Search

A simple and rapid HPLC-MS/MS method was developed and validated for simultaneous measurement of phosphocreatine and its metabolites creatine and creatinine in children's plasma. A 50 μL aliquot of plasma was prepared by protein precipitation with acetonitrile-water (1000 μL, 1:1, v/v) followed by separation on a Hypersil Gold C 18 column (35°C) with gradient mobile phase consisting of 2 mm ammonium acetate aqueous solution (pH 10) and methanol at a flow rate of 0.3 mL/min and analyzed by mass spectrometry in both positive (phosphocreatine) and negative (creatine and creatinine) ion multiple reaction monitoring mode. Good linearity (r > 0.99) was obtained for the three analytes. The intra-day and inter-day values of CV were <5.46% (-13.09% ≤ RE ≤ 2.57%). The average recoveries of the three analytes were 70.9-97.5%. No obvious impact was found for the quantitation of three analytes in normal, hemolyzed and hyperlipemic plasma. In the end, this method was successfully applied to a pharmacokinetic study of phosphocreatine in children (six cases) with viral myocarditis of children after intravenous infusion of 2 g of the test drug. The pharmacokinetc parameters of phosphocreatine/creatine were as follows: t 1/2 0.24/0.83 h, T max 0.49/0.55 h, C max 47.34/59.29 μg/mL, AUC last 17.07/59.63 h μg/mL, AUC inf 17.16/79.01 h μg/mL and MRT 0.29/0.67 h., (© 2019 John Wiley & Sons, Ltd.)

Creatine transporter deficiency (CTD) is caused by a defect in the X-linked creatine transporter SLC6A8 gene leading to severe neurologic and physiologic conditions. Cyclocreatine and phosphocyclocreatine supplementation is seen as a potential treatment, but the presence of these compounds within commercially available dietary supplements presents the risk of self-medication. High-performance liquid chromatography-mass spectrometry (HPLC-MS) is an excellent technique to assess composition of complex amino acid mixtures. Herein, we have developed a facile HPLC-MS method using a cyano column in hydrophilic interaction liquid chromatography (HILIC) mode with isocratic elution over 4 min to identify the main components of two commercially available dietary supplements. The relative standard deviation (RSD) for retention time and extracted ion integrated area are <0.3% and 4%, respectively, showing excellent reproducibility. Cyclocreatine and phosphocyclocreatine were not detectable within the dietary supplements, even at ppm levels, demonstrating the power and importance of the developed HPLC-MS method in analyzing complex mixtures.

Purpose: To determine the exchange parameters for the CEST of phosphocreatine (PCrCEST) in phantoms and to characterize PCrCEST in vivo in the muscle at different saturation powers and magnetic fields., Methods: Exchange parameters were measured in PCr solutions using varying saturation power at 15.2 T. Z-spectra were analyzed using multipool Lorentzian fitting in the hindlimb using various powers at 2 different fields: 9.4 T and 15.2 T. Modulation of PCr signal in PCrCEST and phosphorus MRS was observed in the mouse hindlimb before and after euthanasia., Results: The exchange rate of PCr at physiological pH in phantoms was confirmed to be in a much slower exchange regime compared with Cr: k ex at pH 7.3 and below was less than 400 s -1 . There was insufficient signal for detection of PCrCEST in the brain, but PCrCEST in the hindlimb was measured to be 2.98% ± 0.43 at a B 1 of 0.47 μT at 15.2 T, which is 29% higher than 9.4T values. The value of PCrCEST at a B 1 of 0.71 μT was not significantly different than that measured at a B 1 of 0.47 μT. After euthanasia, PCrCEST signal dropped by 82.3% compared with an 85% decrease in PCr in phosphorus MRS, whereas CrCEST signal increased by 90.6%., Conclusion: The PCrCEST technique has viable sensitivity in the muscle at high fields and shows promise for the study of metabolic dysfunction and cardiac systems., (© 2019 International Society for Magnetic Resonance in Medicine.)

Purpose: To develop a high temporal resolution imaging method that measures muscle-specific phosphocreatine (PCr) resynthesis time constant (τ PCr ) and pH changes in muscles of the lower leg following exercise on a clinical 3T MRI scanner., Methods: We developed a frequency-selective 3D non-Cartesian FLORET sequence to measure PCr with 17-mm nominal isotropic resolution (28 mm actual resolution) and 6-s temporal resolution to capture dynamic metabolic muscle activity. The sequence was designed to additionally collect inorganic phosphate spectra for pH quantification, which were localized using sensitivity profiles of individual coil elements. Nineteen healthy volunteers were scanned while performing a plantar flexion exercise on an in-house developed ergometer. Data were acquired with a dual-tuned multichannel coil array that enabled phosphorus imaging and proton localization for muscle segmentation., Results: After a 90-s plantar flexion exercise at 0.66 Hz with resistance set to 40% of the maximum voluntary contraction, τ PCr was estimated at 22.9 ± 8.8 s (mean ± standard deviation) with statistical coefficient of determination r 2  = 0.89 ± 0.05. The corresponding pH values after exercise were in the range of 6.9-7.1 in the gastrocnemius muscle., Conclusion: The developed technique allows measurement of muscle-specific PCr resynthesis kinetics and pH changes following exercise, with a temporal resolution and accuracy comparable to that of single voxel 31 P-MRS sequences. Magn Reson Med 79:974-980, 2018. © 2017 International Society for Magnetic Resonance in Medicine., (© 2017 International Society for Magnetic Resonance in Medicine.)

Purpose: The metabolites phosphocreatine (PCr), adenosine triphosphate (ATP), and in-organic phosphate (Pi) are biochemically coupled. Their pool sizes, assessed by their magnetization ratios, have been extensively studied and reflect bioenergetics status in vivo. However, most such studies have ignored chemical exchange and T 1 relaxation effects. In this work, we aimed to extend the T1nom method to simultaneously quantify the reaction rate constants as well as phosphorus metabolite pool size ratios under partially relaxed conditions., Materials and Methods: Modified Bloch-McConnell equations were used to simulate the effects of chemical exchanges on T 1 relaxation times and magnetization ratios among PCr, γ-ATP, and Pi. The T1nom method with iteration approach was used to measure both reaction constants and metabolite pool size ratios. To validate our method, in vivo data from rat brains (N = 8) at 9.4 Tesla were acquired under two conditions, i.e., approximately full relaxation (TR = 9 s) and partial relaxation (TR = 3 s). We compared metabolite pool size ratios and reaction constants before and after correcting the chemical exchange and T 1 relaxation effects., Results: There were significant errors in underestimation of PCr/γATP by 12 % (P = 0.03) and overestimation of ATP/Pi ratios by 14 % (P = 0.04) when not considering chemical exchange effects. These errors were minimized using our iteration approach, resulting in no significant differences (PCr/γATP, P = 0.47; ATP/Pi, P = 0.81) in metabolite pool size ratios and reaction constants between the two measurements (i.e., short versus long TR conditions)., Conclusion: Our method can facilitate broad biomedical applications of 31 P magnetization saturation transfer spectroscopy, requiring high temporal and/or spatial resolution for assessment of altered bioenergetics., Level of Evidence: 2 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;47:210-221., (© 2017 International Society for Magnetic Resonance in Medicine.)

The purpose of this work was to develop a 31 P spectroscopic magnetic resonance fingerprinting (MRF) method for fast quantification of the chemical exchange rate between phosphocreatine (PCr) and adenosine triphosphate (ATP) via creatine kinase (CK). A 31 P MRF sequence (CK-MRF) was developed to quantify the forward rate constant of ATP synthesis via CK ( kfCK), the T 1 relaxation time of PCr ( T1PCr), and the PCr-to-ATP concentration ratio ( MRPCr). The CK-MRF sequence used a balanced steady-state free precession (bSSFP)-type excitation with ramped flip angles and a unique saturation scheme sensitive to the exchange between PCr and γATP. Parameter estimation was accomplished by matching the acquired signals to a dictionary generated using the Bloch-McConnell equation. Simulation studies were performed to examine the susceptibility of the CK-MRF method to several potential error sources. The accuracy of nonlocalized CK-MRF measurements before and after an ischemia-reperfusion (IR) protocol was compared with the magnetization transfer (MT-MRS) method in rat hindlimb at 9.4 T (n = 14). The reproducibility of CK-MRF was also assessed by comparing CK-MRF measurements with both MT-MRS (n = 17) and four angle saturation transfer (FAST) (n = 7). Simulation results showed that CK-MRF quantification of kfCK was robust, with less than 5% error in the presence of model inaccuracies including dictionary resolution, metabolite T 2 values, inorganic phosphate metabolism, and B 1 miscalibration. Estimation of kfCK by CK-MRF (0.38 ± 0.02 s -1 at baseline and 0.42 ± 0.03 s -1 post-IR) showed strong agreement with MT-MRS (0.39 ± 0.03 s -1 at baseline and 0.44 ± 0.04 s -1 post-IR). kfCK estimation was also similar between CK-MRF and FAST (0.38 ± 0.02 s -1 for CK-MRF and 0.38 ± 0.11 s -1 for FAST). The coefficient of variation from 20 s CK-MRF quantification of kfCK was 42% of that by 150 s MT-MRS acquisition and was 12% of that by 20 s FAST acquisition. This study demonstrates the potential of a 31 P spectroscopic MRF framework for rapid, accurate and reproducible quantification of chemical exchange rate of CK in vivo., (Copyright © 2017 John Wiley & Sons, Ltd.)

In this study, graphene oxide and creatine phosphate disodium acted as dual template and was employed to synthesize graphene oxide (GO)/hydroxyapatite (HA) hybrids as drug carriers. In the rapid preparation of GO/HA hybrids, creatine phosphate disodium salt (CPDS) severed as a phosphorus source and graphene oxide acted as a template in aqueous solution. The effects of the reaction temperature, time and pH value of the aqueous solution on the morphology of the product were investigated. The result showed that the hydrolysis of CPDS under hydrothermal condition played an important role in the formation of hierarchical hollow GO/HA hybrids. The GO nanosheets provided reactive sites for the binding of HA nanoparticles and absorbing ibuprofen (IBU) molecules. The GO/HA hybrids had ideal sustained drug-release behavior. It indicated that the prepared GO/HA hybrids may be promising materials for applications in biomedical area., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Eukaryotic RNA processing steps during mRNA maturation present the cell with opportunities for gene expression regulation. One such step is the pre-mRNA 3' cleavage reaction, which defines the downstream end of the 3' untranslated region and, in nearly all mRNA, prepares the message for addition of the poly(A) tail. The in vitro reconstitution of 3' cleavage provides an experimental means to investigate the roles of the various multi-subunit cleavage factors. Anion-exchange chromatography is the simplest procedure for separating the core mammalian cleavage factors. Here we describe a method for optimizing the in vitro reconstitution of 3' cleavage activity from the DEAE-sepharose separated HeLa cleavage factors and show how to ensure, or avoid, dependence on creatine phosphate. Important reaction components needed for optimal processing are discussed. We also provide an optimized procedure for preparing small-scale HeLa nuclear extracts from adherent cells for use in 3' cleavage in vitro.

Magnesium phosphate materials have aroused interest of researchers in recent years and are promising for biomedical applications due to their good biocompatibility and biodegradability. In this work, we report the microwave-hydrothermal rapid synthesis of magnesium phosphate pentahydrate nanosheets (MPHSs) using biocompatible creatine phosphate as an organic phosphorus source. This method is facile, rapid, surfactant-free and environmentally friendly. The as-prepared MPHSs have an obvious pH-dependent dissolution performance which can be used as an ideal pH-responsive nanocarrier for drug and gene delivery. Moreover, the MPHSs have a good cytocompatibility and a high ability to promote osteoblast MC-3T3 adhesion and spreading, as well as a relatively high protein adsorption ability using hemoglobin (Hb) as a model protein. Thus, the MPHSs are promising for the applications in biomedical fields such as protein adsorption and bone regeneration., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [(3)H]thymidine or [(3)H]TMP as precursors of [(3)H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [(3)H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [(3)H]TMP produced far less [(3)H]TTP than the amount observed with [(3)H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [(3)H]TTP from [(3)H]TMP was effectively blocked, demonstrating that synthesis of [(3)H]TTP from [(3)H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [(3)H]TMP to [(3)H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [(3)H]thymidine was readily converted to [(3)H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein oxidation has detrimental effects on the brain functioning, which involves inhibition of the crucial enzyme, brain type creatine kinase (CKBB), responsible for the CK/phosphocreatine shuttle system. Here we demonstrate a susceptibility of CKBB to several ordinary stressors. In our study enzymatic activity of purified recombinant brain-type creatine kinase was evaluated. We assayed 30 nMconcentration of CKBB under normal and stress conditions. In the direction of phosphocreatine formation hydrogen peroxide and heat treatments altered CKBB activity down to 26 and 14%, respectively. Also, examination of immunoblotted membrane patterns by SDS-PAGE electrophoresis and western blot analysis showed a decrease in expression levels of intrinsic CKBB enzyme in HeLa andA549 cells. Hence, our results clearly show that cytosolic CKBB is extremely sensitive to oxidative stress and heat induced inactivation. Therefore, due to its susceptibility, this enzyme may be defined as a potential target in brain damage.

Creatine phosphate sodium (CPS) salt is a first-line cardiovascular drug for severe diastolic heart failure. The drug exists in different hydrate forms. The marketed drug form was determined as CPS·4.5H2 O (H1); however, the reference standard was supplied as CPS·6H2 O (H2). In this work, we present two newly identified hydrate forms: a thermodynamically stable low hydrate form, CPS·1.5H2 O (H3), and a pressure-sensitive transit form, CPS·7H2 O (H4). The hydrate forms were discovered through a comprehensive solid-state screening experiment and fully characterized using a range of analytical techniques including X-ray powder diffraction (XRPD), FTIR, Raman spectroscopy, hot-stage microscopy (HSM), thermogravimetric analysis, and differential scanning calorimetry. Stability tests revealed that H3 was the most stable hydrate under thermal stimulation. H4 is a pressure-sensitive hydrate and easily transforms to H2 and then H1 upon grinding. The form transformation process was closely monitored using the HSM, variable-temperature XRPD (VT-XRPD), and VT-Raman spectroscopy techniques. Specifically, the transformation of H4 to H1 is characterized in a single-crystal-to-single-crystal transformation process. The newly discovered hydrate form H3 has superior physicochemical properties than the marketed forms and is worthy of further development., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Phosphocreatine is a phosphorylated creatine molecule synthesized in the liver and transported to muscle cells where it is used for the temporary storage of energy. In Alzheimer's disease, the capture of glucose by cells is impaired, which negatively affects the Krebs cycle, leading to problems with the generation of phosphocreatine. Furthermore, the creatine-phosphocreatine system, regulated by creatine kinase, is affected in the brains of Alzheimer's disease patients. Aluminum ions are associated with Alzheimer's disease. Al(III) decreases cell viability and increases the fluidity of the plasma membrane, profoundly altering cell morphology. In this study, one of the complexes formed by Al(III) and phosphocreatine in aqueous solution was investigated by potentiometry, (31)P and (27)Al NMR, Raman spectroscopy and density functional theory (DFT) calculations. The log KAlPCr value was 11.37±0.03. Phosphocreatine should act as a tridentate ligand in this complex. The (27)Al NMR peak at 48.92ppm indicated a tetrahedral molecule. The fourth position in the arrangement was occupied by a coordinated water molecule. Raman spectroscopy, (31)P NMR and DFT calculations (DFT:B3LYP/6-311++G(**)) indicated that the donor atoms are oxygen in the phosphate group, the nitrogen of the guanidine group and the oxygen of the carboxylate group. Mulliken charges, NBO charges, frontier molecular orbitals, electrostatic potential contour surfaces and mapped electrostatic potential were also examined., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objectives: Retrograde cerebral perfusion (RCP) has been employed to protect the brain during cardiovascular surgery, requiring temporary hypothermic circulatory arrest (HCA). However, the protocol used for RCP remains to be modified if prolonged HCA is expected. The aim of this study was to determine the efficacy of a modified protocol for this purpose., Methods: After establishment of HCA at 15°C, 14 pigs were subjected to 90-min RCP using either the conventional protocol (i.e. alpha-stat strategy, 25-mmHg perfusion pressure and occluded inferior vena cava, Group I, n = 7) or the new protocol (i.e. pH-stat strategy, 40-mmHg perfusion pressure and unoccluded inferior vena cava, Group II, n = 7). After being rewarmed to 37°C, pigs were perfused for another 60 min. Phosphorus-31 magnetic resonance spectroscopy was used to track the changes of brain high-energy phosphates [i.e. adenosine triphosphate and phosphocreatine (PCr)] and intracellular pH (pHi). At the end, brain water content was measured., Results: During RCP, high-energy phosphates decreased in both groups, whereas adenosine triphosphate decreased much faster in Group I (10.4 ± 4.3 vs 30.4 ± 4.4% of the baseline, P = 0.007, 60-min RCP). After rewarming, the recovery of high-energy phosphates and pHi was much slower in Group I (PCr: 55.7 ± 9.1 vs 78.4 ± 5.1% of the baseline, P = 0.046; adenosine triphosphate: 26.6 ± 10.6 vs 64.8 ± 4.6% of the baseline, P = 0.007; pHi: 6.5 ± 0.4 vs 7.1 ± 0.1, P = 0.021 at 30-min normothermic perfusion after rewarming). Brain tissue water content was significantly higher in Group I (81.1 ± 0.4 vs 79.5 ± 0.4%, P = 0.016)., Conclusions: Application of the modified RCP protocol significantly improved cerebral energy conservation during HCA and accelerated energy recovery after rewarming.

Hierarchically nanostructured porous hollow microspheres of hydroxyapatite (HAP) are a promising biomaterial, owing to their excellent biocompatibility and porous hollow structure. Traditionally, synthetic hydroxyapatite is prepared by using an inorganic phosphorus source. Herein, we report a new strategy for the rapid, sustainable synthesis of HAP hierarchically nanostructured porous hollow microspheres by using creatine phosphate disodium salt as an organic phosphorus source in aqueous solution through a microwave-assisted hydrothermal method. The as-obtained products are characterized by powder X-ray diffraction (XRD), Fourier-transform IR (FTIR) spectroscopy, SEM, TEM, Brunauer-Emmett-Teller (BET) nitrogen sorptometry, dynamic light scattering (DLS), and thermogravimetric analysis (TGA). SEM and TEM micrographs show that HAP hierarchically nanostructured porous hollow microspheres consist of HAP nanosheets or nanorods as the building blocks and DLS measurements show that the diameters of HAP hollow microspheres are within the range 0.8-1.5 μm. The specific surface area and average pore size of the HAP porous hollow microspheres are 87.3 m(2) g(-1) and 20.6 nm, respectively. The important role of creatine phosphate disodium salt and the influence of the experimental conditions on the products were systematically investigated. This method is facile, rapid, surfactant-free and environmentally friendly. The as-prepared HAP porous hollow microspheres show a relatively high drug-loading capacity and protein-adsorption ability, as well as sustained drug and protein release, by using ibuprofen as a model drug and hemoglobin (Hb) as a model protein, respectively. These experiments indicate that the as-prepared HAP porous hollow microspheres are promising for applications in biomedical fields, such as drug delivery and protein adsorption., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Tellurium has been used as an industrial component of many alloys and in the electronic industry. Organotellurium compounds can cause poisoning which leads to neurotoxic symptoms such as significant impairment of learning, spatial memory and are potentially neurotoxic to human beings. However, the molecular mechanisms of neurotoxicity of organotellurium compounds are not well understood. Considering that creatine kinase plays a key role in energy metabolism of tissues with intermittently high and fluctuating energy requirements, such as nervous tissue, the main objective of this study was to investigate the mechanisms by which 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one inhibit creatine kinase activity, a key enzyme of energy homeostasis, in the cerebral cortex of 30-day-old Wistar rats. For the kinetic studies, the Lineweaver-Burk plot was used to characterize the mechanisms of enzyme inhibition by 3-butyl-1-phenyl-2-(phenyltelluro)oct-en-1-one. The results suggested that this compound inhibits creatine kinase activity by two different mechanisms: competition with ADP and oxidation of critical sulfhydryl groups for the functioning of the enzyme. The potential for inhibition of creatine kinase to occur in vivo may contribute to the neurotoxicity observed by this organochaocogen., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

A broad spectrum of beneficial effects has been ascribed to creatine (Cr), phosphocreatine (PCr) and their cyclic analogues cyclo-(cCr) and phospho-cyclocreatine (PcCr). Cr is widely used as nutritional supplement in sports and increasingly also as adjuvant treatment for pathologies such as myopathies and a plethora of neurodegenerative diseases. Additionally, Cr and its cyclic analogues have been proposed for anti-cancer treatment. The mechanisms involved in these pleiotropic effects are still controversial and far from being understood. The reversible conversion of Cr and ATP into PCr and ADP by creatine kinase, generating highly diffusible PCr energy reserves, is certainly an important element. However, some protective effects of Cr and analogues cannot be satisfactorily explained solely by effects on the cellular energy state. Here we used mainly liposome model systems to provide evidence for interaction of PCr and PcCr with different zwitterionic phospholipids by applying four independent, complementary biochemical and biophysical assays: (i) chemical binding assay, (ii) surface plasmon resonance spectroscopy (SPR), (iii) solid-state (31)P-NMR, and (iv) differential scanning calorimetry (DSC). SPR revealed low affinity PCr/phospholipid interaction that additionally induced changes in liposome shape as indicated by NMR and SPR. Additionally, DSC revealed evidence for membrane packing effects by PCr, as seen by altered lipid phase transition. Finally, PCr efficiently protected against membrane permeabilization in two different model systems: liposome-permeabilization by the membrane-active peptide melittin, and erythrocyte hemolysis by the oxidative drug doxorubicin, hypoosmotic stress or the mild detergent saponin. These findings suggest a new molecular basis for non-energy related functions of PCr and its cyclic analogue. PCr/phospholipid interaction and alteration of membrane structure may not only protect cellular membranes against various insults, but could have more general implications for many physiological membrane-related functions that are relevant for health and disease.

Ternary systems of Cu(II) with phosphocreatine (PCr) and the polyamines (PAs), ethylenediamine (en), 1,3-diaminopropane (tn), putrescine (Put), spermidine (Spd), and spermine (Spm), were investigated in aqueous solution through potentiometry, ultraviolet-visible, EPR and Raman spectroscopy. The binary complex CuPCr was also studied by Raman spectroscopy, and the calculation of the minimum stabilization energy was done assuming this molecule in aqueous solution. The stability constants of the CuPCrPA ternary complexes were determined by potentiometry (T=25°C, I=0.1 mol L(-1), KNO(3)). The stability order determined was CuPCrSpm>CuPCrSpd>CuPCren>CuPCrtn>CuPCrPut, the same order of the corresponding binary complexes of Cu(II) with these polyamines. The evaluation of intramolecular PA-PCr interactions in protonated and deprotonated species of ternary complexes was carried out using the equation Δlog K=log β(CuPCrPAHq+p)-(log β(CuPAHq)+log β(CuPCrHp)). All of the CuPCrPA ternary complexes have a square planar structure and are bonded to PCr through the nitrogen atom of the guanidine group and the oxygen atom of the phosphate group, and to the PAs through two nitrogen atoms of the amine groups. The structure of the complex CuPCrSpm is planar with distortion towards tetrahedral. Calculation of the minimum stabilization energy for the CuPCr and CuPCrenH complexes confirmed the proposed coordination mode., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: The few studies applying single-voxel ¹H spectroscopy in children and adolescents with bipolar disorder (BD) have reported low N-acetyl-aspartate (NAA) levels in the dorsolateral prefrontal cortex (DLPFC), and high myo-inositol / phosphocreatine plus creatine (PCr+Cr) ratios in the anterior cingulate. The aim of this study was to evaluate NAA, glycerophosphocholine plus phosphocholine (GPC+PC) and PCr+Cr in various frontal cortical areas in children and adolescents with BD. We hypothesized that NAA levels within the prefrontal cortex are lower in BD patients than in healthy controls, indicating neurodevelopmental alterations in the former., Method: We studied 43 pediatric patients with DSM-IV BD (19 female, mean age 13.2 ± 2.9 years) and 38 healthy controls (19 female, mean age 13.9 ± 2.7 years). We conducted multivoxel in vivo ¹H spectroscopy measurements at 1.5 Tesla using a long echo time of 272 ms to obtain bilateral metabolite levels from the medial prefrontal cortex (MPFC), DLPFC (white and gray matter), cingulate (anterior and posterior), and occipital lobes. We used the nonparametric Mann-Whitney U test to compare neurochemical levels between groups., Results: In pediatric BD patients, NAA and GPC+PC levels in the bilateral MPFC, and PCr+Cr levels in the left MPFC were lower than those seen in the controls. In the left DLPFC white matter, levels of NAA and PCr+Cr were also lower in BD patients than in controls., Conclusions: Lower NAA and PCr+Cr levels in the PFC of children and adolescents with BD may be indicative of abnormal dendritic arborization and neuropil, suggesting neurodevelopmental abnormalities., (Copyright © 2011 American Academy of Child and Adolescent Psychiatry. Published by Elsevier Inc. All rights reserved.)

The metabolic phenotype of the failing heart includes a decrease in phosphocreatine and total creatine concentration [Cr], potentially contributing to contractile dysfunction. Surprisingly, in 32- week-old mice over-expressing the myocardial creatine transporter (CrT-OE), we previously demonstrated that elevated [Cr] correlates with left ventricular (LV) hypertrophy and failure. The aim of this study was to determine the temporal relationship between elevated [Cr] and the onset of cardiac dysfunction and to screen for potential molecular mechanisms. CrT-OE mice were compared with wild-type (WT) littermate controls longitudinally using cine-MRI to measure cardiac function and single-voxel (1)H-MRS to measure [Cr] in vivo at 6, 16, 32, and 52 weeks of age. CrT-OE mice had elevated [Cr] at 6 weeks (mean 1.9-fold), which remained constant throughout life. Despite this increased [Cr], LV dysfunction was not apparent until 16 weeks and became more pronounced with age. Additionally, LV tissue from 12 to 14 week old CrT-OE mice was compared to WT using 2D difference in-gel electrophoresis (DIGE). These analyses detected a majority of the heart's metabolic enzymes and identified seven proteins that were differentially expressed between groups. The most pronounced protein changes were related to energy metabolism: alpha- and beta-enolase were selectively decreased (p<0.05), while the remaining enzymes of glycolysis were unchanged. Consistent with a decrease in enolase content, its activity was significantly lower in CrT-OE hearts (in WT, 0.59+/-0.02 micromol ATP produced/microg protein/min; CrT-OE, 0.31+/-0.06; p<0.01). Additionally, anaerobic lactate production was decreased in CrT-OE mice (in WT, 102+/-3 micromol/g wet myocardium; CrT-OE, 78+/-13; p=0.02), consistent with decreased glycolytic capacity. Finally, we found that enolase may be regulated by increased expression of the beta-enolase repressor transcription factor, which was significantly increased in CrT-OE hearts. This study demonstrates that chronically increased myocardial [Cr] in the CrT-OE model leads to the development of progressive hypertrophy and heart failure, which may be mediated by a compromise in glycolytic capacity at the level of enolase., (Published by Elsevier Ltd.)

During low-to-high work transition in adult mammalian heart in vivo the concentrations of free ADP, ATP, PCr (phosphocreatine), P(i) and NADH are essentially constant, in striking contrast with skeletal muscle. The direct activation by calcium ions of ATP usage and feedback activation of ATP production by ADP (and P(i)) alone cannot explain this perfect homoeostasis. A comparison of the response to adrenaline (increase in rate-pressure product and [PCr]) of the intact beating perfused rat heart with the elasticities of the PCr producer and consumer to PCr concentration demonstrated that both the ATP/PCr-producing block and ATP/PCr-consuming block are directly activated to a similar extent during physiological heart activation. Our finding constitutes a direct evidence for the parallel-activation mechanism of the regulation of oxidative phosphorylation in heart postulated previously in a theoretical way.

Proton NMR spectra of freshly isolated human skeletal muscle samples contain creatine and phosphocreatine resonances with distinct chemical shifts that are easily visualized with magic angle spinning (MAS, spinning the sample rapidly at 54.7 degrees with respect to the magnetic field) methods. The identification of the phosphocreatine resonance was based on two findings: that (i) the possible small dipolar coupling does not contribute to line splitting under rapid MAS, and (ii) the 1H signal decreases concurrently with the phosphocreatine resonance observed in 31P NMR experiments. In the MAS 1H spectra, the phosphocreatine resonance remains a singlet with a linewidth of less than 3 Hz. The creatine resonances are split into two peaks with linewidths at half height of approximately 2 and 6 Hz, respectively. The resonance with the broader linewidth represents creatine that is significantly motion-restricted and suggests that a creatine pool in muscle tissue is highly compartmentalized., (Copyright (c) 2008 Wiley-Liss, Inc.)

Incubation of rat brain mitochondria with ubiquitin and ATP followed by subsequent mitochondria sedimentation was accompanied by reduction of ubiquitin content in the supernatant. This decrease was more pronounced in the presence of ATP-regenerating system in the incubation medium (creatine phosphate/creatine phosphokinase). This ubiquitin incorporation into brain mitochondria observed only in the presence of ATP in the incubation medium increased sensitivity of monoamine oxidases (MAO) A and B to proteolytic inactivation by trypsin and papain, respectively. (Ubiquitin did not influence sensitivity of MAO B to trypsin and MAO A to papain). The data obtained suggest that ubiquitin incorporation into rat brain mitochondria increases susceptibility of MAOs to certain exogenous proteases, however, it remains unclear whether these changes stem from direct MAO-ubiquitin conjugation or reflect alterations in the membrane environment of these enzymes.

In this review, we summarize the main structural and functional data on the role of the phosphocreatine (PCr)--creatine kinase (CK) pathway for compartmentalized energy transfer in cardiac cells. Mitochondrial creatine kinase, MtCK, fixed by cardiolipin molecules in the vicinity of the adenine nucleotide translocator, is a key enzyme in this pathway. Direct transfer of ATP and ADP between these proteins has been revealed both in experimental studies on the kinetics of the regulation of mitochondrial respiration and by mathematical modelling as a main mechanism of functional coupling of PCr production to oxidative phosphorylation. In cells in vivo or in permeabilized cells in situ, this coupling is reinforced by limited permeability of the outer membrane of the mitochondria for adenine nucleotides due to the contacts with cytoskeletal proteins. Due to these mechanisms, at least 80% of total energy is exported from mitochondria by PCr molecules. Mathematical modelling of intracellular diffusion and energy transfer shows that the main function of the PCr-CK pathway is to connect different pools (compartments) of ATP and, by this way, to overcome the local restrictions and diffusion limitation of adenine nucleotides due to the high degree of structural organization of cardiac cells.

In vivo magnetic resonance spectroscopy (MRS) studies of glial brain tumours reported that higher grade of astrocytoma is associated with increased level of choline-containing compounds (Cho) and decreased levels of N-acetylaspartate (NAA) and creatine and phosphocreatine (Cr). In this work, we studied the metabolism of glioma tumours by in vitro proton magnetic resonance spectroscopy (1H-MRS). 1H-MR spectra were recorded in vitro from perchloric acid extracts of astrocytoma (WHO II) and glioblastoma multiforme (WHO IV) samples. We observed differences between astrocytoma and glioblastoma multiforme in the levels of Cho, alanine, lactate, NAA, and glutamate/glutamine. In astrocytoma samples, we found higher MR signal of NAA and lower signal of Cho and alanine. MR spectra of glioblastoma samples reported significantly higher levels of lactate and glutamate/glutamine. In contrast, levels of Cr were the same in both tumour types. We also determined NAA/Cr and Cho/Cr ratios in the tumour samples. The NAA/Cr ratio was higher in astrocytomas than in glioblastomas multiforme. Conversely, the Cho/Cr ratio was higher in glioblastoma multiforme. The results indicate that MRS is a promising method for distinguishing pathologies in human brain and for pre-surgical grading of brain tumours.

Six fully conserved arginine residues (R129, R131, R235, R291, R319, and R340) closely grouped in the nucleotide binding site of rabbit muscle creatine kinase (rmCK) were mutated; four to alanine and all six to lysine. Kinetic analyses in the direction of phosphocreatine formation showed that all four alanine mutants led to substantial losses of activity with three (R129A, R131A, and R235A) having no detectable activity. All six lysine mutants retained variable degrees of reduced enzymatic activity. Static quenching of intrinsic tryptophan fluorescence was used to measure the binding constants for MgADP and MgATP. Nucleotide binding was at most only modestly affected by mutation of the arginine residues. Thus, the cluster of arginines seem to be primarily responsible for transition state stabilization which is further supported by the observation that none of the inactive mutants demonstrated the ability to form a transition analogue complex of MgADP.nitrate.creatine as determined by fluorescence quenching assays. As a whole, the results suggest that the most important role these residues play is to properly align the substrates for stabilization of the phosphoryl transfer reaction.

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients., ((c) 2005 Elsevier Ltd. All rights reserved.)

We previously established that activation of the pyruvate dehydrogenase complex (PDC) using dichloroacetate (DCA) reduced the reliance on substrate-level phosphorylation (SLP) at the onset of exercise, with normal and reduced blood flow. PDC activation also reduced fatigue development during contraction with reduced blood flow. Since these observations, several studies have re-evaluated our observations. One study demonstrated a performance benefit without a reduction in SLP, raising a question mark over PDC's role in the regulation of ATP regeneration and our interpretation of fatigue mechanisms. Using a model of muscle contraction similar to the conflicting study (i.e. tetanic rather than twitch stimulation), we re-examined this question. Using canine skeletal muscle, one group was infused with saline while the other was pretreated with 300 mg (kg body mass)(-1) DCA. Muscle biopsies were taken at rest, peak tension (1 min) and after 6 min of tetanic electrical stimulation (75 ms on-925 ms off per second) and blood flow was limited to 25% of normal values observed during contraction. DCA reduced phosphocreatine (PCr) degradation by 40% during the first minute of contraction, but did not prevent the almost complete depletion of PCr stores at 6 min, while muscle fatigue did not differ between the two groups. During intermittent tetanic stimulation PCr degradation was 75% greater than with our previous 3 Hz twitch contraction protocol, despite a similar rate of oxygen consumption at 6 min. Thus, in the present study enhanced acetyl group availability altered the time course of PCr utilization but did not prevent the decline towards depletion. Consistent with our earlier conclusions, DCA pretreatment reduces muscle fatigue only when SLP is attenuated. The present study and our met-analysis indicates that enhanced acetyl group availability results in a readily measurable reduction in SLP when the initial rate of PCr utilization is approximately 1 mmol (kg dry mass)(-1) s(-1) or less (depending on intrinsic mitochondrial capacity). When measured early during an uninterrupted period of muscle contraction, acetyl group availability is likely to influence SLP under any condition where mitochondria are responsible for a significant proportion of ATP regeneration.

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k(cat) = 5.5 s(-1), K(1/2,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-1)s(-1), followed immediately by ATP hydrolysis at 15 s(-1). ATP-dependent Mt.Eg5 dissociation was relatively slow and rate-limiting at 8 s(-1) with mantADP release at 40 s(-1). Surprisingly, Eg5-367 binds microtubules more effectively (11 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,Mt). These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.

Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two loops have been implicated repeatedly. A small domain loop is of length that complements the size of the guanidino substrate, and is located where it could mediate a lock-and-key mechanism. The second loop contacts the substrate with a valine in the methyl-substituted guanidinium of creatine, and with a glutamate in the unsubstituted arginine substrate, leading to the proposal of a discriminating hydrophobic/hydrophilic minipocket. In the present work, chimeric mutants were constructed with creatine kinase loop elements inserted into arginine kinase. Contrary to the prior rationalizations of specificity, most had measurable arginine kinase activity but no creatine kinase activity or enhanced phosphocreatine binding. Guided by structure, additional mutations were introduced in each loop, recovering arginine kinase activities as high as 15% and 64% of wild type, respectively, even though little activity would be expected in the constructs if the implicated sites had dominant roles in specificity. An atomic structure of the mismatched complex of arginine kinase with creatine and ADP indicates that specificity can also be mediated by an active site that allows substrate prealignment that is optimal for reactivity only with cognate substrates and not with close homologs that bind but do not react.

[reaction: see text] Enzymatic production of glycoconjugates is hampered by expensive phosphagens such as acetyl phosphate (AcP) and phosphoenolpyruvate (PEP). Here, we introduce creatine phosphate--creatine kinase system as a novel and practical energy source in carbohydrate synthesis. This system was successfully demonstrated in the production of bioactive oligosaccharides with different sugar nucleotide regeneration systems.

1 Creatine (CR) supplementation augments muscle strength in skeletal muscle cells by increasing intracellular energy pools. However, the effect of CR supplementation on endothelial cells remains to be clarified. 2 In this study, we investigated whether CR supplementation had any anti-inflammatory activity against human pulmonary endothelial cells in culture. 3 We confirmed that supplementation with 0.5 mM CR significantly increased both intracellular CR and phosphocreatine (PC) through a CR transporter while keeping intracellular ATP levels constant independent of CR supplementation and a CR transporter antagonist. 4 In the assay system of endothelial permeability, supplementation with 5 mM CR significantly suppressed the endothelial permeability induced by serotonin and H(2)O(2). 5 In cell adhesion experiments, supplementation with 5 mM CR significantly suppressed neutrophil adhesion to endothelial cells. 6 In the measurement of adhesion molecules, CR supplementation with more than 0.5 mM CR significantly inhibited the expressions of ICAM-1 and E-selectin on endothelial cells, and the inhibition was significantly suppressed by an adenosine A(2A) receptor antagonist. 7 The present study suggests that CR supplementation has anti-inflammatory activities against endothelial cells.

Phosphorus Magnetic Resonance Spectroscopy (31P-MRS) has been utilized to study energy, carbohydrate, and phospholipid metabolism in vitro and in vivo in live tissues non-invasively. Despite its lack of sensitivity, its application has extended to in situ human tissues and organs since proper signal localization was devised. Follow-up of phosphocreatine in neuromuscular diseases and schizophrenia and follow-up of phospholipid-related molecules in tumors are described here to demonstrate the value of 31P-MRS as an imaging technique to determine in vivo markers of disease and in the diagnosis, prognosis, and follow-up of human diseases.

Net phosphocreatine (PCr) resynthesis during muscle contraction is a paradoxical phenomenon because it occurs under conditions of high energy demand. The metabolic underpinnings of this phenomenon were analyzed non-invasively using 31P-magnetic resonance spectroscopy in rat gastrocnemius muscle (n=11) electrically stimulated (7.6 Hz, 6 min duration) in situ under ischemic and normoxic conditions. During ischemic stimulation, [PCr] initially fell to a steady state (9+/-5% of resting concentration) which was maintained for the last 5 min of stimulation, whereas isometric force production decreased to a non-measurable level beyond 3 min. Throughout normoxic stimulation, [PCr] and force production declined to a steady state after respectively 1 min (5+/-3% of resting concentration) and 3.25 min (21+/-8% of initial value) of stimulation. Contrary to the observations under ischemia, a paradoxical net PCr resynthesis was recorded during the last 2 min of normoxic stimulation and was not accompanied by any improvement in force production. These results demonstrate that the paradoxical net PCr resynthesis recorded in contracting muscle relies exclusively on oxidative energy production and could occur in inactivated fibers, similarly to PCr resynthesis during post-exercise recovery.

1. We tested whether the reduction in ATP cost of contraction during in vivo stimulation of rat gastrocnemius muscle was related to fatigue level. 2. Muscles (n = 44) were electrically stimulated to perform 6 min repeated isometric contractions at different frequencies; one non-fatiguing protocol (stimulation at 0.8 Hz) and five fatiguing protocols (2, 3.2, 4, 5.2 and 7.6 Hz) were used. Anaerobic and oxidative ATP turnover rates were measured non-invasively using (31)P-magnetic resonance spectroscopy. 3. At the onset of the stimulation period, no signs of fatigue were measured in the six protocols and ATP cost of contraction did not differ significantly (P = 0.45) among protocols (mean value of 1.76 +/- 0.11 mM (N s)(-1)). 4. For the six protocols, ATP cost of contraction was significantly reduced (P < 0.05) at the end of the stimulation period when compared with the initial value. This reduction did not differ significantly (P = 0.61) among the five fatiguing protocols (averaging 35 +/- 3 % of initial value), whereas isometric force decreased significantly as stimulation frequency increased. No significant correlation (P = 0.87, r(2) = 0.01) was observed between isometric force and ATP cost of contraction at the end of the stimulation period. In addition, this reduction was significantly lower (P < 0.05) for the non-fatiguing protocol (67 +/- 9 % of initial value) when compared with the fatiguing protocols. 5. These results demonstrate that (i) the reduction in ATP cost of contraction during in vivo stimulation of rat gastrocnemius muscle is not related to the fatigue level; (ii) surprisingly, this reduction was significantly larger during the fatiguing protocols compared with the non-fatiguing protocol.

31P saturation-transfer nuclear magnetic resonance spectroscopy was used to study skeletal muscle phosphocreatine (PCr) flux in healthy male volunteers. Data analysis included consideration of effects from incomplete saturation and radiofrequency spillover. Spectra were recorded from the resting gastrocnemius muscle before and after 6 days of creatine monohydrate (Cr-H2O) intake (20 g/day). Parallel to an improved muscle performance during maximal intermittent exercise following Cr-H2O supplementation, the concentration of PCr increased (P=0.01) by 23% (34.9+/-2.8 mmol/l vs. 28.6+/-2.7 mmol/l), whereas other metabolites were unaffected (inorganic phosphate: 4.3+/-1.4 mmol/l, free intracellular Mg(2+): 1.1+/-0.7 mmol/l, cytosolic pH: 7.04+/-0.02). Forward and reverse fluxes through the creatine kinase (CK) reaction did not change significantly from their baseline levels (v(for): 11.8+/-5.4 mmol/l per second vs. 15.3+/-6.8 mmol/l per second, (v(rev): 9.5+/-3.4 mmol/l per second vs. 10.9+/-3.7 mmol/l per second). The rate of PCr resynthesis in resting muscle is not limited by the CK reaction, which is near equilibrium. Consequently, the post-load increase in total creatine has no effect on the unidirectional CK reaction rates.

Sequence homology analysis reveals that arginine-95 is fully conserved in 29 creatine kinases sequenced to date, but fully conserved as a tyrosine residue in 16 arginine kinases. Site-directed mutants of rabbit muscle creatine kinase (rmCK) were prepared in which R95 was replaced by a tyrosine (R95Y), alanine (R95A), or lysine (R95K). Kinetic analysis of phosphocreatine formation for each purified mutant showed that recombinant native rmCK and all R95 mutants follow a random-order, rapid-equilibrium mechanism. However, we observed no evidence for synergism of substrate binding by the recombinant native enzyme, as reported previously [Maggio et al., (1977) J. Biol. Chem. 252, 1202-1207] for creatine kinase isolated directly from rabbit muscle. The catalytic efficiencies of R95Y and R95A are reduced approximately 3000- and 2000-fold, respectively, compared to native enzyme, but that of R95K is reduced only 30-fold. The major contribution to the reduction of the catalytic efficiency of R95K is a 5-fold reduction in the affinity for creatine. This suggests that while a basic residue is required at position 95 for optimal activity, R95 is not absolutely essential for binding or catalysis in CK. R95Y has a significantly lower affinity for creatine than the native enzyme, but it also displays a somewhat lower affinity for MgATP and 100-fold reduction in k(cat). Interestingly, R95A appears to bind either creatine or MgATP first with affinities similar to those for the native enzyme, but it has a 10-fold lower affinity for the second substrate, suggesting that replacement of R95 by an alanine disrupts the active site organization and reduces the efficiency of formation of the catalytically competent ternary complex.

Distribution of total creatine (free creatine + phosphocreatine) between two subcellular macrocompartments--mitochondrial matrix space and cytoplasm--in heart and skeletal muscle cells was reinvestigated by using a permeabilized cell technique. Isolated cardiomyocytes were treated with saponin (50 microg/ml for 30 min or 600 microg/ml for 1 min) to open the outer cellular membrane and release the metabolites from cytoplasm (cytoplasmic fraction, CF). All mitochondrial population in permeabilized cells remained intact: the outer membrane was impermeable for exogenous cytochrome c, the acceptor control index of respiration exceeded 10, the mitochondrial creatine kinase reaction was fully coupled to the adenine nucleotide translocator. Metabolites were released from mitochondrial fraction (MF) by 2-5% Triton X100. Total cellular pool of free creatine + phosphocreatine (69.6 +/- 2.1 nmoles per mg of protein) was found exclusively in CF and was practically absent in MF. When fibers were prepared from perfused rat hearts, cellular distribution of creatine was not dependent on functional state of the heart and only slightly modified by ischemia. It is concluded that there is no stable pool of creatine or phosphocreatine in the mitochondrial matrix in the intact muscle cells, and the total creatine pool is localized in only one macrocompartment--cytoplasm.

Skeletal muscle is a biological structure with a high degree of organization at different spatial levels. This order influences magnetic resonance (MR) in vivo-in particular 1H-spectra-by a series of effects that have very distinct physical sources and biomedical applications: (a) bulk fat (extramyocellular lipids, EMCL) along fasciae forms macroscopic plates, changing the susceptibility within these structures compared to the spherical droplets that contain intra-myocellular lipids (IMCL); this effect leads to a separation of the signals from EMCL and IMCL; (b) dipolar coupling effects due to anisotropic motional averaging have been shown for 1H-resonances of creatine, taurine, and lactate; (c) aromatic protons of carnosine show orientation-dependent effects that can be explained by dipolar coupling, chemical shift anisotropy or by relaxation anisotropy; (d) limited rotational freedom and/or compartmentation may explain differences of 1H-MR-visibility of the creatine/phosphocreatine resonances; (e) lactate 1H-MR resonances are reported to reveal information on tissue compartmentation; (f) transverse relaxation of water and metabolites show multiple components, indicative of intra-, extracellular and/or macromolecular-bound pools, and in addition dipolar or J-coupling lead to a modulation of the signal decay, hindering straightforward interpretation; (g) diffusion weighted 31P-MRS has shown restricted diffusion of phosphocreatine; (h) magnetization transfer (MT) indicates that there is a motionally restricted proton pool in spin-exchange with free creatine; reduced availability or restricted motion of creatine is particularly important for an estimation of ADP from 31P-MR spectra, and in addition MT effects may alter the signal intensity of creatine 1H-resonances following water-suppression pulses; (i) transcytolemmal water-exchange can be studied in 1H-MRS by contrast-agents applied to the extracellular space; (k) transport of glucose across the cell membrane has been studied in diabetes patients using a combination of 13C- and 31P-MRS; and l residual quadrupolar interaction in 23Na MR spectra from human skeletal muscle suggest that sodium ions are bound to ordered muscular structures., (Copyright 2001 John Wiley & Sons, Ltd.)

Human immunodeficiency virus (HIV) infection of the brain causes a complex cascade of cellular events involving several different cell types that eventually leads to neuronal cell death and the manifestation of the AIDS-associated dementia complex (ADC). Upon autopsy HIV-infected individuals show lesions within subcortical regions of the brain, including the cerebellum. Previously we have demonstrated, in primary and cell culture models of rat and human astrocytes, a change in intracellular pH (pH(i)) due to increased Na(+)/H(+) exchange following exposure to inactivated virus or gp120, the major HIV envelope glycoprotein. To further investigate whether any such in vivo pH(i) changes occur in human brains subsequent to HIV infection, we measured the pH(i) of the cerebellum in eight HIV-positive individuals and nine healthy volunteers using (31)P magnetic resonance spectroscopy imaging (MRSI) at high field strength (4.1 T). The results showed a significant difference between the age-adjusted mean pH(i) in the cerebellum in control group and patient groups (7.11 +/- 0.03 vs 7.16 +/- 0.04), and further HIV-infected individuals displayed a significant increase in the number of cerebellar volume elements that were alkaline. We hypothesize that this propensity towards alterations in cerebellar pH(i) may portend later neurological involvement resulting from HIV infection., (Copyright 2001 John Wiley & Sons, Ltd.)

Using the inversion transfer technique, the possible magnetic coupling between water protons and the protons of low-molecular weight metabolites was investigated in human brain and skeletal muscle at 1.5 T. The localized (1)H-MR spectra were recorded at different times after selective inversion of the water resonance. Water inversion led to a significant transient reduction in the signal intensity of the methyl protons of creatine/phosphocreatine, in both tissues. This is indicative of magnetic coupling between the protons of water and those of creatine/phosphocreatine. Neither the choline and N-acetylaspartate protons in brain nor the protons of the trimethylammonium pool in skeletal muscle showed a significant magnetic coupling to mobile water., (Copyright 2001 John Wiley & Sons, Ltd.)

Purpose: Vigabatrin (VGB) is a new antiepileptic drug that increases the human brain gamma-aminobutyric acid (GABA) level by irreversibly inhibiting GABA transaminase. Although some patients respond to VGB with a significant seizure reduction, others do not. The aim of this study was to identify possible responders before or in an early phase of VGB treatment by measuring the GABA and homocarnosine contaminated with macromolecules/creatine and phosphocreatine ratio (GABA+/Cr) signal by means of proton-nuclear magnetic resonance (1H NMR) spectroscopy., Methods: Measurements were performed immediately before and after a titration period of 1 month (2 g/day during the past 2 weeks). A third measurement followed a maintenance period of 3 months (2 or 3 g/day). In 14 patients with drug-resistant temporal lobe epilepsy and 3 patients with occipital lobe epilepsy, GABA+/Cr was measured in the ipsilateral (i.e., epileptogenic) hemisphere and contralateral (i.e., nonepileptogenic) hemisphere in a volume of 8 cm3., Results: Depending on the therapeutic efficacy of VGB, we defined three groups: (a) full responders (n = 7), (b) nonresponders (n = 7), and (c) partial responders (n = 3). The nonresponders had no significant change in the GABA+/Cr signal during the treatment compared with baseline. The full responders had a significant increase of the GABA+/Cr signal during the whole treatment phase and a lower ipsilateral level at baseline. The partial responders had also a lowered ipsilateral GABA+/Cr signal at baseline and an increase during treatment but a decrease when the seizures started again., Conclusions: Responders to VGB could be identified by a lower ipsilateral baseline GABA+/Cr signal and a steeper increase during VGB treatment. However, it was not possible to predict the duration of the response (full versus partial responder) with these criteria.

Signaling by insulin requires autophosphorylation of the insulin receptor kinase (IRK) at Tyr1158, Tyr1162, and Tyr1163. Earlier experiments with (32)P-gamma-ATP indicated that the nonphosphorylated IRK (IRK-0P) is relatively inactive, and crystallographic data indicated that the ATP binding site of IRK-0P is blocked by its activation loop. We now show that phosphocreatine (PCr) in combination with hydrogen peroxide serves as an alternative phosphate donor and that ATP and PCr use distinct binding sites. Whereas phosphorylation of the IRK by ATP is inhibited by the nonhydrolyzable competitor adenylyl-imidodiphosphate, phosphorylation by PCr is enhanced. The IRK mutant Tyr1158Phe showed no phosphorylation with PCr but almost normal phosphorylation with ATP, whereas Tyr1162Phe was phosphorylated well with PCr but less then normal with ATP. 3-Dimensional models of IRK-0P revealed that the conversion of any of the four cysteine residues 1056, 1138, 1234, and 1245 into sulfenic acid produces structural changes that bring Tyr1158 into close contact with Asp1083 and render the well-known catalytic site at Asp1132 and Tyr1162 accessible from a direction that differs from the known ATP binding site. The mutant Cys1138Ala, in contrast, showed relatively inaccessible catalytic sites and weak catalytic activity in functional experiments. Taken together, these findings indicate that 'redox priming' of the IRK facilitates its autophosphorylation by PCr in the activation loop.

Our understanding of O2 uptake (VO2) control mechanisms during exercise may be improved by the simultaneous determination of the kinetics of intramuscular high-energy phosphate turnover and pulmonary VO2. We therefore developed a technique for remote gas-exchange analysis while subjects exercised in a whole body 1.5-T NMR system. Knee-extension exercise was performed against restraining rubber bands in the prone position. Free induction decays were acquired every 1,875 ms by using a transmit-receive coil, which was placed under the quadriceps. This allowed 31P spectra of intramuscular ATP, Pi, and creatine phosphate dynamics to be determined every 15 s. Airflow was measured with a custom-designed turbine and a 45-ft.-long cable to reach the volume-measuring module. This was located in an adjacent radio-frequency-shielded room, as was the respiratory mass spectrometer, which also used a 45-ft.-long sampling line. The respired gas profiles were not discernibly different from those that used the standard inlet; the increase in the delay was readily incorporated into the breathby-breath algorithm, allowing the VO2 kinetics to be determined in concert with those of intramuscular phosphate metabolism.

Here we describe a method for obtaining a ready-to-use stabilized reaction mixture for in vitro translation of mRNA. We also demonstrate the stabilization of a complete translation mixture containing wheat germ extract, amino acids, ATP, GTP, creatine phosphate, creatine kinase, and the reaction buffer by lyophilization in the presence of various sugars. The greatest stabilizing effect is achieved by supplementing the mixture with 10% (mass/volume) trehalose, which is also a unique translation activator, enhancing the translation of various mRNAs. A lyophilized complete translation mixture containing trehalose can be stored at 4-8 degrees C for several months without losing its activity. The mixture can be easily reconstituted by adding an aqueous mRNA solution and retains the potential for reproducible functioning. This allows the employment of such a cell-free translation system for analytical screening of a broad spectrum of compounds inhibiting translation at various stages.

This paper extends the use of principal-component analysis in spectral quantification to the estimation of frequency and phase shifts in a single resonant peak across a series of spectra. The estimated parameters can be used to correct the spectra accordingly, resulting in more accurate peak-area estimation. Further, the removal of the variations in phase and frequency cause by instrumental and experimental fluctuations makes it possible to determine more accurately the remaining variations, which bear biological significance. The procedure is demonstrated on simulated data, a 3D chemical-shift-imaging dataset acquired from a cylinder of inorganic phosphate (Pi), and a set of 736 31P NMR in vivo spectra taken from a kinetic study of rate muscle energetics. In all cases, the procedure rapidly and automatically identifies the frequency and phase shifts present in the individual spectra. In the kinetic study, the procedure is used twice, first to adjust the phase and frequency of a reference peak (phosphocreatine) and then to determine the individual frequencies of the Pi peak in each of the spectra which further can be used for estimation of pH changes during the experiment.

The brain isoform of creatine kinase has been implicated in cellular transformation processes. Cyclocreatine, a creatine kinase substrate analog, was previously shown to be cytotoxic to a broad spectrum of solid tumors. We have synthesized, enzymatically characterized, and evaluated the antitumor activity of a series of substrate analogs of creatine kinase. Using in vitro assays, we demonstrate that several of these analogs are cytotoxic to the human ME-180 cervical carcinoma, the MCF-7 breast adenocarcinoma and the HT-29 colon adenocarcinoma cell lines at low mM concentrations. Analogs that were active in vitro delayed the growth of a subcutaneously implanted rat 13,762 mammary adenocarcinoma. Tumor growth delays of 6-8 days were achieved, which is comparable to effects seen with standard regimens of currently used anticancer drugs. These studies further establish the creatine kinase system as a promising and novel target for anticancer chemotherapy drug design.

Although the exact mechanism(s) responsible for the phosphocreatine/ATP overshoot have not been completely elucidated, our data demonstrate that the overshoot does not stem from reduced myocardial work, and consequently, reduced utilization of phosphocreatine (PCr). Additionally, we highlight a basic difference in the physiologic responses of skeletal and cardial muscle to work demands. By understanding the bioenergetic derangements which accompany reperfusion injury, one may hope to better salvage post-ischemic myocardium.

Maturational changes in intracellular brain phosphocreatine (PCr) transport were investigated using 31P-nuclear magnetic resonance diffusion spectroscopy. The diffusivities of PCr showed significant maturational facilitation in rat brain in vivo. Physicochemical analysis of the cytosol microenvironment as a multicomponent solution, where one of the components is a dilute polymer, indicated that the observed developmental facilitation of PCr diffusivity is likely to be due to a decline in the concentration of the free amino acid taurine. Changes in the concentrations of biopolymers (i.e., proteins or lipids) have only little effect, if any, on PCr diffusivity. PCr diffusivity values of rat brain measured in vivo showed excellent quantitative agreement with the predicted values estimated using a model for multicomponent diffusion. The study confirmed that the taurine/N-acetylaspartate exchange observed during postnatal development of rat brain plays a major, it not unique, role in maturational facilitation of intracellular high-energy phosphate transport.

By sequestering intracellular myoglobin of cardiac muscle cells in the nonfunctioning carboxymyoglobin form, carbon monoxide blocks myoglobin-facilitated diffusion of oxygen, as well as myoglobin-mediated oxidative phosphorylation. Here, we explore the hypothesis that the carbon monoxide blockade of myoglobin function may be responsible at the cellular level for a component of the cardiotoxicity of carbon monoxide observed during exercise. Suspensions of isolated rat cardiac myocytes were held in near steady states of oxygen pressure near the intracellular partial pressure of oxygen of the working heart (2 to 5 torr) and near the end-venous partial pressure of oxygen (20 torr). These suspensions were exposed to CO at low pressure (0.07 to 70 torr; 90 to 90,000 parts per million). The fraction of intracellular carboxymyoglobin, determined spectrophotometrically, was in good agreement with the fraction predicted from the ratio of carbon monoxide partial pressure to oxygen partial pressure. The effects observed were related to the fraction of intracellular myoglobin bound to CO. At physiological oxygen pressures no greater than 5 torr, after sequestration of approximately 50% of the myoglobin, steady-state oxygen uptake decreased significantly and was significantly less than the respiration of cell groups for which the fraction of carboxymyoglobin was 0% to 40%. When respiration is diminished, the rate of aerobic adenosine triphosphate synthesis (oxidative phosphorylation) also decreases. As in the whole heart, cytoplasmic adenosine triphosphate concentration in isolated heart cells is controlled at a constant level by the creatine phosphokinase equilibrium. When adenosine triphosphate utilization is unchanged, a sensitive monitor of the decreased adenosine triphosphate synthesis is the ratio of phophocreatine to adenosine triphosphate. When carboxymyoglobin was at least 40% of the total intracellular myoglobin, we found that the ratio of phosphocreatine to adenosine triphosphate in carbon monoxide-treated heart cells was significantly lower than that in control cells from the same preparation. Thus, we concluded that sequestering intracellular myoglobin as carboxymyoglobin significantly decreased the rate of oxidative phosphorylation of isolated cardiac myocytes. We estimate that intracellular myoglobin-dependent oxidative phosphorylation will be inhibited when approximately 20% to 40% of the arterial hemoglobin in the whole animal is carboxyhemoglobin.