NMR on isolated liver or by HPLC after perchloric acid extraction from the same organ are not significantly different. The total NTP level in normothermic perfused liver is 7.6 +/- 0.2 mumol NTP/g liver dry wt as determined by NMR. In such a liver, ATP + GTP + UTP and ATP contents measured by HPLC are, respectively, 7.9 +/- 1.0 and 6.3 +/- 0.9 mumol/g liver dry wt. This indicates that all NTP is detected by NMR and that a 20% contribution of the signal occurs from UTP + GTP. Under 4-h cold ischemic conditions, NTP visibility remains unchanged, furthermore the UTP + GTP contribution reaches 32% of the whole NTP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMedDate not supplied

Visibility of phospholipids in 31P NMR spectra of human breast tumours in vivo.

The aims of this study were two-fold: to characterize the in vivo 31P NMR spectrum of human breast tumours by identifying the metabolites contributing to each peak; and to demonstrate changes in the detectability of the phosphodiester (PDE) peak at varying field strengths. This was achieved by obtaining 31P spectra from 23 patients in vivo at 1.5 T and also of 11 perchloric acid (PCA) extracts and 3 chloroform-methanol (CM) extracts of tumour biopsy samples at 9.4 T. In spectra acquired in vivo the percentage areas for each peak were: phosphomenoester (PME), 15%; Pi, 11%; PDE 35%; phosphocreatine, 5%; gamma-NTP, 11%; alpha-NTP, 11% and beta-NTP, 12% of the total spectral area. PCA extracts showed the PME peak to be greater than 70% phosphorylethanolamine whilst the PDE peak included almost equal proportions of glycerophosphorylethanolamine and glycerophosphorylcholine. CM extracts of tumours revealed additional metabolites in the PDE region suggesting that the large PDE peak observed in vivo could arise mainly from phospholipids. Spectra of human breast tumour xenografts examined in vivo at both 1.9 T and 9.4 T confirmed that the presence of a large PDE peak in vivo was a function of field strength. Further experiments with microsomal membranes from rat mammary tumours at 1.5 and 9.4 T demonstrated that phospholipids are more clearly visible at the lower field strength due to a substantial decrease in linewidth.

Animals↗
PubMedDate not supplied

Classification of tumour 1H NMR spectra by pattern recognition.

1H spectra of tumours or normal tissues, which include signals from all hydrogen-containing metabolites, are too complex for the human eye to interpret. We have studied 58 1H spectra from perchloric acid extracts of three normal tissues (liver, kidney and spleen) and five rat tumours (GH3 pituitary, fibrosarcoma, Morris Hepatomas 7777 and 9618a and Walker carcinosarcoma). Instead of editing them or quantifying individual metabolites, we have used statistical pattern recognition techniques to classify them into groups. This automatic, objective method differentiated spectra from normal and malignant rat tissue biopsies, and from different types of cancer. It seems likely that this technique can be applied to human tissues and thus used for cancer diagnosis.

Animals↗
PubMedDate not supplied

On the interpretation of proton NMR spectra from brain tumours in vivo and in vitro.

Localized proton NMR spectroscopy in vivo allows focal studies of cerebral metabolites in both man and laboratory animals from image-defined regions as small as 1 mL or 64 microL, respectively. Although brain tumours lead to remarkable spectral alterations relative to normal brain, a number of problems may compromise the interpretation of the results. Potential complications arise from the chosen experimental conditions (method, TE, size and location of volume of interest), from regional metabolic heterogeneity in and around tumours, from differences between human tumours and animal models, and from discrepancies between in vivo and in vitro findings. Strategies and pitfalls are illustrated with use of selected examples from primary brain tumours, a rat tumour model and perchloric acid extracts of resected specimens.

Adult↗
PubMedDate not supplied

Identification of Scyllo-inositol in proton NMR spectra of human brain in vivo.

Scyllo-inositol has been identified in proton NMR spectra of mammalian brain in vitro and in vivo. In contrast to myo-inositol this isomer comprises six equivalent CH protons that yield a singlet resonance at a chemical shift of 3.35 ppm. 1-D and 2-D J-resolved proton NMR studies (7.0 T) of perchloric acid extracts of brain tissues revealed different amounts of scyllo-inositol in man, sheep, cow and rat. Absolute quantification of localized short-echo time proton NMR spectra (2.0 T) of human brain in vivo resulted in scyllo-inositol concentrations of 0.35 +/- 0.06 mM for white matter (n = 25), 0.43 +/- 0.11 mM for grey matter (n = 23) and 0.57 +/- 0.14 mM for cerebellum (n = 10). Evidence for a tight metabolic link to myo-inositol was supported by a simultaneous variation of myo- and scyllo-inositol in patients with various brain diseases.

Animals↗
PubMed1994

Identification of ethanolamine in rat and gerbil brain tissue extracts by NMR spectroscopy.

Some NMR resonances which have previously been observed but not identified in mammalian brain tissue extracts have been shown to arise from ethanolamine. This conclusion is drawn from a systematic study of the perchloric acid extracts of rodent brain tissue in which several NMR experiments were used to assign the peaks unambiguously. The extraction procedure used in this work gave samples with highly reproducible spectra, and ethanolamine was observed in all our extract samples. A localized increase in the concentration of ethanolamine was seen in the spectra of extracts produced from a cerebral infarct induced by occlusion of the middle cerebral artery in the rat.

Animals↗
PubMed1995

Detection and quantitation of phosphorus metabolites in crude tissue extracts by 1H and 31P NMR: use of gradient assisted 1H-31P HMQC experiments, with selective pulses, for the assignment of less abundant metabolites.

The analysis of crude tissue extracts by NMR has proven to be of use in the study of metabolism due to the non-destructive and non-selective character of the technique. Lists of 1H and 31P NMR assignments of phosphorus metabolites in water solution at specified pH and ionic composition are of large general value but their usefulness may be limited when analysing complex mixtures of metabolites at low concentrations. In this work we report on the use of gradient-assisted proton detected multiple quantum 1H and 31P coherence experiments with selective pulses for the rapid and unambiguous assignments of some crowded regions in 1H and 31P spectra of crude extracts from rat liver. The amplitudes of the gradient episodes were calibrated to optimize the coherence transfer pathway between proton and phosphorus, and the delay for the evolution of the long-range coupling was calculated from values of 3JPH and 4JPH ranging from 1.4 to 7.5 Hz. Moreover, a selective 90 degrees Gaussian pulse on the 31P channel was introduced to increase the resolution in the F1-domain and make the method even faster. The procedure was then applied to unambiguously assign the ID 31P and 1H spectra of perchloric acid extracts of rat livers that had been stimulated with phenylephrine, dBcAMP and glucagon and thus detect changes in the concentration of less abundant metabolites such as phosphoenolpyruvate, UDP-glucose and AMP. The fact that the quantification of these metabolites by either 31P and 1H methods lead to different results is discussed, and the use of 1H NMR spectroscopy for the quantification of phosphorus metabolites whose signal are too weak or poorly resolved in a 31P spectrum is proposed.

Adenosine Monophosphate↗
PubMed2003

Biochemical characterization of muscle tissue of limb girdle muscular dystrophy: an 1H and 13C NMR study.

The metabolic differences between the muscle biopsies of patients with limb girdle muscular dystrophy (LGMD) and normal controls were characterized using high-resolution 1H and 13C NMR spectroscopy. In all, 44 metabolites were unambiguously assigned in the perchloric acid extracts of skeletal muscle tissue, using 2D double quantum filtered (DQF COSY), total correlation (TOCSY), and 1H/13C heteronuclear multiple quantum coherence (HMQC) spectroscopy. The concentrations of glycolytic substrate, glucose (p=0.03), gluconeogenic amino acids, glutamine (p=0.02) and alanine (p=0.009) together with glycolytic product, lactate (p=0.04), were found to be significantly lowered in LGMD patients as compared with controls. The reduction in the concentration of glucose may be attributed to the decrease in the concentration of gluconeogenic amino acids in the degenerated muscle. Reduction in the rate of anaerobic glycolysis and lowered substrate concentration appear to be the possible reasons for the decrease in the concentration of lactate. A significant reduction in the concentration of choline in LGMD patients was also observed compared with controls. Lower concentration of choline may be the result of decreased rate of membrane turnover in LGMD patients. The data presented here provide an insight into the potentials of in-vitro NMR spectroscopy in the study of muscle metabolism.

Amino Acids↗
PubMedDate not supplied

Involvement of brain lactate in neuronal metabolism.

The involvement of brain lactate in neuronal metabolism was analyzed by ex vivo NMR spectroscopy with rats under the effects of pentobarbital, alphachloralose or morphine, which were infused with a solution of either [1-(13)C]glucose+lactate or glucose+[3-(13)C]lactate for 20 min. Electroencephalogram recordings indicated different brain electrical activity levels under the three drugs with a clear distinction between pentobarbital, on the one hand, and alphachloralose and morphine on the other. Labeling of metabolites in brain perchloric acid extracts and of blood glucose and lactate was determined by (13)C- and/or (1)H-observed/(13)C-edited-NMR spectroscopy. The following were found: (i) the ratio between glutamate C3 and C4 (13)C-enrichments increased from pentobarbital to alphachloralose and morphine whatever the labeled precursor, indicating a link between metabolic and electrical activity; (ii) under glucose+[3-(13)C]lactate infusion, alanine C3 and acetyl-CoA C2 enrichments were higher than that of lactate C3, revealing the occurrence of an isotopic dilution of the brain exogenous lactate (arising from blood) by lactate from brain (endogenous lactate); the latter was synthesized from glycolysis in a compartment other than the neurons; (iii) the contributions of labeled glucose and lactate to acetyl-CoA C2 enrichment indicated that the involvement of blood glucose relative to that of blood lactate to brain metabolism was correlated with brain activity. It can therefore be concluded that the brain electrical activity-dependent increase in the contribution of blood glucose relative to that of blood lactate to brain metabolism occurred partly via the increase in the metabolism of lactate generated from astrocytic glycolysis. This conclusion supports the hypothesis of an astrocyte-neuron lactate shuttle component in the coupling mechanism between cerebral activity and energy metabolism.

Animals↗
PubMed2004

Brain GABA editing by localized in vivo (1)H magnetic resonance spectroscopy.

Editing of GABA by (1)H MRS in a specific brain area is a unique tool for in vivo non-invasive investigation of neurotransmission disorders. Selective GABA detection is achieved using sequences based on double quantum coherence (DQC). Our pulse sequence makes accurate measurements without artefacts due to spatial localization. The sequence was tested on a phantom solution. The effect of vigabatrin, a specific inhibitor of GABA transaminase, was measured in rat brain and GABA detection was performed in vivo in monkey brain using this procedure. Rats were split into two groups. In the control group, the rats had access to water and, in the other group (vigabatrin, VGB, rats), animals were allowed free access to drinking water containing vigabatrin. After 3 weeks of treatment, rats were anesthetized for in vivo NMR spectroscopy investigation. At the end of the experiment, brains were quickly removed, freeze-clamped and extracted with 4% perchloric acid. One part of the acid extract was used for GABA concentrations assessment by ion exchange chromatography with ninhydrin detection. The second was used for high-resolution NMR analysis. By chromatography measurements, the GABA concentration was 1.23+/-0.06 micromol/g for controls, while for vigabatrin-treated rats the GABA concentration was 4.89+/-1.60 micromol/g. The NMR in vivo results were closely correlated with the NMR ex vivo (r=0.99, p<0.01) and chromatography results (r=0.98, p<0.01). The correlation between ex vivo results and chromatography results was also high (r=0.99, p<0.001). This pulse sequence performed GABA editing from a 376 microl voxel located on the right basal ganglia area in a non-human primate brain. This in vivo GABA editing scheme can thus be proposed for accurate measurement of brain GABA concentrations.

Administration, Oral↗
PubMed2005

MRS reveals additional hexose N-acetyl resonances in the brain of a mouse model for Sandhoff disease.

Sandhoff disease, one of several related lysosomal storage disorders, results from the build up of N-acetyl-containing glycosphingolipids in the brain and is caused by mutations in the genes encoding the hexosaminidase beta-subunit. Affected individuals undergo progressive neurodegeneration in response to the glycosphingolipid storage. (1)H magnetic resonance spectra of perchloric acid extracts of Sandhoff mouse brain exhibited several resonances ca 2.07 ppm that were not present in the corresponding spectra from extracts of wild-type mouse brain. High-performance liquid chromatography and mass spectrometry of the Sandhoff extracts post-MRS identified the presence of N-acetylhexosamine-containing oligosaccharides, which are the likely cause of the additional MRS resonances. MRS of intact brain tissue with magic angle spinning also showed additional resonances at ca 2.07 ppm in the Sandhoff case. These resonances appeared to increase with disease progression and probably arise, for the most part, from the stored glycosphingolipids, which are absent in the aqueous extracts. Hence in vivo MRS may be a useful tool for detecting early-stage Sandhoff disease and response to treatment.

Animals↗
PubMed1994

Magnetic resonance spectroscopy detects metabolic differences between seven Dunning rat prostate tumor sublines with different biological behavior.

In this study, it was investigated whether prostate tumor biological parameters correlate with metabolic profiles. 1H and 31P magnetic resonance spectra were acquired from perchloric acid extracts of seven Dunning R-3327 prostate tumor sublines. Several metabolic ratios, for example, phosphocholine/total phosphate, choline/total creatine, and inositol/total creatine, did not correlate specifically with one biological characteristics but, based on each of these ratios, the well-differentiated, nonmetastatic, and hormone-dependent sublines could be discriminated from the poorly differentiated or anaplastic, metastatic, and hormone-independent sublines. The glycerophosphoethanolamine/total phosphate, glycerophosphocholine/total phosphate, and phosphocreatine/total phosphate ratios correlated with differentiation grade, and the differences in glycerophosphorylglycerol/total phosphate ratio between metastatic and nonmetastatic sublines was highly significant. No correlation for hormonal sensitivity with any of the metabolites measured could be found, neither by 31P nor by 1H MRS.

Animals↗
PubMed1975

Incorporation of 5-lododeoxyuridine into the DNA of mouse embryos: its relation to embryotoxicity.

Pregnant female ICR mice were administered, ip, either a trace (200 muCi/kg) or teratogenic (200 muCi + 300 mg/kg) dose of [6(-3)H] 5-iododeoxyuridine (IdU) on day 10 of gestation. Maternal liver, spleen, intestine, and kidneys, and placentas and embryos were removed at various time intervals after injection, weighed, and homogenized in cold 0.5 m perchloric acid. The half-lives of IdU-derived nucleotides in the acid-soluble fraction ranged from 31-46 min (trace) to 57-131 min (teratogenic) for the tissues analyzed. [3H]IdU was incorporated into the DNA of all mitotically active tissues after both dosages. The presence of the label in iodouracil was demonstrated by thin-layer chromatography of DNA bases extracted from maternal spleen and embryo. Growth of embryos following injection on day 10 resulted in decreased 3H-specific activity in the DNA fraction and concomitant retention of total activity. It is suggested that the previously demonstrated embryotoxicity of IdU is related to its retention at its presumed intracellular site of action.

Animals↗
PubMed1993

The assay of S-D-lactoylglutathione in biological systems.

A procedure for the assay of S-D-lactoylglutathione, the physiological intermediate of the glyoxalase system, in biological systems is described, together with sample storage, sample processing, and statistical evaluation. Specimen data are presented. S-D-Lactoylglutathione was assayed by reverse-phase high-performance liquid chromatography (HPLC) with spectrophotometric detection of the thiolester chromophore at 233 nm. The biological sample was deproteinized with perchloric acid and partially purified by strong anion-exchange solid-phase extraction prior to HPLC. The limit of detection was 3.7 nmol, the recovery 49 +/- 4%, and the intra- and interbatch coefficients of variance 0.7 and 12%, respectively. The concentration of S-D-lactoylglutathione in whole blood from normal control human subjects was (mean +/- SD, nmol/ml whole blood) 16.5 +/- 4.4 (n = 8), and from diabetic patients 21.2 +/- 9.2 (n = 25), which is a significant increase (P < 0.05) from normal controls. The assay of S-D-lactoylglutathione is of increasing interest in studies of the elevation of glyoxalase metabolites in diabetes mellitus and in investigations of the antiproliferative activity of S-D-lactoylglutathione.

Animals↗
PubMed1993

Methods for liquid- and solid-state CP-MAS NMR spectroscopy of untreated tissue biopsies.

We describe a method for NMR analysis of rapidly cooled or frozen biopsies and report its use on rat liver. Since the metabolic state of the biopsies can be expected to reflect the in vivo state, the method may be used as an alternative to the traditional examination of a perchloric acid extract of the biopsy. Perfusion-cooling of rat liver was applied as an efficient means of preserving the metabolic state. This method ensures very rapid cooling without interruption of the oxygen supply to the liver, and the results show that biopsies taken subsequently maintain energy metabolites near in vivo concentrations for at least 40-60 min. High resolution, natural abundance 13C liquid-state NMR spectroscopy could be carried out within this time frame on the untreated biopsy. In addition, the biopsy was frozen for 13C cross polarization-magic angle spinning solid-state spectroscopic examination, which was carried out at -40, -100, and -150 degrees C. The solid-state spectra allowed analysis of the relative glycogen content of the intact liver tissue, which showed good correlation with chemically measured glycogen on the same samples. Furthermore it was observed that the C1-carbon of glycogen in all liver samples splits into two resonances (5.4 ppm apart) in the solid state but not in the liquid state. This suggests that two conformational states of glycogen are populated, with rapid equilibration in the liquid state but no equilibration in the frozen state.

Adenosine Diphosphate↗