Citrate Formation Research Articles

13C Stable Isotope Tracing Reveals Distinct Fatty Acid Oxidation Pathways in Proliferative vs. Oxidative Cells.

The TCA cycle serves as a central hub to balance catabolic and anabolic needs of the cell, where carbon moieties can either contribute to oxidative metabolism or support biosynthetic reactions. This differential TCA cycle engagement for glucose-derived carbon has been extensively studied in cultured cells, but the fate of fatty acid (FA)-derived carbons is poorly understood. To fill the knowledge gap, we have developed a strategy to culture cells with long-chain FAs without altering cell viability. By tracing 13C-FA we show that FA oxidation (FAO) is robust in both proliferating and oxidative cells while the metabolic pathway after citrate formation is distinct. In proliferating cells, a significant portion of carbon derived from FAO exits canonical TCA cycle as citrate and converts to unlabeled malate in cytosol. Increasing FA supply or b-oxidation does not change the partition of FA-derived carbon between cytosol and mitochondria. Oxidation of glucose competes with FA derived carbon for the canonical TCA pathway thus promoting FA carbon flowing into the alternative TCA pathway. Moreover, the coupling between FAO and the canonical TCA pathway changes with the state of oxidative energy metabolism.

The Effects of Sodium Silicate and Sodium Citrated on the Properties and Structure of Alkali-Activated Foamed Concrete

Alkali-activated slag cementitious (AASC) foamed concrete (FC) has presented challenges such as rapid setting time and poor working performance. The use of sodium citrate (Na3Cit) as a retarding agent can improve the workability and microstructure of AASC foamed concrete. The effects of the dosage, modulus of water glass (WG, the main component is Na2O·nSiO2), and retarding agent on the properties and structure of FC were studied in this paper. The results indicated that using a water binder ratio of 0.4, WG with a modulus of 1.2, and an additional amount of 15% and 0.5% of Na3Cit, the prepared FC had a flowability of 190 mm. Its initial and final setting times were 3.7 h and 35.3 h. Its 7 d and 28 d compressive strengths reached 1.1 MPa and 1.5 MPa, respectively. After hardening, the pore walls were dense and consistent in size, with few larger pores and nearly spherical shapes. The addition of Na3Cit resulted in the formation of calcium citrate, which adsorbed onto the slag surface. This hindered the initial dissolution of the slag, reduced the number of hydration products produced, and decreased the early strength. With increasing curing time, the slag in the FC mixture dissolved further. This led to the decomposition of a portion of calcium citrate and the release of Ca2+. The Ca2+ reacted with [Si(OH)4]4− and [Al(OH)4]−, creating more C-(A)-S-H gel. This gel filled the voids in the FC and repaired any defects on the pore walls. Ultimately, this process increased the compressive strength of the FC in the later stages.

Elucidating the Mechanism of Tetrahydrofuran-Diol Formation through Os(VI)-Catalyzed Oxidative Cyclization of 5,6-Dihydroxyalkenes Ligated by Citric Acid.

A computational study is reported here on the mechanism of tetrahydrofuran (THF)-diol formation from the Os(VI)-catalyzed oxidative cyclization of 5,6-dihydroxyalkene ligated with citric acid and in the presence of Bro̷nsted acid. Initiated by Os(VI) dioxo citrate formation, coordination of co-oxidant pyridine-N-oxide (PNO) and protonation of its oxo group generate the active catalyst. The catalytic cycle commences through successive steps, including dihydroxyalkene addition to the active catalyst in a concerted mechanism to form hexacoordinated alkoxy-protonated PNO-complexed Os(VI) bisglycolate as a turnover-limiting step (TLS), cyclization to Os(IV) THF-diolate, reoxidation to Os(VI) THF-diolate, and hydrolysis via a dissociative mechanism to furnish the THF-diol and regenerate the active species, sustaining the catalytic cycle through an Os(VI)/Os(IV) cycle. Despite the overall exergonic nature of catalytic cycle (ΔGrcycle = -45.0 kcal/mol), the TLS is accelerated by the formation of an open-valence 16-electron Os(VI) intermediate but decelerated by the undesired formation of a saturated/hexacoordinate 18-electron Os(VI) intermediate. Bro̷nsted acid plays crucial roles in the formation of Os(VI) citrate and the active catalyst, impediment of the second cycle, and the cyclization step. Additionally, besides its role as a co-oxidant, and in the presence of acid, PNO is found to assist the insertion of dihydroxyalkene and, importantly, in releasing the THF-diol to regenerate the active intermediate.

Complexation of Boronic Acid with Chiral α-Hydroxycarboxylic Acids and the Ability of the Complexes to Catalyze α-Hydroxycarboxylic Acid Esterification.

The complexation of boric acid (BA) with various α-hydroxycarboxylic acids (HCAs) was examined by analyzing the change in the optical rotation after the addition of BA to aqueous HCA solutions, and the catalytic properties of the complexes were examined by catalyzing the esterification of the HCAs. The absolute values of the optical rotation of the HCAs increased with increasing BA-to-HCA molar ratio, and the rate of change of the optical rotation gradually decreased as the BA-to-HCA molar ratio increased, reaching a minimum value at a molar ratio of approximately three. As a catalyst, BA could catalyze the acetylation of hydroxyl groups in addition to the esterification of HCAs. Compared to the conventional synthesis routes of ATBC and ATOC, a synthesis route with BA as the catalyst allowed for a lower frequency of catalyst separation and replacement while providing light-colored products. BA could catalyze the formation of triethyl citrate, and the yield of triethyl citrate reached 93.8%. BA could also catalyze the reaction between malic acid and pinene to produce borneol malate. After saponification of borneol malate, borneol was obtained with a yield of 39%.

Primary amine citrate-based supramolecular designer solvent: Preconcentration of ochratoxin A for determination in foods by liquid chromatography

BackgroundSupramolecular solvents are nanostructured liquids that are separated from colloidal solutions of amphiphilic compounds as a result of self-assembly of amphiphiles and coacervation under changing conditions. They are considered to be designer solvents as their properties can be tailored to a specific analytical task by controlling the conditions of their formation (amphiphile, coacervation inducer, medium, concentration of components). The discovery of new extraction systems based on supramolecular solvents and their application to relevant analytical tasks are of great importance for the advancement of environmentally-friendly sample preparation. ResultsA novel green liquid-phase microextraction approach involving in situ formation of 1-octylamine citrate followed by preconcentration of ochratoxin A from aqueous extract of food sample in supramolecular solvent droplets was developed. The extraction system was carefully characterized. The density of the solvent allowed it to be to retrieved from the extraction system by its solidification. The alkaline nature of the obtained extract allowed the use of acetic acid for its dissolution instead of more toxic organic solvents followed by high-performance liquid chromatography with fluorometric detection. An excellent extraction recovery of 99 % and a satisfactory enrichment factor of 8.3 were achieved. The limit of detection was 0.5 μg kg−1, while the calibration plot was linear over the range of 1.5–50 μg kg−1. Cereal and roasted coffee bean samples were successfully analyzed with a relative bias less than 20 %. SignificanceIn the present work, a phenomenon of supramolecular solvent formation based on primary amine citrate was presented for the first time. Tetrabutylammonium bromide was investigated as a coacervation agent in an extraction system, and possible interactions responsible for its ability to induce phase separation in a micellar solution of primary amine citrate were described. The critical micelle concentration of 1-octylamine citrate in aqueous solution of tetrabutylammonium bromide was firstly determined.

Abstract P2184: Isocitrate Dehydrogenase 1 Regulates Cardiac Metabolic Adaptation During Oncometabolic Stress

Cardiovascular disease and cancer are the two leading causes of morbidity and mortality in the world. Metabolic dysregulation of cancer cells extends beyond the tumor microenvironment and increases risk for cardiovascular diseases. One common somatic mutation in cancer cells affects isocitrate dehydrogenase (IDH) 1 and 2, which catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate in the cytosol and mitochondria, respectively. IDH1 and 2 mutations cause the production of the oncometabolite D-2-hydroxyglutarate (D2-HG) which allosterically inhibits alpha-ketoglutarate dehydrogenase (alpha-KGDH) and is associated with reduced cardiac contractile function. We combined stable isotope tracer studies with computational modeling to study the fundamental role of IDH isoforms for cardiac adaptation during oncometabolic stress. We uncovered an unexpected cardiac phenotype that expands the role of IDH1 in the heart beyond oxidative metabolism. Using mass spectrometry-based metabolomics, we quantified the stable isotopomer distributions from glucose and glutamine in perfused working rat hearts and isolated adult ventricular cardiomyocytes. Our analysis revealed that defective mitochondrial metabolism causes redirection of carbon flux oxidative towards reductive pathways. Reductive carboxylation of alpha-KGDH increases glutamine uptake and glutamine-derived citrate formation in both working rat heart perfusions and cultured adult mouse ventricular cardiomyocytes. To identify which IDH isoform is responsible for redirecting carbon flux, we developed knockout models of IDH1, 2, and 3 in adult mouse ventricular cardiomyocytes. Loss of IDH1 expression impaired reductive formation of citrate and caused functional defects in cardiomyocytes. Lastly, epigenetic analyses of histone marks revealed that IDH1 causes widespread alterations in histone acetylation and tri-methylation. Our results highlight a novel role for IDH1 in cardiac metabolism and transcriptional control of metabolic adaptation in response to tumor-mediated stress and provide evidence of how reductive-citrate formation may induce epigenetic modifications in the heart.

DETERMINATION OF THE COMPOSITION AND STABILITY CONSTANT OF COBALT (II) POLYLIGAND CITRATE-MOLYBDATE COMPLEXES

The complex formation of cobalt (II) with citrate (cit3–) and molybdate (MoO42–) ions in 0.3 mol·l-1 Na2SO4 solution at 20°C was studied by spectrophotometric method. It has been established that in a citrate-molybdate electrolyte at pH 9.0, cobalt (II) ions form, depending on the ratio of the equilibrium concentrations of ligands: molybdate [Co(MoO4)2]2–, citrate [Co(cit)2]4– and polyligand complexes [Co(cit)m(MoO4)n]+2-(3m+2n). The composition of [Co(cit)(MoO4)]3– polyligand complexes was determined, the equilibrium constant Ke of the reaction of their formation and their stability constant (lgβMLX = 5.86) The dependence of the degree of formation of molybdate, citrate and polyligand complexes of cobalt (II) in citrate-molybdate electrolyte on the logarithm of the ratio of equilibrium concentrations of ligands was calculated.

A Mechanochemical Route to Magnesium/Calcium Citrate Formation for Chrysotile/Asbestos Cement Correct Disposal as Agricultural Input

A Mechanochemical Route to Magnesium/Calcium Citrate Formation for Chrysotile/Asbestos Cement Correct Disposal as Agricultural Input

Thermodynamic Study of Leaching Conditions of Galena with Citrate Ions and Hydrogen Peroxide as Oxidizing Agent.

Galena is the most important mineral for lead production, as it is the main source of lead in the world. Currently, the concentrates of this mineral are mainly treated using pyrometallurgical methods, creating several environmental problems, such as the generation of toxic and greenhouse gases. In addition, these processes involve high energy consumption, which limits their applicability. Hydrometallurgical routes are proposed as alternative processes for obtaining some metals such as silver, copper, gold, etc. The drawback of these processes is that the minerals tend to be passive in aqueous media. To mitigate this issue, researchers have used extreme conditions of pressure and temperature (6 atm. and 155 °C) or the use of very corrosive conditions. In this sense, the use of complexing agents that dissolve the metals of interest has been proposed. Citrate ion is one of the most promising complexing agents for galena leaching, obtaining high percentages of dissolution in relatively short times. Unfortunately, there has not been enough investigation about the concentration optimization of the complexing in the pH range from 5 to 9. In this sense, thermodynamic diagrams, such as the Pourbaix diagrams, are very useful for this purpose. Therefore, in this work, the effects of pH and temperature on the leaching of galena in citrate ion solutions are studied thermodynamically and experimentally. The experimental work was carried out with pure galena samples with a particle size of +149 - 74 µm (-100 + 200 mesh). The results show that higher recoveries were obtained working at a pH of 8 and at temperatures of 30 and 40 °C. The thermodynamic and experimental data demonstrated that the existence of an optimal concentration of citrate ion, due the extraction of lead from galena, has a greater reaction rate at a relatively low initial concentration of 0.3 M. This is due the formation of the complex lead citrate 1 (Pb(cit)-).

DETERMINATION OF POLYLIGAND COMPLEXES OF COBALT (II) WITH CITRATE AND PYROPHOSPHATE IONS

In the work it is shown by the spectrophotometry method that depending on the concentration ratio of ligands [PPi4–]/[Cit3–] in the pyrophosphate-citrate electrolyte, cobalt (II) ions form not only citrate [Co(Cit)2]4– and pyrophosphate [Co(PPi)2]6–, but also polyligand complexes [Co(PPi)m(Cit)n]+2–(4m+3n). The composition of polyligand complexes [Co(PPi)Cit]5– was determined, and the equilibrium constant of the reaction of their formation and the constant of their stability were calculated (pβ=8.47). The dependence of the degree of formation of citrate, polyligand, and pyrophosphate complexes of cobalt (II) in the pyrophosphate-citrate electrolyte on the logarithm of the ratio of equilibrium concentrations of ligands is calculated.

New aspects of the photodegradation of iron(III) citrate: spectroscopic studies and plant-related factors

Iron (Fe) is an essential cofactor for all livings. Although Fe membrane transport mechanisms often utilize FeII, uncoordinated or deliberated ferrous ions can initiate Fenton reactions. FeIII citrate complexes are among the most important complexed forms of FeIII especially in plants that, indeed, can undergo photoreduction. Since leaves as photosynthetic organs of higher plants are generally exposed to illumination in daytime, photoreaction of ferric species may have biological relevance in iron metabolism, the relevance of which is poorly understood. In present work FeIII citrate transformation during the photodegradation in solution and after foliar application on leaves was studied by Mössbauer analysis directly. To obtain irradiation time dependence of the speciation of iron in solutions, four model solutions of different pH values (1.5, 3.3, 5.5, and 7.0) with Fe to citrate molar ratio 1:1.1 were exposed to light. Highly acidic conditions led to a complete reduction of Fe together with the formation of FeII citrate and hexaaqua complexes in equal concentration. At higher pH, the only product of the photodegradation was FeII citrate, which was later reoxidized and polymerized, resulting in the formation of polynuclear stable ferric compound. To test biological relevance, leaves of cabbage were treated with FeIII citrate solution. X-ray fluorescence imaging indicated the accumulation of Fe in the treated leaf parts. Mössbauer analysis revealed the presence of several ferric species incorporated into the biological structure. The Fe speciation observed should be considered in biological systems where FeIII citrate has a ubiquitous role in Fe acquisition and homeostasis.Graphical abstract

EXPERIMENTAL STUDY OF FURCATION AREA AND PROSPECTS OF ITS COMPLEX TREATMENT

Relevance. The issue of quality endodontic treatment of teeth with complicated caries is still known as requiring thoughtful research. A significant percentage of periodontitis occurs in the area of furcations of multi-rooted teeth, sometimes alone, but more often as combined with apical periodontitis. Among the number of unsolved tasks, there is question about furcation areas and in particularly their antiseptic treatment and obturation in the complex treatment of chronic apical periodontitis of multi-rooted teeth.
 Aim: to investigate the histo-morphological features of the furcation area of the first, second molars of the upper and lower jaws and, according to the results of the experimental study, to develop a scheme of complex treatment of chronic apical periodontitis of multi-rooted teeth.
 Materials and methods. Ten first and second molars of both jaws not affected by fluorosis and removed according to orthodontic or surgical indications in patients aged from 25 to 50 years, were used for this study. In the first stage, transverse and longitudinal sections of the molars were made using diamond-coated separation disks. Using an orthopaedic tip, the first half of the molars were cut longitudinally in the mesio-distal direction with separation disks, under water cooling. The other half of the molars were cut transversely along the neck of the tooth and histochemically stained. In the second stage of the study, we used the experimental application of photodynamic therapy for antiseptic treatment of furcation channels and sealing them with citrate buffer. For this purpose, the next two first and two second mandibular molars were selected, respectively dissected in two planes. Samples were fixed in a vertical position; cotton rolls soaked with 2% methylene blue were placed onto the bottom of each half for 30 s., 1 min., 5 min., and 24 h. In this study we used light binocular stereoscopic microscope MBS-9; histochemical dyes: CHIC-thionine, ammonia silver solution "Argentat", reducing agent (hydroquinone), methylene blue, device "UFL - 122" company "Lux Dent", citrate buffer solution and white clay. The results were recorded with a digital camera mounted on a microscope tube.
 Results and discussion. The results of histochemical examination of ten molars showed the presence of furcation channels in four teeth. Two samples were stained with CHIC-thionine, the others were stained with two-component "Argenate". The selected samples were dissected in the sagittal plane through the area of localization of the furcation channels and studied under the microscope (magnification: 8x32). In teeth dissected in the transverse plane, the crown and mouth pulp were instrumentally removed without dilation of the root canals and stained accordingly.
 Thus, when modelling the stage of photodynamic treatment using a 2% solution of methylene blue, there is a significant filling of furcation channels and root canals along their entire length. The photosensitizer 2% methylene blue at the exposure of 30s demonstrates the highest efficiency at the minimum staining of surrounding fabrics. The microscopy was performed to confirm the ability of the photosensitizer to penetrate into the furcation channels at х8 – х32 magnification.
 After the experimental filling of furcation channels and root canals, we can see clear white stripes along their entire length on the dentin surface of the longitudinal section of the root; these stripes are obliquely directed towards the canal surface and consist of numerous dentinal tubules impregnated with citrate buffer. Numerous thin white lines are clearly visible on the surface of the section that run in parallel, tangentially through almost the entire thickness of the root wall and end up their course in the area around the peripulpal dentin. This indicates the formation of calcium citrate in the root canal that tightly covers its entire surface impregnating the dentinal tubules and completely fills them along their entire length.
 Therefore, the experimental model of using 2% methylene blue photosensitizer at a 30 s. exposure for photodynamic therapy and citrate buffer for obturating furcation and root canals can be applied in the treatment of patients with chronic apical periodontitis and lesions of the furcation area.
 Conclusions. Thus, morphological studies of the first and second molars of both jaws have shown the presence of furcation channels, which anatomical structure is quite complex and demanding in terms of their antiseptic treatment and filling. Therefore, we developed new effective for disinfection and obturation techniques including applying photodynamic therapy for disinfection of furcation channels and citrate buffer for their obturation.

Structural basis of the cooperative activation of type II citrate synthase (HyCS) from Hymenobacter sp. PAMC 26554

Citrate synthase (CS) catalyzes the formation of citrate and coenzyme A from acetyl-CoA and oxaloacetate. CS exists in two forms: type I and type II. We determined the citrate-bound crystal structure of type II CS from the Hymenobacter sp. PAMC 26554 bacterium (HyCS; isolated from Antarctic lichen). Citrate molecules bound to a cleft between the large and small domains of HyCS. Structural comparison of HyCS with other type II CSs revealed that type II CSs have a highly conserved flexible hinge region (residues G264–P265 in HyCS), enabling correct positioning of active site residues. Notably, the catalytic His266 residue of HyCS interacted with Trp262 in the inactive (unliganded open) state of other type II CSs, whereas the His266 residue moved to the active site via a small-domain swing motion, interacting with the bound citrate in the closed conformation of HyCS. However, type I CSs lack this tryptophan residue and face-to-edge interactions. Thus, type II CSs might have a unique domain-motion control mechanism enabling a tight allosteric regulation. An activity assay using a W262A mutant showed a Hill coefficient of 2.4; thus, the interaction between Trp262 and His266 was closely related to the positive cooperative ligand binding of type II CS.

One-pot synthesis of highly magnetic and stable citrate coated superparamagnetic iron oxide nanoparticles by modified coprecipitation method

Highly stable superparamagnetic iron oxide nanoparticles (SPION) prepared through a modified one-pot coprecipitation technique using citrate as a surfactant and capping agent. The result of the as-synthesized citrate–SPION via a one-pot technique is compared with peptized SPION synthesized via the coprecipitation method. Formation of citrate–SPION is characterized using transmission electron microscopy, x-ray diffractometer (XRD), Fourier transform infrared, zeta potential, and vibrating sample magnetometer. XRD result reveals that the citrate does not affect the crystalline structure and purity of the SPION. Zeta potential of the citrate–SPION and the peptized SPION is −44 mV and 38.4 mV, respectively. Similarly, the saturation magnetization values of citrate–SPION and peptized SPION are 53.9 emu g−1 and 43.8 emu g−1, respectively. The results show that the citrate–SPION prepared by modified a one pot technique has a higher saturation magnetization and is more stable than the peptized SPION.

Abstract 308: Reductive Carboxylation Contributes to Cardiac Adaptation in Response to the Oncometabolite D2-hydroxyglutarate

Cancer cells rewire metabolism to support tumor growth and proliferation. In isocitrate dehydrogenase (IDH) 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. D2-HG causes inhibition of alpha-ketoglutarate dehydrogenase (AKGDH), which is associated with reduced cardiac contractile function. How tumor cells influence the metabolism of cardiomyocytes remains mostly unknown. Specific cancer cells use glutamine-dependent reductive carboxylation to circumvent defective mitochondrial metabolism by producing citrate and acetyl-CoA for lipid synthesis, which tumors require for growth. Here, we explore the hypothesis that inhibition of AKGDH by the oncometabolite D2-HG increases glutamine-dependent reductive carboxylation in the heart. We combined ex vivo rat heart perfusions with mass-spectrometry-based stable isotope tracer studies and in silico metabolic flux analysis. In response to D2-HG-mediated inhibition of AKGDH, we observed an increased reductive carboxylation of alpha-ketoglutarate to citrate rather than oxidative decarboxylation. This pathway increases glutamine uptake and glutamine-derived citrate formation in both working rat heart perfusions and cultured adult mouse ventricular cardiomyocytes. When we perfused rat hearts with 13C-labelled D2-HG, we observed a similarly increased formation of citrate. To identify which IDH isoform is responsible for redirecting carbon flux, we modulated IDH1, 2, and 3 in adult mouse ventricular cardiomyocytes using siRNAs. Reduced expression of IDH1 impaired reductive formation of citrate. Importantly, we observed a significant correlation between reductive citrate formation and epigenetic modifications of histones, including increased histone 3 lysine 9 acetylation and di-methylation. To explore these observations, we conducted ChIP-sequencing and identified distinct transcriptional remodeling. Taken together, we demonstrate how oncometabolic stress in the heart causes redirection of central carbon metabolism via reductive carboxylation, and provide evidence of how reductive-citrate formation may induce epigenetic modifications in the heart.

Inhibition of the ketolytic acetyl CoA supply to tumors could be their “Achilles heel”

Inhibition of the ketolytic acetyl <scp>CoA</scp> supply to tumors could be their “Achilles heel”

Development of Morphology Control Method of Coating Layer Utilizing the Ligand Interaction between Iron Particle Surface and Buffer Solution

In recent years, with the progress of functional material development technology, our familiar products in electronic devices, such as smartphones and conductive materials, are dramatically improved in performance. In these functional material, various particles which surface were coated by various inorganic films using dry PVD processes, CVD processes and wet alcohol sol-gel processes, to show novel and characteristic properties. Among these coating methods, the inorganic film coating process of an aqueous solution process has been energetically researched, since it is environmental friendly and easiness to industrialize. For example, Kishimoto et,.al reported that uniformly coating method of titanium dioxide film onto iron particle surfaces by using a buffer solution as a mother liquid. Synthesized TiO2 coated iron particles shows the optical interference effects by alternately laminating several films having gaps of refractive indexes, and as a result, the powder color was changed from gray (the color of original iron powder) to blight blue and purple, etc.1 However, the control of the solid-liquid interface in this process is conplicated, and the slight changes in the surface condition of the substrate and/or its composition results in the formation of island-like structure or flat film structure rather than uniform and homogeneous coating. On the contrary, it means that, by the precise control between the solid-liquid interfaces, various coating morphology, for example, nano-dot coating structure using different substances, can be freely synthesized. Therefore, in our research, we try to develop a method to control the morphology of TiO2 particles formed on the iron particle surface by controlling the interaction with the ligand which is made possible by focusing on the correlation between the iron particle surface and the buffer solution. Carbonyl iron powder (BASF EM grade, median diameter: 4-5 μm), and Clark& Lubs buffer solution (H3BO3-KCl-NaOHaq, pH 9, 0.4 M) were used. As for the carbonyl iron powder, those degassed to 6.0 × 10−1 Pa in a vacuum degassing device and those not degassed were used. The carbonyl iron powder and the buffer solution were mixed in equal amounts and heated in a water bath to a solution temperature of 80℃ for 1 hour. Fines were removed from the supernatant after solution standing and the total dissolved iron was measured with ICP-OES (Optima8300, PerkinElmer). In addition, H3BO3-Na3(C3H5O(COO)3,) -Na3PO4・12H2O (pH9)was used for comparison with Clark&Lubs buffer solution. Furthermore, in order to specify a ligand, ESI-TOF-MS analysis was performed using BRUKER micrOTOFII-EST. Tetra-isopropoxy titanium (C14H28O4Ti), hydrogen peroxide water, and ammonia water were used as the titanium oxide film raw materials in the subsequent film coating step. The temperature was optimized and film coating was performed so that the peroxidated titanium bonds to the substrate surface. The amount of iron dissolved in the Clark&Lubs buffer solution was 1.7 mg/L, while that for iron particles before degassing was 0.6 mg/L, from the ICP-OES analysis results. In the reaction of iron particles in the buffer solution prepared for comparison, the color of the solution in the case of the iron particles before degassing exhibited a green color, which indicating ferrous citrate formation for about 12 hours, then it was changed into an orange color, which indicating ferric citrate formation. On the other hand, in the case of the iron powder after degassing, the color of the solution were changed into orange. From this results, it was suggested that the form of dissolved iron eluted divalent iron ion, and it changed into trivalent by dissolved oxygen2. From the results of ESI-TOF-MS analysis, multiple isotope peaks of the ligand, which caused the iron particle-buffer solution reaction. These results suggests that the morphology of the coating film can be controlled by regulating the iron complex species. Details of other results will be present in our presentation. 1) WO/2003/031683, POWDER COATED WITH TITANIA FILM AND METHOD FOR PRODUCTION THEREOF. 2)A.J.Francis and C.J.Dodge, Applied and Environmental Microbiology, vol59, No.1, 109-113(1993)

Assay of Orphenadrine Citrate in Pharmaceuticals via Extraction-Spectrophotometric Method

An extraction-spectrophotometric technique has been applied and approved for the estimation of orphenadrine citrate in the presence of paracetamol in a binary synthetic mixture and in combined drugs. The procedure is built on the formation of a soluble red colour orphenadrine citrate – eriochrom black T (EBT) ion pair complex at pH 1.40, while the paractamol not paired. The produced red colour ion-pair complex was extracted with chloroform and showed maximum absorption at 509 nm. For quantitative evaluation Beer’s law applied to plot the absorbance against concentration, the relation was a linear in the concentration range of 0.10-6.00 µg/ mL with the molar absorptivity 4.4025 x104 L / mol. cm. The limit of detection and limit quantification were 0.024 µg/ mL, and 0.100 µg/ mL respectively. The intra-assay precision evaluated in terms of % relative standard deviation (RSD) (< 2%). And accuracy was validated with % recovery (98.8-102.5%). The results showed that orphenadrine citrate could be determined successfully in the combined tablet without interference by paracetamol and other common co-formulated substance.

Abstract 476: Cardiac Adaptation to the Oncometabolite D-2-hydroxyglutarate is Driven by Alpha-Ketoglutarate and Acetyl-CoA

Metabolic reprogramming is a hallmark in both cancer and heart failure. Mutations of the isocitrate deyhydrogenase (IDH) 1 and 2 cause metabolic dysfunction in cancer cells through overproduction of the oncometabolite D-2-hydroxyglutarate (D2-HG) and are associated with cardiomyopathy. We recently discovered that alpha-Ketoglutarate dehydrogenase inhibition by D2-HG redirects Krebs cycle flux. This implies a central role for IDH and ATP citrate lyase (ACL) in regulating reductive formation of citrate and histone acetylation in response to mitochondrial impairment in heart and skeletal muscle. Elucidating how metabolic rewiring promotes changes in gene expression and remodeling in heart muscle holds the promise for development of metabolic strategies to support the failing heart. We tested whether modulation of ACL activity reverses D2-HG-mediated metabolic changes using adult rat ventricular cardiomyocytes and L6 myocytes. The ACL inhibitor BMS303141 (BMS) decreased ATP provision in cultured myocytes in a concentration-dependent manner. There was an inverse relation between alpha-KG and ACL activities. Conversely, co-culture with both BMS (0.5 μM) and D2-HG (1 mM) increased ATP provision suggesting that ACL inhibition in presence of D2-HG may be beneficial for energy provision. Next, we conducted isolated working rat heart perfusions with BMS (0.5 μM) and/or D2-HG (1 mM). Cardiac power rapidly declined (by 25%) in the presence of BMS or D2-HG. Simultaneous perfusion with D2-HG and BMS improved cardiac power, suggesting that ACL inhibition protects the heart from metabolic dysfunction by D2-HG. Further, D2-HG elevation mediated structural remodeling in the heart by activating authophagy through increased acetylation of p300, increased phosphorylation of AMPK, and a corresponding decrease in activation and phosphorylation of mTOR. Parallel tracer studies using labeled glucose and glutamine allowed us to conduct computational flux rate analysis by applying the metabolic network CardioNet. We identified major metabolic pathways that are up- and downregulated by D2-HG. Our findings suggest an “oncometabolic axis” in the heart and underscore the potential application of ACL inhibitors to protect the heart from failing.

Reductive carboxylation supports redox homeostasis during anchorage-independent growth.

Epithelial cells receive growth and survival stimuli through their attachment to an extracellular matrix (ECM)1. Overcoming the addiction to ECM-induced signals is required for anchorage-independent growth, a property of most malignant cells2. Detachment from ECM is associated with enhanced reactive oxygen species (ROS) due to altered glucose metabolism2. Here we identify an unconventional pathway that supports redox homeostasis and growth during adaptation to anchorage independence. We observed that detachment from monolayer culture and growth as anchorage-independent tumor spheroids was accompanied by changes in both glucose and glutamine metabolism. Specifically, oxidation of both nutrients was suppressed in spheroids, whereas reductive formation of citrate from glutamine was enhanced. Reductive glutamine metabolism was highly dependent on cytosolic isocitrate dehydrogenase-1 (IDH1), because the activity was suppressed in cells homozygous null for IDH1 or treated with an IDH1 inhibitor. This activity occurred in absence of hypoxia, a well-known inducer of reductive metabolism. Rather, IDH1 mitigated mitochondrial ROS in spheroids, and suppressing IDH1 reduced spheroid growth through a mechanism requiring mitochondrial ROS. Isotope tracing revealed that in spheroids, isocitrate/citrate produced reductively in the cytosol could enter the mitochondria and participate in oxidative metabolism, including oxidation by IDH2. This generates NADPH in the mitochondria, enabling cells to mitigate mitochondrial ROS and maximize growth. Neither IDH1 nor IDH2 was necessary for monolayer growth, but deleting either one enhanced mitochondrial ROS and reduced spheroid size, as did deletion of the mitochondrial citrate transporter protein. Together, the data indicate that adaptation to anchorage independence requires a fundamental change in citrate metabolism, initiated by IDH1-dependent reductive carboxylation and culminating in suppression of mitochondrial ROS.