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Although several drugs are available to treat recurrences of human epithelial ovarian cancer (EOC), clinical responses often remain short lived and lead to only marginal improvements in patients' survival. One of the new drugs proposed for recurrent platinum-resistant EOC patients is trabectedin (Trab), a marine-derived antitumor agent initially isolated from the tunicate Ecteinascidia turbinata and currently produced synthetically. Predictive biomarkers of therapy response to this drug and the potential use of non-invasive functional MRI and MRS approaches for an early assessment of Trab efficacy have not yet been evaluated, although they might be relevant for improving the clinical management of EOC patients. In the present work we combined functional and spectroscopic magnetic resonance technologies, such as in vivo diffusion-weighted MRI and 1 H MRS, with ex vivo high resolution MRS (HR-MRS) metabolomic analyses, with the aim of identifying new pharmacodynamic markers of Trab effectiveness on well characterized, highly aggressive human SKOV3.ip (a HER2-enriched cell variant derived from SKOV3 cells) EOC xenografts. In vivo treatment with Trab (three consecutive weekly 0.2 mg/kg i.v. injections) resulted in the following: (1) a significant reduction of in vivo tumor growth, along with the formation in cancer lesions of diffuse hyper-intense areas detected by T 2 -weighted MRI and attributed to necrosis, in agreement with histopathology findings; (2) significant increases in the apparent diffusion coefficient mean and median values versus saline-treated control tumors; and (3) a significant reduction in the choline-containing metabolites' signal detected by quantitative in vivo MRS. Multivariate and quantitative HR-MRS analyses on ex vivo tissue samples revealed Trab-induced alterations in phospholipid and glucose metabolism identified as a decrease in phosphocholine and an increase in lactate. Collectively, these data identify Trab-induced functional MRI and MRS alterations in EOC models as a possible basis for further developments of these non-invasive imaging methods to improve the clinical management of EOC patients., (© 2018 John Wiley & Sons, Ltd.)

Background: Limited knowledge is available on alterations induced by cytostatic drugs on magnetic resonance spectroscopy (MRS) and imaging (MRI) parameters of human cancers, in absence of apoptosis or cytotoxicity. We here investigated the effects of a cytostatic cisplatin (CDDP) treatment on (1)H MRS and MRI of HER2-overexpressing epithelial ovarian cancer (EOC) cells and in vivo xenografts., Methods: High-resolution MRS analyses were performed on in vivo passaged SKOV3.ip cells and cell/tissue extracts (16.4 or 9.4 T). In vivo MRI/MRS quantitative analyses (4.7 T) were conducted on xenografts obtained by subcutaneous implantation of SKOV3.ip cells in SCID mice. The apparent diffusion coefficient (ADC) and metabolite levels were measured., Results: CDDP-induced cytostatic effects were associated with a metabolic shift of cancer cells towards accumulation of MRS-detected neutral lipids, whereas the total choline profile failed to be perturbed in both cultured cells and xenografts. In vivo MRI examinations showed delayed tumour growth in the CDDP-treated group, associated with early reduction of the ADC mean value., Conclusion: This study provides an integrated set of information on cancer metabolism and physiology for monitoring the response of an EOC model to a cytostatic chemotherapy, as a basis for improving the interpretation of non-invasive MR examinations of EOC patients.

Magnetic resonance imaging (MRI) and spectroscopy (MRS) offer powerful approaches for detecting physiological and metabolic alterations in malignancies and help investigate underlying molecular mechanisms. Research on epithelial ovarian carcinoma (EOC), the gynaecological malignancy with the highest death rate characterised by frequent relapse and onset of drug resistance, could benefit from application of these molecular imaging approaches. In this study, MRI/MRS were used to characterise solid tumour models obtained by subcutaneous (s.c.) or intraperitoneal (i.p.) implantation of human SKOV3.ip cells in severe combined immunodeficiency (SCID) mice. In vivo MRI/MRS, ex vivo magic-angle-spinning (MAS), and in vitro (1)H-NMR measurements were carried out at 4.7 T, 9.4 T, and 9.4/16.5 T, respectively. MRI evaluation was performed by T1-, T2-, and diffusion-weighted (DW) multislice spin-echo imaging. The in vivo (1)H spectra of all tumour models showed a prominent resonance of total choline-containing metabolites (tCho). Quantitative in vivo MRS of both i.p. and s.c. SKOV3.ip xenografts showed that the mean tCho content was in the 2.9-4.5 mM range, with a mean PCho/tCho ratio of 0.99 ± 0.01 [23 examinations, 14-34 days post injection (dpi)], in good agreement with ex vivo and in vitro analyses. Myo-inositol ranged between 11.7 and 17.0 mM, with a trend towards higher values in i.p. xenografts at 14-16 dpi. The average apparent diffusion coefficient (ADC) values of SKOV3.ip xenografts [1.64 ± 0.11 (n = 9, i.p.) and 1.58 ± 0.03 x10(-3) mm(2)/s (n = 7, s.c.)] were in agreement with values reported for tumours from patients with EOC, while the mean vascular signal fraction (VSF) was lower (≤ 4%), probably due to the more rapid growth of preclinical models. Both s.c. and i.p. xenografts are valuable preclinical models for monitoring biochemical and physiopathological changes associated with in vivo EOC tumour growth and response to therapy, which may serve as the basis for further clinical development of noninvasive MR approaches., (Copyright © 2011 John Wiley & Sons, Ltd.)

Objectives: To assess the clinical feasibility of 3-dimensional (3D) proton magnetic resonance spectroscopy (MRS) of ovarian masses at 1.5 T., Materials and Methods: We prospectively evaluated 16 patients with 23 ovarian masses using contrast-enhanced magnetic resonance imaging and 3D chemical shift imaging MRS (time of reception/time of echo = 700/135 ms, number of excitations = 6, interpolated voxel = 5 × 5 × 5 mm(3), water and fat suppression). Spectral editing consisted of water reference, filtering, zero-filling, Fourier transformation, frequency shift, automatic baseline and phase correction, and curve fitting. The volume of interest was placed to encompass both solid and cystic tumor components as well as apparently healthy pelvic tissues. The presence of a choline peak at 3.14 to 3.34 ppm was considered as a marker of malignancy. All patients underwent surgery and histopathological evaluation., Results: Of 23 masses, 19 were malignant and the remaining 4 benign lesions were a fibrothecoma, an endometriosis, a cyst, and a cystadenofibroma. A choline peak was detected in 17/19 malignant tumors (sensitivity 89%), absent in 2 G1 tumors. It was visible in 16 solid components of 19 malignant tumors (in one of them, a choline peak was detected only in the cystic component, in 6 in both solid and cystic components). The choline peak was absent in 20/21 apparently healthy pelvic tissues, with a very low choline peak being detected in one intraperitoneal fluid collection with malignant cells at cytologic analysis; 3/4 benign tumors showed a choline peak (overall specificity 21/25 = 84%). A significant difference between the mean choline peak integral detected within the solid component and that within the cystic component was observed (P = 0.002). No correlation between the choline peak integral and the tumor size was found (r = 0.120, P = 0.615)., Conclusions: 3D MRS of ovarian masses is clinically feasible at 1.5 T. This opens new research strategies for early diagnosis of ovarian cancer.

Objective: The purpose of our study was to use the total choline-containing compound (tCho) peak integral as a marker of malignancy in breast MR spectroscopy (MRS)., Subjects and Methods: Forty-eight single-voxel water- and fat-suppressed 1.5-T MRS measurements were performed in 42 patients, obtaining both absolute tCho peak integral and tCho peak integral normalized for the volume of interest (VOI). Our reference standard was histology for lesions with BI-RADS category 4 and 5 and histology or at least a 2-year follow-up for findings with BI-RADS 2 and 3 and normal glands. Receiver operating characteristic (ROC) analysis, Mann-Whitney U test, and Spearman's rank correlation were used., Results: Three of 48 measurements (6%) failed. Of the remaining 45 spectra, 18 nonmalignant tissues showed no tCho peak, eight nonmalignant tissues showed a tCho peak integral from 0.99 to 9.03 arbitrary units (AU), and 19 malignant lesions showed a tCho peak integral from 1.26 to 19.80 AU. The diameter of nonmalignant tissues was 16.9 +/- 7.4 mm; that of malignant lesions was 15.3 +/- 6.9 mm (p = 0.308). At ROC analysis, the optimal threshold was 1.90 AU for absolute tCho peak, with 0.895 (17/19) sensitivity, 0.923 (24/26) specificity, and an AUC (area under the curve) of 0.917 (95% CI, 0.822-1.000); the optimal threshold was 0.85 AU/mL for the normalized tCho peak integral with 0.842 (16/19) sensitivity, 0.885 (23/26) specificity, and an AUC of 0.941 (0.879-1.000) (p = 0.470). A negative correlation (p = 0.011) was found between the VOI and the normalized tCho peak integral of malignant tissues., Conclusion: Breast MRS using tCho peak integral reaches a high level of diagnostic performance.

This literature review assesses the clinical potential of proton ((1)H) magnetic resonance spectroscopy (MRS) of breast lesions. We here illustrate the basic principles of spectrum acquisition for volumes of interest, determined on the basis of dynamic magnetic resonance imaging (MRI) and of MRS postprocessing. We discuss the criteria for interpreting the spectrum with particular reference to the metabolic significance of the peak of total choline containing compounds at 3.2 ppm, a marker that is correlated with malignancy. We then summarise the findings obtained in lesion characterisation (with a possible gain in specificity with respect to dynamic MRI), the assessment of the effects of neoadjuvant chemotherapy and the correlation reported at high-field between the tumour tissue concentration of choline-containing compounds and the presence of lymph node metastases. Lastly, we outline the clinical use of this technique as the final phase of a complete breast MR examination after intravenous administration of paramagnetic contrast material for the dynamic study, with reference to its use by radiologists dedicated to breast imaging.

Ovarian carcinomas represent a major form of gynaecological malignancies, whose treatment consists mainly of surgery and chemotherapy. Besides the difficulty of prognosis, therapy of ovarian carcinomas has reached scarce improvement, as a consequence of lack of efficacy and development of drug-resistance. The need of different biochemical and functional parameters has grown, in order to obtain a larger view on processes of biological and clinical significance. In this paper we report novel metabolic features detected in a series of different human ovary carcinoma lines, by (1)H NMR spectroscopy of intact cells and their extracts. Most importantly, a new ovarian adenocarcinoma line CABA I, showed strong signals in the spectral region between 3.5 and 4.0 p.p.m., assigned for the first time to the polyol sorbitol (39+/-11 nmol/10(6) cells). (13)C NMR analyses of these cells incubated with [1-(13)C]-D-glucose demonstrated labelled-sorbitol formation. The other ovarian carcinoma cell lines (OVCAR-3, IGROV 1, SK-OV-3 and OVCA432), showed, in the same spectral region, intense resonances from other metabolites: glutathione (up to 30 nmol/10(6) cells) and myo-inositol (up to 50 nmol/10(6) cells). Biochemical and biological functions are suggested for these compounds in human ovarian carcinoma cells, especially in relation to their possible role in cell detoxification mechanisms during tumour progression.

Protons J-coupled to 13C were selectively detected in the mouse head by in vivo 1H NMR imaging based on Twin Spin Echo DOuble Resonance (T-SEDOR) excitation. This pulse sequence combines a good chemical specificity with high sensitivity, requires no solvent pre-saturation and is well adapted to the imaging modality. 1H T-SEDOR maps of the mouse head allowed detection of areas of preferential accumulation of 13C-enriched compounds, upon repeated injections of uniformly 13C-labelled glucose, which induced hyperglycemia. The results demonstrated the feasibility, both in time scale and metabolite concentration, of applying T-SEDOR MRI for in vivo mapping brain areas characterized by enhanced rates of glucose uptake and/or accumulation of its metabolites.

The C6 methylene protons were selectively detected in (1)H-NMR spectra of intact glioma cells incubated with 6-(13)C-D-2-deoxyglucose (6-(13)C-2dG), a (13)C-enriched glucose analog that is suitable for monitoring glucose utilization in brain tumors. Spectral editing via (1)H-(13)C scalar coupling was performed with twin spin-echo double resonance (T-SEDOR), a pulse sequence which combines chemical specificity and high sensitivity, requires no solvent pre-saturation, and can easily be adapted to imaging protocols. This work demonstrates the suitability of the pulse sequence for monitoring 6-(13)C-2dG uptake in living cells in vitro, in spite of line-broadening and the occurrence of other strong signals in the spectral region of interest (3.5-4.4 ppm)., (Copyright 2000 John Wiley & Sons, Ltd.)

By utilizing achievements and results of two previous concerted research projects on magnetic resonance imaging and spectroscopy (MRS), the EU BIOMED 1 Concerted Action on "Cancer and brain disease characterization and therapy assessment by quantitative MRS" was specifically aimed at: 1) developing at a multicentre level harmonized methodologies and protocols for quantitative and reproducible MRS measurements, as a basis for validating these procedures in well controlled clinical and experimental conditions; and 2) providing multicentre critical reviews on the present understanding of the significance of MRS parameters as possible new markers of diagnosis, prognosis and response to therapy. The programme comprised the following main areas of collaborative research and multicentre evaluation: a) development of methods and protocols for quality assessment, calibration and absolute metabolite quantification in in vivo localized, volume-selective MRS; b) design and validation of a new method for assessing localization performance in spectroscopic imaging (MRSI); c) interlaboratory comparison of different methods of signal processing and data analysis, for improving signal quantification in vivo and in vitro MRS spectra; d) quality assessment of high resolution MRS analyses of biological fluids; e) protocol for assembling a pilot data base of MR spectra of tumour extracts for pattern recognition analysis; f) multicentre review on evaluation of the significance of MRS parameters in monitoring lipid metabolism and function in cancer; and g) multicentre review on evaluation of drug pharmacokinetics and metabolism using MRS. The main results and conclusions of four multi-centre trials on items (a), (b) and (c), which involved 24 teams, are reported in the accompanying papers of this series.

The difference between the experimental and theoretical spatial response function (SRF) of a narrow tube with water is used for a localization test for magnetic resonance spectroscopic imaging (MRSI). From this difference a quantitative performance parameter is derived for the relative amount of signal within a limited region in the field of view. The total signal loss by the MRSI experiment and eddy currents is described by a parameter SL derived from the signal intensities of two echoes. Results of a European multi-centre trial show that this approach is suited for assessment of MRSI localization performance.

We have performed a multicentre trial to assess the performance of three techniques for absolute quantification of cerebral metabolites using in vivo proton nuclear magnetic resonance (NMR). The techniques included were 1) an internal water standard method, 2) an external standard method based on phantom replacement, and 3) a more sophisticated method incorporating elements of both the internal and external standard approaches, together with compartmental analysis of brain water. Only the internal water standard technique could be readily implemented at all participating sites and gave acceptable precision and interlaboratory reproducibility. This method was insensitive to many of the experimental factors affecting the performance of the alternative techniques, including effects related to loading, standing waves and B1 inhomogeneities; and practical issues of phantom positioning, user expertise and examination duration. However, the internal water standard method assumes a value for the concentration of NMR-visible water within the spectroscopic volume of interest. In general, it is necessary to modify this assumed concentration on the basis of the grey matter, white matter and cerebrospinal fluid (CSF) content of the volume, and the NMR-visible water content of the grey and white matter fractions. Combining data from 11 sites, the concentrations of the principal NMR-visible metabolites in the brains of healthy subjects (age range 20-35 years) determined using the internal water standard method were (mean+/-SD): [NAA]=10.0+/-3.4 mM (n=53), [tCho]=1.9+/-1.0 mM (n=51), [Cr + PCr]=6.5+/-3.7 mM (n=51). Evidence of system instability and other sources of error at some participating sites reinforces the need for rigorous quality assurance in quantitative spectroscopy.

High resolution 1H MRS studies report increased mobile neutral lipid (MNL) signals in transformed and malignant as well as in some in vitro cultured embryonic cells. Nature, subcellular localization and biological function of MNL are still under debate. This work was aimed at assessing alterations induced in MNL signals of NIH-3T3 mouse embryo fibroblasts by transformation with human HJ-ras oncogene. Lower MRS-visible MNL levels were unexpectedly detected in ras-transformed, in vivo tumorigenic fibroblasts, with respect to their untransformed and non-tumorigenic parental cells. MRS, gas chromatography and chemical analysis on cells and their lipid extracts indicated that these spectral differences could hardly be attributed to different triacylglycerol, free fatty acids and total cholesterol levels or to changes in the fatty acyl degree of unsaturation and average chain length. Additional, possibly more relevant mechanisms of regulation of MNL mobility may implicate the extensive morphogenetic changes and reorganization of cytoskeleton components (notably actin) associated with ras-transformation.

The increasing sensitivity of neuro-imaging in the diagnosis of brain expanding lesions is not directly related to biopathological specificity and new technological approaches are under study. In particular Magnetic Resonance Spectroscopy (MRS) allows evaluation of some biochemical pathways whose metabolic alterations may be correlated with the nature and malignancy grading of primary brain tumours. In the present study the author performed an in vitro high field 1H MRS (9.4 and 14.1 T) analysis of specimens obtained from stereotactic biopsy or microsurgical removal of primary brain tumours. Different samples derived from heterogeneous areas and/or infiltrated perilesional regions were examined. This study was principally focused on malignancy grading of gliomas and its correlation with the ratio (R) between the resonance band arising from choline containing compounds (between 3.14 and 3.35 ppm) and the total creatine signal (3.0 ppm). Analyses allowed significant discrimination between astrocytomas (R = 2.4 +/- 0.6) and glioblastoma (GBM) (R = 4.4 +/- 1.3) [p < 0.002]; however the results did not allow discrimination between differentiated and anaplastic astrocytomas. The GBM showed the largest spread of values corresponding to their higher level of tissue heterogeneity and de-differentiation. Studies on non astrocytic brain tumours indicated that even higher R values were exhibited by oligodendrogliomas, even in well differentiated forms (p < 0.02 with respect to GBM). Moreover, preliminary observations indicated that signals arising from other metabolites may also contribute to a differential diagnosis of different oncotypes. Among these glycine appears particularly relevant, since higher levels were measured for this amino acid in GBM with respect to both astrocytomas and oligodendrogliomas.

A protocol has been developed for quality assessment in in vivo NMR spectroscopy (MRS) on whole body and animal systems. The protocol, which has been evaluated in a series of international trials, defines procedures, objects and substances to evaluate the localisation of common slice and volume selection sequences, and to assess other relevant aspects of system performance, including: signal-to-noise; signal loss; relation between signal strength, concentration and volume; selective suppression of spectral lines; spectrometer stability.

By enabling noninvasive measurements of tissue biochemistry, nuclear magnetic resonance spectroscopy (MRS) provides a unique means of characterizing tissues. Differences in equipment, techniques, and methodology between different laboratories cause major difficulties when comparing results, whether from measurements of tissue metabolism, or from the effects of different therapies. This is of concern in critically evaluating work from different laboratories and centres, causing potential difficulties in reproducing results, limiting the establishment of MRS as a standard method of diagnosis and of characterising disease and response to therapy in the laboratory and clinic. It also poses particular problems in establishing the multicentre clinical trials of MRS that are now required to provide adequate statistical power in confirming the encouraging preliminary clinical observations. These difficulties arise principally from imperfect localization of signal from selected regions of interest in the body, and from the subsequent analyses of the MRS spectra. Improvement is possible by establishing agreed procedures for test measurements and for data analysis, and by using appropriate test objects and test substances to establish the quality of measurements. A concerted research project on characterisation of biological tissues by NMR, principally concerned with MR imaging (MRI), was activated in 1984 by the European Economic Community as part of its third Medical and Health Research Programme, under the auspices of the Biomedical Engineering Concerted Actions' Committee (COMAC-BME). In 1988, this project was prolonged for 5 years, when the programme was expanded to encompass MRS.(ABSTRACT TRUNCATED AT 250 WORDS)

This paper reports the results of multicentre studies aimed at designing, constructing, and evaluating prototype test objects for performance assessment in small-bore MRS systems, by utilizing the test protocols already proposed by the EEC COMAC-BME Concerted Action for clinical MRS equipment. Three classes of test objects were considered: (1) a multicompartment test object for 31P MRS measurements performed with slice-selective sequences; (2) a two-compartment test object for volume-selection 1H MRS; and (3) two-compartment test objects for assessing the performance of experimental systems using ISIS as volume localization sequence in 31P MRS. The results suggested the interest of adopting some of these prototypes for improving the comparison of spectroscopy data obtained from different sites, for providing useful means of quality assurance in experimental MRS, and facilitating the validation of new localization sequences.

Based on the requirements of test protocols developed to evaluate clinical MRS single slice and volume localisation sequences, two clinical test objects, STO1 and STO2 have been developed. The properties of a range of potential construction materials have been assessed, demonstrating that the water/Perspex interface produced minimum susceptibility effects. The design of the objects has been evaluated in trials on different magnetic resonance instruments, with size and loading being adjusted to allow use on currently available equipment. Appropriate test solutions for 31P and 1H measurements have been developed and their properties evaluated.

The recent technological developments in nuclear magnetic resonance (NMR) have made it possible to extend the use of this methodology to the formation of body images (MRI), as well as to the in vivo detection of metabolites in tissues and organs, by means of localized magnetic resonance spectroscopy (MRS). After presenting a brief semi-classical introduction to the NMR phenomenon, this article illustrates the principles of MRI, starting from phase encoding to the most commonly used pulse sequences. The main techniques of localized MRS are then introduced, with particular attention to their major advantages and limitations. The last session is devoted to the schematic description of a system for magnetic resonance imaging and spectroscopy.

This chapter summarizes requirements, rationale and main steps in the recent evolution of protocols adopted at international level for quality controls in clinical magnetic resonance imaging (MRI) and spectroscopy (MRS). An accurate assessment of instrumental performance in MRI and MRS is essential for enhancing the clinical value of these methodologies, both in terms of anatomical and physio-pathological information. In the field of MRI, a series of Concerted Actions, carried out in the frame of the medical and health research programmes of the European Economic Communities (EEC), led to the definition of standardized methodologies and to the construction of five test objects for assessing the quality of fourteen instrumental parameters, inherent to: signal uniformity and linearity; geometry; slice position and thickness; spatial resolution; signal-to-noise and contrast-to-noise ratios; T1 and T2 precision and accuracy. The first part of this paper devotes particular attention to the protocols adopted by the EEC Concerted Actions and by the National Electrical Manufacturers Association (NEMA, US), for assessing signal uniformity, geometric distortion and signal-to-noise ratio in MRI. A brief description is also given of the rationale and protocols for measuring other MRI parameters. The second part is devoted to describe eleven parameters of instrumental performance, two test objects and the measuring protocols recently proposed by the EEC Concerted Actions for quality control in single-volume localized MRS, following their validation in a series of experimental multi-centre trials.

This review summarizes experimental bases, limitations and rationale of guide-lines and recommendations adopted by national health authorities for the safety of patients exposed to the magnetic fields utilized during magnetic resonance (MR) examinations: static magnetic field (B0); time-varying magnetic field gradients (dB/dt) and radiofrequency magnetic fields. Although the mechanisms of interaction of these fields with the living matter are today sufficiently understood to identify the main categories of potentially associated health hazards, epidemiological studies and dosimetric concepts are still far from the levels of definition of protection standards adopted in the field of ionizing radiations. The definition of safety thresholds and levels of exposure to magnetic fields during MR examinations has evolved in the last decade and the matter is still under study today. This article summarizes the main steps in this continuous updating process and discusses limitations and criteria, which recently suggested the adoption in some Countries (including Italy) of a two-tier system for the levels of exposure of patients to the magnetic fields generated by clinical MR equipment.

The multi-parameter dependence of magnetic resonance (MR) images allows a unique flexibility of soft tissue contrast and gives access to peculiar sources of in vivo tissue characterization, mainly associated with magnetic relaxation properties. However, MR methodologies have not yet expressed their full potential in terms of tissue characterization for several reasons: a) problems of quality control and quantitation have generally not been addressed by centers using MRI equipment, nor in most of the published literature; and b) data scattering of quantitative measurements obtained from tissues in vitro and in vivo appear to be a major factor in inhibiting or limiting the clinical utility of MRI, for a possible in vivo characterization of pathological tissues. An international project, aimed at evaluating the clinical significance of tissue characterization by MR, was activated in 1984 by the Biomedical Engineering Advisory Committee of the European Communities (EC COMAC-BME) within the 3rd EC Medical and Health Research Programme (MHRP). The scientific achievements of this first project (Magn. Reson. Imaging, 6:171-222; 1988) represented the basis for launching and performing a second Concerted Action, in the frame of the 4th MHRP (1988-1992). Main areas of research of this second project were: a) development of standard methodologies for quantitative measurements of MR parameters and correlation with histo- and physiopathology; b) performance assessment and calibration of MR clinical equipment; c) harmonization of test procedures with other centers and industry; and d) pilot multi-center collections of data.(ABSTRACT TRUNCATED AT 250 WORDS)

Based on an international "Interlaboratory Comparison of Protocol Trials for In Vitro Studies by NMR," the EEC COMAC-BME concerted action on "Identification and Characterization of Biological Tissue by NMR" started collection of a data bank for in vitro relaxation time data of normal and pathological tissues. Relaxation time measurements were performed in a frequency range from 10-90 MHz and in a temperature range from 3 to 40 degrees C; data analysis was done using single exponential fitting routines. Specimens were taken from different organs or tissues (n = 19) of various species (n = 5) and both sexes. More than 900 relaxation time data from 12 participating groups were collected. However, for quantitative analysis we concentrated on liver tissue (287 entries). Main results are the high reproducibility of data from different centers, allowing quantification of temperature dependence of T1 and T2, respectively, in fresh mouse (3.9 and 0.41 msec/degrees C), rat (2.1 and 0.24 msec/degrees C), and hamster (2.6 and 0.22 msec/degrees C) liver. Further, a highly significant species difference between mouse and rat liver, not depending on water content, has been established. We discuss results quantitatively as well as consequences for in vitro protocols and future multicenter data collection.

The flux of 13C-labeled glucose through the Embden-Meyerhof and pentose phosphate pathways was studied by 13C NMR in intact erythrocytes isolated from normal subjects or from patients suffering of glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) deficiency. Similar rates of glucose catabolism and similar fluxes of the 13C-label into 2,3-bisphosphoglycerate and lactate were found, under basal conditions, in normal and in G6PD-deficient erythrocytes incubated in the presence of either [1-13C]- or D[6-13C]glucose. Exposure to oxidative stress by preincubation with tert-butylhydroperoxide induced in normal, but not in G6PD-deficient erythrocytes, a significant enhancement of glucose consumption, as well as a substantial reduction in 13C-label transfer from C1-glucose into lactate. It was also possible, by 31P NMR, to evaluate the conversion of 2-deoxyglucose to its phosphate-containing metabolites. The oxidation and subsequent decarboxylation of 2-deoxyglucose-6-phosphate was assessed in reconstituted systems and could subsequently be evidenced also in ethanolic extracts from normal (but not from G6PD-deficient) erythrocytes which had been exposed to oxidative stress. The results indicate that, in terms of glucose flux through the glycolytic pathway, there is little or no difference between normal and G6PD-deficient erythrocytes, regardless of previous exposure to oxidative stress. Faster consumption of either glucose or 2-deoxyglucose is induced, only in normal cells, by treatment with tert-butylhydroperoxide, essentially as a consequence of the activation of the pentose-phosphate pathway.

1H NMR spin-lattice relaxation times (T1) were measured in vitro and in vivo in Friend leukemia cell tumors during subcutaneous tumor growth in syngeneic mice and after in vivo administration of either purified murine interferon alpha/beta (IFN) or recombinant tumor necrosis factor alpha (TNF). Untreated tumors exhibited monoexponential T1 relaxation independently of tumor age at least until Day 16 after implantation. Histological examinations showed that under these conditions tumors were highly homogeneous and substantially free of necrotic areas. Peritumoral administrations of either IFN or TNF did not significantly alter the tumor relaxation properties at early stages of inhibition of tumor growth. The longitudinal relaxation decay became instead clearly biexponential at later stages (more than 7 days of IFN treatment or 2 days after TNF administration). While the T1 relaxation behavior could be unequivocally correlated with the presence of necrotic areas in these tumors, it could not be considered as an early marker of the altered growth capability, induced by administration of either IFN or TNF.

Recent technological developments extended the use of nuclear magnetic resonance to clinical imaging (MRI), as well as to in vivo metabolic studies on tissues and organs by means of localized spectroscopy (MRS). The quantitative determination of absolute metabolic concentrations by in vivo MRS still represents a challenge for technological efforts and biochemical investigations. In fact, the different methodologies today available for spectral acquisition from localized regions within a body organ or pathological lesion should ideally lead to a complete suppression of signals from surrounding tissues, without any appreciable signal loss from the volume of interest. Accurate assessment of deviations of the real from the ideal case represents an essential requirement for either relative or absolute quantification of in vivo localized MR spectra. A review will be presented of the most utilized MRS methodologies, together with a discussion on their potentialities and limitations. Techniques have been classified according to the use of either r.f. (B1) and/or static magnetic field (B0) gradients. The last session will be devoted to the presentation of some results obtained in our laboratory on the use of a particular test-object (constructed at the Deft Institute of Technology) for assessing signal localization efficiency provided by a surface coil in a small scale MRS/MRI equipment (for in vivo biochemical studies on small animals). The surface coil was used in conjunction with a number of sequence (t1-90 degrees-acq; 1-D "chemical shift imaging"; "depth") of either square or adiabatic r.f. pulses. The results allow the selection, on a quantitative basis, of the most appropriate pulse sequence(s) to be used for metabolic studies on superficial experimental tumours (implanted s.c. in small animals), according to the mean area and thickness of the neoplastic lesion. The latter parameters can be non-invasively assessed by previous MRI analysis of the tumour. This study was carried out in the frame of the EC COMAC-BME Concerted Research Project on Tissue Characterization by MRS and MRI.

NMR offers, in the complex proton relaxation properties of biological tissues, a unique and potentially powerful tool of tissue characterization. The difficulties encountered so far in comparing the results of in vitro and in vivo NMR studies carried out in different laboratories inhibit NMR methodologies from fully expressing their content of physiopathological information, as well as their potential for therapeutic monitoring. Moreover, obtaining accurate data on proton relaxation in vivo requires accurate location of the anatomical region under study. The acquisition of acceptable information about location and effects is dependent on the establishment of agreed procedures for tests, as well as on the use of appropriate test objects and test substances. A concerted research project entitled "Identification and Characterization of Biological Tissues by NMR" was activated in 1984 by the European Economic Community as part of the Third Medical Research Program, under the auspices of the Biomedical Engineering Concerted Action Committee. A series of papers is presented here which illustrates objectives and scientific programs of this project, the protocols adopted for multi-center comparison of in vitro and in vivo studies, and the results of validating trials, as well as problems of reference substances.

This review article outlines some potentials and limits of the recent application of high resolution Nuclear Magnetic Resonance technique--coupled to the Fourier transformation methods--to the study of biological membranes. Molecular arrangement and dynamical structure characters can be assessed at the level of individual chemical groups in lipid bilayer regions of natural and model membranes, through the determination of physical parameters like chemical shifts, spin-lattice (T1) and spin-spin (T2) nuclear magnetic relaxation times. The results of some significant experiments carried out on single-wall lecithin vesicles as well as on intact natural membranes, are summarized and discussed. Useful information can be obtained on the lipid fatty-acid chains thermal transition, by comparing two lecithin vesicles of the same size, formed by the same host lecithin, but incorporated with different molecular components. In particular, T1 and T2 measurements, interpreted in terms of a two- (or more-) correlation time theoretical models, are able to demonstrate different degrees of motional anisotropy in bilayers formed by mixed lecithins or by mixtures of lecithin and fatty acids, possessing moderately different chain lengths [13]. Chromophore-containing molecules, like chlorophyll [12] or fluorescent probes [14] can be located, within few Angstroms, in a lipid bilayer through proton chemical shift measurements; in addition the perturbation of the lipid membrane structure, as induced by the incorporated probe, is assessed mainly in terms of the intramolecular dynamical structure of the host lecithin molecules, by means of T1 and linewidth studies. The comparison of the n.m.r. relaxation behaviour in intact membranes and in vesicles formed by their extracted lipids may, finally, provide indirect information on the lipid-protein intermolecular interactions and relative mobility, besides indicating the intramolecular mobility characters of the lipid bilayer regions of the membrane.

Abstract: Administration of methylphenidate (MPH, Ritalin®) to children affected by attention deficit hyperactivity disorder (ADHD) is an elective therapy, which however raises concerns for public health, due to possible persistent neuro-behavioral alterations. We investigated potential long-term consequences at adulthood of MPH exposure during adolescence, by means of behavioral and brain MRS assessment in drug-free state. Wistar adolescent rats (30- to 44-day-old) were treated with MPH (0 or 2 mg/kg once/day for 14 days) and then left undisturbed until adulthood. Levels of impulsive behavior were assessed in the intolerance-to-delay task: Food-restricted rats were tested in operant chambers with two nose-poking holes, delivering one food pellet immediately, or five pellets after a delay whose length was increased over days. MPH-exposed animals showed a less marked shifting profile from the large/late to the small/soon reward, suggesting reduced basal levels of impulsivity, compared to controls. In vivo MRI-guided 1H MRS examinations at 4.7 T in anaesthetised animals revealed long-term biochemical changes in the dorsal striatum (STR), nucleus accumbens (NAcc), and prefrontal cortex (PFC) of MPH-exposed rats. Notably, total creatine and taurine, metabolites respectively involved in bioenergetics and synaptic efficiency, were up-regulated in the STR and conversely down-regulated in the NAcc of MPH-exposed rats. A strong correlation was evident between non-phosphorylated creatine in the STR and behavioral impulsivity. Moreover, unaltered total creatine and increased phospho-creatine/creatine ratio were detected in the PFC, suggesting improved cortical energetic performance. Because of this enduring rearrangement in the forebrain function, MPH-exposed animals may be more efficient when faced with delay of reinforcement. In summary, MPH exposure during adolescence produced enduring MRS-detectable biochemical modifications in brain reward-related circuits, which may account for increased self-control capacity of adult rats. [Copyright &y& Elsevier]