Effect of fructooligosaccharide on endothelial function in CKD patients: a randomized controlled trial.

BACKGROUND: Microbiota-derived uremic toxins have been associated with inflammation that could corroborate with endothelial dysfunction (ED) and increase cardiovascular risk in patients with chronic kidney disease (CKD). This trial aimed to evaluate the effect of the prebiotic fructooligosaccharide (FOS) on endothelial function and arterial stiffness in nondialysis CKD patients. METHODS: In a double-blind controlled trial, 46 nondiabetic CKD patients were randomized to receive 12 g/day of FOS or placebo (maltodextrin) for 3 months. Total p-cresyl sulfate (PCS) and indoxyl sulfate by high-performance liquid chromatography, urinary trimethylamine N-oxide by mass spectrometry, C-reactive protein, interleukin-6 (IL-6), serum nitric oxide and stroma-derived factor-1 alfa were measured at baseline and at the end of follow-up; endothelial function was assessed through flow-mediated dilatation (FMD) and arterial stiffness by pulse wave velocity (PWV). RESULTS: The mean (± standard deviation) age of the study participants was 57.6 ± 14.4 years, with an estimated glomerular filtration rate of 21.3 ± 7.3 mL/min/1.73 m2. During the follow-up, regarding the inflammatory markers and uremic toxins, there was a significant decrease in IL-6 levels (3.4 ± 2.1 pg/mL versus 2.6 ± 1.4 pg/mL; P = 0.04) and a trend toward PCS reduction (55.4 ± 38.1 mg/L versus 43.1 ± 32.4 mg/L, P = 0.07) only in the prebiotic group. Comparing both groups, there was no difference in FMD and PWV. In an exploratory analysis, including a less severe ED group of patients (FMD ≥2.2% at baseline), FMD remained stable in the prebiotic group, while it decreased in the placebo group (group effect P = 0.135; time effect P = 0.012; interaction P = 0.002). CONCLUSIONS: The prebiotic FOS lowered circulating levels of IL-6 in CKD patients and preserved endothelial function only in those with less damaged endothelium. No effect of FOS in arterial stiffness was observed.

Separation of carbohydrates on electrophoresis microchips with controlled electrolysis.

This study reports the separation of fructose, galactose, glucose, lactose and sucrose on glass microchip electrophoresis (ME) devices using a microfluidic platform adapted with external reservoirs for controlling the electrolysis phenomenon. The connections between external reservoirs and microfluidic platform were performed by saline bridges created using silicone tubing filled with BGE. The separation conditions were optimized and the best results were achieved using a BGE containing 75 mmol/L NaOH and 15 mmol/L trisodium phosphate. Electrophoretic separations were monitored using a capacitively coupled contactless conductivity detection system. The controlled electrolysis has successfully allowed the application of a higher voltage on the separation channel promoting the baseline separation of five carbohydrates within 180 s with great run-to-run repeatability (RSD < 1%). The achieved efficiencies ranged from 45 000 ± 6000 to 70 000 ± 3000 plates/m demonstrating a performance better than ME devices without controlled electrolysis. The proposed system offered good linearity from 1 to 10 mmol/L and LODs between 150 and 740 µmol/L. The use of external tubes for controlling the electrolysis phenomenon on ME devices has solved common problems associated to run-to-run repeatability and analytical reliability required for routine and quantitative analysis.

Phytochemical characterization of the Vochysia rufa (Vochysiaceae) extract and its effects on oxidative stress in the pancreata of streptozotocin-induced diabetic rats.

Aqueous extract of macerated Vochysia rufa stem bark has been commonly used in the treatment of diabetes. Therefore, we evaluated the antihyperglycemic and antioxidant effects of an extract of V. rufa on the pancreata of streptozotocin (STZ)-induced diabetic rats. Animals received one of the following treatments daily by oral gavage: water (diabetic-control), V. rufa extract (diabetic-V. rufa), or glibenclamide (diabetic-GBD). Total antioxidant capacity; levels of thiobarbituric acid reactive substances, reduced glutathione, and sulfhydryls; and superoxide dismutase, catalase, and glutathione peroxidase (GPx) activities were measured in the pancreas. Biochemical analysis of serum total cholesterol and fractions, triglycerides, creatinine, urea, acid uric, ALP, γ-GT, AST, and ALT was performed, and pancreatic ß-cells positive for insulin were evaluated by immunohistochemistry. Rats treated with extract exhibited a decrease in fasting blood glucose compared with levels in diabetic control rats. GPx activity and sulfhydryl levels were significantly lower in diabetic-V. rufa rats compared with those of diabetic-control rats. V. rufa extract acted to normalize the biochemical alterations found in diabetic rats (diabetic-controls), as demonstrated by increases in urea, HDL, ALP, AST, and ALT. Reduction in blood glucose was independent of an increase in insulin. The V. rufa extract was found to be composed of free sugars (inositol, galactose, glucose, mannose, sucrose, arabinose, and ribose) as the main metabolites. Thus, aqueous extract of the stem bark of V. rufa is capable of reducing blood glucose, resulting in an antioxidant effect on the pancreatic tissue of STZ-diabetic rats.

Determination of Inorganic Ion Profiles of Illicit Drugs by Capillary Electrophoresis.

A portable capillary electrophoresis instrument with dual capacitively coupled contactless conductivity detection (C4 D) was used to determine the inorganic ionic profiles of three pharmaceutical samples and precursors of two illicit drugs (contemporary samples of methylone and para-methoxymethamphetamine). The LODs ranged from 0.10 µmol/L to 1.25 µmol/L for the 10 selected cations, and from 0.13 µmol/L to 1.03 µmol/L for the eight selected anions. All separations were performed in less than 6 min with migration times and peak area RSD values ranging from 2 to 7%. The results demonstrate the potential of the analysis of inorganic ionic species to aid in the identification and/or differentiation of unknown tablets, and real samples found in illicit drug manufacture scenarios. From the resulting ionic fingerprint, the unknown tablets and samples can be further classified.

Formation of isomers of anionic hemiesters of sugars and carbonic acid in aqueous medium.

Hemiesters of carbonic acid can be freely formed in aqueous media containing HCO3(-)/CO2 and mono- or poly-hydroxy compounds. Herein, (13)C NMR spectroscopy was used to identify isomers formed in aqueous solutions of glycerol (a prototype compound) and seven carbohydrates, as well as to estimate the equilibrium constant of formation (Keq). Although both isomers are formed, glycerol 1-carbonate corresponds to 90% of the product. While fructose and ribose form an indistinct mixture of isomers, the anomers of d-glucopyranose 6-carbonate correspond to 74% of the eight isomers of glucose carbonate that were detected. The values of Keq for the disaccharides sucrose (4.3) and maltose (4.2) are about twice the values for the monosaccharides glucose (2.0) and fructose (2.3). Ribose (Keq = 0.89)-the only sugar without a significant concentration of a species containing a -CH2OH group in an aqueous solution-resulted in the smallest Keq. On the basis of the Keq value and the concentrations of HCO3(-) and glucose in blood, one can anticipate a concentration of 2-4 µmol L(-1) for glucose 6-carbonate, which corresponds to ca. of 10% of its phosphate counterpart (glucose 6-phosphate).

Fast production of microfluidic devices by CO2 laser engraving of wax-coated glass slides.

Glass is one of the most convenient materials for the development of microfluidic devices. However, most fabrication protocols require long processing times and expensive facilities. As a convenient alternative, polymeric materials have been extensively used due their lower cost and versatility. Although CO2 laser ablation has been used for fast prototyping on polymeric materials, it cannot be applied to glass devices because the local heating causes thermal stress and results in extensive cracking. A few papers have shown the ablation of channels or thin holes (used as reservoirs) on glass but the process is still far away from yielding functional glass microfluidic devices. To address these shortcomings, this communication describes a simple method to engrave glass-based capillary electrophoresis devices using standard (1 mm-thick) microscope glass slides. The process uses a sacrificial layer of wax as heat sink and enables the development of both channels (with semicircular shape) and pass-through reservoirs. Although microscope images showed some small cracks around the channels (that became irrelevant after sealing the engraved glass layer to PDMS) the proposed strategy is a leap forward in the application of the technology to glass. In order to demonstrate the capabilities of the approach, the separation of dopamine, catechol and uric acid was accomplished in less than 100 s.

Analysis of ecstasy tablets using capillary electrophoresis with capacitively coupled contactless conductivity detection.

A method for the identification of 3,4-methylenedioxymethamphetamine (MDMA) and meta-chlorophenylpiperazine (mCPP) was developed employing capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C(4) D). Sample extraction, separation, and detection of "Ecstasy" tablets were performed in <10 min without sample derivatization. The separation electrolyte was 20 mm TAPS/Lithium, pH 8.7. Average minimal detectable amounts for MDMA and mCPP were 0.04 mg/tablet, several orders of magnitude lower than the minimum amount encountered in a tablet. Seven different Ecstasy tablets seized in Rio de Janeiro, Brazil, were analyzed by CE-C(4) D and compared against routine gas chromatography-mass spectrometry (GC-MS). The CE method demonstrated sufficient selectivity to discriminate the two target drugs, MDMA and mCPP, from the other drugs present in seizures, namely amphepramone, fenproporex, caffeine, lidocaine, and cocaine. Separation was performed in <90 sec. The advantages of using C(4) D instead of traditional CE-UV methods for in-field analysis are also discussed.

Getting started with open-hardware: development and control of microfluidic devices.

Understanding basic concepts of electronics and computer programming allows researchers to get the most out of the equipment found in their laboratories. Although a number of platforms have been specifically designed for the general public and are supported by a vast array of on-line tutorials, this subject is not normally included in university chemistry curricula. Aiming to provide the basic concepts of hardware and software, this article is focused on the design and use of a simple module to control a series of PDMS-based valves. The module is based on a low-cost microprocessor (Teensy) and open-source software (Arduino). The microvalves were fabricated using thin sheets of PDMS and patterned using CO2 laser engraving, providing a simple and efficient way to fabricate devices without the traditional photolithographic process or facilities. Synchronization of valve control enabled the development of two simple devices to perform injection (1.6 ± 0.4 µL/stroke) and mixing of different solutions. Furthermore, a practical demonstration of the utility of this system for microscale chemical sample handling and analysis was achieved performing an on-chip acid-base titration, followed by conductivity detection with an open-source low-cost detection system. Overall, the system provided a very reproducible (98%) platform to perform fluid delivery at the microfluidic scale.

Characterization of microchip electrophoresis devices fabricated by direct-printing process with colored toner.

This paper reports for the first time the use of colored toner to produce polyester toner (PT) ME devices. Colored PT devices were designed in drawing software and printed on a polyester film using a color laser printer with 3600 dpi resolution. The colored toner is composed of a copolymer mixture (styrene and acrylate), wax, silicon dioxide, and pigments. The presence of silica in the toner composition has enhanced the EOF magnitude and improved the analytical performance. For a pH range between 2 and 12, the EOF measured on a magenta PT chip, for example, ranged from 3.8 to 5.8 (× 10(-4) cm(2) V(-1) s(-1) ). Typical separations of inorganic cations (K(+) , Na(+) , and Li(+) ) were used as model system to investigate the analytical feasibility of the proposed devices. The repeatability for the migration times of all analytes exhibited RSD values lower than 1% (n = 10). The separation efficiencies found on colored PT devices ranged from 10 000 to 49 000 plates/m, which means between 7 and 23% of the maximum theoretical efficiency on this microfluidic platform (1.85 × 10(5) plates/m). The improvements achieved on the proposed devices are associated with the small additional amount of silica on the toner composition as well as the printing of channels with smoother surfaces and better uniformity when compared to the conventional PT chips printed with monochromatic laser printers.

Simultaneous determination of diclofenac and its common counter-ions in less than 1 minute using capillary electrophoresis with contactless conductivity detection.

This paper presents a method for fast and simultaneous determination of diclofenac (DCF) and its common counter-ions (potassium, sodium, and diethylammonium) using CE with capacitively coupled contactless conductivity detection (CE-C(4) D). On the basis of a single electropherogram (about 50 s), the proposed method allows the determination of the stoichiometry, absolute quantification and evaluation of the degradation degree of the active pharmaceutical ingredient (DCF). A linear working range from 100 to 500 µmol/L was obtained for all analytes in an equimolar TRIS/TAPS (10 mmol/L) solution as the background electrolyte as well as adequate LOD (7, 6, 7, and 10 µmol/L for K(+) , Na(+) , diethylammonium, and DCF, respectively). The proposed method was applied to the analysis of pharmaceutical formulations (tablets and spray form) with similar results to those achieved by HPLC (DCF) or flame photometry (K and Na) at a 95% confidence level.

Capillary electrophoresis with capacitively coupled contactless conductivity detection applied to the quantitation and to the determination of physical-chemical properties of peroxycarboxylates in aqueous medium.

CE with C4D (CE-C4D) was successfully applied to the investigation of performate, peracetate, and perpropionate in aqueous medium. Ionic mobilities, diffusion coefficients, and hydrodynamic radii were obtained for the first time for these species. CE-C4D was also used to estimate the pKa values of the peroxycarboxylic acids. Because the peroxycarboxylates (POCs) undergoes hydrolysis while migrating, a simple calibration curve cannot be used for quantitation. Thus, an indirect calibration approach was used. The new method was used to monitor the formation of peroxycarboxylic acids from hydrogen peroxide and the carboxylic acid as well as to the quantitation of peracetic acid in a commercial sample. The CE-C4D method compares favorably with the conventional titration method because of the possibility of speciation of the POC, the low sample consumption, and the low LOD (14, 8, and 24 µmol/L for performate, peracetate, and perpropionate, respectively). Although POCs are structural isomers of monoalkyl carbonates, they have greater hydrodynamic radii, which suggests that the positions of the oxygen atoms in the molecules have a direct impact in the charge density and consequently on the hydration atmosphere.

Quantification of terbinafine in pharmaceutical tablets using capillary electrophoresis with contactless conductivity detection and batch injection analysis with amperometric detection.

Terbinafine hydrochloride (TerbHCl) is an allylamine derivative with fungicidal action, especially against dermatophytes. Different analytical methods have been reported for quantifying TerbHCl in different samples. These procedures require time-consuming sample preparation or expensive instrumentation. In this paper, electrochemical methods involving capillary electrophoresis with contactless conductivity detection, and amperometry associated with batch injection analysis, are described for the determination of TerbHCl in pharmaceutical products. In the capillary electrophoresis experiments, terbinafine was protonated and analyzed in the cationic form in less than 1 min. A linear range from 1.46 to 36.4 µg mL(-1) in acetate buffer solution and a detection limit of 0.11 µg mL(-1) were achieved. In the amperometric studies, terbinafine was oxidized at +0.85 V with high throughput (225 injection h(-1)) and good linear range (10-100 µmol L(-1)). It was also possible to determine the antifungal agent using simultaneous conductometric and potentiometric titrations in the presence of 5% ethanol. The electrochemical methods were applied to the quantification of TerbHCl in different tablet samples; the results were comparable with values indicated by the manufacturer and those found using titrimetry according to the Pharmacopoeia. The electrochemical methods are simple, rapid and an appropriate alternative for quantifying this drug in real samples.

On the formation of carbonate adducts of fatty alcohols, sterols, and sugars in biological conditions.

The formation and properties of carbonate adducts of some organic hydroxy compounds in aqueous medium were investigated. Fatty alcohols and sugars were chosen as representative classes of biological interest, and the medium was carbonated aqueous solution with pH ranging from 3.0 to 8.3. Capillary electrophoresis with two capacitively coupled contactless conductivity detectors (C4 Ds) was used for quantitation and to obtain the mobility of the monoalkyl carbonates (MACs), which were used to determine the equilibrium and kinetic constants of the reaction as well as the diffusion coefficients. For increasing chain length of the alcohols, the equilibrium constant tends to the unit, which suggests that fatty alcohols can form the corresponding MACs. The formation of MACs for cyclohexanol and cyclopentanol also suggest the existence of similar species for sterols. Carbonate adducts of fructose, glucose, and sucrose were also detected, which suggests that these counterparts of the well-known phosphates can also occur in the cytosol. Our calculations suggest that one in 1000 to one in 10,000 molecules of these hydroxy compounds would be available as the corresponding MAC in such a medium. Experiments carried out at pH values less than 3.0 showed that there is a catalytic effect of hydronium on the interconversion of bicarbonate and a MAC. Taking into account the great number of hydroxy compounds similar to the ones investigated and that bicarbonate is ubiquitous in living cells, one can anticipate the existence of a whole new class of carbonate adducts of these metabolites.

Environmental formaldehyde analysis by active diffusive sampling with a bundle of polypropylene porous capillaries followed by capillary zone electrophoretic separation and contactless conductivity detection.

Compared to other volatile carbonylic compounds present in outdoor air, formaldehyde (CH(2)O) is the most toxic, deserving more attention in terms of indoor and outdoor air quality legislation and control. The analytical determination of CH(2)O in air still presents challenges due to the low-level concentration (in the sub-ppb range) and its variation with sampling site and time. Of the many available analytical methods for carbonylic compounds, the most widespread one is the time consuming collection in cartridges impregnated with 2,4-dinitrophenylhydrazine followed by the analysis of the formed hydrazones by HPLC. The present work proposes the use of polypropylene hollow porous capillary fibers to achieve efficient CH(2)O collection. The Oxyphan fiber (designed for blood oxygenation) was chosen for this purpose because it presents good mechanical resistance, high density of very fine pores and high ratio of collection area to volume of the acceptor fluid in the tube, all favorable for the development of air sampling apparatus. The collector device consists of a Teflon pipe inside of which a bundle of polypropylene microporous capillary membranes was introduced. While the acceptor passes at a low flow rate through the capillaries, the sampled air circulates around the fibers, impelled by a low flow membrane pump (of the type used for aquariums ventilation). The coupling of this sampling technique with the selective and quantitative determination of CH(2)O, in the form of hydroxymethanesulfonate (HMS) after derivatization with HSO(3)(-), by capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C(4)D) enabled the development of a complete analytical protocol for the CH(2)O evaluation in air.

Purification, characterization and molecular cloning of the major chitinase from Tenebrio molitor larval midgut.

Insect chitinases are involved in degradation of chitin from the exoskeleton cuticle or from midgut peritrophic membrane during molts. cDNAs coding for insect cuticular and gut chitinases were cloned, but only chitinases from moulting fluid were purified and characterized. In this study the major digestive chitinase from T. molitor midgut (TmChi) was purified to homogeneity, characterized and sequenced after cDNA cloning. TmChi is secreted by midgut epithelial cells, has a molecular weight of 44 kDa and is unstable in the presence of midgut proteinases. TmChi shows strong substrate inhibition when acting on umbelliferyl-derivatives of chitobio- and chitotriosaccharides, but has normal Michaelis kinetics with the N-acetylglucosamine derivative as substrate. TmChi has very low activity against colloidal chitin, but effectively converts oligosaccharides to shorter fragments. The best substrate for TmChi is chitopentaose, with highest k(cat)/K(M) value. Sequence analysis and chemical modification experiments showed that the TmChi active site contains carboxylic groups and a tryptophane, which are known to be important for catalysis in family 18 chitinases. Modification with p-hidroximercuribenzoate of a cysteine residue, which is exposed after substrate binding, leads to complete inactivation of the enzyme. TmChi mRNA encodes a signal peptide plus a protein with 37 kDa and high similarity with other insect chitinases from family 18. Surprisingly, this gene does not encode the C-terminal Ser-Thr-rich connector and chitin-binding domain normally present in chitinases. The special features of TmChi probably result from its adaptation to digest chitin-rich food without damaging the peritrophic membrane.

Determination of salbutamol in syrups by capillary electrophoresis with contactless conductivity detection (CE-C(4)D).

This paper describes the separation and quantification of salbutamol in pharmaceutical products (salbutamol syrups) by capillary electrophoresis (CE) with contactless conductivity detection (C(4)D). The system was studied by micellar electrokinetic capillary chromatography (MEKC) and free solution capillary electrophoresis (FSCE), being the latter chosen in function of best resolution and sensitivity in comparison with the MEKC method. CE-C(4)D was applied to analysis of salbutamol in syrups utilizing 1.0 x 10(-2) molL(-1) acetic acid/sodium acetate buffer (pH 4.9) as running electrolyte. Tetraethylammonium (TEA) solution was used as internal standard. The results obtained include a linear dynamic range from 7. x 10(-5) to 3.0 x 10(-4) molL(-1) and good repeatability (R.S.D.=4.7% for n=10 for a 7.0 x 10(-5) molL(-1) salbutamol solution). The detection limit was calculated as 1.0 x 10(-5) molL(-1) and the limit of quantification was estimated as 3.3 x 10(-5) molL(-1). For syrups analysis the reproducibility presented deviations between 1.5% and 2.5% (three different days) obtained for measurements in triplicate.

Microfluidic devices obtained by thermal toner transferring on glass substrate.

A new process for the manufacture of microfluidic devices based on deposition of laser-printing toner on glass substrates is described. It is an alternative method to the toner on polyester film (toner-polyester) one, previously introduced. Commercial laser printers cannot print directly on glass, thus the toner must first be printed on a special paper and then transferred by heating under pressure to the glass surface. Although this procedure is more complex than the toner-polyester one, it can be repeated several times, yielding multiple toner layers. Even without special alignment equipment, up to four layers could be satisfactorily piled up. Characterization tests revealed that the toner-glass devices have similar behavior as toner-polyester ones regarding the toner layer porosity. The main advantages of the toner-glass technology are improved mechanical stability, possibility of multiple toner layers, augmented electroosmotic flow (EOF), and improved heat transfer. On the other hand, toner adhesion to glass is weaker than to polyester, which limits the device lifetime and usable liquid media. The measured EOF mobility (3.5 x 10(-4) cm2.V(-1).s(-1) for pH 7) suggests that it is mainly determined by the glass surface, being little influenced by the toner walls. Microchip electrophoresis with contactless conductivity detection and photometric detection were implemented using toner-glass devices.

Electrophoresis microchip fabricated by a direct-printing process with end-channel amperometric detection.

We describe the development of an electrophoresis microchip fabricated by a direct-printing process, based on lamination of printed polyester films with end-channel amperometric detection. The channel structures are defined by polyester (base and cover) and by a toner layer (walls). The polyester-toner devices presented an electroosmotic flow (EOF) magnitude of approximately 10(-5) cm2 V(-1) s(-1), which is generated by a polymeric mixture of the toner and polyester composition. The microelectrodes used for detection were produced combining this laser-printer technology to compact discs. The performance of this device was evaluated by amperometric detection of iodide and ascorbate. The detection limits found were 500 nmol.L(-1) (135 amol) and 1.8 micromol.L(-1) (486 amol) for iodide and ascorbate, respectively.

Disposable twin gold electrodes for amperometric detection in capillary electrophoresis.

We describe a simple and easy way to construct gold microelectrodes for amperometric detection in capillary electrophoresis (CE). The gold microelectrodes, in single or twin sets, were obtained from recordable compact discs (gold-sputtered type), which present highly reproducible surface characteristics. The performance of these electrodes was evaluated by using a home-made CE equipment. The basic steps for the electrode construction are: drawing on a microcomputer; laser printing of the design on wax paper; heat-transfer of the toner onto the gold surface of a peeled recordable compact disc (CD-R); etching of the gold layer from unprinted regions; removal of the toner with a solvent; sealing of unused electrode areas with varnish. One electrode at a time was connected to a potentiostat (or two, to a bipotentiostat) and operated in a wall jet configuration relative to the CE capillary outlet. The amperometric signals were integrated for quantification purposes. Repetitive injections (n = 10) of a mixture containing iodide, ascorbic acid, dipyrone, and acetaminophen (20, 200, 500, and 100 microM), presented relative standard deviations of 2.9, 4.5, 6.1, and 4.0%, respectively. For these analytes, the detection limits (S/N = 3, 30 s of 100 mm hydrodynamic injection) were 0.1, 0.5, 3.1, and 1.1 microM, respectively.

Determination of mono- and disaccharides by capillary electrophoresis with contactless conductivity detection.

The separation and detection of common mono- and disaccharides by capillary electrophoresis (CE) with contactless conductivity detection (CCD) is presented. At high values of pH, the sugars are converted to anionic species that can be separated by CE and indirectly detected by CCD. The main anionic species present in the running electrolytes are hydroxide and phosphate, which have greater mobility than the ionized sugars, and, thus, the indirect detection is possible. The method was applied to analysis of glucose, fructose, and sucrose in soft drinks, isotonic beverages, fruit juice, and sugarcane spirits. Galactose was used as internal standard in all cases. Plate numbers range from ca. 70,700 to 168,200 and the limits of detection from 13 to 31 microM.