Speciation of copper(II) complexes in an ionic liquid based on choline chloride and in choline chloride/water mixtures

A deep-eutectic solvent with the properties of an ionic liquid is formed when choline chloride is mixed with copper(II) chloride dihydrate in a 1:2 molar ratio. EXAFS and UV-vis-near-IR optical absorption spectroscopy have been used to compare the coordination sphere of the cupric ion in this ionic liquid with that of the cupric ion in solutions of 0.1 M of CuCl(2)·2H(2)O in solvents with varying molar ratios of choline chloride and water. The EXAFS data show that species with three chloride ions and one water molecule coordinated to the cupric ion as well as species with two chloride molecules and two water molecules coordinated to the cupric ion are present in the ionic liquid. On the other hand, a fully hydrated copper(II) ion is formed in an aqueous solution free of choline chloride, and the tetrachlorocuprate(II) complex forms in aqueous choline chloride solutions with more than 50 wt % of choline chloride. In solutions with between 0 and 50 wt % of choline chloride, mixed chloro-aquo complexes occur. Upon standing at room temperature, crystals of CuCl(2)·2H(2)O and of Cu(choline)Cl(3) formed in the ionic liquid. Cu(choline)Cl(3) is the first example of a choline cation coordinating to a transition-metal ion. Crystals of [choline](3)[CuCl(4)][Cl] and of [choline](4)[Cu(4)Cl(10)O] were also synthesized from molecular or ionic liquid solvents, and their crystal structures were determined.     

On the catalytic mechanism of choline oxidase

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with betaine aldehyde as an intermediate. In this study, primary deuterium and solvent kinetic isotope effects have been used to elucidate the mechanism for substrate oxidation by choline oxidase using both steady-state kinetics and rapid kinetics techniques. The D(kcat/Km) value with 1,2-[2H4]-choline at saturating oxygen concentration was independent of pH in the range between 6.5 and 10, with a value of approximately 10.6, indicating that CH bond cleavage is not masked by other titratable kinetic steps belonging to the reductive half-reaction. In agreement with this conclusion, a Dkred value of approximately 8.9 was determined at pH 10 for the anaerobic reduction of the flavin by choline, irrespective of whether aqueous or deuterated solvent was used. At pH 10, both the D2(O)(kcat/Km) and the D2(O)kred values were not different from unity with choline or 1,2-[2H4]-choline, while the Dkcat and D2(O)kcat values were 7.3 and 1.1, respectively. The kcat and kred values were 133 s(-1) and 135 s(-1) with betaine aldehyde and 60 s(-1) and 93 s(-1) with choline. These data are consistent with a chemical mechanism in which the choline hydroxyl proton is not in flight in the transition state for CH bond cleavage and with chemical steps of flavin reduction by choline and betaine aldehyde being rate limiting for the overall turnover of the enzyme.     

A sensitive choline biosensor using Fe3O4 magnetic nanoparticles as peroxidase mimics

A sensitive choline biosensor using Fe(3)O(4) magnetic nanoparticles and a choline oxidase modified gold electrode was developed. Fe(3)O(4) magnetic nanoparticles as peroxidase mimics used in the choline biosensor can not only improve the sensitivity of the response signal, but also possess the favorable properties of stability, magnetic separation and easy preparation, etc. When using the reduction currents of square wave voltammetry as the detection signals, the interferences of ascorbic acid and uric acid to the choline biosensor can be reduced effectively. The reduction currents of the square wave voltammetry were increased with the logarithm values of the choline chloride concentration (from 10(-9) to 10(-2) M), the detection limit was estimated to be 0.1 nM (S/N = 3). This choline biosensor also exhibited favorable selectivity and stability in the determination of choline chloride.     

Synthesis and Evaluation of GdIII‐Based Magnetic Resonance Contrast Agents for Molecular Imaging of Prostate‐Specific Membrane Antigen

Magnetic resonance (MR) imaging is advantageous because it concurrently provides anatomic, functional, and molecular information. MR molecular imaging can combine the high spatial resolution of this established clinical modality with molecular profiling in vivo. However, as a result of the intrinsically low sensitivity of MR imaging, high local concentrations of biological targets are required to generate discernable MR contrast. We hypothesize that the prostate‐specific membrane antigen (PSMA), an attractive target for imaging and therapy of prostate cancer, could serve as a suitable biomarker for MR‐based molecular imaging. We have synthesized three new high‐affinity, low‐molecular‐weight Gd^III‐based PSMA‐targeted contrast agents containing one to three Gd^III chelates per molecule. We evaluated the relaxometric properties of these agents in solution, in prostate cancer cells, and in an in vivo experimental model to demonstrate the feasibility of PSMA‐based MR molecular imaging.     

The potential for practical improvements in cancer diagnostics by mathematically-optimized magnetic resonance spectroscopy

The fast Padé transform (FPT) has been benchmarked as a stable, high-resolution processor. In this paper, the performance of the FPT is examined for in vitro magnetic resonance (MR) spectroscopic data associated with ovarian, breast and prostate cancer as well as benign or normal tissue. We also examine how the FPT handles in vivo MR spectroscopic (MRS) time signals from human brain encoded by high field and clinical (1.5 T) scanners. Salient comparisons are made with the conventional data analysis through the fast Fourier transform (FFT). Separation of noise from genuine signal is carried out with a view to practical applications. Compared to the FFT, the fast Padé transform provided markedly improved resolution of total shape spectra from encoded in vivo time signals from healthy human brain and for in vitro data associated with ovarian cancer. Evidence is presented as to why it is necessary to go beyond MR total shape spectra to calculate metabolite concentrations. It is shown that error spectra, while necessary, are insufficient for accurate assessment of MR data. Two examples from oncology are given to illustrate this point: (1) a marker of breast cancer, phosphocholine, is detected on the component shape spectra, but not on the total shape spectrum, (2) diagnostically important multiplet resonances in prostate cancer spectra can only be detected on the component shape spectra, but not on the total shape spectrum. The FPT provides accurate calculation of metabolite concentrations based on in vitro MR data from three diagnostic problems in clinical oncology: (1) malignant and benign ovarian lesions, (2) breast cancer, fibroadenoma and normal breast tissue and (3) prostate cancer tissue, healthy glandular and stromal prostate tissue. Practical implementation of signal-noise separation is demonstrated for MR time signals encoded in vivo from the human brain on a clinical (1.5 T) scanner. Some 23 stable resonances are thereby identified and quantified. These results provide the basis for the needed next steps: to extensively apply the FPT to in vivo time signals encoded mainly on clinical scanners from e.g. brain tumors, breast, ovary and prostate cancers as well as from benign and normal tissue. The overall goal is that this practical approach through mathematical optimization enables Padé-based MRS to soon be implemented in clinical oncology, including target planning, post-radiotherapeutic follow-up and other aspects of radiation therapy.     

Exact quantification of time signals from magnetic resonance spectroscopy by the fast Padé transform with applications to breast cancer diagnostics

Mathematical modeling via the fast Padé transform (FPT) is applied according to experimental NMR data encoded from (a) normal, non-infiltrated breast tissue, (b) benign pathology (fibroadenoma) and (c) malignant breast tissue. At a partial signal length N _P  = 1500, the FPT provided exact reconstruction of all the input spectral parameters for the time signals corresponding to the normal, benign as well as to the malignant lesions. The converged parametric results remained stable at longer signal lengths. The Padé absorption spectra yielded unequivocal resolution of all the extracted physical metabolites, even of those that were nearly completely overlapping (phosphocholine and phosphoethanolamine at 3.22 ppm). The capacity of the FPT to resolve and precisely quantify the physical resonances as encountered in normal versus benign versus malignant breast is demonstrated. In particular, the FPT unambiguously delineated and quantified diagnostically important metabolites such as lactate, as well as choline, phosphocholine and glycerophosphocholine that are very closely overlapping and may represent MR-retrievable molecular markers of breast cancer. This was achieved by the FPT without any fitting or numerical integration of peak areas. We conclude that these advantages of the FPT could be of definite benefit for breast cancer diagnostics via NMR and that this line of investigation should continue with encoded data from benign and malignant breast tissue, in vitro and in vivo. We anticipate that Padé-optimized MRS will reduce the false positive rates of MR-based modalities and further improve their sensitivity. Once this is achieved, and given that MR entails no ionizing radiation, new possibilities for screening/early detection open up, especially for risk groups, e.g. Padé-optimized MRS could be used with greater surveillance frequency among younger women with high breast cancer risk.     

Oxygen- and temperature-dependent kinetic isotope effects in choline oxidase: correlating reversible hydride transfer with environmentally enhanced tunneling

Choline oxidase catalyzes the flavin-linked oxidation of choline to glycine betaine, with betaine aldehyde as intermediate and oxygen as electron acceptor. Here, the effects of oxygen concentration and temperature on the kinetic isotope effects with deuterated choline have been investigated. The D(kcat/Km) and Dkcat values with 1,2-[(2)H4]-choline were pH-independent at saturating oxygen concentrations, whereas they decreased at high pH to limiting values that depended on oxygen concentration at < or = 0.97 mM oxygen. The kcat/Km and kcat pH profiles had similar patterns reaching plateaus at high pH. Both the limiting kcat/Km at high pH and the pKa values were perturbed to lower values with choline and < or = 0.25 mM oxygen. These data suggest that oxygen availability modulates whether the reduced enzyme-betaine aldehyde complex partitions forward to catalysis rather then reverting to the oxidized enzyme-choline alkoxide species. At saturating oxygen concentrations, the D(kcat/Km) was 10.6 +/- 0.6 and temperature independent, and the isotope effect on the preexponential factors (A(H)'/A(D)') was 14 +/- 3, ruling out a classical over-the-barrier behavior for hydride transfer. Similar enthalpies of activation (deltaH(double dagger)) with values of 18 +/- 2 and 18 +/- 5 kJ mol(-1) were determined with choline and 1,2-[(2)H4]-choline. These data suggest that the hydride transfer reaction in which choline is oxidized by choline oxidase occurs quantum mechanically within a preorganized active site, with the reactive configuration for hydride tunneling being minimally affected by environmental vibrations of the reaction coordinate other than those affecting the distance between the donor and acceptor of the hydride.     

离子色谱法测定饲料中氯化胆碱和三甲胺的含量

建立了离子色谱法测定饲料中氯化胆碱含量及鉴别饲料中氯化胆碱及掺假物三甲胺的方法。选用IonPac CS12阳离子交换色谱柱(250 mm×4 mm i.d.)和8.5 mmol/L H2SO4淋洗液,抑制型电导检测,在16 min内分离测定了包括胆碱和三甲胺在内的8种阳离子。胆碱和三甲胺的最小检出限分别为0.1 mg/L和0.05 mg/L。方法回收率为99.25%~102.5%。该方法具有灵敏度高、选择性强、操作简单等优点。     

Amperometric determination of choline and acetylcholine with enzymes immobilized in a photocross-linkable polymer

Choline and acetylcholine sensors were prepared by using choline oxidase and acetylcholinesterase, entrapped in photocross- linkable poly(vinyl alcohol) bearing styrylpyridinium (PVA-SbQ). The measurements were based on the detection of hydrogen peroxide liberated by an enzyme reaction (choline oxidase) or two sequential enzyme reactions (acetylcholine esterase and choline oxidase). The determination range for choline was 2.5-2-150 αmol 1^-1 and for acetylcholine 20-2-750 αmol 1^-1. The response times were 2-2-4 min. The immobilized enzyme membranes stored in a dry state were very stable and no loss of activity was observed after storage for 60 days.     

Multifunctional Core–Shell Upconverting Nanoparticles for Imaging and Photodynamic Therapy of Liver Cancer Cells

Lanthanide‐doped upconversion nanoparticles (UCNPs) have attracted considerable attention for their application in biomedicine. Here, silica‐coated NaGdF₄:Yb,Er/NaGdF₄ nanoparticles with a tetrasubstituted carboxy aluminum phthalocyanine (AlC₄Pc) photosensitizer covalently incorporated inside the silica shells were prepared and applied in the photodynamic therapy (PDT) and magnetic resonance imaging (MRI) of cancer cells. These UCNP@SiO₂(AlC₄Pc) nanoparticles were uniform in size, stable against photosensitizer leaching, and highly efficient in photogenerating cytotoxic singlet oxygen under near‐infrared (NIR) light. In vitro studies indicated that these nanoparticles could effectively kill cancer cells upon NIR irradiation. Moreover, the nanoparticles also demonstrated good MR contrast, both in aqueous solution and inside cells. This is the first time that NaGdF₄:Yb,Er/NaGdF₄ upconversion‐nanocrystal‐based multifunctional nanomaterials have been synthesized and applied in PDT. Our results show that these multifunctional nanoparticles are very promising for applications in versatile imaging diagnosis and as a therapy tool in biomedical engineering.     

Bienzyme Electrode Compatible Behavior for Water-Soluble and-Insoluble Substrates

Abstract

A bienzymatic sensing layer containing two enzymes able to work sequentially, choline oxidase (ChOD) and phospholipase D (PLaseD), was used to design an electrochemical biosensor for the detection of either a water-soluble (choline) or insoluble (phosphatidylcholine) substrate. A photocrosslinkable polymer, poly(vinyl alcohol) bearing styrylpyridinium groups (PVA-SbQ), was used as host-matrix for enzyme immobilization. Controlled amounts of PVA-SbQ and of the two enzymes were directly coated on a platinum disk, then photopolymerized. The compatibility of working conditions for choline and phosphatidylcholine detection in the presence of Triton X-100 and CaCl₂ was investigated. The effect of the activity ratio PLaseD / ChOD on the sensor performance was determined. The sensitivities to choline and to phosphatidylcholine were 18 mA.1mol^?1 and 0.66 mA.1.mol^?1 respectively, the detection limit being 1.5.10^?8 M for choline and 1.5.10^?6 M for phosphatidylcholine. The linear range extended up to ca. 10^?4 M for choline and ca. 2.10^?5 M for phosphatidylcholine and the response time was close to 30 seconds for choline and ca. 2 min for phosphatidylcholine.     

Diol lipids

Conclusions We synthesized some diol analogs of phosphatidylcholine that contained an unsaturated aliphatic acid moiety, and specifically: 2-oleoyloxyethyl choline phosphate, 3-oleoyloxypropyl choline phosphate, 4-oleoyl-oxybutyl choline phosphate, 5-oleoyloxypentyl choline phosphate, and 6-oleoyloxyhexyl choline phosphate.See [1] for Communication 22.Translated from Izvestiya Akademii NaukSSSR, Seriya Khimicheskaya, No. 2, pp. 410–414, February, 1973.     

Biochemical sniffer with choline oxidase for measurement of choline vapour

An enzyme-based gas sensor (bio-sniffer) for choline vapour was fabricated and tested. The bio-sniffer was constructed using a Clark-type dissolved oxygen electrode and an enzyme (choline oxidase) immobilized membrane. This bioelectronic device measures choline concentration by the oxygen consumption induced by an enzyme reaction of choline oxidase. As the assessment of sensor performances, the calibration curves for choline in the liquid and gas phases were investigated, respectively. The responses of the bioelectronic device to choline solutions of various concentrations were related within a range from 5.00 to 700 μmol·L⁻¹ with a correlation coefficient of 0.999. On the other hand, the bio-sniffer for choline vapour was placed into a gas-measuring chamber and calibrated using gas detection tubes. The calibration range was 1.00–30.0 ppm (correlation coefficient: 0.996). The response time for choline vapour was approximately 15% slower than that of biosensor for choline solution. These results indicate that the bio-sniffer is useful to monitor colourless and odourless choline gas released from coating compositions including choline. Correspondence: Kohji Mitsubayashi, Department of Biomedical Devices and Instrumentation, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan     

Determination of choline in milk and infant formulas by enzymatic analysis: collaborative study

A collaborative study was conducted on a coupled enzymatic-spectrophotometric method for the determination of choline in infant formula and milk powders. Twenty-nine laboratories participated in the analysis of 8 blind duplicates over the range of 45-175 micrograms/100 g sample. After the combined acid hydrolysis-phospholipase release of choline, incubation with choline oxidase in the presence of peroxidase and phenol generates a quinoneimine chromophore with 4-aminoantipyrine. Following raw data screening, overall mean RSDR was estimated at 5.14% (range, 4.26-6.34%) with a HORRAT value of 0.91 (range, 0.76-1.00) and an overall mean RSDr:RSDR value of 0.53 (range, 0.42-0.74). The method was also compared with alternative independent analytical techniques for several of the collaborative study samples.     

Analysis of acetylcholine and choline in microdialysis samples by liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionisation tandem mass spectrometric (LC/ESI-MS/MS) method was developed for the analysis of acetylcholine and choline in microdialysis samples. A Ringer's solution that contains high (150 mM) concentrations of inorganic salts was used to extract acetylcholine and choline from a rat or mouse brain. The separation of acetylcholine, choline, an internal standard acetyl-beta-methylcholine, endogenous compounds and inorganic cations was achieved with hydrophilic interaction chromatography using a diol column. The eluent consisted of 20 mM ammonium formate (pH 3.3) and acetonitrile (20:80) which is favourable for the ESI process. Limits of detection (signal-to-noise (S/N) ratio = 3) of 0.02 nM (0.2 fmol) for acetylcholine and 1 nM (10 fmol) for choline were observed using standards diluted in Ringer's solution. A good linearity was obtained from the limit of quantitation: 0.1 nM (S/N ratio = 10) to 50 nM (r = 0.999) for acetylcholine and within the concentration range of 100-3500 nM (r = 0.998) for choline. The between-day repeatability of the method was good; RSD was 3.1% at 1 nM level of acetylcholine and 3.5% at 1000 nM level of choline. The recoveries for addition of 1 or 2.5 nM acetylcholine and 0.2 or 1 microM choline in microdialysis balancing samples were between 93 and 101% indicating that no suppressing endogenous compounds were co-eluting with acetylcholine or choline. The developed method was applied to the analysis of microdialysis balancing samples collected from rat and mouse brains.     

Mn~(2+)-doped ZrO_2@PDA nanocomposite for multimodal imaging-guided chemo-photothermal combination therapy

Developing low toxicity and multifunctional theranostic nanoplatform is the key for precise cancer diagnosis and treatment.Herein,an inorganic-organic hybrid nanocomposite is designed by modifying zirconium dioxide(ZrO_2) with polydopamine(PDA) followed by doping Mn~(2+) ions and functionalizing with Tween 20(Tween-ZrO_2@PDA-Mn~(2+)) for multimodal imaging and chemo-photothermal combination therapy.The as-prepared nanocomposite exhibits good biocompatibility in vitro and in vivo.Specifically,it can be employed as a multifunctional platform not only for computed tomography(CT)imaging and T_1-weighted magnetic resonance(MR) imaging,but also for efficient chemotherapeutic drug doxorubicin hydrochloride(DOX) loading.Importantly,because of the pronounced photothermal conversion performance and controllable DOX release ability triggered by the near-infrared(NIR)irradiation and acidic pH,the synergistic effect between photothermal the rapy and chemotherapy results in an enhanced cancer treatment efficacy in vivo.Our work provides a high-performance inorganicorganic hybrid nanotheranostic platform for chemo-photothermal cancer therapy guided by CT and MR imaging.     

Evaluation of the inhibition of choline uptake in synaptosomes by capillary electrophoresis with electrochemical detection

A direct method for evaluating choline uptake by the high-affinity choline transport system in synaptosomes was developed using capillary electrophoresis (CE) with electrochemical (EC) detection. On-column EC detection of choline and the internal standard, butyrylcholine, was accomplished with a 25 microm platinum electrode modified with the enzymes, choline oxidase and acetylcholinesterase. Choline uptake was evaluated as a function of choline concentration and a KM value of 1.7 microM was determined. The method was also used to evaluate a new class of redox affinity inhibitors of choline transport. In particular, the effectiveness of 3-[(trimethylammonio)methyl]catechol (TMC) as an inhibitor of choline uptake was examined independently and relative to the inhibition of the well-known inhibitor of choline transport, hemicholinium-3. The IC50 and KI for TMC were determined to be 30 microM and 14 microM, respectively. The combination of the selectivity and sensitivity afforded by CEEC provides a relatively straightforward approach for monitoring choline transport in synaptosomes.     

Unequivocal disentangling genuine from spurious information in time signals: clinical relevance in cancer diagnostics through magnetic resonance spectroscopy

Pole-zero cancellation in the polynomial quotient of the fast Padé transform (FPT) is shown to clearly and unequivocally distinguish spurious resonances from the true metabolites, in the presence of random Gauss-distributed zero mean noise in a magnetic resonance (MR) spectrum. Thus, by applying the principle of Froissart doublets within the FPT, noise as typically encountered in clinically encoded MR-time signals, is completely separated from the genuine metabolic information. The basis for the unprecedented algorithmic success of the FPT for processing of MR signals is explained within the framework of quantum mechanics. The clear, direct and immediate importance of these findings is reviewed with respect to clinical oncology. Besides confirming the high resolution and stability of the FPT in general studies of MR total shape spectra, this superior resolution performance of the FPT has also been confirmed with respect to data directly derived from malignant and benign ovarian samples. Not only does the FPT markedly enhance resolution of MR spectra compared to the conventional Fourier analysis, but it also yields the unequivocal, exact parametric data needed to reconstruct the metabolite concentrations which characterize ovarian cancer and distinguish this from non-malignant lesions. These features of the FPT are deemed to be of critical benefit to ovarian cancer diagnostics via MRS, in particular for early detection, a goal which has thus far been elusive, but achievement of which would definitely confer a major survival advantage. It is anticipated that MRS via Padé processing will reduce the false positive rates of MR-based modalities and, moreover, will further improve the sensitivity of these methods. Once this is achieved, and given that all MR-based diagnostic methods are free from ionizing radiation, new possibilities for cancer screening and early detection would emerge, especially for risk groups, e.g., the application of Padé-optimized MRS in younger women at high risk for breast and/or ovarian cancer.     

Enzymic method for the spectrophotometric determination of choline in liquor.

A sensitive spectrophotometric method for the determination of choline in liquor is described. The method involves the conversion of choline into formaldehyde by sequential enzymic reactions (choline oxidase and catalase), followed by the formation of a chromophore with 4-aminopent-3-en-2-one. The calibration graph was linear in the range 0.4-15 micrograms cm-3 of choline. The relative standard deviation at 5 micrograms cm-3 of choline was 1.3%. There was no interference from most of the common ingredients of liquor. More than 95% of choline added at two levels was recovered from real samples. The method is simple, and the detection limit was 2 micrograms g-1 when 5 g of sample were assayed.     

On-line biosensors for simultaneous determination of glucose, choline, and glutamate integrated with a microseparation system

An effective microseparation system integrated with ring-disc electrodes and two microfluidic devices was fabricated for in vivo determination using a microdialysis pump. The major interference of ascorbic acid (AA) was excluded by direct oxidation with ascorbate oxidase. Glucose, glutamate, and choline were successfully determined simultaneously through the biosensors modified with a bilayer of osmium-poly(4-vinylpyridine)gel-horseradish peroxidase (Os-gel-HRP)/glucose oxidase (GOD), glutamate oxidase (GlutaOD) or choline oxidase (ChOD). To stabilize the biosensors, 0.2% polyethylenimine (PEI) was mixed with the oxidases. The cathodic currents of glucose, glutamate, and choline biosensors started to increase after the standard solutions were injected into the microseparation system. The on-line biosensors show a wide calibration range (10(-7)-10(-5) mol/L) with a detection limit of 10(-8) mol/L at the working potential of -50 mV. The variations of glucose, glutamate, and choline were determined simultaneously in a free moving rat when we perfused the medial frontal cortex with 100 micro mol/L N-methyl-D-aspartate (NMDA) solution, which is the agonist of the NMDA receptor.