Efficient and fast detection of uric acid based on a colorimetric sensing method.

The uric acid (UA) level is an important physiological indicator of the human body, and its abnormality can lead to a series of diseases. Therefore, the immediate detection of uric acid concentration has broad application prospects. Commonly used methods for the analysis of uric acid include chromatography, high-performance capillary electrophoresis and electrochemical methods. However, these methods have the disadvantages of cumbersome sample pre-treatment, high cost, time-consuming, and the need for experimental instruments and professional operators, which are extremely unfavorable for the detection of uric acid and the diagnosis of related diseases in resource-limited areas. In this study, a portable visualization method was developed for the detection of uric acid using hydrogen peroxide (H2O2) test strips. Uric acid enzyme specifically catalyzes the oxidation of uric acid to produce H2O2, which causes a significant change in the color of the H2O2 test strip. The response has good linearity in the range of 1 ∼ 50 µg mL-1. Thus, it provides a simple, rapid, and cost-effective visualized bioassay for uric acid.

Tumour lysis syndrome.

Tumour lysis syndrome (TLS) represents a critical oncological emergency characterized by extensive tumour cell breakdown, leading to the swift release of intracellular contents into the systemic circulation, outpacing homeostatic mechanisms. This process results in hyperuricaemia (a by-product of intracellular DNA release), hyperkalaemia, hyperphosphataemia, hypocalcaemia and the accumulation of xanthine. These electrolyte and metabolic imbalances pose a significant risk of acute kidney injury, cardiac arrhythmias, seizures, multiorgan failure and, rarely, death. While TLS can occur spontaneously, it usually arises shortly after the initiation of effective treatment, particularly in patients with a large cancer cell mass (defined as ≥500 g or ≥300 g/m2 of body surface area in children). To prevent TLS, close monitoring and hydration to improve renal perfusion and urine output and to minimize uric acid or calcium phosphate precipitation in renal tubules are essential. Intervention is based on the risk of a patient of having TLS and can include rasburicase and allopurinol. Xanthine, typically enzymatically converted to uric acid, can accumulate when xanthine oxidases, such as allopurinol, are administered during TLS management. Whether measurement of xanthine is clinically useful to optimize the use of allopurinol or rasburicase remains to be determined.

Benchmarking predictive methods for small-angle X-ray scattering from atomic coordinates of proteins using maximum likelihood consensus data.

Stimulated by informal conversations at the XVII International Small Angle Scattering (SAS) conference (Traverse City, 2017), an international team of experts undertook a round-robin exercise to produce a large dataset from proteins under standard solution conditions. These data were used to generate consensus SAS profiles for xylose isomerase, urate oxidase, xylanase, lysozyme and ribonuclease A. Here, we apply a new protocol using maximum likelihood with a larger number of the contributed datasets to generate improved consensus profiles. We investigate the fits of these profiles to predicted profiles from atomic coordinates that incorporate different models to account for the contribution to the scattering of water molecules of hydration surrounding proteins in solution. Programs using an implicit, shell-type hydration layer generally optimize fits to experimental data with the aid of two parameters that adjust the volume of the bulk solvent excluded by the protein and the contrast of the hydration layer. For these models, we found the error-weighted residual differences between the model and the experiment generally reflected the subsidiary maxima and minima in the consensus profiles that are determined by the size of the protein plus the hydration layer. By comparison, all-atom solute and solvent molecular dynamics (MD) simulations are without the benefit of adjustable parameters and, nonetheless, they yielded at least equally good fits with residual differences that are less reflective of the structure in the consensus profile. Further, where MD simulations accounted for the precise solvent composition of the experiment, specifically the inclusion of ions, the modelled radius of gyration values were significantly closer to the experiment. The power of adjustable parameters to mask real differences between a model and the structure present in solution is demonstrated by the results for the conformationally dynamic ribonuclease A and calculations with pseudo-experimental data. This study shows that, while methods invoking an implicit hydration layer have the unequivocal advantage of speed, care is needed to understand the influence of the adjustable parameters. All-atom solute and solvent MD simulations are slower but are less susceptible to false positives, and can account for thermal fluctuations in atomic positions, and more accurately represent the water molecules of hydration that contribute to the scattering profile.

Arginine-Rich Peptide-Rhodium Nanocluster@Reduced Graphene Oxide Composite as a Highly Selective and Active Uricase-like Nanozyme for the Degradation of Uric Acid and Inhibition of Urate Crystal.

Metal nanozymes have offered attractive opportunities for biocatalysis and biomedicine. However, fabricating nanozymes simultaneously possessing highly catalytic selectivity and activity remains a great challenge due to the lack of three-dimensional (3D) architecture of the catalytic pocket in natural enzymes. Here, we integrate rhodium nanocluster (RhNC), reduced graphene oxide (rGO), and protamine (PRTM, a typical arginine-rich peptide) into a composite facilely based on the single peptide. Remarkably, the PRTM-RhNC@rGO composite displays outstanding selectivity, activity, and stability for the catalytic degradation of uric acid. The reaction rate constant of the uric acid oxidation catalyzed by the PRTM-RhNC@rGO composite is about 1.88 × 10-3 s-1 (4 µg/mL), which is 37.6 times higher than that of reported RhNP (k = 5 × 10-5 s-1, 20 µg/mL). Enzyme kinetic studies reveal that the PRTM-RhNC@rGO composite exhibits a similar affinity for uric acid as natural uricase. Furthermore, the uricase-like activity of PRTM-RhNC@rGO nanozymes remains in the presence of sulfur substances and halide ions, displaying incredibly well antipoisoning abilities. The analysis of the structure-function relationship indicates the PRTM-RhNC@rGO composite features the substrate binding site near the catalytic site in a confined space contributed by 2D rGO and PRTM, resulting in the high-performance of the composite nanozyme. Based on the outstanding uricase-like activity and the interaction of PRTM and uric acid, the PRTM-RhNC@rGO composite can retard the urate crystallization significantly. The present work provides new insights into the design of metal nanozymes with suitable binding sites near catalytic sites by mimicking pocket-like structures in natural enzymes based on simple peptides, conducing to broadening the practical application of high-performance nanozymes in biomedical fields.

Atavistic strategy for the treatment of hyperuricemia via ionizable liposomal mRNA.

Hyperuricemia is associated with an increased risk of gout, hypertension, diabetes, and cardiovascular diseases. Most mammals maintain normal serum uric acid (SUA) via urate oxidase (Uox), an enzyme that metabolizes poorly-soluble UA to highly-soluble allantoin. In contrast, Uox became a pseudogene in humans and apes over the long course of evolution. Here we demonstrate an atavistic strategy for treating hyperuricemia based on endogenous expression of Uox in hepatocytes mediated by mRNA (mUox) loaded with an ionizable lipid nanoparticle termed iLAND. mUox@iLAND allows effective transfection and protein expression in vitro. A single dose of mUox@iLAND lowers SUA levels for several weeks in two female murine models, including a novel long-lasting model, which is also confirmed by metabolomics analysis. Together with the excellent safety profiles observed in vivo, the proposed mRNA agent demonstrates substantial potential for hyperuricemia therapy and the prevention of associated conditions.

Metal-ligand dual-site single-atom nanozyme mimicking urate oxidase with high substrates specificity.

In nature, coenzyme-independent oxidases have evolved in selective catalysis using isolated substrate-binding pockets. Single-atom nanozymes (SAzymes), an emerging type of non-protein artificial enzymes, are promising to simulate enzyme active centers, but owing to the lack of recognition sites, realizing substrate specificity is a formidable task. Here we report a metal-ligand dual-site SAzyme (Ni-DAB) that exhibited selectivity in uric acid (UA) oxidation. Ni-DAB mimics the dual-site catalytic mechanism of urate oxidase, in which the Ni metal center and the C atom in the ligand serve as the specific UA and O2 binding sites, respectively, characterized by synchrotron soft X-ray absorption spectroscopy, in situ near ambient pressure X-ray photoelectron spectroscopy, and isotope labeling. The theoretical calculations reveal the high catalytic specificity is derived from not only the delicate interaction between UA and the Ni center but also the complementary oxygen reduction at the beta C site in the ligand. As a potential application, a Ni-DAB-based biofuel cell using human urine is constructed. This work unlocks an approach of enzyme-like isolated dual sites in boosting the selectivity of non-protein artificial enzymes.

Multimodal smart systems reprogramme macrophages and remove urate to treat gouty arthritis.

Gouty arthritis is a chronic and progressive disease characterized by high urate levels in the joints and by an inflammatory immune microenvironment. Clinical data indicate that urate reduction therapy or anti-inflammatory therapy alone often fails to deliver satisfactory outcomes. Here we have developed a smart biomimetic nanosystem featuring a 'shell' composed of a fusion membrane derived from M2 macrophages and exosomes, which encapsulates liposomes loaded with a combination of uricase, platinum-in-hyaluronan/polydopamine nanozyme and resveratrol. The nanosystem targets inflamed joints and promotes the accumulation of anti-inflammatory macrophages locally, while the uricase and the nanozyme reduce the levels of urate within the joints. Additionally, site-directed near-infrared irradiation provides localized mild thermotherapy through the action of platinum and polydopamine, initiating heat-induced tissue repair. Combined use of these components synergistically enhances overall outcomes, resulting in faster recovery of the damaged joint tissue.

Uricase biofunctionalized plasmonic sensor for uric acid detection with APTES-modified gold nanotopping.

Current uric acid detection methodologies lack the requisite sensitivity and selectivity for point-of-care applications. Plasmonic sensors, while promising, demand refinement for improved performance. This work introduces a biofunctionalized sensor predicated on surface plasmon resonance to quantify uric acid within physiologically relevant concentration ranges. The sensor employs the covalent immobilization of uricase enzyme using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) crosslinking agents, ensuring the durable adherence of the enzyme onto the sensor probe. Characterization through atomic force microscopy and Fourier transform infrared spectroscopy validate surface alterations. The Langmuir adsorption isotherm model elucidates binding kinetics, revealing a sensor binding affinity of 298.83 (mg/dL)-1, and a maximum adsorption capacity of approximately 1.0751°. The biofunctionalized sensor exhibits a sensitivity of 0.0755°/(mg/dL), a linear correlation coefficient of 0.8313, and a limit of detection of 0.095 mg/dL. Selectivity tests against potentially competing interferents like glucose, ascorbic acid, urea, D-cystine, and creatinine showcase a significant resonance angle shift of 1.1135° for uric acid compared to 0.1853° for interferents at the same concentration. Significantly, at a low uric acid concentration of 0.5 mg/dL, a distinct shift of 0.3706° was observed, setting it apart from the lower values noticed at higher concentrations for all typical interferent samples. The uricase enzyme significantly enhances plasmonic sensors for uric acid detection, showcasing a seamless integration of optical principles and biological recognition elements. These sensors hold promise as vital tools in clinical and point-of-care settings, offering transformative potential in biosensing technologies and the potential to revolutionize healthcare outcomes in biomedicine.

Gut microbiota dysbiosis in hyperuricaemia promotes renal injury through the activation of NLRP3 inflammasome.

BACKGROUND: The prevalence of hyperuricaemia (HUA), a metabolic disorder characterized by elevated levels of uric acid, is on the rise and is frequently associated with renal injury. Gut microbiota and gut-derived uremic toxins are critical mediators in the gut-kidney axis that can cause damage to kidney function. Gut dysbiosis has been implicated in various kidney diseases. However, the role and underlying mechanism of the gut microbiota in HUA-induced renal injury remain unknown. RESULTS: A HUA rat model was first established by knocking out the uricase (UOX). HUA rats exhibited apparent renal dysfunction, renal tubular injury, fibrosis, NLRP3 inflammasome activation, and impaired intestinal barrier functions. Analysis of 16S rRNA sequencing and functional prediction data revealed an abnormal gut microbiota profile and activation of pathways associated with uremic toxin production. A metabolomic analysis showed evident accumulation of gut-derived uremic toxins in the kidneys of HUA rats. Furthermore, faecal microbiota transplantation (FMT) was performed to confirm the effects of HUA-induced gut dysbiosis on renal injury. Mice recolonized with HUA microbiota exhibited severe renal injury and impaired intestinal barrier functions following renal ischemia/reperfusion (I/R) surgery. Notably, in NLRP3-knockout (NLRP3-/-) I/R mice, the deleterious effects of the HUA microbiota on renal injury and the intestinal barrier were eliminated. CONCLUSION: Our results demonstrate that HUA-induced gut dysbiosis contributes to the development of renal injury, possibly by promoting the production of gut-derived uremic toxins and subsequently activating the NLRP3 inflammasome. Our data suggest a potential therapeutic strategy for the treatment of renal diseases by targeting the gut microbiota and the NLRP3 inflammasome. Video Abstract.

Immunogenicity-masking delivery of uricase against hyperuricemia and gout.

Improving the activity of uricase and lowering its immunogenicity remain significant challenges in the enzyme replacement management of hyperuricemia and related inflammatory diseases. Herein, an immunogenicity-masking strategy based on engineered red blood cells (RBCs) was developed for effective uricase delivery against both hyperuricemia and gout. The dynamic membrane of RBCs enabled high resistance to protease inactivation and hydrogen peroxide accumulation. Benefiting from these advantages, a single infusion of RBC-loaded uricase (Uri@RBC) performed prolonged blood circulation and sustained hyperuricemia management. Importantly, RBCs masked the immunogenicity of uricase, leading to the maintenance of UA-lowering performance after repeated infusion through reduced antibody-mediated macrophage clearance. In an acute gout model, Uri@RBC profoundly alleviated joint edema and inflammation with minimal systemic toxicity. This study supports the employment of immunogenicity-masking tools for efficient and safe enzyme delivery, and this strategy may be leveraged to improve the usefulness of enzyme replacement therapies for managing a wide range of inflammatory diseases.

Maximization of uricase production in a column bioreactor through response surface methodology-based optimization.

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme that is widely exploited for diagnostic and treatment purposes in medicine. This study focuses on producing recombinant uricase fromE. coliBL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through the Plackett-Burman design from eight different variables and were further optimized by the central composite design of the response surface methodology (RSM). The selected variables included the inoculum size (%v/v), isopropylß-d-1-thiogalactopyranoside (IPTG) concentration (mM) and the initial pH of the culture medium. The activity of uricase, the final optical density at 600 nm wavelength (OD600) and the final pH were considered as the responses of this optimization and were modeled. As a result, activity of 5.84 U·ml-1and a final OD600of 3.42 were obtained at optimum conditions of 3% v/v inoculum size, an IPTG concentration of 0.54 mM and a pH of 6.0. By purifying the obtained enzyme using a Ni-NTA agarose affinity chromatography column, 165 ± 1.5 mg uricase was obtained from a 600 ml cell culture. The results of this study show that BCBs can be a highly effective option for large-scale uricase production.

Crystal Facet Controlled Metal-Support Interaction in Uricase Mimics for Highly Efficient Hyperuricemia Treatment.

The effect of strong metal-support interaction (SMSI) has never been systematically studied in the field of nanozyme-based catalysis before. Herein, by coupling two different Pd crystal facets with MnO2, i.e., (100) by Pd cube (Pdc) and (111) by Pd icosahedron (Pdi), we observed the reconstruction of Pd atomic structure within the Pd-MnO2 interface, with the reconstructed Pdc (100) facet more disordered than Pdi (111), verifying the existence of SMSI in such coupled system. The rearranged Pd atoms in the interface resulted in enhanced uricase-like catalytic activity, with Pdc@MnO2 demonstrating the best catalytic performance. Theoretical calculations suggested that a more disordered Pd interface led to stronger interactions with intermediates during the uricolytic process. In vitro cell experiments and in vivo therapy results demonstrated excellent biocompatibility, therapeutic effect, and biosafety for their potential hyperuricemia treatment. Our work provides a brand-new perspective for the design of highly efficient uricase-mimic catalysts.

Real-World Effectiveness of Pegloticase Associated With Use of Concomitant Immunomodulatory Therapy.

OBJECTIVE: The objective of this study was to ascertain pegloticase persistence and adverse events associated with concomitant immunomodulatory drug treatment in patients with gout. METHODS: We conducted a retrospective analysis of patients with gout using the American College of Rheumatology's Rheumatology Informatics System for Effectiveness registry from January 2016 through June 2020. The first pegloticase infusion defined the index date. Based on concomitant immunomodulatory drug treatment, we identified three exposure groups: (1) immunomodulatory drug initiators (patients initiating an immunomodulatory prescription ±60 days from the index date), (2) prevalent immunomodulatory drug recipients (patients receiving their first immunomodulatory drug prescription >60 days before the index date with at least one prescription within ±60 days of the index date), and (3) immunomodulatory nonrecipients (patients receiving pegloticase without concomitant immunomodulatory drugs). We calculated the proportion of patients who achieved serum urate levels ≤6 mg/dL and who had laboratory abnormalities (white blood cell count <3.4 x 109/L, platelet count <135,000, hematocrit level <30%, alanine aminotransferase or aspartate aminotransferase level ≥1.5 times the upper limit normal value) within 180 days after the index date. Cox regression analyzed time to pegloticase discontinuation, controlling for potential confounders. RESULTS: We identified 700 pegloticase recipients (91 immunomodulatory drug initiators, 33 prevalent immunomodulatory drug recipients, and 576 nonrecipients), with a median follow-up of 14 months. Immunomodulatory drug recipients were less likely to discontinue pegloticase. The adjusted hazard ratios of pegloticase discontinuation associated with concomitant immunomodulatory drug initiation and prevalent treatment were 0.52 (95% confidence interval [CI] 0.37-0.75) and 0.69 (95% CI 0.42-1.16), respectively. Laboratory abnormalities were uncommon (<5%) and were not higher in concomitant immunomodulatory drug treatment. CONCLUSION: Consistent with clinical trials, results from this large observational registry suggest that concomitant immunomodulatory drug treatment improves pegloticase persistence.

Rationalization design, soluble expression and PEG modification of highly active recombinant human-porcine uricase mutant protein.

Uric acid is the end product of purine metabolism in humans due to inactivation of the uricase determined by the mutated uricase gene. Uricase catalyzes the conversion of uric acid into water-soluble allantoin that is easily excreted by the kidneys. Hyperuricemia occurs when the serum concentration of uric acid exceeds its solubility (7 mg/dL). However, modifications to improve the uricase activity is under development for treating the hyperuricemia. Here we designed 7 types of human-porcine chimeric uricase by multiple sequence comparisons and targeted mutagenesis. An optimal human-porcine chimeric uricase mutant (uricase-10) with both high activity (6.33 U/mg) and high homology (91.45 %) was determined by enzyme activity measurement. The engineering uricase was further modified with PEGylation to improve the stability of recombinant protein drugs and reduce immunogenicity, uricase-10 could be more suitable for the treatment of gout and hyperuricemia theoretically.

Flow injection amperometric uric acid biosensor based on AuNPs-GO-CS porous composite cryogel coated on PB-PEDOT:PSS modified screen-printed carbon electrode.

An enzymatic amperometric uric acid (UA) biosensor was successfully developed by modifying a screen-printed carbon electrode (SPCE) with Prussian blue-poly(3,4-ethylene dioxythiophene) polystyrene sulfonate composite (PB-PEDOT:PSS). The modified SPCE was coated with gold nanoparticles-graphene oxide-chitosan composite cryogel (AuNPs-GO-CS cry). Uricase (UOx) was directly immobilized via chemisorption on AuNPs. The nanocomposite was characterized by scanning electron microscopy, transmission electron microscopy, ultraviolet-visible spectroscopy, and Fourier transform infrared spectroscopy. The electrochemical characterization of the modified electrode was performed by cyclic voltammetry and electrochemical impedance spectroscopy. UA was determined using amperometric detection based on the reduction current of PB which was correlated with the amount of H2O2 produced during the enzymatic reaction. Under optimal conditions, the fabricated UA biosensor in a flow injection analysis (FIA) system produced a linear range from 5.0 to 300 µmol L-1 with a detection limit of 1.88 µmol L-1. The proposed sensor was stable for up to 221 cycles of detection and analysis was rapid (2 min), with good reproducibility (RSDs < 2.90 %, n = 6), negligible interferences, and recoveries from 94.0 ± 3.9 to 101.1 ± 2.6 %. The results of UA detection in blood plasma were in agreement with the enzymatic colorimetric method (P > 0.05).

Discovery of digallic acid as XOD/URAT1 dual target inhibitor for the treatment of hyperuricemia.

The development of XOD/URAT1 dual target inhibitors has emerged as a promising therapeutic strategy for the management of hyperuricemia. Here, through virtual screening, we have identified digallic acid as a novel dual target inhibitor of XOD/URAT1 and subsequently evaluated its pharmacological properties, pharmacokinetics, and toxicities. Digallic acid inhibited URAT1 with an IC50 of 5.34 ± 0.65 µM, which is less potent than benzbromarone (2.01 ± 0.36 µM) but more potent than lesinurad (10.36 ± 1.23 µM). Docking and mutation analysis indicated that residues S35, F241 and R477 of URAT1 confer a high affinity for digallic acid. Digallic acid inhibited XOD with an IC50 of 1.04 ± 0.23 µM. Its metabolic product, gallic acid, inhibited XOD with an IC50 of 0.91 ± 0.14 µM. Enzyme kinetic studies indicated that both digallic acid and gallic acid act as mixed-type XOD inhibitors. It shares the same binding mode as digallic acid, and residues E802, R880, F914, T1010, N768 and F1009 contribute to their high affinity. The anion group (carboxyl) of digallic acid contribute significantly to its inhibition activity on both XOD and URAT1 as indicated by docking analysis. Remarkably, at a dosage of 10 mg/kg in vivo, digallic acid exhibited a stronger urate-lowering and uricosuric effect compared to the positive drug benzbromarone and lesinurad. Pharmacokinetic study indicated that digallic acid can be hydrolyzed into gallic acid in vivo and has a t1/2 of 0.77 ± 0.10 h. Further toxicity evaluation indicated that digallic acid exhibited no obvious renal toxicity, as reflected by CCK-8, biochemical analysis (CR and BUN) and HE examination. The findings of our study can provide valuable insights for the development of XOD/URAT1 dual target inhibitors, and digallic acid deserves further investigation as a potential anti-hyperuricemic drug.

Raising serum uric acid with a uricase inhibitor worsens PKD in rat and mouse models.

Humans are predisposed to gout because they lack uricase that converts uric acid to allantoin. Rodents have uricase, resulting in low basal serum uric acid. A uricase inhibitor raises serum uric acid in rodents. There were two aims of the study in polycystic kidney disease (PKD): 1) to determine whether increasing serum uric acid with the uricase inhibitor, oxonic acid, resulted in faster cyst growth and 2) to determine whether treatment with the xanthine oxidase inhibitor, oxypurinol, reduced the cyst growth caused by oxonic acid. Orthologous models of human PKD were used: PCK rats, a polycystic kidney and hepatic disease 1 (Pkhd1) gene model of autosomal recessive PKD (ARPKD) and Pkd1RC/RC mice, a hypomorphic Pkd1 gene model. In PCK rats and Pkd1RC/RC mice, oxonic acid resulted in a significant increase in serum uric acid, kidney weight, and cyst index. Mechanisms of increased cyst growth that were investigated were proinflammatory cytokines, the inflammasome, and crystal deposition in the kidney. Oxonic acid resulted in an increase in proinflammatory cytokines in the serum and kidney in Pkd1RC/RC mice. Oxonic acid did not cause activation of the inflammasome or uric acid crystal deposition in the kidney. In Pkd1RC/RC male and female mice analyzed together, oxypurinol decreased the oxonic acid-induced increase in cyst index. In summary, increasing serum uric acid by inhibiting uricase with oxonic acid results in an increase in kidney weight and cyst index in PCK rats and Pkd1RC/RC mice. The effect is independent of inflammasome activation or crystal deposition in the kidney.NEW & NOTEWORTHY This is the first reported study of uric acid measurements and xanthine oxidase inhibition in polycystic kidney disease (PKD) rodents. Raising serum uric acid with a uricase inhibitor resulted in increased kidney weight and cyst index in Pkd1RC/RC mice and PCK rats, elevated levels of proinflammatory cytokines in the serum and kidney in Pkd1RC/RC mice, and no uric acid crystal deposition or activation of the caspase-1 inflammasome in the kidney.

Bioinspired mp20 mimicking uricase in ZIF-8: Metal ion dependent for controllable activity.

Mini protein mimicking uricase (mp20) has shown significant potential as a replacement for natural enzymes in the development of uric acid biosensors. However, the design of mp20 has resulted to an inactive form of peptide, causing of loss their catalytic activity. Herein, this paper delineates the impact of various metal cofactors on the catalytic activity of mp20. The metal ion-binding site prediction and docking (MIB) web server was employed to identify the metal ion binding sites and their affinities towards mp20 residues. Among the tested metal ions, Cu2+ displayed the highest docking score, indicating its preference for interaction with Thr16 and Asp17 residues of mp20. To assess the catalytic activity of mp20 in the presence of metal ions, uric acid assays was monitored using a colorimetric method. The presence of Cu2+ in the assays promotes the activation of mp20, resulting in a color change based on quinoid production. Furthermore, the encapsulation of the mp20 within zeolitic imidazolate framework-8 (ZIF-8) notably improved the stability of the biomolecule. In comparison to the naked mp20, the encapsulated ZIFs biocomposite (mp20@ZIF-8) demonstrates superior stability, selectivity and sensitivity. ZIF's porous shells provides excellent protection, broad detection (3-100 µM) with a low limit (4.4 µM), and optimal function across pH (3.4-11.4) and temperature (20-100°C) ranges. Cost-effective and stable mp20@ZIF-8 surpasses native uricase, marking a significant biosensor technology breakthrough. This integration of metal cofactor optimization and robust encapsulation sets new standards for biosensing applications.

Tumor lysis syndrome in induction therapy for acute myeloid leukemia before the rasburicase era.

Guidelines recommend rasburicase for high-risk patients to prevent tumor lysis syndrome (TLS). However, little information is available on the incidence and outcome of TLS in AML patients. We analyzed 145 patients with AML who underwent induction therapy before the approval of rasburicase to evaluate the incidence of TLS and the necessity of rasburicase as prophylaxis. Three patients had already developed clinical TLS (CTLS) at diagnosis of AML, and another three developed CTLS after the initiation of chemotherapy. In patients without TLS at diagnosis of AML, the risk for developing TLS was classified as high in 44 patients, intermediate in 41 and low in 57, according to the current guidelines. Allopurinol alone was administered to prevent hyperuricemia in all patients. All three patients who developed CTLS after diagnosis of AML were at high risk of TLS, and had elevated serum creatinine levels and a WBC count greater than 200,000 per microliter at diagnosis of AML. Allopurinol may be insufficient to prevent TLS in high-risk patients with renal dysfunction at diagnosis of AML, especially those with a high tumor burden and a WBC count of 200,000 or more, which indicates that prophylactic administration of rasburicase should be considered.

Colorimetric and fluorometric determination of uric acid by a suspension-based assay using enzyme-immobilized micro-sized particles.

Daily monitoring of serum uric acid levels is very important to provide appropriate treatment according to the constitution and lifestyle of individual hyperuricemic patients. We have developed a suspension-based assay to measure uric acid by adding a sample solution to the suspension containing micro-sized particles immobilized on uricase and horseradish peroxidase (HRP). In the proposed method, the mediator reaction of uricase, HRP, and uric acid produces resorufin from Amplex red. This resorufin is adsorbed onto enzyme-immobilized micro-sized particles simultaneously with its production, resulting in the red color of the micro-sized particles. The concentration of resorufin on the small surface area of the microscopic particles achieves a colorimetric analysis of uric acid with superior visibility. In addition, ethanol-induced desorption of resorufin allowed quantitative measurement of uric acid using a 96-well fluorescent microplate reader. The limit of detection (3σ) and RSD (n = 3) were estimated to be 2.2 × 10-2 µg/mL and ≤ 12.1%, respectively. This approach could also be applied to a portable fluorometer.