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PubMed2002

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.

Animals↗
PubMed1987

Mobile phospholipid signals in NMR spectra of cultured human adenocarcinoma cells.

An intense peak is visible in the phosphodiester region of the 31P NMR spectra of human colon adenocarcinoma HCT-8R cells. A signal at the same frequency, partially deriving from mobile phospholipids, is also observable in the spectra of total membranes and of perchloric acid extracts prepared from the same cells. The phosphodiester signals of glycerophosphorylcholine, glycerophosphorylethanolamine, and glycerophosphorylserine resonate at lower fields with respect to this broader peak and appear only in aged samples. Enzymatic treatments of the extracts would indicate that ribonucleic acid also contributes to the broad phosphodiester signal. Moreover, 1H NMR spectra show the presence of lipid structures, characterized by high mobility, in the cell and in the membrane samples, similar to what was already observed in other tumor cells and tissues.

Adenocarcinoma↗
PubMed1988

31P NMR spectroscopy of the in vivo metabolism of an intracerebral glioma in the rat.

The in vivo high-energy phosphorus metabolic profile and pH of an experimental intracerebral C6 glioma in rats was examined using surface coil 31P NMR spectroscopy. Initially, phosphorus-containing metabolites of the glioma were characterized by in vivo 31P surface coil spectroscopy of subcutaneously implanted tumors and by high-resolution NMR studies of perchloric acid (PCA) extracts of both freeze-clamped subcutaneous tumor tissue and cultured cells. These studies demonstrated that the C6 glioma has reduced levels of phosphocreatine (PCr) compared to the levels found in normal rat brain. Thus, reduced spectral PCr levels were useful as a metabolic indicator for monitoring the spatial selectivity of tumor metabolism distinct from that of adjacent normal brain tissue. To maximize 31P NMR signals from intracerebral tumors, tumor cells were stereotaxically placed superficially in the brain. Proton magnetic resonance imaging (1H MRI) was used to determine the size and location of the resultant brain tumors in order to preselect rats with large superficial tumors for spectroscopic study. 31P NMR spectra of the glioma tumors revealed a consistent reduction in the PCr/ATP ratio, an increase in the Pi/ATP ratio, and a slightly increased tissue pH. No correlation was found between levels of Pi/ATP and tumor pH in subcutaneous or intracerebral gliomas and the amount of necrosis as determined histologically. This study demonstrates that phosphorus metabolites of an experimental brain tumor in the rat can be monitored in vivo with minimal contributions from adjacent normal brain tissue metabolites using surface coil 31P NMR spectroscopy.

Adenosine Triphosphate↗
PubMed1989

Pathways for the synthesis of sorbitol from 13C-labeled hexoses, pentose, and glycerol in renal papillary tissue.

Suspensions of rabbit renal papillary tissue were incubated with D-[6-13C]glucose, D-[1-13C]fructose, D-[1-13C]ribose, and [2-13C]glycerol. The perchloric acid extracts of the above incubations were investigated with 13C NMR spectroscopy. All 13C-labeled substrates give rise to 13C-labeled D-sorbitol. D-[6-13C]Glucose and D-[1-13C]fructose are converted directly into D-sorbitol via the aldose reductase and sorbitol dehydrogenase pathway, respectively, whereas D-[1-13C]ribose and [2-13C]glycerol give rise to labeling of the D-glyceraldehyde pool which on its turn causes a labeling of D-sorbitol. Label exchanges observed from incubations with glycerol and D-ribose indicate that the pentose shunt plays a role in this synthesis of D-sorbitol.

Animals↗
PubMed1989

Intrauterine fetal brain NMR spectroscopy: 1H and 31P studies in rats.

Fetal brain metabolism was investigated in utero noninvasively using multinuclear nuclear magnetic resonance spectroscopy in rats at two representative prenatal stages: early (17-18 days) and late (20-21 days) stages. Phosphorus-31 (31P) spectroscopy revealed that phosphocreatine is significantly lower in the early stage and increases to the level of early neonates by the late prenatal stage. Intracellular pH at the early stage was found to be strikingly high (7.52 +/- 0.21) and decreased to a level similar to that of neonates by the late stage (7.29 +/- 0.07). Phosphomonoester levels at both stages were similar to the values reported for early neonates. Water-suppressed proton (1H) spectroscopy demonstrated a distinctive in vivo fetal brain spectral pattern characterized by low levels of N-acetyl aspartate and high levels of taurine. High-resolution proton spectroscopy and homonuclear chemical-shift correlate spectroscopy of brain perchloric acid extracts confirmed these in vivo findings. In vitro 31P spectroscopy of acidified chloroform methanol extracts showed the characteristic membrane phospholipid profiles of fetal brain. The phosphatidylethanolamine (PE)-to-phosphatidylcholine (PC) ratio (PE/PC) did not show significant changes between the two stages at 0.40 +/- 0.11, a value similar to that of early neonates.

Animals↗
PubMed1990

A 13C NMR study of the application of [U-13C]succinate for metabolic investigations in rabbit renal proximal convoluted tubular cells.

The fate of the 13C label arising from [U-13C]succinate in rabbit renal proximal convoluted tubular (PCT) cells, incubated with [U-13C]succinate, was investigated by 13C NMR spectroscopy of the perchloric acid extracts of the cell pellets. The 13C label of [U-13C]succinate was predominantly metabolized into glutamate, glutamine, and into glucose, lactate, and alanine, which is the consequence of a carbon efflux of the label from the Krebs cycle at the stage of alpha-ketoglutarate and oxaloacetate, respectively.

Animals↗
PubMed1991

1H and 31P NMR and HPLC studies of mouse L1210 leukemia cell extracts: the effect of Au(I) and Cu(I) diphosphine complexes on the cell metabolism.

The effect of the antitumor complex [Au(dppe)2]Cl (where dppe is Ph2P(CH2)2PPh2) on the overall metabolism of cultured mouse L1210 leukemia cells was investigated by comparing 1H and 31P NMR spectra of perchloric acid extracts of cells incubated for 1 h in the presence and absence of 2 microM [Au(dppe)2]Cl. There were marked (ca. two-fold) increases in the levels of lactate and almost all detectable amino acids suggesting a drug-induced increase in the rate of glycolysis and inhibition of protein synthesis. The levels of taurine and phosphorylcholine were significantly decreased and 31P NMR spectra revealed a depletion of nucleoside triphosphates (NTP). The effect on nucleotide metabolism was investigated further by separating purine and pyrimidine nucleotides and precursors by anion-exchange HPLC. NTP levels were depleted by ca. 70-90% and there was a ca. three- to four-fold increase in nucleoside di- and monophosphates. The effect is postulated to be the result of uncoupling of mitochondrial oxidative phosphorylation. The Cu(I) complex [Cu(Ph2PCH = CHPPh2)2]Cl produced a similar effect on the cellular metabolism but was more potent. The water-soluble complex [Cu(Ph2P(CH2)PEt2)2]Cl caused the accumulation of cellular amino acids at a concentration that did not significantly deplete ATP levels.

Animals↗
PubMed1991

In vivo 13CNMR spectroscopy of glucose metabolism of RIF-1 tumors.

An efficient method for measuring in vivo 13C NMR spectra of tumors has been developed and employed to monitor glucose metabolism in radiation-induced fibrosarcomas (RIF-1) subcutaneously implanted in C3H/HeN mice. [1-13C]Glucose was injected directly into the tumors at a dose of 1 g/kg body wt. Spectra were obtained with a Bruker AM 360-WB spectrometer (8.4 T/8.9 cm bore) employing a homebuilt probe equipped with a four-turn solenoidal coil (1.5 cm outer diameter) for detection of 13C signals and a Helmholtz coil (two 3-cm turns separated by a 3-cm gap, oriented orthogonally to the 13C coil) for 1H decoupling. In addition to the natural abundance 13C resonances of the tumors, signals were detected from the alpha- and beta-anomers of labeled glucose. Within 15 min following injection of labeled glucose [3-13C]lactate and [3-13C]alanine were detected. Lactate labeling approached steady state levels within about 50 min after glucose injection: in contrast, alanine labeling increased continuously over the duration of the experiment (70 min). Sixty minutes after glucose injection, the ratio of the intensity of [3-13C]lactate to the principal lipid methylene resonance (30 ppm from external tetramethylsilane), which served as an internal intensity reference, was correlated with tumor size, whereas the corresponding ratio of the [3-13C]alanine resonance was not. Labeling of glutamate was below the level of detection in the in vivo spectra; however, labeling of C4-glutamate at a level approximately 50-fold lower than the level of [3-13C]lactate was detected in perchloric acid extracts. Incorporation of 13C label into C2- and C3-glutamate and C2-lactate was also observed.

Animals↗
PubMed1991

The effects of chronic ethanol consumption on the intact rat liver studied by in vivo 31P NMR spectroscopy.

In vivo 31P nuclear magnetic resonance (NMR) spectroscopy provides unique opportunities to study the biochemistry of an organ within the intact animal in a totally non-invasive way. We have used in vivo and in vitro 31P NMR spectroscopy to study steady state changes in the major phosphorus-containing metabolites of the rat liver in control and chronically ethanol-treated rats. Chronic (4 month) ethanol treatment caused a statistically significant increase in the inorganic phosphate and phosphodiester resonances of rat liver in in vivo 31P NMR spectra relative to pair-fed control rats. Phosphomonoester and adenosine 5'-triphosphate resonances, as well as intracellular pH, were not appreciably altered. The effects of chronic ethanol treatment were particularly apparent in the response of the liver to a metabolic challenge of glycerol. Glycerol is phosphorylated almost exclusively in the liver and metabolized predominately via glycolysis and gluconeogenesis. Our in vivo 31P NMR results after administration of glycerol showed a significant increase in the phosphomonoester resonance in the liver of chronic ethanol-treated rats but not for their pair-fed controls. In vitro 31P NMR studies of perchloric acid extracts of liver showed that the increase was due to an accumulation of sn-glycerol 3-phosphate. This effect is due to the NAD(+)-dependent glycerol 3-phosphate dehydrogenase step being inhibited in the chronic ethanol-treated rats. This glycerol test may be useful in assessing the ability of the liver to rapidly regenerate NAD+ in situ and may be a more sensitive indicator of redox imbalance than steady state ratios of redox pairs (e.g., lactate/pyruvate).

Adenosine Triphosphate↗
PubMed1992

Differentiation of human prostate cancer from benign hypertrophy by in vitro 1H NMR.

In vitro 1H NMR spectra were acquired for perchloric acid extracts of tissue samples of human prostate. Seven patients were diagnosed with prostate cancer, 13 with benign prostatic hypertrophy, and 3 with both conditions. Statistically significant differences between the cancer and benign groups were seen for the metabolite peak area ratios of citrate, creatine, and phosphorylcholine to alanine, and citrate to glutamate. There was no correlation of Gleason grade with any of the ratios measured for the cancer samples. Spectra from different sections of large tumors often yielded substantially different area ratios, confirming the heterogeneous nature of these prostate tumors.

Alanine↗
PubMed1992

31P NMR studies of resting zone cartilage from growth plate.

31P NMR of superfused resting cartilage demonstrated the presence of phosphocreatine in chondrocytes. Changes in pH and in the NTP level were followed during carbon source starvation. From 31P spectra of perchloric acid extracts, phosphoethanolamine, phosphocholine, and the corresponding glycerol diesters were identified as the major phosphomonoester and phosphodiester components.

Animals↗
PubMed1992

An investigation of tumor 1H nuclear magnetic resonance spectra by the application of chemometric techniques.

1H nuclear magnetic resonance (NMR) spectra of tumors and normal tissue include signals from all hydrogen-containing metabolites and can therefore be considered multicomponent multivariate mixtures. We have obtained 1H spectra from perchloric acid extracts of three normal tissues (liver, kidney, and spleen) and five rat tumors (GH3 prolactinoma, Morris hepatomas 7777 and 9618a, LBDS1 fibrosarcoma, and Walker 256 carcinosarcoma). We have applied several different chemometric methods to analyze the data. First, we used principal component analysis, cluster analysis, and an optimized artificial neural network to develop a classification rule from a training set of samples of known origin or class. The classification rule was then assessed using a set of unknown samples. We were able to successfully determine the class of each unknown sample. Second, we used the chemometric techniques of factor analysis followed by target testing to investigate the underlying biochemical differences that are detected between the classes of samples.

Adenocarcinoma↗
PubMed1994

In vivo and in vitro 31P magnetic resonance spectroscopic studies of the hepatic response of healthy rats and rats with acute hepatic damage to fructose loading.

The hepatic response to a fructose challenge for control rats, and rats subjected to an acute sublethal dose of carbon tetrachloride (CCl4) or bromobenzene (BB), was compared using dynamic in vivo 31P MRS. Fructose loading conditions were used in which control rats showed only a modest increase in hepatic phosphomonoester (PME), and a small decrease in ATP, Pi, and intracellular pH after fructose administration. Both CCl4 and BB-treated rats showed a much greater fructose-induced accumulation of PME than did controls. Trolox C, a free radical scavenger, prevented most of this PME increase. BB-treated rats, given sufficient time to recover from the hepatotoxic insult, responded to the fructose load similarly to controls. Liver aldolase activities of control, toxicant-treated rats, and toxicant plus Trolox C-treated rats correlated inversely with PME accumulation after fructose loading (correlation coefficient: -0.834, P < 0.05). Perchloric acid extracts of rat livers studied by in vitro 31P MRS confirmed that the PME accumulation after fructose loading is mainly due to an increase in fructose 1-phosphate. These studies are consistent with the aldolase-catalyzed cleavage of fructose 1-phosphate being rate-limiting in hepatic fructose metabolism, and that the CCl4 and BB treatment modify and inactivate the aldolase enzyme.

Adenosine Triphosphate↗
PubMed1994

Quantitative proton spectroscopy of canine brain: in vivo and in vitro correlations.

Quantitative, single-voxel proton NMR spectroscopy of normal brain was performed in five adult beagle dogs using the cerebral water signal as an internal intensity reference. The same brain regions were then rapidly isolated and frozen using a pneumatic biopsy drill, perchloric acid extracted, and analyzed by biochemical assay and high-resolution NMR spectroscopy. The concentrations of the major resonances in the in vivo and in vitro spectra were compared, and good agreement was found between the different measurements. The in vivo spectra contained three peaks at 3.21, 3.04, and 2.02 ppm, which are usually assigned to trimethylamines (TMA), creatines, and N-acetyl derivatives (NAc), which corresponded to be the following metabolite concentration values: 1.7 +/- 0.6, 7.7 +/- 2.1, and 10.9 +/- 2.7 mumol/g wet weight respectively. In vitro, the following metabolite concentrations were measured: glycerophosphocholine (GPC) 1.3 +/- 0.2, phosphocholine (PC) 0.5 +/- 0.1, phosphocreatine (PCr) 2.6 +/- 0.4, creatine (Cr) 5.9 +/- 1.4, and N-Acetyl aspartate (NAA) 8.9 +/- 1.8 mumol/g wet weight. Therefore, the 3.21 ppm resonance observed in the in vivo spectrum is predominantly GPC and PC in a ratio of 2.6:1, the 3.04 ppm resonance is Cr and PCr in a ratio of 2.3:1, and the 2.02 ppm resonance is predominantly (approximately 80%) NAA with small contributions from N-acetylaspartylglutamate (NAAG) and glutamate. The data presented here validate the technique of water referencing as a simple and convenient means of quantitating single-voxel in vivo proton NMR spectra of the brain.

Animals↗
PubMed1994

31P NMR assessment of orthotopic liver rejection in a rat model.

31P NMR spectroscopy was used serially to study rejecting (ACl-->LEW) and nonrejecting (ACl-->ACl) orthotopic liver transplants in rats. Recipients were evaluated on post-transplant days 1, 3, 5, 7, 9, and 11. The relative changes in phosphomonoester (PME), inorganic phosphate (Pi), high-energy phosphates and pH were studied. The earliest significant difference between the rejecting and nonrejecting groups was a decrease in the alpha-NTP peak area on Day 5. This was followed by significant decreases in beta-NTP and pH, and increases in PME and Pi on Day 7. High-resolution 31P NMR spectra of perchloric acid extracts demonstrated the PME increase to be due mainly to elevated phosphoethanolamine. Using the parameter (Pi + PME)/(alpha + beta + gamma-NTP), rejecting livers were distinguished from nonrejecting livers at a moderate stage of rejection.

Animals↗
PubMed1996

Simultaneous extraction of cellular lipids and water-soluble metabolites: evaluation by NMR spectroscopy.

A method for simultaneous extraction of lipids and water-soluble metabolites from a single cell sample was developed and optimized for NMR spectroscopy. Intermediary metabolites in cultured M2R mouse melanoma cells and changes therein in response to challenge with melanotropin were studied by 31P and 13C NMR. Cells were extracted with methanol, chloroform, and water (1:1:1, v/v/v). The contents of the chloroform and methanol-water phases were separated and quantitatively recovered. The contents of the upper and lower phases compared well with the homologous fractions obtained by perchloric acid and Folch's lipid extraction methods. The pH of the extracts remained within the physiologic range, eliminating potential deleterious effect on cellular metabolites. The water phase contained minimal amounts of salts, making these extracts amenable to subsequent analytical procedures. Obtaining lipid- and water-soluble metabolites from the same sample enables characterization of metabolic pathways that bridge the two cellular components in a quantitative manner.

Animals↗
PubMed1996

5-Fluorouracil metabolite patterns in viable and necrotic tumor areas of murine colon carcinoma determined by 19F NMR spectroscopy.

High-resolution 19F NMR spectroscopy at 9.4 T was used to study the difference in the metabolite pattern of 5-fluorouracil (5-FU) between viable and necrotic tissues of C38 murine colon tumors grown in C57BI/6 mice. Studies were performed on perchloric acid extracts of these tumor fractions after 5-FU treatment. The 19F nuclear magnetic resonance spectra exhibited resonances representing 5-FU, the catabolites alpha-fluoro-beta-ureidopropionic acid and alpha-fluoro-beta-alanine, as well as several fluoronucleotide anabolites. The absolute concentrations of anabolites and catabolites and the anabolite-to-catabolite ratio were significantly lower in the necrotic fraction than in the viable tumor fraction 50 min after administration of 5-FU, whereas the absolute concentration of 5-FU was the same. Therefore, in 5-FU metabolism studies with NMR spectroscopy, it is important to consider the necrotic contribution to the tumor volume.

Animals↗
PubMed1997

Low visibility of lactate in excised rat muscle using double quantum proton spectroscopy.

Lactate NMR visibility was investigated in excised rat muscle at 3 T by comparing the concentration determined in situ by double quantum (DQ) proton spectroscopy (150 ms effective echo time) to the concentration measured in vitro from perchloric acid extracts of the same muscle samples. After 1-2 h of ischemia, lactate NMR visibility was 32 +/- 3% (+/- SE, n = 9), and was only 21 +/- 1% (n = 6) after 10-12 h. Muscle lactate T2 was 140 +/- 11 ms and 184 +/- 6 ms, respectively. All potential mechanisms of DQ lactate signal attenuation (B0 and B1 inhomogeneity, DQ transverse relaxation, diffusion) were examined, and accounted for when necessary. A significant increase in lactate NMR visibility was demonstrated using a shorter effective echo time (79 ms) DQ editing sequence. These results are interpreted as reflecting muscle lactate compartmentation between a long T2 pool predominantly detected by DQ spectroscopy, and a short T2 pool virtually invisible with longer echo time NMR techniques.

Animals↗