Chemical exchange saturation transfer imaging of phosphocreatine in the muscle.

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: kex 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 B1 of 0.47 µT at 15.2 T, which is 29% higher than 9.4T values. The value of PCrCEST at a B1 of 0.71 µT was not significantly different than that measured at a B1 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.

Simultaneous quantitative analysis of phosphocreatine, creatine and creatinine in plasma of children by HPLC-MS/MS method: Application to a pharmacokinetic study in children with viral myocarditis.

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 C18 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: t1/2 0.24/0.83 h, Tmax 0.49/0.55 h, Cmax 47.34/59.29 µg/mL, AUClast 17.07/59.63 h µg/mL, AUCinf 17.16/79.01 h µg/mL and MRT 0.29/0.67 h.

Dynamic phosphocreatine imaging with unlocalized pH assessment of the human lower leg muscle following exercise at 3T.

PURPOSE: To develop a high temporal resolution imaging method that measures muscle-specific phosphocreatine (PCr) resynthesis time constant (τPCr ) 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, τPCr was estimated at 22.9 ± 8.8 s (mean ± standard deviation) with statistical coefficient of determination r2 = 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.

Rapid and simultaneous measurement of phosphorus metabolite pool size ratio and reaction kinetics of enzymes in vivo.

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 T1 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 T1 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 T1 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.

31 P magnetic resonance fingerprinting for rapid quantification of creatine kinase reaction rate in vivo.

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 T1 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 T2 values, inorganic phosphate metabolism, and B1 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.

Heart mitochondrial TTP synthesis and the compartmentalization of TMP.

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.

Hydroxyapatite hierarchically nanostructured porous hollow microspheres: rapid, sustainable microwave-hydrothermal synthesis by using creatine phosphate as an organic phosphorus source and application in drug delivery and protein adsorption.

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.

Mice over-expressing the myocardial creatine transporter develop progressive heart failure and show decreased glycolytic capacity.

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.

Physiological heart activation by adrenaline involves parallel activation of ATP usage and supply.

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.

Facile High-Performance Liquid Chromatography Mass Spectrometry Method for Analysis of Cyclocreatine and Phosphocyclocreatine in Complex Mixtures of Amino Acids.

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.

The creatine kinase phosphotransfer network: thermodynamic and kinetic considerations, the impact of the mitochondrial outer membrane and modelling approaches.

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.

Graphene oxide and creatine phosphate disodium dual template-directed synthesis of GO/hydroxyapatite and its application in drug delivery.

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.

Optimizing In Vitro Pre-mRNA 3' Cleavage Efficiency: Reconstitution from Anion-Exchange Separated HeLa Cleavage Factors and from Adherent HeLa Cell Nuclear Extract.

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.

Transition state stabilization by six arginines clustered in the active site of creatine kinase.

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.

Magnesium phosphate pentahydrate nanosheets: Microwave-hydrothermal rapid synthesis using creatine phosphate as an organic phosphorus source and application in protein adsorption.

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.

Metabolic underpinnings of the paradoxical net phosphocreatine resynthesis in contracting rat gastrocnemius muscle.

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.

Creatine kinase: a role for arginine-95 in creatine binding and active site organization.

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.

Cysteamine prevents and reverses the inhibition of creatine kinase activity caused by cystine in rat brain cortex.

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.

In vitro study of astrocytic tumour metabolism by proton magnetic resonance spectroscopy.

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.

Analysis of creatine kinase activity with evaluation of protein expression under the effect of heat and hydrogen peroxide.

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.