In Search of Effective UiO-66 Metal-Organic Frameworks for Artificial Kidney Application.

The removal of uremic toxins from patients with acute kidney injury is a key issue in improving the quality of life for people requiring peritoneal dialysis. The currently utilized method for the removal of uremic toxins from the human organism is hemodialysis, performed on semipermeable membranes where the uremic toxins, along with small molecules, are separated from proteins and blood cells. In this study, we describe a mixed-linker modulated synthesis of zirconium-based metal-organic frameworks for efficient removal of uremic toxins. We determined that the efficient adsorption of uremic toxins is achieved by optimizing the ratio between -amino functionalization of the UiO-66 structure with 75% of -NH2 groups within organic linker structure. The maximum adsorption of hippuric acid and 3-indoloacetic acid was achieved by UiO-66-NH2 (75%) and by UiO-66-NH2 (75%) 12.5% HCl prepared by modulated synthesis. Furthermore, UiO-66-NH2 (75%) almost completely adsorbs 3-indoloacetic acid bound to bovine serum albumin, which was used as a model protein to which uremic toxins bind in the human body. The high adsorption capacity was confirmed in recyclability test, which showed almost 80% removal of 3-indoloacetic acid after the third adsorption cycle. Furthermore, in vitro cytotoxicity tests as well as hemolytic activity assay have proven that the UiO-66-based materials can be considered as potentially safe for hemodialytic purposes in living organisms.

Antifungal compound, methyl hippurate from Bacillus velezensis CE 100 and its inhibitory effect on growth of Botrytis cinerea.

Botrytis cinerea, the causal agent of gray mold is one of the major devastating fungal pathogens that occurs in strawberry cultivation and leads to massive losses. Due to the rapid emergence of resistant strains in recent years, an ecofriendly disease management strategy needs to be developed to control this aggressive pathogen. Bacillus velezensis CE 100 exhibited strong antagonistic activity with 53.05% against B. cinerea by dual culture method. In the present study, 50% of culture filtrate supplemented into PDA medium absolutely inhibited mycelial growth of B. cinerea whereas the highest concentration (960 mg/L) of different crude extracts including ethyl acetate, chloroform, and n-butanol crude extracts of B. velezensis CE 100, strongly inhibited mycelial growth of B. cinerea with the highest inhibition of 79.26%, 70.21% and 69.59% respectively, resulting in severe damage to hyphal structures with bulging and swellings. Hence, the antifungal compound responsible was progressively separated from ethyl acetate crude extract using medium pressure liquid chromatography. The purified compound was identified as methyl hippurate by nuclear magnetic resonance and mass spectrometry. The inhibitory effect of methyl hippurate on both spore germination and mycelial growth of B. cinerea was revealed by its dose-dependent pattern. The spore germination rate was completely restricted at a concentration of 3 mg/mL of methyl hippurate whereas no mycelial growth was observed in agar medium supplemented with 4 mg/mL and 6 mg/mL of methyl hippurate by poisoned food method. Microscopic imaging revealed that the morphologies of spores were severely altered by long-time exposure to methyl hippurate at concentrations of 1 mg/mL, 2 mg/mL and 3 mg/mL and hyphae of B. cinerea were severely deformed by exposure to methyl hippurate at concentrations of 2 mg/mL, 4 mg/mL and 6 mg/mL. No significant inhibition on tomato seed germination was observed in treatments with methyl hippurate (2 mg/mL) for both 6 h and 12 h soaking period as compared to the controls. Based on these results, B. velezensis CE 100 could be considered a potential agent for development of environmentally friendly disease control strategies as a consequence of the synergetic interactions of diverse crude metabolites and methyl hippurate.

Preparation of chitosan-modified magnetic Schiff base network composite nanospheres for effective enrichment and detection of hippuric acid and 4-methyl hippuric acid.

Chitosan-modified magnetic Schiff base network composite nanospheres (Fe3O4@SNW@Chitosan) were prepared for the enrichment and detection of hippuric acid (HA) and 4-methyl hippuric acid (4-MHA) via magnetic solid phase extraction (MSPE) connected with HPLC. The SNW was one of the covalent organic framework, which constructed through covalent bonds, shown comprising solvent stability, low density and accessible pores. The obtained Fe3O4@SNW@Chitosan has many merits as a magnetic sorbent, including a hydrophilic surface, uniform pore size, unique ordered channel structure, and superparamagnetism. The favourable linearity of this MSPE-HPLC method was in the range of 1-1000 µg L-1, and LODs of HA and 4-MHA were 0.3 µg L-1 and 0.2 µg L-1, respectively. The recoveries in urine samples were range from 95.3 to 109.0 % with the RSD less than 9.6 %. When employed for the enrichment of HA and 4-MHA, Fe3O4@SNW@Chitosan exhibited great potential as a candidate for preconcentration.

Chitosan tailor-made membranes as biopolymeric support for electromembrane extraction.

A chitosan membrane composed by 60% (w/w) chitosan and 40% (w/w) Aliquat®336 has been proposed as a new biopolymeric support for electromembrane extraction. The new support has been characterized by Scanning Electron Microscopy, resulting a 30-35 µm thickness. Amoxicillin, nicotinic acid, hippuric acid, salicylic acid, anthranilic acid, ketoprofen, naproxen and ibuprofen have been successfully extracted using the proposed support. Better enrichment factors were obtained for the acidic polar analytes than for the non-steroidal anti-inflammatory compounds (ranging from 118 for hippuric acid and 20 for ibuprofen). Electromembrane extraction was developed applying a DC voltage of 100 V, 1-octanol as supported liquid membrane and 20 min of extraction. The target analytes have also been satisfactorily extracted from human urine samples, providing high extraction efficiencies. The chitosan membrane is presented as a promising alternative for supporting liquid membrane compared to commonly used materials for this purpose.

Increasing the removal of protein-bound uremic toxins by liposome-supported hemodialysis.

Protein-bound uremic toxins (PBUTs) accumulate at high plasma levels and cause various deleterious effects in end-stage renal disease patients because their removal by conventional hemodialysis is severely limited by their low free-fraction levels in plasma. Here, we assessed the extent to which solute removal can be increased by adding liposomes to the dialysate. The uptake of liposomes by direct incubation in vitro showed an obvious dose-response relationship for p-cresyl sulfate (PCS) and indoxyl sulfate (IS) but not for hippuric acid (HA). The percent removal of both PCS and IS but not of HA was gradually increased with the increased concentration of liposomes in a rapid equilibrium dialysis setup. In vitro closed circulation showed that adding liposomes to the dialysate markedly increased the dialysances of PBUTs without greatly altering that of urea and creatinine. In vivo experiments in uremic rats demonstrated that adding liposomes to the dialysate resulted in higher reduction ratios (RRs) and more total solute removal (TSR) for several PBUTs compared to the conventional dialysate, which was approximately similar to the addition of bovine serum albumin to the dialysate. These findings highlight that as an adjunct to conventional hemodialysis, addition of liposomes to the dialysate could significantly improve the removal of protein-bound uremic solutes without greatly altering the removal of small, water-soluble solutes.

Use of Polymer Inclusion Membranes (PIMs) as support for electromembrane extraction of non-steroidal anti-inflammatory drugs and highly polar acidic drugs.

The use of polymer inclusion membranes (PIMs) as support of 1-octanol liquid membrane in electromembrane extraction (EME) procedure is proposed. Synthesis of PIMs were optimized to a composition of 29% (w/w) of cellulose triacetate as base polymer and 71% (w/w) of Aliquat®336 as cationic carrier. Flat PIMs of 25µm thickness and 6mm diameter were used. EME protocol was implemented for the simultaneous extraction of four non-steroidal anti-inflammatory drugs (NSAIDs) (salicylic acid, ketoprofen, naproxen and ibuprofen) and four highly polar acidic drugs (anthranilic acid, nicotinic acid, amoxicillin and hippuric acid). Posterior HPLC separation of the extracted analytes was developed with diode array detection. Recoveries in the 81-34% range were obtained. EME procedure was applied to human urine samples.

Carbon Adsorbents With Dual Porosity for Efficient Removal of Uremic Toxins and Cytokines from Human Plasma.

The number of patients with chronic kidney disease increases while the number of available donor organs stays at approximately the same level. Unavoidable accumulation of the uremic toxins and cytokines for these patients comes as the result of malfunctioning kidneys and their high levels in the blood result in high morbidity and mortality. Unfortunately, the existing methods, like hemodialysis and hemofiltration, provide only partial removal of uremic toxins and/or cytokines from patients' blood. Consequently, there is an increasing need for the development of the extracorporeal treatments which will enable removal of broad spectrum of uremic toxins that are usually removed by healthy kidneys. Therefore, in this work we developed and tested ordered mesoporous carbons as new sorbents with dual porosity (micro/meso) that provide selective and efficient removal of a broad range of uremic toxins from human plasma. The new sorbents, CMK-3 are developed by nanocasting methods and have two distinct pore domains, i.e. micropores and mesopores, therefore show high adsorption capacity towards small water soluble toxins (creatinine), protein-bound molecules (indoxyl sulfate and hippuric acid), middle molecules (ß-2-microglobulin) and cytokines of different size (IL-6 and IL-8). Our results show that small amounts of CMK-3 could provide selective and complete blood purification.

New nanostructured support for carrier-mediated electromembrane extraction of high polar compounds.

A new support has been proposed to be used for carrier-mediated electromembrane extraction purposes. The new support (Tiss®-OH) is a 100µm thickness sheet nanofiber membrane manufactured by electrospinning and composed by acrylic nanofibers. It has been used in an electromembrane extraction (EME) combined with a HPLC procedure using diode array detection. The proposed method has been used for the extraction of four high polarity acidic compounds: nicotinic acid, amoxicillin, hippuric acid and salicylic acid. Analytes were extracted from an aqueous sample solution (pH 4) (donor phase) using a Tiss®-OH sheet that supports a 5% (w/v) Aliquat®336 in 1-octanol liquid membrane. Aqueous solution (pH 6) was used as acceptor phase. The electrical field was generated from a d.c. electrical current of 100V through two spiral shaped platinum wires placed into donor and acceptor phases. Analytes were extracted in 10min with recoveries in the 60-85% range. The proposed EME procedure has been successfully applied to the determination of the target analytes in human urine samples.

Heterogeneous electrochemical immunoassay of hippuric acid on the electrodeposited organic films.

By directly coordinating hippuric acid (HA) to the ferrate (Fe) as an electron transfer mediator, we synthesized a Fe-HA complex, which shows a good electrochemical signal and thus enables the electrochemical immunoanalysis for HA. We electrodeposited organic films containing imidazole groups on the electrode surface and then bonded Ni ion (positive charge) to induce immobilization of Fe-HA (negative charge) through the electrostatic interaction. The heterogeneous competitive immunoassay system relies on the interaction between immobilized Fe-HA antigen conjugate and free HA antigen to its antibody (anti-HA). The electric signal becomes weaker due to the hindered electron transfer reaction when a large-sized HA antibody is bound onto the Fe-HA. However, in the presence of HA, the electric signal increases because free HA competitively reacts with the HA antibody prior to actual reaction and thus prevents the HA antibody from interacting with Fe-HA at the electrode surface. This competition reaction enabled an electrochemical quantitative analysis of HA concentration with a detection limit of 0.5 µg mL(-1), and thus allowed us to develop a simple and rapid electrochemical immunosensor.

Hippuric acid as a significant regulator of supersaturation in calcium oxalate lithiasis: the physiological evidence.

At present, the clinical significance of existing physicochemical and biological evidence and especially the results we have obtained from our previous in vitro experiments have been analyzed, and we have come to the conclusion that hippuric acid (C6H5CONHCH2COOH) is a very active solvent of Calcium Oxalate (CaOX) in physiological solutions. Two types of experiments have been discussed: clinical laboratory analysis on the urine excretion of hippuric acid (HA) in patients with CaOX lithiasis and detailed measurements of the kinetics of the dissolution of CaOX calculi in artificial urine, containing various concentrations of HA. It turns out that the most probable value of the HA concentration in the control group is approximately ten times higher than the corresponding value in the group of the stone-formers. Our in vitro analytical measurements demonstrate even a possibility to dissolve CaOX stones in human urine, in which increased concentration of HA have been established. A conclusion can be that drowning out HA is a significant regulator of CaOX supersaturation and thus a regulation of CaOX stone formation in human urine. Discussions have arisen to use increased concentration of HA in urine both as a solubilizator of CaOX stones in the urinary tract and on the purpose of a prolonged metaphylactic treatment.

Mixed matrix hollow fiber membranes for removal of protein-bound toxins from human plasma.

In end stage renal disease (ESRD) waste solutes accumulate in body fluid. Removal of protein bound solutes using conventional renal replacement therapies is currently very poor while their accumulation is associated with adverse outcomes in ESRD. Here we investigate the application of a hollow fiber mixed matrix membrane (MMM) for removal of these toxins. The MMM hollow fiber consists of porous macro-void free polymeric inner membrane layer well attached to the activated carbon containing outer MMM layer. The new membranes have permeation properties in the ultrafiltration range. Under static conditions, they adsorb 57% p-cresylsulfate, 82% indoxyl sulfate and 94% of hippuric acid from spiked human plasma in 4 h. Under dynamic conditions, they adsorb on average 2.27 mg PCS/g membrane and 3.58 mg IS/g membrane in 4 h in diffusion experiments and 2.68 mg/g membrane PCS and 12.85 mg/g membrane IS in convection experiments. Based on the dynamic experiments we estimate that our membranes would suffice to remove the daily production of these protein bound solutes.

Achieving greater selectivity for the analysis of o-, m-, p-methylhippuric acids in workers' urine by ultra performance liquid chromatography coupled with tandem mass spectrometry.

A selective method analyzing separately o-, m- and p-methylhippuric acid isomers in workers' urine samples has been developed using ultra performance liquid chromatography coupled with tandem mass spectrometry. Chromatographic separation has been optimized to resolve the three isomers at baseline. Combined with this optimal separation, the mass spectrometer allowed rapid switching from MRM scan to full scan and product ion scan within the chromatographic peak. This feature allowed the retention of analyte chemical structure information for the three methylhippuric acid isomers in parallel with the simultaneous acquisition of quantitative data. Such an approach is unequaled for the reliability of the data generated and it can be applied to each isomer separately. The method was adjusted to a dynamic range between 0.2mM and 8.12mM for o-methylhippuric acid and p-methylhippuric acid, and between 0.41mM and 16.23mM for m-methylhippuric acid in order to cover the biological exposure index. A negligible matrix effect was observed with the conditions used. Also, intra-day and inter-day precisions were both <6% for all the concentration levels tested and the accuracy was evaluated at 97±4%. The inclusion of simultaneous full scan acquisitions did not prevent the robustness of the quantitative data. The method applied to the determination of inter-laboratory proficient test samples led to results in the tolerated range. Moreover, urine samples from workers were robustly quantified and the MHA levels were all below the biological exposure index reference value.

Analysis method of the angiotensin-I converting enzyme inhibitory activity based on micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method was developed for estimating the angiotensin-I converting enzyme (ACE) inhibitory activity by separating the hippuric acid liberated in the ACE reaction mixture in the presence of an inhibitor, captopril. The hippuric acid was successfully separated and detected by MEKC with a 25 mM sodium dodecyl sulfate solution in a 25 mM phosphate-50 mM borate buffer at pH 7.0; the total analysis took about 5 min. A good linear relationship was observed between the inhibitor and the peak area of hippuric acid release. No significant difference in the ACE inhibitory activity (IC50) of captopril (an antihypertensive medicine) or autolyzed-mushrooms (functional foods) was observed between the conventional method and the MEKC method. The MEKC method was found to be a useful technique for a rapid assay of the ACE inhibitory activity.

Characterization of p-hydroxy-hippuric acid as an inhibitor of Ca2+-ATPase in end-stage renal failure.

Characterization of p-hydroxy-hippuric acid as an inhibitor of Ca2+-ATPase in end-stage renal failure. In patients with end-stage renal failure (ESRF), disturbances of Ca2+ metabolism are common. Besides hormonal changes, inhibition of cellular Ca2+-ATPase was postulated to contribute to uremic toxicity. We purified a potent inhibitor of the Ca2+-ATPase from the ultrafiltrate of patients with ESRF by multiple steps of high-performance liquid chromatography to homogeneity, and identified the isolated inhibitor by mass spectrometric methods as p-hydroxy-hippuric acid. The enzyme used for the Ca2+-ATPase assay system was isolated from red blood cells by cross-flow filtration. The activity of the Ca2+-ATPase was measured spectrophotometrically as the difference in hydrolysis of adenosine 5'-triphosphate (ATP) in the presence and absence of Ca2+ with different concentrations of ATP and p-hydroxyhippuric acid. The Ca2+-ATPase was found to be inhibited by p-hydroxy-hippuric acid at a concentration above 11.7 micromol/L. p-Hydroxyhippuric acid inhibited the erythrocyte Ca2+-ATPase by reducing Vmax and increasing the Km value. The EC50 (log mol/L; mean +/- SEM) for p-hydroxy-hippuric acid was calculated as 4.82 +/- 0.14. In conclusion, p-hydroxy-hippuric acid may play a role in disturbed Ca2+ metabolism in end-stage renal failure.

Sensitive method for quantitation of angiotensin-converting enzyme (ACE) activity in tissue.

A novel sensitive and specific method for the measurement of tissue angiotensin-converting enzyme (ACE) activity utilizing HPLC is described. ACE activity was determined in detergent-extracted canine hearts utilizing the synthetic ACE-specific substrate hippuryl histidyl leucine (HHL), both in the presence and the absence of the site-specific inhibitor captopril. Tissue ACE activity was quantitated from the moles of hippuric acid (HA) formed, in time-fixed assays, utilizing HPLC separation of HA from HHL and UV-spectrophotometry for quantitation of HA as in the standard Cushman and Cheung assay (Cushman DW and Cheung HS, Biochem Pharmacol 20: 1637-1648, 1971). Separation of HA from HHL was performed by reverse phase HPLC on a phenyl silica gel column with an eluent consisting of 20% acetonitrile in 0.1 M aqueous ammonium phosphate buffer, pH 6.8. After the standard liquid/liquid extraction procedure with ethyl acetate, HPLC analysis revealed the presence of unreacted substrate, HHL, in amounts comparable to the product of interest, HA, in the final assay; moreover, the amount of HA formed did not fall completely to zero in the presence of captopril. Regional studies of canine cardiac ACE activity utilizing the HPLC-based assay and the standard assay method showed a significantly higher ACE activity in the right ventricle compared with the left ventricle (2.37 +/- 0.7 vs 1.24 +/- 0.18 mU/g, P < 0.05 [N = 6], respectively) in the HPLC-based assay, but no difference in right and left ventricular ACE activities by the standard assay (0.25 +/- 0.08 vs 0.31 +/- 0.09 mU/g [N = 6], respectively). Kinetic studies utilizing the HPLC-based assay coupled with the use of captopril showed Km (1.34 +/- 0.08 mM) and Vmax (36.8 +/- 11.5 x 10(-10) M/min) values in agreement with those in the literature. Our results demonstrate that the application of HPLC to the standard Cushman and Cheung assay improves the sensitivity and specificity of the standard assay and enables the use of much smaller amounts (approximately 4 vs approximately 400 mg for the Cushman and Cheung assay) of tissue for ACE activity assay.

Isolation and chemical identification of inhibitors of plasma ligand binding.

The binding by serum albumin of many drugs and endogenous metabolites is impaired in humans and animals with renal failure. Unknown solute(s) retained in renal failure have been extracted from uremic fluids. When added to normal plasma they induce a similar binding defect. Similar activity can be extracted from normal urine. We have devised a series of extraction and purification techniques that yielded three binding inhibitory ligands from normal human urine in sufficient quantity and of a high degree of purity. Rigorous methods have been applied to determine chemical identity of the ligands. Purification steps consisted of: adsorption at pH 3.0 to polystyrene-divinylbenzene resin (XAD-2); elution from the resin with methanol followed by drying and solution in dilute formic acid; passage through SP-Sephadex to remove cations, especially yellow-brown pigments; adsorption to the anion exchanger QAE-Sephadex, and separation into three zones of inhibitory activity with a formic acid gradient; purification to homogeneity with C-8 or C-18 silica reversed-phase chromatography. Using this isolation procedure, followed by mass spectroscopy and nuclear magnetic resonance spectroscopy, we have shown that the binding inhibitory activity is due not to one ligand, but to a family of aromatic acids. To date hippurate, beta-(m-hydroxyphenyl)-hydracrylate and p-hydroxyphenylacetate have been identified as binding inhibitors. Other active ligands remain to be identified.

Rapid assay of human plasma carboxypeptidase N by high-performance liquid chromatographic separation of hippuryl-lysine and its product.

A rapid and sensitive method for measuring carboxypeptidase N (CPN) activity in human plasma is described. The procedure is based on the hydrolysis of a high-specificity/low-affinity substrate, hippuryl-L-lysine, to its products hippuric acid and lysine. The substrate and product are separated quantitatively by high-performance liquid chromatography in less than 10 min following a minimal sample preparation time. The advantages of this method over previous ones are discussed and data are presented demonstrating the reliability of this method for the routine clinical determination of CPN activity.

Isolation and chemical characterization of 2-hydroxybenzoylglycine as a drug binding inhibitor in uremia.

An organic compound that inhibits drug binding in uremia has been isolated from the sera of chronic renal failure patients, and its chemical structure has been determined. Addition of the compound to normal human sera in vitro resulted in drug binding defects similar to those seen in uremia. The purification of this substance was accomplished by n-butyl chloride extraction of acidified (pH 3.0) uremic sera followed by column chromatography, thin-layer chromatography, and paper electrophoresis. From analytical studies including ultraviolet and fluorescence spectroscopy, gas chromatography, chemical ionization and electron impact mass spectrometry, and proton nuclear magnetic resonance spectroscopy, the chemical structure of the uremic binding inhibitor was deduced to be 2-hydroxybenzoylglycine. This confirms the hypothesis that the drug binding defect in uremia is due to the accumulation of endogenous metabolic products rather than an intrinsic structural defect in albumin.