DESCRIPTIF DU TRAVAIL DE RECHERCHE
L’IRM (Imagerie par Résonance Magnétique) et la SRM (Spectroscopie par Résonance Magnétique) sont deux techniques basées sur le même phénomène : la RMN (Résonance Magnétique Nucléaire). Elles sont développées sur le même appareil : un spectromètre dont l’élément principal est un aimant supraconducteur. Ce sont des techniques proches de la RMN chère aux chimistes, à laquelle on ajoute la localisation de la mesure à l’aide d’impulsions de gradients de champ magnétique. L’IRM permet d’accéder à la structure interne d’un être vivant, la SRM donne accès à la Biochimie des métabolites en solution. Ce sont des techniques d’investigation des milieux vivant qui présentent par rapport aux multiples autres techniques, deux gros avantages : elles sont atraumatiques et non-invasives. A tel point qu’elles sont classées, d’un point de vue éthique, dans les méthodes substitutives. Mais elles présentent également un très gros défaut : elles sont peu sensibles !
Pour compenser la faible sensibilité de la mesure, nous développons nos recherches sur un appareil à haut champ (pour l’IRM) (aimant horizontal 9,4 Teslas, voir ci-dessous). L’IRM et la SRM à ce niveau de champ nécessitent le développement de méthodologies spécialisées. Une partie de notre travail consiste donc à développer de nouvelles techniques spécifiques pour des applications en Biologie, il s’agit alors de progresser en IRM/SRM pour progresser en Biologie intégrative. L’IRM/SRM à haut champ constitue également le moyen de créer de nouvelles techniques qui seront appliquées en IRM/SRM hospitalière.
Nous avons accès, pour des durées limitées, au spectromètre à aimant vertical wide-bore 17,6 Teslas installé au laboratoire CEMHTI, proche du CBM. Ce champ magnétique exceptionnel pour l’IRM (quelques appareils de ce type en Europe) nous permet d’accéder à une sensibilité également exceptionnelle. Les travaux à un tel champ magnétique posent des problèmes physiques importants qui font parties de nos recherches fondamentales.
Le développement remarquable de l’IRM ne serait pas complet sans l’apparition d’agents de contraste spécifiques. Contrairement aux autres techniques, l’IRM ne nécessite pas l’injection d’agents exogènes. Par contre, l’utilisation de ces traceurs amène un accroissement spectaculaire de la richesse des informations recueillies. Par exemple, on peut identifier et localiser une enzyme particulière à l’intérieur d’un être vivant. L’équipe "Complexes métalliques pour applications biomédicales" dirigée par Eva Jakab-Toth développe de nouveaux agents de contraste (dits "intelligents"). Parallèlement, nous développons les techniques IRM nécessaires, en particulier à haut champ, au recueil de l’information générée par ces molécules.
Comme toutes les méthodes physiques, l’IRM/SRM nécessite désormais une évolution des équipes jusqu’alors spécialisées en méthodologie vers la biologie. Nous associons donc désormais à l’imagerie, la notion de signal avec l’électrophysiologie. La plasticité cellulaire résulte en effet des changements dans le temps des propriétés de traitement de l’information d’un réseau neuronal et est à la base de la consolidation des phénomènes mnésiques. Nous y associons la Spectroscopie par Résonance Magnétique afin d’évaluer les substrats énergétiques disponibles et leur utilisation par les neurones sur des modèles souris modèles de trisomie 21 afin de mieux comprendre à l’échelle cellulaire et moléculaire les relations et les interactions entre certains gènes du chromosome humain 21 et les troubles de l’apprentissage et de la mémoire.
Nous accompagnons aussi la très forte tendance actuelle en IRM consistant à investir le domaine de la génétique, en accueillant une spécialiste de ce domaine et en ouvrant une toute nouvelle voie de recherche en nous investissant dans l’IRM de la Drosophile. L’un des objectifs de l’imagerie moléculaire est, en effet, d’accéder à « l’imagerie de l’expression des gènes ». La drosophile, est un modèle de choix pour les études génétiques mais reste un défi pour l’IRM en raison de la taille de l’insecte (<2mm). Il s’agit de développer un système complet de détection d’enzymes spécifiques in vivo.
SRM hétéronucléaire 1H-13C bivoxel chez le rat et la souris
SRM 2D 1H in vivo
IRM du disque intervertébral chez le lapin
Angiographie chez la souris (tumeurs, infections)
IRM du poumon
Etude des modifications du métabolisme cérébral dans la trisomie 21
Spectromètre-imageur à aimant horizontal 9,4 Teslas (Résonance du proton 1H : 400MHz, diamètre du puits d’accès : 20cm).
Nous avons accès au spectromètre à aimant vertical wide-bore 17,6 Teslas (Résonance du proton 1H : 750 MHz, diamètre du puits d’accès : 8 cm). Il s’agit du champ le plus élevé disponible en France pour l’IRM, actuellement. Seuls quelques appareils de ce type sont utilisés pour l’IRM dans le monde.
Un nouvel spectromètre imageur à aimant horizontal 7 Teslas (diamètre du puits d’accès : 16cm) est arrivé en 2011
Poste double-patch clamp
Etuves pour élevage de drosophiles
Equipements de biologie moléculaire : électrophorèse ADN et protéines, thermocycleur, centrifugeuses.....
SRM hétéronucléaire 1H-13C bivoxel
SRM 1D et 2D 1H in vivo
Analyse d’image par analyse de texture
Electronique HF pour la conception et la réalisation de bobines pour l’IRM et la SRM.
Double patch clamp
Immunoprécipitation de la chromatine pontée (X-ChIP), co-immunoprécipitation (co-IP)
Amplification par PCR
Extraction d’ADN et d’ARN
Elevage et croisement de drosophiles,
Séquences de SRM 2D homo et hétéronucléaires mono et bivoxels avec applications chez des modèles de souris de trisomie 21.
Imagerie de l’expression des gènes (drosophile, rat et souris).
Microscopie IRM en hyper-champ (17,6 Teslas) avec applications à l’expression des gènes chez la drosophile
Développement des techniques CSI (Chemical Shift Imaging) à haut champ
SRM en hyperchamp (17,6 Teslas)
En collaboration avec l’équipe "Complexes métalliques pour applications biomédicales" dirigée par Eva Jakab-Toth : Séquences d’impulsions adaptées à l’utilisation des agents de contraste de type "PARACEST" à haut champ
En collaboration avec l’équipe "Composés de lanthanides luminescents pour applications bioanalytiques et imagerie" dirigée par Stéphane Petoud, nous voulons aller plus loin que l’IRM seule et développer de l’imagerie bimodale qui associe dans une même expérience l’IRM et l’imagerie optique.
En collaboration avec l’équipe "Reconnaissance cellulaire : Lectines endogènes et glycobiologie" dirigée par Claudine Kiéda : développement de l’angiographie IRM pour l’étude de la néoangiogénèse tumorale.
Imagerie bimodale (IRM/TEP) → PROJET EQUIPEX
Légende des images
Image n°1 : Image de microscopie par résonance Magnétique (MRM) de drosophile réalisée à 17.6T (A : coupe axiale de drosophile, B : images après reconstruction 3D).
Images n°2 : Angiographie par Résonance Magnétique (ARM) par la méthode du Time Of Flight(TOF) réalisée sur le cerveau d’une souris. Seule la vascularisation intra cérébrale apparaît en hyper signal
Image n°3 : Coupe coronale de cerveau de rat réalisée à 9.4T et spectre 1H (filtré 13C) réalisé dans la zone sélectionnée (carré noir) après 1 heure de perfusion par du glucose marqué 13C.
Image n°4 : La drosophile, modèle d’étude de la dégénérescence musculaire : a) Phénotype ailes relevées d’un mutant up. b) Image IRM des muscles du thorax d’un mutant up.
Images n°5 : A : Courant post-synaptique des neurones épineux de taille moyenne avant (courbe noire) et 20 min après (courbe rouge) un protocole stimulation haute fréquence (SHF), chez des souris 2N et Ms4Yah.
B : Evolution au cours du temps de l’amplitude des courants post-synaptiques avant et après une stimulation haute fréquence.
Charpentier, G. Louat, F. Bonmatin, J. M. Marchand, P. A. Vanier, F. Locker, D. Decoville, M. (2014)
Lethal and sublethal effects of imidacloprid, after chronic exposure, on the insect model Drosophila melanogasterEnvironmental science and technology (201) 48 (7) 4096-4102 - doi : 10.1021/es405331c
Neonicotinoids are subjected to vigilance because of environmental contaminations and deleterious effects on bees. Imidacloprid (IMI) is one of the most representative insecticides of this family. At chronic exposure, concentration-effect relationships are non linear. An insect model should allow a better description of this toxicity. We compared the lethal concentration 50% (LC50) of IMI for a Drosophila-field strain, after acute and chronic exposure. Relative to the acute LC50, the chronic LC50 was lowered by a factor of 29 for males (1.3 mM/45 muM), 52 for larvae (157 muM/3 muM) and more than 172 for females (>3.1 mM/18 muM). Chronic exposure also revealed significant lethal and sublethal effects, at concentrations 3-5 orders of magnitude lower than the chronic LC50. Mean mortalities reached 28% (at 3.91 nM) and 27% (at 39.1 nM) for females and males, respectively. Fecundity decreased of 16% at 1.96 nM. Mating increased of 30% at 0.391 nM. The LOEC (lowest observed effect concentration : 0.391 nM) was 46 000 times lower than the chronic LC50 for males ; it was 115 000 times lower than the chronic LC50 for females. This study illuminates effects that neonicotinoids can induce at very low concentrations. This is of particular interest for nontarget insects and for insect dependent species.
In Vivo 9.4T MRI and 1H MRS for Evaluation of Brain Structural and Metabolic Changes in the Ts65Dn Mouse Model for Down SyndromeWorld Journal of Neuroscience (2014) 4, 152-163 - doi : 10.4236/wjns.2014.42018
In the present study we investigated structural and metabolic modifications of the brain in the Ts65Dn mouse model of Down syndrome(DS)using both in vivo magnetic resonance imaging(MRI)and proton magnetic resonance spectroscopy(MRS). MRI was performed for further texture analysis and changes in texture parameters, including mean grey levels, contrast and homogeneity, and they were found in Ts65Dn compared to diploid littermates (2n). These phenotypic changes were different in the hippocampus and cerebellum, since in Ts65Dn mean grey levels increased in the cerebellum and decreased in the hippocampus. In addition, proton NMR spectra revealed differences in metabolite ratios. Levels of N-acetylaspartate(NAA)and glutamate(Glu), were lower compared to total creatine levels (CX), in the Ts65Dn brain. However, the most striking finding was an increase in the concentration of myo-inositol(Ins)and choline(Cho)in the hippocampus, whereas the Ins concentration was reduced in the cerebellum. Overall, these data illustrate that MRI and MRS are valuable assesment tools sufficiently sensitive to detect associated changes in different brain areas, thus providing new insight into the causative role of dosage-sensitive genes in the Ts65Dn DS mouse model.
Developmental molecular and functional cerebellar alterations induced by PCP4/PEP19 overexpression : Implications for Down syndromeNeurobiology of Disease 63 92-106 - doi : 10.1016/j.nbd.2013.11.016
PCP4/PEP19 is a modulator of Ca2 +-CaM signaling. In the brain, it is expressed in a very specific pattern in postmitotic neurons. In particular, Pcp4 is highly expressed in the Purkinje cell, the sole output neuron of the cerebellum. PCP4, located on human chromosome 21, is present in three copies in individuals with Down syndrome (DS). In a previous study using a transgenic mouse model (TgPCP4) to evaluate the consequences of 3 copies of this gene, we found that PCP4 overexpression induces precocious neuronal differentiation during mouse embryogenesis. Here, we report combined analyses of the cerebellum at postnatal stages (P14 and adult) in which we identified age-related molecular, electrophysiological, and behavioral alterations in the TgPCP4 mouse. While Pcp4 overexpression at P14 induces an earlier neuronal maturation, at adult stage it induces increase in cerebellar CaMK2alpha and in cerebellar LTD, as well as learning impairments. We therefore propose that PCP4 contributes significantly to the development of Down syndrome phenotypes through molecular and functional changes.
Palomo J., Fauconnier M., Coquard L., Gilles M., Meme S., Szeremeta F., Fick L., Franetich J.F., Jacobs M., Togbe D., Beloeil J.C., Mazier D., Ryffel B. and Quesniaux V.F. (2013)
Type I interferons contribute to experimental cerebral malaria development in response to sporozoite or blood-stage Plasmodium berghei ANKAEuropean Journal of Immunology 43 (10) 2683-2695 - doi : 10.1002/eji.201343327
Cerebral malaria is a severe complication of Plasmodium falciparum infection. Although T-cell activation and type II IFN-gamma are required for Plasmodium berghei ANKA (PbA)-induced murine experimental cerebral malaria (ECM), the role of type I IFN-alpha/beta in ECM development remains unclear. Here, we address the role of the IFN-alpha/beta pathway in ECM devel-opment in response to hepatic or blood-stage PbA infection, using mice deficient for types I or II IFN receptors. While IFN-gammaR1(-)/(-) mice were fully resistant, IFNAR1(-)/(-) mice showed delayed and partial protection to ECM after PbA infection. ECM resistance in IFN-gammaR1(-)/(-) mice correlated with unaltered cerebral microcirculation and absence of ischemia, while WT and IFNAR1(-)/(-) mice developed distinct microvascular pathologies. ECM resistance appeared to be independent of parasitemia. Instead, key mediators of ECM were attenuated in the absence of IFNAR1, including PbA-induced brain sequestration of CXCR3(+)-activated CD8(+) T cells. This was associated with reduced expression of Granzyme B, IFN-gamma, IL-12Rbeta2, and T-cell-attracting chemokines CXCL9 and CXCL10 in IFNAR1(-)/(-) mice, more so in the absence of IFN-gammaR1. Therefore, the type I IFN-alpha/beta receptor pathway contributes to brain T-cell responses and microvascular pathology, although it is not as essential as IFN-gamma for the development of cerebral malaria upon hepatic or blood-stage PbA infection.
In preclinical research, genetic studies have made considerable progress as a result of the development of transgenic animal models of human diseases. Consequently, there is now a need for higher resolution MRI to provide finer details for studies of small animals (rats, mice) or very small animals (insects). One way to address this issue is to work with high-magnetic-field spectrometers (dedicated to small animal imaging) with strong magnetic field gradients. It is also necessary to develop a complete methodology (transmit/receive coil, pulse sequence, fixing system, air supply, anesthesia capabilities, etc.). In this study, we developed noninvasive protocols, both in vitro and in vivo (from coil construction to image generation), for drosophila MRI at 9.4 T. The 10*10*80-μm resolution makes it possible to visualize whole drosophila (head, thorax, abdomen) and internal organs (ovaries, longitudinal and transverse muscles, bowel, proboscis, antennae and optical lobes). We also provide some results obtained with a Drosophila model of muscle degeneration. This opens the way for new applications of structural genetic modification studies using MRI of drosophila.
Almhdie-Imjabber A., Ferrer-Villena J.M., Harba R., Lédée R., Léger C., Lopes-Pereira P. and Même S. (2012)
In this paper, a semi automatic method is proposed for the segmentation of mice cerebral structures (brain, cerebellum and hippocampus) in MR images. First, a Chan-Vese method is applied on the axial images to segment the brain volume. The method takes into account the special shape of the brain mice. Second, variational atlases are constructed by manual segmentation of various MRI brain images of reference and Trisomy 21 mice. These atlases are then registered on true data to assist the Chan-Vese segmentation of the cerebellum and the hippocampus. This semi automatic method makes that human intervention is limited and the tedious manual handling is greatly reduced. Results have shown that the brain volumes estimated by the method are identical to expert manually estimated volumes. The new method was used in the analysis of the cerebral malformations linked to Trisomy 21 : no significant difference of the cerebral structures between Trisomy 21 mice and the control ones was found.
Functionalized single-walled carbon nanotubes containing traces of iron as new negative MRI contrast agents for in vivo imagingContrast Media & Molecular Imaging 7 (2) 153-159
Single-walled carbon nanotubes (SWCNTs) containing traces of iron oxide were functionalized by noncovalent lipid-PEG or covalent carboxylic acid function to supply new efficient MRI contrast agents for in vitro and in vivo applications. Longitudinal (r1) and transversal (r2) water proton relaxivities were measured at 300 ?MHz, showing a stronger T2 feature as an MRI contrast agent (r2/r1 ?= ?190 for CO2H functionalisation). The r2 relaxivity was demonstrated to be correlated to the presence of iron oxide in the SWNT-carboxylic function COOH, in comparison to iron-free ones. Biodistribution studies on mice after a systemic injection showed a negative MRI contrast in liver, suggesting the presence of the nanotubes in this organ until 48 ?h after i.v. injection. The presence of carbon nanotubes in liver was confirmed after ex vivo carbon extraction. Finally, cytotoxicity studies showed no apparent effect owing to the presence of the carbon nanotubes. The functionalized carbon nanotubes were well tolerated by the animals at the dose of 10 ?mu g ?g-1 body weight.
A Th1 response is required for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The role of pro-Th1 IL-12 in malaria is complex and controversial. In this study, we addressed the role of IL-12R beta 2 in ECM development. C57BL/6 mice deficient for IL-12R beta 2, IL-12p40, or IL-12p35 were analyzed for ECM development after blood-stage PbA infection in terms of ischemia and blood flow by noninvasive magnetic resonance imaging and angiography, T cell recruitment, and gene expression. Without IL-12R beta 2, no neurologic sign of ECM developed upon PbA infection. Although wildtype mice developed distinct brain microvascular pathology, ECM-resistant, IL-12R beta 2-deficient mice showed unaltered cerebral microcirculation and the absence of ischemia after PbA infection. In contrast, mice deficient for IL-12p40 or IL-12p35 were sensitive to ECM development. The resistance of IL-12R beta 2-deficient mice to ECM correlated with reduced recruitment of activated T cells and impaired overexpression of lymphotoxin-alpha, TNF-alpha, and IFN-gamma in the brain after PbA infection. Therefore, IL-12R beta 2 signaling is essential for ECM development but independent from IL-12p40 and IL-12p35. We document a novel link between IL-12R beta 2 and lymphotoxin-alpha, TNF-alpha, and IFN-gamma expression, key cytokines for ECM pathogenesis.
Mouchel-Vielh, E., Rougeot, J., Decoville, M. & Peronnet, F. (2011)
The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development.Bmc Developmental Biology 11 (1) 17
Background : Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here.
Results : Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK) in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation.
MRI, MRS and chemistry Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) are two techniques derived from NMR, which is well known by chemists. If MRI is the technique of choice for the non-invasive and atraumatic exploration of living beings, it is now moving towards obtaining additional information of biochemical nature, we can speak of "molecular imaging" and even of "imaging of gene expression". These developments require the creation of contrast agents specific of the enzymes of interest and that is where the chemistry is essential. In SRM, the role of the chemist is to transform high resolution NMR techniques to make them compatible with the very special characteristics of localized NMR of living environments. In this article, we will discuss the growing importance of chemistry in the spectacular development of MRI and MRS.
Lamiable, O., Rabhi, M., Peronnet, F., Locker, D. & Decoville, M. (2010)
Même, W., Vandecasteele, M., Glowinski, J., Giaume, C. & Venance, L. (2009)
MRI Characterization of Structural Mouse Brain Changes in Response to Chronic Exposure to the Glufosinate Ammonium Herbicide.Toxicol. Sci. 111, 321-330. Doan, B.T., Autret, G., Mispelter, J., Meric, P., Meme, W., Montecot-Dubourg, C., Correze, J.L., Szeremeta, F., Gillet, B. & Beloeil, J.C. (2009)
Simultaneous two-voxel localized H-1-observed C-13-edited spectroscopy for in vivo MRS on rat brain at 9.4 T : Application to the investigation of excitotoxic lesions.J. Magn. Reson. 198, 94-104.
Togbe, D., de Sousa, P., Fauconnier, M., Boissay, V., Fick, L., Scheu, S., Pfeffer, K., Menard, R., Grau, G., Doan, B., Beloeil, J., Renia, L., Hansen, A., Ball, H., Hunt, N., Ryffel, B. & Quesniaux, V. (2008)
Both functional LTbeta receptor and TNF receptor 2 are required for the development of experimental cerebral malaria.PLOS One 3, e2608. Pisani, E., Tsapis, N., Galaz, B., Santin, M., Berti, R., Taulier, N., Kurtisovski, E., Lucidarme, O., Ourevitch, M., Doan, B.T., Beloeil, J.C., Gillet, B., Urbach, W., Bridal, S.L. & Fattal, E. (2008)
Perfluorooctyl Bromide Polymeric Capsules as Dual Contrast Agents for Ultrasonography and Magnetic Resonance Imaging.Adv. Funct. Mater. 18, 2963-2971. Noury, F ; Mispelter, J ; Szeremeta, F ; Même, S ; Doan, BT ; Beloeil, JC (2008)
MRI methodological development of intervertebral disc degeneration : a rabbit in vivo study at 9.4 TMagn. Reson. Imaging 26 (10) 1421-1432 Chauvin, T ; Durand, P ; Bernier, M ; Meudal, H ; Doan, BT ; Noury, F ; Badet, B ; Beloeil, JC ; Tóth, E (2008)
Detection of enzymatic activity by PARACEST MRI : a general approach to target a large variety of enzymesAngew. Chem. Int. Edit. 47 (23) 4370 -4372 Calas, AG ; Richard, O ; Même, S ; Beloeil, JC ; Doan, BT ; Gefflaut, T ; Même, W ; Crusio, WE ; Pichon, J ; Montécot, C (2008)
Chronic exposure to glufosinate-ammonium induces spatial memory impairments, hippocampal MRI modifications and glutamine synthetase activation in miceNeurotoxicology 29 (4)740-747 Loureiro de Sousa, P ; Livramento, JB ; Helm, L ; Merbach, AE ; Même, W ; Doan, BT ; Beloeil, JC ; Prata, MIM ; Santos, AC ; Geraldes CFGC ; Tóth, E (2008)
In vivo MRI assessment of a novel GdIII-based contrast agent designed for high magnetic field applicationsContrast Media and Molecular Imaging 3 (2) 78-85
Gd(3)L is a trinuclear Gd(3+) complex of intermediate size, designed for contrast agent applications in high field magnetic resonance imaging (H(12)L is based on a trimethylbenzene core bearing three methylene-diethylenetriamine- N,N,N’’,N’’-tetraacetate moieties). Thanks to its appropriate size, the presence of two inner sphere water molecules and a fast water exchange, Gd(3)L has remarkable proton relaxivities at high magnetic field (r(1) = 10.2 vs 3.0 mM(-1) s(-1) for GdDOTA at 9.4 T, 37 degrees C, in H(2)O). Here we report an in vivo MRI feasibility study, complemented with dynamic gamma scintigraphic imaging and biodistribution experiments using the (153)Sm-enriched analog. MRI experiments were performed at 9.4 T in mice with Gd(3)L and the commercial contrast agent gadolinium(III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (GdDOTA). Gd(3)L was well tolerated by the animals at the dose of 8 micromol Gd kg(-1) body weight. Dynamic contrast enhanced (DCE) images showed considerably higher signal enhancement in the kidney medulla and cortex after Gd(3)L injection than after GdDOTA injection at an identical dose. The relaxation rates, DeltaR(1), were calculated from the IR TrueFISP data. During the excretory phase, the DeltaR(1) for various tissues was similar for Gd(3)L and GdDOTA, when the latter was injected at a three-fold higher dose (24 vs 8 micromol Gd kg(-1) body weight). These results point to an approximately three times higher in vivo relaxivity (per Gd) for Gd(3)L relative to GdDOTA, thus the ratio of the relaxivities of the two compounds determined in vitro is retained under in vivo conditions. They also indicate that the two inner sphere water molecules per Gd in Gd(3)L are not substantially replaced by endogenous anions or other donor groups under physiological conditions. Gd(3)L has a pharmacokinetics typical of small, hydrophilic complexes, involving fast renal clearance and no retention in the blood pool. The dynamic gamma scintigraphic studies and the biodistribution experiments performed in Wistar rats with (153)Sm-enriched (*)Sm(3)L are also indicative of a fast elimination via the kidneys.
Noncovalent functionalization of carbon nanotubes with amphiphilic Gd3+ chelates : Toward powerful T-1 and T-2 MRI contrast agentsNano Letters 8 232-236
An amphiphilic gadolinium (III) chelate (GdL) was synthesized from commercially available stearic acid. Aqueous solutions of the complex at different concentrations (from 1 mM to 1 mu M) were prepared and adsorbed on multiwalled carbon nanotubes. The resulting suspensions were stable for several days and have been characterized with regard to magnetic resonance imaging (MRI) contrast agent applications. Longitudinal water proton relaxivities, r(1), have been measured at 20, 300, and 500 MHz. The r(1) values show a strong dependence on the GdL concentration, particularly at low field.
A yeast artificial chromosome (YAC) transgenic murine model of partial trisomy 21 overexpressing five human genes-including DYRK1A, which encodes a serine threonine kinase involved in cell cycle control-has been shown to present an increase in brain weight. We analyzed this new phenotype by measuring total and regional brain volumes at different ages, using a 7 Tesla magnetic resonance imaging volumetric approach. Volumetric measurements showed a total volume increase of 13.6% in adult mice. Changes in brain morphogenesis were already visible at a very early postnatal stage (postnatal days 2-7). Region-specific changes were characterized from postnatal day 15 to 5 months. These results, made it possible to define region-specific effects of DYRK1A overexpression, with the strongest increase seen in the thalamus-hypothalamus area (24%).
Pasco, A ; Lemaire, L ; Franconi, F ; Lefur, Y ; Noury, F ; Saint-André, JP ; Benoît, JP ; Cozzone, PJ ; Le Jeune, JJ (2007)
Perfusional deficit and the dynamics of cerebral edemas in experimental traumatic brain injury using perfusion and diffusion-weighted magnetic resonance imagingJ. Neurotrauma 24 1321 -1330 Magnol, L ; Chevallier, MC ; Nalesso, V ; Retif, S ; Fuchs, H ; Klempt, M ; Pereira, P ; Riottot, M ; Andrzejewski, S ; Doan, BT ; Panthier, JJ ; Puech, A ; Beloeil, JC ; de Angelis, MH ; Herault, Y (2007)
Background : The Kit gene encodes a receptor tyrosine kinase involved in various biological processes including melanogenesis, hematopoiesis and gametogenesis in mice and human. A large number of Kit mutants has been described so far showing the pleiotropic phenotypes associated with partial loss-of-function of the gene. Hypomorphic mutations can induce a light coat color phenotype while complete lack of KIT function interferes with embryogenesis. Interestingly several intermediate hypomorphic mutations induced in addition growth retardation and post-natal mortality.
In vivo brain edema classification : New insight offered by large b-value diffusion-weighted MR imagingJournal of Magnetic Resonance Imaging 25 (1) 26-31
Purpose : To asses the role of large b-value diffusion weighted imaging (DWI) in the characterization of the physicochemical properties ot the water in the brain edema under expeirmental and clinical conditions. Materials and Methods : Vasogenic brain edema was induced in mice by means of cold injury. A total of 17 patients wtih extensive peritumoral brain edema were also investigated. The longitudinal relaxation time(T-1) and apparent diffusion coefficient (D) were measured in the edematous area both in humans and in mice. D was calculated by using both mono- (D-mono) and biexponential (D-fast an D-slow) approaches in the low and overall range of b-values, respectively. The D values were correlated with the T-1 values. Results : A strong linear correlation was found between T-1 and D-mono in vasogenic brain edema, both in humans and in mice. After breakdown of D-mono into fast and slow diffusing components, only Dfast exhibited a strong correlation with T-1 : D-slow was unchanged in vasogenic brain edema. Conclusion : Large b-value DWI can furnish a detailed characterization of vasogenic brain edema, and may provide a quantitative approach for the differentiation of edema types on the basis of the physicochemical properties of the water molecules. Application of the DWI method may permit prediction and follow-up of the effects of antiedematous therapy.
Rouach N., Pébay A., Même W., Cordier J., Ezan P., Chalal Y., Etienne E., Giaume C & Tencé M. (2006)
Sphingosine-1-phosphate inhibits gap junctional communication in astrocytes : Involvement of Gi and Rho GTPase/ROCK.European Journal of Neuroscience 23 : 1453 - 1464 Rouach, N., Pébay, A., Meme, S., Cordier, J., Ezan, P., Chalal, Y., Etienne, E., Giaume, C. & Tencé, M. (2006)
Sphingosine-1-phosphate inhibits gap junctional communication in astrocytes : Involvement of Gi and Rho GTPase/ROCKEurop. J. Neurosci. 23, 1453-1464. Même, W., Calvo, C., Froger, N., Ezan, P., Amigou, E., Koulakoff, A. & Giaume, C. (2006)
Pro-inflammatory cytokines released from microglia inhibit gap junctions in astrocytes : potentiation by ß-amyloid.FASEB J 20, 494-496. Léone, C., Le Pavec, G., Même, W., Porcheray, F., Samah, B., Dormont, D. & G., G. (2006)
Recent studies have shown that cell migration can be monitored in vivo by magnetic resonance imaging after intracellular contrast agent incorporation. This is due to the dephasing effect on proton magnetization of the local magnetic field created by a labelled cell. Anionic iron oxide nanoparticles (AMNP) are among the most efficient and non-toxic contrast agents to be spontaneously taken up by a wide variety of cells.
In vivo cellular imaging of lymphocyte tracking by MRI : A tumor model approach to cell-based anticancer therapyMagnetic Resonance in Medicine 56 (3) 498-508
The aim of this study was to demonstrate the feasibility of in vivo cell tracking to monitor anticancer cell therapy by means a high-resolution noninvasive MRI method. Ovalbumin-specifi splenocytes (OT-1) labeled with anionic ?-Fe2O3 superparamagnetic iron oxide (SPIO) nanoparticles were adoptively transferred into C57BL/6 mice with growing ovalbumin-expressing tumors. OT-1 cells were tracked in vivo by 7 T MRI 24, 48, an 72 hr after they were injected. The results showed significant negative enhancement of the spleen at 24 hr, and of the tumor at 48 and 72 hr, after labeled cell injection.
Study of a potential inhibitor of acetylcholinesterase using UV spectrophotometry, NMR spectroscopy and molecular modelingJournal of Physical Organic Chemistry 19 (2) 148-156
1-[6-(Acridine-9-carbonyloxy)hexyl]pyridinium chloride (1) was synthesized and studied as a potential inhibitor of acetylcholinesterase (AChE), which is frequently involved in Alzheimer’s disease. UV spectrophotometry showed that 1 is a reversible and competitive inhibitor of AChE (K-i approximate to 2 x 10(-7) M). NMR (TrNOESY) showed that 1, bonded to AChE, maintains an extended form that allows hydrophobic interactions to occur between the aliphatic chain and the deep and narrow gorge of AchE and favors interactions between the acridine group and the catalytic and anionic subsites situated at the bottom of the gorge, and also between the pyridinium ring and the peripheral site. A more detailed picture of the structure of the complex was obtained by combining NMR structural data and molecular modeling (docking, dynamics simulation and energy calculations). Copyright (c) 2006 John Wiley & Sons, Ltd.
In vivo imaging of small animals is a rapidly developing field. However, the potential of global imaging of infectious processes in animal models remains poorly explored. We used magnetic resonance imaging (MRI) to follow the development and regression of inflammatory lesions caused by infection by Klebsiella pneumoniae in mouse lungs. A virulent strain caused an intense inflammation within 2 days in the whole lungs, while an avirulent strain did not show significant changes. Mice infected with the virulent strain and subsequently treated with antibiotics presented a severe inflammation localized mainly in the left lung that disappeared after a week. The lesions observed by MRI correlated with the damage seen by histological analysis and a 3D representation of the tissue allowed better visualization of the development and healing of inflammatory lesions. MRI thus represents a powerful technique to study in vivo the interactions between a pathogen and its host in real time.
Preclinical metabolic changes in mouse prion diseases detected by H-1-nuclear magnetic resonance spectroscopyNeuroreport 17 (1) 89-93
Magnetic resonance spectroscopy studies in animal models of prion disease are very few and concern terminal stages of infection. In order to study earlier stages of the disease, we used in vivo magnetic resonance spectroscopy in a mouse model of scrapie and, for the first time, in mice infected with a bovine spongiform encephalopathy strain. In bovine spongiform encephalopathy-infected mice, we observed an increase in myo-inositol preceding clinical signs by 20 days, followed by a decrease in N-acetylaspartate at advanced stages. In scrapie-infected mice, changes in N-acetylaspartate and myo-inositol were detected at the beginning of the symptomatic phase. These results show that magnetic resonance spectroscopy is a valuable tool for detecting subtle metabolic changes associated to gliosis and neuronal dysfunction in prion diseases.
We show here, for the first time, that two neutral polymers may completely associate together in water to spontaneously form supramolecular nanoassemblies (nanogels) of spherical shape. The cohesion of these stable structures of about 200 nm is based upon a "lock and key" mechanism : inclusion complexes are formed between the hydrophobic alkyl chains grafted on a polysaccharide (dextran) and the molecular cavities contained in a poly-cyclodextrin polymer. Production yields reached 95%. It was established that all the alkyl chains were included within the cyclodextrins’ cavities in these nanoassemblies. The multivalent character of the interactions between the two polymers ensures the stability of the nanoassemblies. Moreover, empty cyclodextrin units remained accessible for the inclusion of compounds of interest such as benzophenon or tamoxifen. (c) 2006 Elsevier B.V. All rights reserved.
Ducher, G ; Meme, S ; Magni, C ; Viala, J ; Chappard, C ; Courteix, D ; Benhamou, C (2005)
Effects of long-term tennis playing on bone gross geometry at the distal radius : A quantitative magnetic resenance imaging study.Journal of Bone and Mineral Research 20 (9) S135-S135 Suppl. 1 Ducher, G ; Courteix, D ; Meme, S ; Magni, C ; Viala, JF ; Benhamou, CL (2005)
Bone geometry in response to long-term tennis playing and its relationship with muscle volume : A quantitative magnetic resonance imaging study in tennis playersBone 37 (4) 457-466
The benefit of impact-loading activity for bone strength depends on whether the additional bone mineral content (BMC) accrued at loaded sites is due to an increased bone size, volumetric bone mineral density (vBMD) or both. Using magnetic resonance imaging (MRI) and dual energy X-ray absorptiometry (DXA), the airn of this study was to characterize the geometric changes of the dominant radius in response to long-term tennis playing and to assess the influence of muscle forces on bone tissue by investigating the muscle-bone relationship. Twenty tennis players (10 men and 10 women, mean age : 23.1 +/- 4.7 years, with 14.3 +/- 3.4 years of playing) were recruited. The total bone volume, cortical volume, sub-cortical volume and muscle volume were measured at both distal radii by MRI. BMC was assessed by DXA and was divided by the total bone volume to derive vBMD.
Analysis of Trabecular Bone Texture on Magnetic Resonance Imaging of the Distal Radius : Side-to-side Differences in Tennis Players.Journal of Bone and Mineral Research 19 Su 228 P.S260 Suppl. 1 Autret, G ; Liger-Belair, G ; Nuzillard, JM ; Parmentier, M ; de Montreynaud, AD ; Jeandet, P ; Doan, BT ; Beloeil, JC (2005)
Use of magnetic resonance spectroscopy for the investigation of the CO2 dissolved in champagne and sparkling wines : a nondestructive and unintrusive methodAnalytica Chimica Acta 535 (1-2) 73-78
It is demonstrated in this paper that an imaging spectrometer with the C-13 Magnetic Resonance Spectroscopy (MRS) technique is an original unintrusive and nondestructive method, perfectly adapted to determine the amount of CO2 dissolved in a closed bottle of Champagne or Sparkling wine as well as the self-diffusion of CO2 in this matrix. Two Champagnes and one Sparkling wine were analyzed on a 7T imaging spectrometer. It was necessary to use an imaging spectrometer with a large available bore to insert the bottle, localize the measurement and carry out H-1 and C-13 spectroscopy with a good sensitivity. In the C-13 spectra, CO2 and alcohol signals are well separated. By use of the quantity of ethanol acting as an internal reference, it was possible to deduce the CO2 concentration. We determined the relaxation parameter T-1 of CO2. Thanks to diffusion spectroscopy, the diffusion rate of the CO2 in the closed bottle was also calculated. The method can easily be extended to study the CO2 content in closed bottles of other sparkling drinks, like beers, sodas, and fizzy waters. (c) 2004 Elsevier B.V All rights reserved.
The in vivo spectrum of regenerating muscles shows a specific cross-correlation signal assigned to the (n-3) fatty acyl chain, which peaks during the myoblast fusion phase. In order to identify the origin of this signal and to take all the lipid metabolites into account, we investigated the degeneration-regeneration process by H-1 2D NMR of lipid muscle extracts. We observed an increase in the total amount of lipids during the regeneration process, although the lipid profile did not show any drastic change during this process. The changes in the NMR signal observed in vivo and, in particular, the appearance of the specific (n-3) fatty acyl chain signal appears to arise from mobile lipid compartments located in fusing cells. (c) 2005 Published by Elsevier B.V.
Herlidou, G ; Grebe, R ; Grados, F ; Lecuyer, N ; Fardellone, F ; Meyer, ME (2004)
Influence of age and osteoporosis on calcaneus trabecular bone structure. A preliminary in vivo MRI study by quantitative texture analysis.Magnetic Resonance Imaging 22(2) 237-243
Recent developments in high-resolution MR imaging techniques have opened up new perspectives for structural characterization of trabecular bone by non-invasive methods. In this study, 3-D MR imaging was performed on 17 healthy volunteers and 6 osteoporotic patients. Two different MR sequences were used to evaluate the impact on MR acquisition on texture analysis results. Images were analyzed with four automated methods of texture analysis (grey level histogram, cooccurrence, runlength and gradient matrices) enabling quantitative analysis of grey level intensity and distribution within three different regions of interest (ROI).
Localized in vivo NMR spectroscopy, chemical shift imaging or multi-voxel spectroscopy are potentially useful tools in small animals that are complementary to MRI, adding biochemical information to the mainly anatomical data provided by imaging of water protons. However the contribution of such methods remains hampered by the low spectral resolution of the in vivo I D spectra. Two-dimensional methods widely developed for in vitro studies have been proposed as suitable approaches to overcome these limitations in resolution.
The existence of biexponential signal decay in magnetic resonance diffusion-weighted imaging appears to be independent of compartmentalizationMagnetic Resonance in Medicine 51 (2) 278-285
It is generally believed that the apparent diffusion coefficient (ADC) changes measured by diffusion-weighted imaging (DWI) in brain pathologies are related to alterations in the water compartments. The aim of this study was to elucidate the role of compartmentalization in DWI via biexponential analysis of the signal decay due to diffusion. DWI experiments were performed on mouse brain over an extended range of b-values (up to 10000 mm(-2) s) under intact, global ischemic, and cold-injury conditions. DWI was additionally applied to centrifuged human erythrocyte samples with a negligible extracellular space. Biexponential signal decay was found to occur in the cortex of the intact mouse brain.
CK flux or direct ATP transfer : Versatility of energy transfer pathways evidenced by NMR in the perfused heartMolecular and Cellular Biochemistry 256 (1-2) 43-58
How the myocardium is able to permanently coordinate its intracellular fluxes of ATP synthesis, transfer and utilization is difficult to investigate in the whole organ due to the cellular complexity. The adult myocardium represents a paradigm of an energetically compartmented cell since 50% of total CK activity is bound in the vicinity of other enzymes ( myofibrillar sarcolemmal and sarcoplasmic reticulum ATPases as well as mitochondrial adenine nucleotide translocator, ANT). Such vicinity of enzymes is well known in vitro as well as in preparations of skinned fibers to influence the kinetic properties of these enzymes and thus the functioning of the subcellular organelles. Intracellular compartmentation has often been neglected in the NMR analysis of CK kinetics in the whole organ. It is indeed a methodological challenge to reveal subcellular kinetics in a working organ by a global approach such as NMR.
Herlidou, S ; Constans, JM ; Carsin, B ; Oliviï¿1/2, D ; Eliat, PA ; Nadal-Desbarats, EL ; Gondry, C ; Le Rumeur, E ; Idy-Peretti, I ; de Certaines, JD (2003)
Texture analysis was performed in three different MRI units on T1 and T2-weighted MR images from 10 healthy volunteers and 63 patients with histologically confirmed intracranial tumors. The goal of this study was a multicenter evaluation of the usefulness of this quantitative approach for the characterization of healthy and pathologic human brain tissues (white matter, gray matter, cerebrospinal fluid, tumors and edema). Each selected brain region of interest was characterized with both its mean gray level values and several texture parameters.
Differential MRI diagnosis between brain abscesses and necrotic or cystic brain tumors using the Apparent Diffusion Coefficient and Normalized Diffusion-WeightedMagnetic Resonance Imaging 21 (6) 645-650
Magnetic Resonance Diffusion-Weighted Imaging (DWI) has been reported to be helpful for the differential diagnosis between abscesses and cystic/necrotic brain tumors. However the number of patients is still limited, and the sensitivity and specificity of the method remain to be confirmed. The primary purpose of this study was to investigate a larger sample of patients, all investigated under the same experimental conditions, in order to obtain statistically significant data. Moreover, there is no consensus about the appropriate values of b required to use to make an accurate diagnosis from DWI.
It is important to obtain high resolution images of joints for the study of disease, especially in rodent experimental models. We optimized H-1 magnetic resonance imaging three-dimensional sequences at 7 T, with lipid signal suppression, and T-1 and T-2 measurements for in vivo experiments on rat joints, in order to assess the effectiveness of high-field MRI. The method was validated by applying it to the early diagnosis of arthritis. We studied the progress of rheumatoid arthritis in an arthritic rat model. We observed the rats’ knees for 21 days after inducing arthritis. The images acquired over one hour had a high resolution of 1.75 x 10(-3) mm(3), (105 x 105 x 145 mum(3)) which allowed us to spot the early stages of joint degeneration, such as bone erosion, and to observe an apparent ’MRI’ loss of cartilage thickness, attributed to dehydration of the cartilage tissue. The MR images obtained during the early stages of rheumatoid arthritis enabled us to study joint changes accurately before any histological signs of attack were visible. Copyright (C) 2003 John Wiley Sons, Ltd.
Correia, I ; Bezzenine, N ; Ronzani, N ; Platzer, N ; Beloeil, JC ; Doan, BT (2002)
Study of inclusion complexes of acridine with ß- and (2,6-di-O-methyl)-ß-cyclodextrin by use of solubility diagrams and NMR spectroscopyJournal of Physical Organic Chemistry 15 (9) 647-659
Previous molecular modeling studies, in our laboratory, have shown that some esters of type RCOO(CH2)(n)C5H5N+Cl- are potentially active against Alzheimer’s disease. We have also demonstrated that acridine, which has strong anticholinesterase activity appears to be a suitable R substituent. The main obstacle to the possible pharmaceutical application of these compounds is their limited solubility in water, which is due to the poor aqueous solubility of acridine itself (0.26 mM). Inclusion complexation with cyclodextrins may overcome this problem.
Follow-up by one- and two-dimensional NMR of plasma from pigs with ischemia-induced acute liver failure treated with a bioartificial liverNMR in Biomedicine 15 (6) 393-403
Hepatic encephalopathy may occur following acute hepatic failure (AHF), which results in the release of toxic compounds from the injured liver. These compounds, which induce cerebral edema, are not well characterized, yet. The aim of this study was to evaluate the potential interest of NMR spectroscopy in the follow-up of different plasma compounds in pigs with ischemia-induced fulminant hepatic failure treated or not with a bioartificial liver (BAL), which has been previously shown to improve the neurological status of the animals.
The purpose of this study was to acquire a localized 2D (two-dimensional) H-1 correlation spectrum. in a volume of interest reasonably small, and within an experiment time compatible with clinical applications. A modified PRESS technique has been used. The last 180degrees pulse of the PRESS sequence has been converted into a 90degrees pulse for both refocusing and coherence transfer. 2D correlation spectroscopy was performed on healthy volunteers in a clinical magnet, at 3 T, within 34 min, for a voxel size of 27 cm(3). This result makes it possible to consider clinical applications. (C) 2002 Elsevier Science B.V. All rights reserved.
Mahmoud-Ghoneim, D ; de Certaines, JD ; Herlidou, S ; Rolland, Y ; Manière, A (2001)
The combination of localized 2D H-1 MR correlation spectroscopy and Hadamard encoding allows the simultaneous acquisition of multiple volumes of interest without an increase in the experimental duration, compared to single-voxel acquisition. In the present study, 2D correlation spectra were acquired simultaneously within 20 to 40 min in two voxels located in each hemisphere of the rat brain. An intervoxel distance of 20% of the voxel size was sufficient to limit spatial contamination. The following cerebral metabolites gave detectable crosspeaks : N-acetylaspartate, the glutamate/glutamine pool, aspartate, phosphoethanolamine, glucose, glutathione, taurine, myo-inositols, lactate, threonine, ? -aminobutyric acid, and alanine. Most of the metabolites were measured without contamination of other resonances. (C) 2001 Academic Press.
In this study, skeletal muscle degeneration-regeneration, induced by notexin (a myotoxic substance) injection is studied by Magnetic Resonance Imaging (MRI) and by histological cuts (hematoxylin/eosin and Evans blue colorations). Comparison bem een MR images and histological cuts results (necrosis, myoblasts replication and fusion) permits MR images interpretation. From day 0 to day 13 after notexin injection, injected zone images are very different from those realized in healthy muscles. Preliminary localized 1D H-1-MRS (Magnetic Resonance Spectroscopy) study is also reported. (C) 2001 Academie des sciences/Editions scientifiques et medicales Elsevier SAS.
Cardiac creatine kinase metabolite compartments revealed by NMR magnetization transfer spectroscopy and subcellular fractionationBiochemistry 40 (7) 2129-2137
In the perfused rat heart NMR inversion transfer revealed the existence of a compartment of ATP not exchanging through creatine kinase (CK), as demonstated by an apparent discrepancy between the forward (F-f) and reverse (F-r) CK flux if this compartment was neglected in the analysis [Joubert et al, (2000) Biophys. J. 79, 1-13], To localize this compartment, CK fluxes were measured by inversion of PCr (inv-PCr) or ? ATP (inv-ATP), and the distribution of metabolites between mitochondria and cytosol was studied by subcellular fractionation. Physiological conditions were designed to modify the concentration and distribution of CK metabolites (control, adenylate depletion, inhibition of respiration, KCl arrest). Depending on cardiac activity, mitochondrial ATP (mito-ATP) assessed by fractionation varied from 11% to 30% of total ATP. In addition, the apparent flux discrepancy increased together with mito-ATP (F-f/F-r ranged from 0.85 to 0.50 in inv-PCr and from 1.13 to 1.88 in inv-ATP), Under conditions masking the influence of the ATP-P-i exchange on CK flux, the ATP compartment could be directly quantified by the apparent flux discrepancy ; its size was similar to that of mito-ATP measured by fractionation. Thus NMR inversion technique is a potential tool to assess metabolite compartmentation in the whole organ.
Wrynn, AS ; Franconi, F ; de Certaines, JD ; Herlidou, S ; Gandon, JM ; Leonard, BE (2000)
Evidence for myocardial ATP compartmentation from NMR inversion transfer analysis of creatine kinase fluxesBiophysical Journal 79 (1) 1-13
The interpretation of creatine kinase (CK) flux measured by P-31 NMR magnetization transfer in vivo is complex because of the presence of competing reactions, metabolite compartmentation, and CK isozyme localization. In the isovolumic perfused rat heart, we considered the influence of both ATP compartmentation and ATP-P-i exchange on the forward (F-f : PCr —> ATP) and reverse (F-r) CK fluxes derived from complete analysis of inversion transfer. Although F-f should equal F-r because of the steady state, in both protocols when PCr (inv-PCr) or ATP (inv-ATP) was inverted and the contribution of ATP-P-i was masked by saturation of P-i (sat-P-i), F-f/F-r significantly differed from 1 (0.80 +/- 0.06 or 1.32 +/- 0.06, respectively, n = 5). These discrepancies could be explained by a compartment of ATP (f(ATP)) not involved in CK. Consistently, neglecting ATP compartmentation in the analysis of CK in vitro results in an underestimation of F-f/F-r for inv-PCr and its overestimation for inv-ATP. Both protocols gave access to f(ATP) if the system was adequately analyzed. The fraction of ATP not involved in CK reaction in a heart performing medium work amounts to 20-33% of cellular ATP. Finally, the data suggest that the effect of sat-P-i might not result only from the masking of ATP-P-i exchange.