Publications

Purpose: To develop a diffusion-tensor-imaging (DTI) protocol that is sensitive to the complex diffusion and perfusion properties of the healthy and malignant pancreas tissues. Materials and Methods: Twenty-eight healthy volunteers and nine patients with pancreatic-ductal-adenocacinoma (PDAC), were scanned at 3T with T2-weighted and DTI sequences. Healthy volunteers were also scanned with multi-b diffusion-weighted-imaging (DWI), whereas a standard clinical protocol complemented the PDAC patients' scans. Image processing at pixel resolution yielded parametric maps of three directional diffusion coefficients lambda 1, lambda 2, lambda 3, apparent diffusion coefficient (ADC), and fractional anisotropy (FA), as well as a lambda 1-vector map, and a main diffusion-direction map. Results: DTI measurements of healthy pancreatic tissue at b-values 0,500 s/mm(2) yielded: lambda 1=(2.65 +/- 0.35) x 10(-3), lambda 2 = (1.87 +/- 0.22) x 10(-3), lambda 3=(1.20 +/- 0.18) x 10(-3), ADC=(1.91 +/- 0.22) x10(-3) (all in mm(2)/s units) and FA=0.38 +/- 0.06. Using b-values of 100,500 s/mm(2) led to a significant reduction in lambda 1, lambda 2, lambda 3 and ADC (p

Purpose: To investigate the parameters obtained with magnetic resonance (MR) diffusion-tensor imaging (DTI) of the breast throughout the menstrual cycle phases, during lactation, and after menopause, with and without hormone replacement therapy (HRT). Materials and Methods: All protocols were approved by the internal review board, and signed informed consent was obtained from all participants. Forty-five healthy volunteers underwent imaging by using T2-weighted and DTI MR sequences at 3 T. Premenopausal volunteers (n = 16) underwent imaging weekly, four times during one menstrual cycle. Postmenopausal volunteers (n = 19) and lactating volunteers (n = 10) underwent imaging once. The principal diffusion coefficients (lambda 1, lambda 2, and lambda 3), apparent diffusion coefficient (ADC), fractional anisotropy (FA), and maximal anisotropy (lambda 1-lambda 3) were calculated pixel by pixel for the fibroglandular tissue in the entire breast. Results: In all premenopausal volunteers, the DTI parameters exhibited high repeatability, remaining almost equal along the menstrual cycle, with a low mean within-subject coefficient of variance of lambda 1, lambda 2, lambda 3, and ADC (1%-2% for all) and FA (5%), as well as a high intraclass correlation of 0.92-0.98. The diffusion coefficients were significantly lower (a) in the group without HRT use as compared with the group with HRT use (P

The metabolic status of muscle changes according to the energetic demands of the organism. Two key regulators of these changes include exercise and insulin, with exercise eliciting catabolic expenditure within seconds and insulin enabling anabolic energy investment over minutes to hours. This study explores the potential of time-resolved hyperpolarized dynamic C-13 spectroscopy to characterize the in vivo metabolic phenotype of muscle during functional and biochemical insulin-induced stimulation of muscle. Using [C-13(1)] pyruvic acid as a tracer, we find that despite the different time scales of these forms of stimulation, increases in pyruvate label transport and consumption and concomitant increases in initial rates of the tracer metabolism to lactate were observed for both stimuli. By contrast, rates of tracer metabolism to labeled alanine increased incrementally for insulin but remained unchanged following exercise-like muscle stimulation. Kinetic analysis revealed that branching of the hyperpolarized [C-13] pyruvate tracer between lactate and alanine provides significant tissue-specific biomarkers that distinguish between anabolic and catabolic fates in vivo according to the routing of metabolites between glycolytic and amino acid pathways.

Metabolic fluxes can serve as specific biomarkers for detecting malignant transformations, tumor progression, and response to microenvironmental changes and treatment procedures. We present noninvasive hyperpolarized C-13 NMR investigations on the metabolic flux of pyruvate to lactate, in a well-controlled injection/perfusion system using T47D human breast cancer cells. Initial rates of pyruvate-to-lactate conversion were obtained by fitting the hyperpolarized C-13 and ancillary P-31 NMR data to a model, yielding both kinetic parameters and mechanistic insight into this conversion. Transport was found to be the rate-limiting process for the conversion of extracellular pyruvate to lactate with K-m = 2.14 +/- 0.03 mM, typical of the monocarboxylate transporter 1 (MCT1), and a V-max = 27.6 +/- 1.1 fmol.min(-1).cell(-1), in agreement with the high expression level of this transporter. Modulation of the environment to hypoxic conditions as well as suppression of cells' perfusion enhanced the rate of pyruvate-to-lactate conversion, presumably by up-regulation of the MCT1. Conversely, the addition of quercetin, a flavonoidal MCT1 inhibitor, markedly reduces the apparent rate of pyruvate-to-lactate conversion. These results suggest that hyperpolarized C-13(1)-pyruvate may be a useful magnetic resonance biomarker of MCT regulation and malignant transformations in breast cancer.

NMR experiments devised to aid in analyses Of tissues include magnetization transfer (MT), which can highlight the signals of biological macromolecules through cross-relaxation and/or chemical exchange processes with the bulk (1)H water resonance, and high-resolution magic-angle-spinning (HRMAS) methods, akin to those used in solid-state NMR to introduce additional spectral resolution via the averaging of spin anisotropies. This paper explores the result of combining these methodologies, and reports on MT "z-spectroscopy" between water and cell components in excised tissues under a variety of HRMAS conditions. Main features arising from the resulting (1)H "MTMAS" experiments include strong spinning sideband manifolds centered at the liquid water shift, high-resolution isotropic features coinciding with aliphatic and amide proton resonances, and a second sideband manifold arising as spinning speeds are increased. Interpretations are given for the origin of these various features, including simulations shedding further light onto the nature of MT NMR signals observed for tissue samples. Concurrently, histological examinations are reported validating the limits of HRMAS NMR procedures to the analysis of tissue samples preserved in a number of different ways. (C) 2009 Elsevier Inc. All rights reserved.

Novel estrogen-conjugated pyridine-containing Gd(III) and Eu(Ill) contrast agents (EPTA-Gd/Eu) were designed and effectively synthesized. Convenient to administration and MRI experiments, both EPTA-Gd and EPTA-Eu are soluble in water. The EPTA-Gd selectively binds with a micromolar affinity to the estrogen receptor and induces proliferation of human breast cancer cells. The EPTA-Gd is not lethal and does not cause any adverse effects when administrated intravenously. It enhances T-1 and T-2 nuclear relaxation rates of water and serves as a selective contrast agent for localizing the estrogen receptor by MRI.

Metastatic spread to regional lymph nodes is one of the earliest events of tumor cell dissemination and presents a most significant prognostic factor for predicting survival of cancer patients. Real-time in vivo imaging of the spread of tumor cells through the lymphatic system can enhance our understanding of the metastatic process. Herein, we describe the use of in vivo fluorescence microscopy imaging to monitor the progression of lymph node metastasis as well as the course of spontaneous metastasis through the lymphatic system of orthotopic MDA-MB-231 human breast cancer tumors in severe combined immunodeficient mice. High-resolution noninvasive visualization of metastasizing cancer cells in the inguinal lymph nodes was achieved using cells expressing high levels of red fluorescent protein. Sequential imaging of these lymph nodes revealed the initial invasion of the tumor cells through the lymphatic system into the subcapsular sinuses followed by intrusion into the parenchyma of the nodes. FITC-dextran injected i.d. in the tumor area enabled simultaneous tracking of lymphatic vessels, labeled in green, and disseminated red cancer cells within these vessels. Fast snapshots of spontaneously metastasizing cells in the lymphatic vessels monitored the movement of a few tumor cells and the development of clumps clustered at lymphatic vessel junctions. Quantification of high interstitial fluid pressure (IFP) in the tumors and fast drainage rates of the FITC-dextran into the peritumoral lymphatic vessels suggested an IFP-induced intravasation into the lymphatic system. This work presents unprecedented live fluorescence images that may help to clarify the steps occurring in the course of spontaneous lymphogenic metastasis.

We report on a short host defense-like peptide that targets and arrests the growth of aggressive and hormone-resistant primary human prostate and breast tumors and prevents their experimental and spontaneous metastases, respectively, when systemically inoculated to immuodeficient mice. These effects are correlated with increased necrosis of the tumor cells and a significant decrease in the overall tumor microvessel density, as well as newly formed capillary tubes and prostate-specific antigen secretion (in prostate tumors). Growth inhibition of orthotopic tumors derived from stably transfected highly fluorescent human breast cancer cells and prevention of their naturally occurring metastases were visualized in real time by using noninvasive whole-body optical imaging. The exclusive selectivity of the peptide towards cancer derives from its specific binding to surface phosphatidylserine and the killing of the cancer cells via cytoplasmic membrane depolarization. These data indicate that membrane disruption can provide a therapeutic means of inhibiting tumor growth and preventing metastases of various cancers.

The clinical detection of evolving acute tubular necrosis ( ATN) and differentiating it from other causes of renal failure are currently limited. The maintenance of the corticomedullary sodium gradient, an indicator of normal kidney function, is presumably lost early in the course of ATN. Herein, sodium magnetic resonance imaging ( Na-23 MRI) was applied to study the early alteration in renal sodium distribution in rat kidneys 6 h after the induction of ATN. Three-dimensional gradient echo sodium images were recorded at 4.7 T with high spatial resolution. ATN was produced by the administration of radiologic contrast medium, combined with inhibition of nitric oxide and prostaglandin synthesis. The sodium images revealed that the sham-controlled kidney exhibited a linear increase in sodium concentration along the corticomedullary axis of 30 +/- 2 mmol/l/mm, resulting in an inner medulla to cortex sodium ratio of 4.3 +/- 0.3 ( n = 5). In the ATN kidney, however, the cortico-outer medullary sodium gradient was reduced by 21% ( P

The cholinergic system is an important modulatory neurotransmitter system in the brain. Changes in acetylcholine concentration have been previously determined directly in animal models and human brain biopsy specimens, and indirectly, by the effects of drugs, in living humans. Here, we developed a method for direct determination of acetylcholine synthesis in living brain tissue. The method is based on administration of choline, enriched with carbon-13 (stable isotope) in the two methylene positions, and detection of labeled acetylcholine and all other metabolic fates of choline, by carbon-13 magnetic resonance spectroscopy. We tested this method in rat brain slices and found it to be specific for acetylcholine synthesis in both the cortex and hippocampus. This method is potentially useful as a research tool for exploring the cholinergic system role in cognitive processes and memory storage as well as in diseases in which the malfunction of the cholinergic system has been implicated. (c) 2005 Elsevier B.V. All rights reserved.

The parameters that characterize the intricate water diffusion in tumors may serve to reveal their distinct pathology. Specifically, the application of diffusion magnetic resonance imaging (MRI) can aid in characterizing breast cancer, as well as monitoring response to therapy. We present here a non-invasive, quantitative MRI investigation, at high spatial resolution, of water diffusion in hormonal dependent MCF7 breast tumors implanted orthotopically in immunodeficient mice. Distinctive MRI protocols were designed in this study, utilizing a broad range of diffusion times and diffusion gradient strengths. Application of these protocols allowed water diffusion in the tissue extracellular and intracellular compartments to be distinguished, and the effect of restricted diffusion and water exchange on the water diffusion in these compartments to be evaluated. Pixel-by-pixel analysis yielded parametric maps of the estimated volume fraction and apparent diffusion coefficient of each compartment. The diffusion of the water in the extracellular microenvironment was approximately two fold slower than that of free water, and in the intracellular compartment was about one order of magnitude slower than that of free water and demonstrated restriction of water diffusion at long diffusion times. Mapping of the water fraction in each compartment was further employed to monitor changes during tumor progression and to assess tumor response to hormonal manipulation with a new antiestrogenic drug, tamoxifen methiodide (TMI). It was found that, in parallel to the growth arrest by this drug, the volume fraction of the slowly diffusing water increased, suggesting a TMI-induced cell swelling. This study can serve as a basis for extending diffusion breast MRI in the clinical setting. Copyright (C) 2004 John Wiley Sons, Ltd.

Angiogenic activity and formation of a vascular network facilitate tumor perfusion and play a critical role in tumor growth and metastasis. Tumor vasculature may be visualized by means of parametric imaging of specific morphological and physiological characteristics that collectively describe its properties. In this review, we describe advanced magnetic resonance imaging (MRI) techniques that have been developed in order to image and quantify the distribution of tumor vasculature throughout the tumor and characterize its function. These techniques-have been used to monitor changes in the magnetic resonance signal intensity of tissue water hydrogens generated by intrinsic effects, as well as by exogenous contrast agents administered into the blood circulation. We further describe specific applications of magnetic resonance imaging using a contrast agent, gadolinium diethylene triamine penta-acetic acid (GdDTPA), which has long been approved for clinical use. Examples include studies of the vascular properties of breast cancer tumors and metastases in animal models, as well as of breast cancer vasculature in patients. We also discuss the use of MRI to improve breast cancer diagnosis in humans by quantifying the permeability of the tumor vasculature. By m aximizing, the spatial resolution of the images in both animal and human studies, the capacity of magnetic resonance imaging to enhance our understanding of the processes regulating tumor angiogenesis, and improve the diagnosis of cancer, could be clearly demonstrated.

Proton magnetic resonance spectroscopy has been increasingly utilized in brain research to monitor non-invasively metabolites such as N-acetyl aspartate (NAA), creatine (Cr) and choline (Cho). We present here studies of the effect of aging on the ratios of these metabolites measured in the rat brain in vivo and on choline transport and lipid synthesis in rat brain slices, in vitro. The in vivo studies indicated that the ratios of Cho/NAA and Cho/Cr increased in the aged hippocampus, whereas the ratio of Cr/NAA was similar in the aged and adult hippocampus. These three ratios remained similar in the cortex of adult and aged rats. The in vitro studies revealed that in the aged cortex and the aged hippocampus the activity of the low-affinity choline uptake increased, possibly compensating for a decrease in the high-affinity uptake activity and the rate of choline diffusion. The incorporation of choline into phospholipids exhibited high and low affinity kinetics which were not modified by aging. (C) 2002 Elsevier Science B.V. All rights reserved.

The development and growth of solid tumors rely on a process known as perfusion which allows for the delivery and clearance of nutrients through their vasculature. The classical approach of studying tumor vasculature by histologic staining of endothelial cells provides a measure of microvessel density in areas of high vascularization (so-called "hot spots"). More advanced high-resolution, parametric, contrast-enhanced MRI techniques enable quantitative assessment of the vascular distribution and function over the entire tumor. Hence, MRI overcomes the intratumoral variation of the histological method. Clinical testing of the MRI approach, that evaluates the permeability and surface area of the tumor vasculature, demonstrates improved accuracy of breast cancer diagnosis.

The present investigation was performed to elucidate the role of purine nucleotides as potential indicators of chemosensitivity of malignant tumors. Drug-sensitive (s) and -resistant (r) tumor cell lines grown as monolayers (s: T47D, MCF-7 wild-type; r: NCI/ADR-RES, MCF-7/MDR) or as multicellular spheroids (T47D; NCI/ADR-RES) were exposed to 0.1, 1.0, and 10.0 muM Doxorubicin for up to 24 h. Purine nucleotides were assayed using HPLC and with some selected spheroids using imaging bioluminescence. The data show that in the time frame of the experiments reproducible and statistically significant changes in the nucleotides only occur at the highest drug concentration investigated. Under these conditions and using monolayer cultures, Doxorubicin caused a significant increase in ATP and GTP in sensitive but not in resistant cancer cells. Consequently, this differential change may be exploited for drug sensitivity testing in vitro. Doxorubicin exposure to spheroids was associated with significant increases in ATP and GTP in both sensitive and resistant variants. However, the kinetic of the changes in GTP was largely different between T47D and NCI/ADR-RES spheroids with a long-lasting, almost 3-fold elevation and a smaller, relatively short transient increase in GTP, respectively. Supplementing experiments with Doxorubicin treatment under inhibition of oxidative phosphorylation with Oligomycin abolished the drug-induced ATP and GTP peaks at persistent increases in ADP and AMP. Assuming that the spheroids may represent the in vivo situation to a better degree than monolayer cultures, experimental in vivo studies should clarify whether kinetic changes in GTP could be used as differential markers for the chemosensitivity of solid tumors. The experiments using Oligomycin support the hypothesis that purine nucleotides may be recycled from DNA fragments that result from the interaction of the drug with the DNA strands.

Parametric analysis of breast MRI provides unique mapping of pathophysiological characteristics that cannot be obtained by standard conventional MRI. We describe in this review methods based on intrinsic contrast and tissue elasticity as well as methods that use external paramagnetic contrast agents and follow the time evolution of contrast. Processing of the raw data, frequently kith new mathematical models and algorithms, yielded calculated parametric images that may help improve the non-invasive detection and diagnosis of breast cancer.

The gene for the liver-type subunit of phosphofructokinase (PFKL) resides on chromosome 21 and is overexpressed in Down syndrome (DS) patients. Transgenic PFKT, (Tg-PFKL) mice with elevated levels of PFKL were used to determine whether, as in DS, overexpression of PFKT, was also associated with altered sugar metabolism. We found that Tg-PFKL mice had an abnormal glucose metabolism with reduced clearance rate from blood and enhanced metabolic rate in brain. Transgenic-PFKL mice exhibited elevated activity of phosphofructokinase in both blood and brain, as compared to control non-transgenic (ntg) mice. Following glucose infusion, the rate of glucose clearance from the blood of Tg-PFKL mice was significantly slower than that of control ntg mice, although the basal blood glucose levels were similar. However, unlike the slower rate of glucose metabolism in blood, the initial rate of glucose utilization in the brain of the transgenic mice, was 58% faster than in control ntg mice. This was determined by infusion of [1-(13)C]-glucose followed by in vivo nuclear magnetic resonance (NMR) measurements of brain glucose metabolism. The faster utilization of glucose in Tg-PFKL brain is similar to the increased rate of cerebral glucose metabolism found in the brain of young adult DS patients, which may play a role in the etiology of their cognitive disabilities. (C) 1998 Elsevier Science B.V. All rights reserved.

Variations in the cellular volume fraction and in the microvascular permeability of MCF7 human breast tumors were used to assess response to tamoxifen. These pathophysiological features were mapped by applying the three-time-point, contrast-enhanced, high resolution magnetic resonance imaging method (H, Degani et at, Nat, Med,, 3: 780-782, 1997), Short-term treatment with tamoxifen caused a highly significant increase in the fraction of pixels displaying intermediate contrast agent clearance pattern and a significant increase in the fraction of pixels displaying high rate of contrast agent entrance. These changes resulted from a marked rise in the extracellular volume fraction, indicating increased necrosis, and from an augmentation in the microvascular permeability, predominantly in the vicinity of the high extracellular volume fraction areas, as a result of stress-induced angiogenesis.

Tumor necrosis factor alpha (TNF) is a cytokine that is cytocidal for certain tumor cells and induces necrotic and apoptotic forms of cell death. Flow cytometry and transmission electron microscopy analysis demonstrated that in human breast cancer cells (MCF7) TNF induces cell cycle arrest in G(0)+G(1)/S, accompanied by apoptosis. P-31 and C-13 NMR spectroscopy was applied to study cellular metabolism of MCF7 cells during TNF-induced signal to apoptosis. Deuterated choline and H-2 NMR spectroscopy were utilized to monitor the kinetics of the rate limiting reactions in phosphocholine metabolism. The NMR measurements revealed that immediately after administration of TNF, choline transport was inhibited by 52 +/- 6%. Later (similar to 15 h), the activity of phosphocholine :cytidine triphosphate cytidylyltransferase, a key enzyme in the biosynthesis of phosphatidylcholine, was enhanced two-fold. These two opposing changes led to a decrease in the level of phosphocholine. Throughout these changes the energetic state of the cells, determined by the level of nucleoside triphosphates and the rate of glucose metabolism via glycolysis, remained constant. The results indicate that TNF specifically modulates the kinetics of membrane-bound enzymes of the rate determining steps in phosphatidylcholine biosynthesis, possibly as part of early events involved in apoptosis. (C) 1998 Elsevier Science B.V.

A modified Prony method (MPM) was applied to analyze the main signals present in spatially resolved P-31 NMR spectra of MCF7 breast tumors implanted in nude mice, First, the method was tested on synthetic data to establish its limits of reliability, Its performance with respect to peak identification and quantification of signal intensities was then exploited on data from three implanted tumors during hormonal manipulation with estrogen and the antiestrogenic drug tamoxifen, The phosphomonoester peak was resolved into phosphocholine (PC) and phosphoethanolamine (PE), Treatment with tamoxifen led to a significant reduction in the PE to PE+PC peak amplitude ratio in the tumors under consideration, MPM analysis also revealed the presence of two different inorganic phosphate pools: a larger acidic pool and a smaller alkaline pool during estrogen-induced growth and the reverse during tumor regression.

Magnetic resonance imaging (MRI) is a noninvasive method that reveals anatomical details in vivo and detects lesions for diagnosis. Although standard breast MRI cannot clearly delineate breast cancer, contrast-enhanced MRI enables the detection of breast masses with high sensitivity(1,2). Dynamic studies demonstrated that malignant lesions were characterized by a faster signal enhancement rate than benign ones(3). Dynamic MRI of human breast cancer in mice revealed high heterogeneity in the distribution of contrast-enhanced curves and derived pathophysiological features, indicating the importance of high spatial resolution(4,5). With clinical MRI, it is difficult to achieve simultaneously high spatial and temporal resolution. In previous dynamic studies, the emphasis was on high temporal resolution and mainly empiric analyses(6-12). We describe here a new model-based method that optimizes spatial resolution by using only three time points, and yet characterizes tumor heterogeneity in terms of microvascular permeability and extracellular fraction. Mapping these pathophysiological features may aid diagnosis and prognosis assessment, while the high spatial resolution may improve the capacity to detect smaller lesions. The method was tested in human breast tumors implanted in mice and in a limited number of benign and malignant breast lesions of patients.

The mechanism of contrast enhancement of tumors using magnetic resonance imaging was investigated in MCF7 human breast cancer implanted in nude mice. Dynamic contrast-enhanced images recorded at high spatial resolution were analyzed by an Image analysis method based on a physiological model, which included the blood circulation, the tumor, the remaining tissues, and clearance via the kidneys. This analysis enabled us to map in rapidly enhancing regions within the tumor, the capillary permeability factor (capillary permeability times surface area per voxel volume) and the fraction of leakage space correlation of these maps with T-2-weighted spin echo Images, with histopathology, and with immunohistochemical staining of endothelial cells demonstrated the presence of dense permeable microcapillaries in the tumor periphery and in intratumoral regions that sur rounded necrotic loci, The high leakage from the intratumoral permeable capillaries indicated an Induction of a specific angiogenic process associated with stress conditions that cause necrosis, This induction was augmented in tumors responding to tamoxifen treatment, Determination of the distribution and extent of this stress-induced angiogenic activity by contrast-enhanced MRI might be of diagnostic and of prognostic value.

The mechanism and kinetics of choline transport and phosphorylation in MCF7 human breast cancer cells was studied by P-37, C-13 and H-2 NMR, applying the zero trans method. Choline was transported by a Michaelis-Menten like mechanism with a maximum transport rate T-max = 13.5 +/- 2.6 nmol/hour/mg protein (3.06 +/- 0.6 fmol/cell/hour) and choline concentration at half maximal transport rate of K-t = 46.5 +/- 2.8 mu M. The rate of choline phosphorylation was more than two orders of magnitude faster than the rate of its transport (T-max) maintaining the ratio [phosphocholine]/[choline] higher than 100. The results demonstrated enhanced choline transport and choline kinase activity in breast cancer cells.

Prony's method, successfully used in processing NMR signals, performs poorly at low signal-to-noise ratios. To overcome this problem, a statistical approach has been adopted by using Prony's method as a sampling device from the distribution associated with the true spectrum. Specifically, Prony's method is applied for each regression order p and number of data points n, both considered in a suitable range, and the estimates of frequencies, amplitudes, and decay factors are pooled separately. A histogram of the pooled frequencies is computed and, looking at the histogram, a lower and an upper frequency bound for each line of interest is determined. All frequency estimates in each of the determined intervals as well as associated decay factors and amplitudes are considered to be independent normal variates. A mean value and a corresponding 95% confidence interval are computed for each parameter. P-31 NMR signals from MCF7 human breast cancer cells, inoculated into athymic mice and which developed into tumors, have been processed with traditional methods and with this modified Prony's method. The main components of the phosphomonoester peak, namely those deriving from phosphorylcholine and phosphorylethanolamine, are always well resolved with this new approach and their relative amplitudes can be consequently evaluated. Peak intensities of these two signals show different behavior during treatment of tumors with the antiestrogenic drug tamoxifen. The results of this new approach are compared with those obtainable with traditional techniques. (C) 1994 Academic Press,Inc.

Alterations in the energy state and glucose metabolism of hippocampal slices exposed to high extracellular K+ ([K+]o) were monitored using P-31 and C-13 NMR spectroscopy. Slices were perfused (37-degrees-C) continuously within the NMR spectrometer and tissue viability and metabolic activity were maintained for at least 18 h. P-31 spectra showed that upon exposure to 40 mM [K+]o, there was a rapid compromise in tissue energetics where, by 15 min of exposure, the ratio of phosphocreatine and of nucleoside triphosphates to inorganic phosphate (extra- and intracellular) decreased 30-50% relative to pre-exposure values. This was accompanied by a pH decrease of almost-equal-to 0.3 units in both the intra and extracellular environments. A lower but stable energy state was reached at almost-equal-to 15 min of exposure and full recovery was observed by 30 min following the removal of high [K+]o. Utilizing C-13 NMR in the presence of [1-C-13]glucose, an immediate and dramatic acceleration in tissue glycolysis was observed when slices were exposed to 40 mM [K+]o: the rates of both [1-C-13]glucose consumption and [3-C-13]lactate synthesis increased by almost-equal-to 20 fold. By 60 min following the removal of high-[K+]o, pre-exposure rates of tissue glycolysis were restored. The results indicated that the rapid and dramatic induction of energy production via glycolysis probably accounts for the ability of hippocampal slices to maintain viability and recuperate from brief but intense depolarizing conditions which are reminiscent of seizure states in vivo.

Keywords: Endocrinology & Metabolism; Obstetrics & Gynecology

The effects of 17-beta-estradiol versus tamoxifen on the growth and metabolism of MCF7 human breast cancer cells, in culture and in tumors implanted in nude mice, were studied by P-31 and C-13 nuclear magnetic resonance spectroscopy and by proton magnetic resonance imaging. In culture, the content of the phosphate metabolites including nucleoside triphosphates (NTP), phosphomonoesters, phosphodiesters and inorganic phosphate (Pi) were not affected by tamoxifen treatment. However, in the presence of estrogen the rate of glucose consumption and lactate production via glycolysis (270 and 280 fmol/cell . h, respectively) were twice that of tamoxifen treated cells. Estrogen rescue of tamoxifen treated cells indicated that glycolysis induction occurs at the early stages of the hormonal response. The in vivo studies included recording of proton images that provided an accurate measure of tumor size and distribution of tumor cells, necrotic regions and stromal tissue. Tamoxifen caused enhanced necrosis extending from the center of the tumor during the first two days of treatment (12 h to 6 days). This was followed by growth of reparative tissue along with tumor regression. Tamoxifen also modified the content of the phosphate metabolites, increasing markedly (P

P-31- and C-13-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as large (300-mu-m) spheroids. Spheroids were perfused inside the spectrometer with 1,2-C-13-labeled choline or ethanolamine (0.028 mM) and the buildup of labeled phosphorylcholine (PC) or phosphorylethanolamine (PE) was monitored. To analyze the NMR kinetic data, it was assumed that each signal represents a weighted average of signal from the proliferating and non-proliferating compartments of the large spheroid. The average ATP pool size was 4 +/- 1 fmol/cell compared to 8 +/- 1 fmol/cell in small (150-mu-m) proliferating spheroids (P

Phosphorous 31 (P-31) nuclear magnetic resonance (NMR) spectra were recorded from perchloric acid extracts of benign and malignant breast tumors. The spectra were correlated with the histopathologic diagnosis and the steroid receptor status of the tumor. Higher relative content of the lipid-derived metabolite glycerolphosphoethanolamine (GPE), the high-energy nucleoside phosphates (nucleoside-diphosphate [NDP], nucleoside-triphosphate [NTP]) and sugar esters of uridine diphosphate (UDPS) appeared in the carcinomas. Malignant tumors also showed a lower ratio of phosphoethanolamine to phosphocholine (PE/PC) than benign conditions. Lower content of the lipid-derived metabolite glycerolphosphocholine (GPC) and high content of the high-energy compound phosphocreatine (PCr) were associated with malignant tumors having high content of estrogen receptors (ER). High PCr content was also associated in the carcinomas with high progesterone receptors (PgR) content. In the benign tumors NDP and NTP were higher in tumors with high PgR content. The authors suggest that P-31 magnetic resonance spectroscopy (MRS) of the breast can provide additional variables to diagnose malignancy, and when combined with magnetic resonance imaging (MRI), invasive procedures may be avoided. It also seems that levels of PCr and GPC obtained from the spectra can serve as markers to hormonal receptor status of breast carcinomas, and may be used in addition to the ER and PgR content to improve prediction of the response to hormonal therapy. Additional development requires in situ MRI and MRS combined studies.

Polyphosphate synthesis and the state of the intracellular polyphosphates in the unicellular green alga Dunaliella salina were studied using in vivo P-31-NMR spectroscopy. By perfusing phosphate-depleted algal cells trapped inside agarose beads with orthophosphate (P(i)) containing medium, we were able to follow the process of polyphosphate synthesis in whole, living cells. The results suggest that, in Dunaliella, low molecular weight, probably cyclic, polyphosphate intermediates are synthesized from P(i), and are then condensed to high molecular weight polymers. Studies of the intracellular organization of the polyphosphates by electron microscopy and solid-state NMR techniques indicate that most of these polymers are stored in the cell in a soluble form, and not in solid-like structures.

The effects of 17 beta-estradiol treatment versus tamoxifen on the metabolism of human breast cancer T47D-clone 11 cells were studied by noninvasive 31P and 13C nuclear magnetic resonance techniques. 31P nuclear magnetic resonance spectra revealed differences between estrogen and tamoxifen treated cells. The steady state content of phosphorylcholine and of the nucleoside diphosphates was higher in the tamoxifen treated cells by 33 and 140%, respectively, relative to estrogen treated cells. The intracellular pH of 7.2 and the content of the nucleoside triphosphates, Pi, phosphocreatine, glycerolphosphorylcholine, and glycerolphosphorylethanolamine and uridine diphosphoglucose remained the same in both treatments. Glucose utilization and subsequent lactate, glutamate, alanine, and glycerol 3-phosphate synthesis were monitored on line following administration of specifically labeled [13C]glucose. In estrogen treated cells the rate of lactate production via glycolysis was 560 fmol/cell/h and the initial rate of 13C labeling of the glutamate pool via the Krebs cycle was 6.8 fmol/cell/h. In the tamoxifen treated cells these rates were 2-fold lower, at 250 and 2.9 fmol/cell/h for lactate and glutamate labeling, respectively. In estrogen treated cells, the calculated content of glutamate (19 fmol/cell), alanine (11 fmol/cell), and glycerol 3-phosphate (8 fmol/cell) was higher than in tamoxifen treated cells, where only glutamate labeling was detected (13 fmol/cell). The observed differences in the in vivo kinetics of glucose metabolism may provide a sensitive measure for detecting the response of human breast cancer cells to estrogen versus tamoxifen treatments.