A metabolomics perspective reveals the mechanism of the uric acid-lowering effect of Prunus salicina Lindl. cv. "furong" polyphenols in hypoxanthine and potassium oxybate-induced hyperuricemic mice.

The incidence of hyperuricemia (HUA) shows a gradually increasing trend towards affecting younger individuals, and it can significantly harm the overall health status of the body. Based on a metabolomics perspective, this study reveals the mechanism of the uric acid-lowering action of Prunus salicina Lindl. cv. "furong" polyphenols (PSLP) on a hyperuricemia mouse model induced by hypoxanthine and potassium oxybutyrate. The results demonstrate that PSLP comprise an effective treatment strategy for reducing the levels of serum uric acid (SUA), serum creatinine (SCr) and blood urea nitrogen (BUN) in HUA mice (p < 0.05), wherein the maximum decrease rates are up to 44.50%, 29.46%, and 32.95%, respectively. PSLP are observed to exert a pronounced inhibitory effect on the activities of xanthine oxidase (XOD) and adenosine deaminase (ADA) in the livers of HUA mice, with reductions of up to 16.36% and 20.13%, respectively. These findings illustrate that PSLP exert a significant uric acid-lowering effect. Subsequent metabolomic analysis of mouse serum identified 28 potential biomarkers for hyperuricemia, whose levels were markedly diminished by PSLP. This process involved alterations in purine, glycine, the pentose phosphate pathway, and galactose metabolism. Twenty-eight potential biomarkers were identified for hyperuricemia by subsequent metabolomic analysis of mouse serum, whose levels were markedly reversed by PSLP intervention. The regulation of HUA by PSLP involved alterations in purine metabolism, glycerolipid metabolism, the pentose phosphate pathway, and galactose metabolism. The mechanism of PSLP ameliorated hyperuricemia might be attributed to reduction of the level of the uric acid precursor ribose-5-phosphate in the pentose phosphate pathway, the inhibition of the activities of uric acid synthase XOD and ADA in purine metabolism, and reduction of the synthesis of the end product uric acid. This study provides a theoretical basis for the development of functional foods based on PSLP, which can potentially reduce uric acid levels.

Hydrogen-Rich Gas Enhanced Sprint-Interval Performance: Metabolomic Insights into Underlying Mechanisms.

(1) Background: The diversity of blood biomarkers used to assess the metabolic mechanisms of hydrogen limits a comprehensive understanding of its effects on improving exercise performance. This study evaluated the impact of hydrogen-rich gas (HRG) on metabolites following sprint-interval exercise using metabolomics approaches, aiming to elucidate its underlying mechanisms of action. (2) Methods: Ten healthy adult males participated in the Wingate Sprint-interval test (SIT) following 60 min of HRG or placebo (air) inhalation. Venous blood samples were collected for metabolomic analysis both before and after gas inhalation and subsequent to completing the SIT. (3) Results: Compared with the placebo, HRG inhalation significantly improved mean power, fatigue index, and time to peak for the fourth sprint and significantly reduced the attenuation values of peak power, mean power, and time to peak between the first and fourth. Metabolomic analysis highlighted the significant upregulation of acetylcarnitine, propionyl-L-carnitine, hypoxanthine, and xanthine upon HRG inhalation, with enrichment pathway analysis suggesting that HRG may foster fat mobilization by enhancing coenzyme A synthesis, promoting glycerophospholipid metabolism, and suppressing insulin levels. (4) Conclusions: Inhaling HRG before an SIT enhances end-stage anaerobic sprint capabilities and mitigates fatigue. Metabolomic analysis suggests that HRG may enhance ATP recovery during interval stages by accelerating fat oxidation, providing increased energy replenishment for late-stage sprints.

Significance and amplification methods of the purine salvage pathway in human brain cells.

Previous studies suggest that uric acid or reactive oxygen species, products of xanthine oxidoreductase (XOR), may associate with neurodegenerative diseases. However, neither relationship has ever been firmly established. Here, we analyzed human brain samples, obtained under protocols approved by research ethics committees, and found no expression of XOR and only low levels of uric acid in various regions of the brain. In the absence of XOR, hypoxanthine will be preserved and available for incorporation into the purine salvage pathway. To clarify the importance of salvage in the brain, we tested using human-induced pluripotent stem cell-derived neuronal cells. Stable isotope analyses showed that the purine salvage pathway was more effective for ATP synthesis than purine de novo synthesis. Blood uric acid levels were related to the intracellular adenylate pool (ATP + ADP + AMP), and reduced levels of this pool result in lower uric acid levels. XOR inhibitors are related to extracellular hypoxanthine levels available for uptake into the purine salvage pathway by inhibiting the oxidation of hypoxanthine to xanthine and uric acid in various organs where XOR is present and can prevent further decreases in the intracellular adenylate pool under stress. Furthermore, adding precursors of the pentose phosphate pathway enhanced hypoxanthine uptake, indicating that purine salvage is activated by phosphoribosyl pyrophosphate replenishment. These findings resolve previous contradictions regarding XOR products and provide new insights into clinical studies. It is suggested that therapeutic strategies maximizing maintenance of intracellular adenylate levels may effectively treat pathological conditions associated with ischemia and energy depletion.

Metabolic signatures and risk of sarcopenia in suburb-dwelling older individuals by LC-MS-based untargeted metabonomics.

Background: Untargeted metabonomics has provided new insight into the pathogenesis of sarcopenia. In this study, we explored plasma metabolic signatures linked to a heightened risk of sarcopenia in a cohort study by LC-MS-based untargeted metabonomics. Methods: In this nested case-control study from the Adult Physical Fitness and Health Cohort Study (APFHCS), we collected blood plasma samples from 30 new-onset sarcopenia subjects (mean age 73.2 ± 5.6 years) and 30 healthy controls (mean age 74.2 ± 4.6 years) matched by age, sex, BMI, lifestyle, and comorbidities. An untargeted metabolomics methodology was employed to discern the metabolomic profile alterations present in individuals exhibiting newly diagnosed sarcopenia. Results: In comparing individuals with new-onset sarcopenia to normal controls, a comprehensive analysis using liquid chromatography-mass spectrometry (LC-MS) identified a total of 62 metabolites, predominantly comprising lipids, lipid-like molecules, organic acids, and derivatives. Receiver operating characteristic (ROC) curve analysis indicated that the three metabolites hypoxanthine (AUC=0.819, 95% CI=0.711-0.927), L-2-amino-3-oxobutanoic acid (AUC=0.733, 95% CI=0.598-0.868) and PC(14:0/20:2(11Z,14Z)) (AUC= 0.717, 95% CI=0.587-0.846) had the highest areas under the curve. Then, these significant metabolites were observed to be notably enriched in four distinct metabolic pathways, namely, "purine metabolism"; "parathyroid hormone synthesis, secretion and action"; "choline metabolism in cancer"; and "tuberculosis". Conclusion: The current investigation elucidates the metabolic perturbations observed in individuals diagnosed with sarcopenia. The identified metabolites hold promise as potential biomarkers, offering avenues for exploring the underlying pathological mechanisms associated with sarcopenia.

De novo and salvage purine synthesis pathways across tissues and tumors.

Purine nucleotides are vital for RNA and DNA synthesis, signaling, metabolism, and energy homeostasis. To synthesize purines, cells use two principal routes: the de novo and salvage pathways. Traditionally, it is believed that proliferating cells predominantly rely on de novo synthesis, whereas differentiated tissues favor the salvage pathway. Unexpectedly, we find that adenine and inosine are the most effective circulating precursors for supplying purine nucleotides to tissues and tumors, while hypoxanthine is rapidly catabolized and poorly salvaged in vivo. Quantitative metabolic analysis demonstrates comparative contribution from de novo synthesis and salvage pathways in maintaining purine nucleotide pools in tumors. Notably, feeding mice nucleotides accelerates tumor growth, while inhibiting purine salvage slows down tumor progression, revealing a crucial role of the salvage pathway in tumor metabolism. These findings provide fundamental insights into how normal tissues and tumors maintain purine nucleotides and highlight the significance of purine salvage in cancer.

Green manufacturing of a hypoxanthine enzyme sensor for fish freshness based on modified nitrocellulose surface with chito-oligosaccharide.

Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 µM; the linear range is 0.05-0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness.

Allopurinol Disrupts Purine Metabolism to Increase Damage in Experimental Colitis.

Inflammatory bowel disease (IBD) is marked by a state of chronic energy deficiency that limits gut tissue wound healing. This energy shortfall is partially due to microbiota dysbiosis, resulting in the loss of microbiota-derived metabolites, which the epithelium relies on for energy procurement. The role of microbiota-sourced purines, such as hypoxanthine, as substrates salvaged by the colonic epithelium for nucleotide biogenesis and energy balance, has recently been appreciated for homeostasis and wound healing. Allopurinol, a synthetic hypoxanthine isomer commonly prescribed to treat excess uric acid in the blood, inhibits the degradation of hypoxanthine by xanthine oxidase, but also inhibits purine salvage. Although the use of allopurinol is common, studies regarding how allopurinol influences the gastrointestinal tract during colitis are largely nonexistent. In this work, a series of in vitro and in vivo experiments were performed to dissect the relationship between allopurinol, allopurinol metabolites, and colonic epithelial metabolism and function in health and during disease. Of particular significance, the in vivo investigation identified that a therapeutically relevant allopurinol dose shifts adenylate and creatine metabolism, leading to AMPK dysregulation and disrupted proliferation to attenuate wound healing and increased tissue damage in murine experimental colitis. Collectively, these findings underscore the importance of purine salvage on cellular metabolism and gut health in the context of IBD and provide insight regarding the use of allopurinol in patients with IBD.

High-Accuracy Renal Cell Carcinoma Discrimination through Label-Free SERS of Blood Serum and Multivariate Analysis.

Renal cell carcinoma (RCC) represents the sixth most frequently diagnosed cancer in men and is asymptomatic, being detected mostly incidentally. The apparition of symptoms correlates with advanced disease, aggressive histology, and poor outcomes. The development of the Surface-Enhanced Raman Scattering (SERS) technique opened the way for investigating and detecting small molecules, especially in biological liquids such as serum or blood plasma, urine, saliva, and tears, and was proposed as a simple technique for the diagnosis of various diseases, including cancer. In this study, we investigated the use of serum label-free SERS combined with two multivariate analysis tests: Principal Component Analysis combined with Linear Discriminate Analysis (PCA-LDA) and Supported Vector Machine (SVM) for the discrimination of 50 RCC cancer patients from 45 apparently healthy donors. In the case of LDA-PCA, we obtained a discrimination accuracy of 100% using 12 principal components and a quadratic discrimination function. The accuracy of discrimination between RCC stages was 88%. In the case of the SVM approach, we obtained a training accuracy of 100%, a validation accuracy of 92% for the discrimination between RCC and controls, and an accuracy of 81% for the discrimination between stages. We also performed standard statistical tests aimed at improving the assignment of the SERS vibration bands, which, according to our data, are mainly due to purinic metabolites (uric acid and hypoxanthine). Moreover, our results using these assignments and Student's t-test suggest that the main differences in the SERS spectra of RCC patients are due to an increase in the uric acid concentration (a conclusion in agreement with recent literature), while the hypoxanthine concentration is not statistically different between the two groups. Our results demonstrate that label-free SERS combined with chemometrics holds great promise for non-invasive and early detection of RCC. However, more studies are needed to validate this approach, especially when combined with other urological diseases.

Analysis of Metabolites in Gout: A Systematic Review and Meta-Analysis.

(1) Background: Many studies have attempted to explore potential biomarkers for the early detection of gout, but consistent and high levels of evidence are lacking. In this study, metabolomics was used to summarize the changes of metabolites in the literature and explore the potential value of metabolites in predicting the occurrence and development of gout. (2) Methods: We searched the databases including the EMBASE, the Cochrane Library, PubMed, Web of Science, VIP Date, Wanfang Data, and CNKI, and the screening was fulfilled on 30 July 2022. The records were screened according to the inclusion criteria and the risk of bias was assessed. Qualitative analysis was performed for all metabolites, and meta-analysis was performed for metabolite concentrations using random effects to calculate the Std mean difference and 95% confidence interval. (3) Results: A total of 2738 records were identified, 33 studies with 3422 participants were included, and 701 metabolites were identified. The qualitative analysis results showed that compared with the healthy control group, the concentration of 56 metabolites increased, and 22 metabolites decreased. The results of the meta-analysis indicated that 17 metabolites were statistically significant. (4) Conclusions: Metabolites are associated with gout. Some specific metabolites such as uric acid, hypoxanthine, xanthine, KYNA, guanosine, adenosine, creatinine, LB4, and DL-2-Aminoadipic acid have been highlighted in the development of gout.

Two dimensional iron metal-organic framework nanosheet with peroxidase-mimicking activity for colorimetric detection of hypoxanthine related to shrimp freshness.

Two dimensional iron metal-organic framework nanosheet (2D Fe MOF) was facilely synthesized at room temperature by simple stirring of iron salts and terephthalic acid ligand in a mixed solution containing triethylamine. Its morphology and structure were fully characterized by TEM, AFM, XPS and TEM element mapping. Then, its peroxidase-mimicking activity was studied by using H2O2 and 3, 3', 5, 5'- tetramethylbenzidine as substrate. Km and Vmax of 2D Fe MOF towards H2O2 were 0.02 mM and 2.08 × 10-8 M s-1, respectively. Through the formation of cascade reaction between xanthine oxidase and 2D Fe MOF, a visual method for hypoxanthine (Hx) detection was constructed to evaluate aquatic products freshness. After effective validation, this method presented wide linear range (5.0-500.0 µM), low limit of detection (3.29 µM), satisfied accuracy (recovery of 94.78-99.85%), and good selectivity. By using this method, Hx content in shrimp samples at different storage time were determined.

Enzymatic determination of hypoxanthine in fish samples as a freshness indicator using the CUPRAC colorimetric sensor.

Fish consumption is essential for a healthy diet. However, all seafood including fish are susceptible to deterioration unless properly preserved. Controlling the freshness of fresh or packaged fish is a challenging issue for the food industry in terms of human health and shelf life determination. One of the main indicators showing the freshness of fish is undoubtedly the amount of hypoxanthine (Hx). As soon as the organism dies, Hx begins to be released with the cessation of ATP synthesis and shows a gradual increase over time. Therefore, Hx determination is an important indicator in the control of fish freshness. Based on this fact, a colorimetric method for the enzymatic determination of Hx using the CUPRAC (Cupric ion Reducing Antioxidant Capacity) sensor was developed. Uric acid (UA) and H2O2 are enzymatically produced by xanthine oxidase (XOD) from Hx, and both products respond to the CUPRAC reagent to produce the cuprous neocuproine (Cu(I)-Nc) chromophore chelate formed in situ on a Nafion anionic membrane on which the cationic Cu(II)-Nc complex was fixed. Hx was measured at different time intervals in the meat samples taken from sea bass (Dicentrarchus labrax), which was left to stand at room temperature for a time period between 0 and 24 h; the level of spoilage was determined from the coloration of the CUPRAC membrane sensor (via absorbance measurement at 450 nm). It was observed that there was a linear increase in the amount of Hx during the measurement period. The method was optimized for Hx determination, verified with interference analysis and standard additions to real samples, and validated against HPLC. The linear detection range of the developed method for Hx was 2.0-32.0 µM with an LOD of 0.79 µM, and early stages of fish degradation could be detected at several nanomoles of Hx per gram of fish meat. The proposed method was demonstrated to have distinct superiority over many recent colorimetric sensors of fish freshness in regard to its lower LOD for Hx, wider linear range, capability to cope with interferents (including biologically important antioxidants, such as cysteine, reduced glutathione, ascorbic acid, UA and α-tocopherol) and applicability to real samples.

Semi-mechanistic Modeling of Hypoxanthine, Xanthine, and Uric Acid Metabolism in Asphyxiated Neonates.

BACKGROUND AND OBJECTIVE: Previously, we developed a pharmacokinetic-pharmacodynamic model of allopurinol, oxypurinol, and biomarkers, hypoxanthine, xanthine, and uric acid, in neonates with hypoxic-ischemic encephalopathy, in which high initial biomarker levels were observed suggesting an impact of hypoxia. However, the full pharmacodynamics could not be elucidated in our previous study. The current study included additional data from the ALBINO study (NCT03162653) placebo group, aiming to characterize the dynamics of hypoxanthine, xanthine, and uric acid in neonates with hypoxic-ischemic encephalopathy. METHODS: Neonates from the ALBINO study who received allopurinol or placebo mannitol were included. An extended population pharmacokinetic-pharmacodynamic model was developed based on the mechanism of purine metabolism, where synthesis, salvage, and degradation via xanthine oxidoreductase pathways were described. The initial level of the biomarkers was a combination of endogenous turnover and high disease-related amounts. Model development was accomplished by nonlinear mixed-effects modeling (NONMEM®, version 7.5). RESULTS: In total, 20 neonates treated with allopurinol and 17 neonates treated with mannitol were included in this analysis. Endogenous synthesis of the biomarkers reduced with 0.43% per hour because of precursor exhaustion. Hypoxanthine was readily salvaged or degraded to xanthine with rate constants of 0.5 1/h (95% confidence interval 0.33-0.77) and 0.2 1/h (95% confidence interval 0.09-0.31), respectively. A greater salvage was found in the allopurinol treatment group consistent with its mechanism of action. High hypoxia-induced initial levels of biomarkers were quantified, and were 1.2-fold to 2.9-fold higher in neonates with moderate-to-severe hypoxic-ischemic encephalopathy compared with those with mild hypoxic-ischemic encephalopathy. Half-maximal xanthine oxidoreductase inhibition was achieved with a combined allopurinol and oxypurinol concentration of 0.68 mg/L (95% confidence interval 0.48-0.92), suggesting full xanthine oxidoreductase inhibition during the period studied. CONCLUSIONS: This extended pharmacokinetic-pharmacodynamic model provided an adequate description of the complex hypoxanthine, xanthine, and uric acid metabolism in neonates with hypoxic-ischemic encephalopathy, suggesting a positive allopurinol effect on these biomarkers. The impact of hypoxia on their dynamics was characterized, underlining higher hypoxia-related initial exposure with a more severe hypoxic-ischemic encephalopathy status.

Hypoxanthine is a pharmacodynamic marker of ischemic brain edema modified by glibenclamide.

Brain edema after a large stroke causes significant morbidity and mortality. Here, we seek to identify pharmacodynamic markers of edema that are modified by intravenous (i.v.) glibenclamide (glyburide; BIIB093) treatment. Using metabolomic profiling of 399 plasma samples from patients enrolled in the phase 2 Glyburide Advantage in Malignant Edema and Stroke (GAMES)-RP trial, 152 analytes are measured using liquid chromatography-tandem mass spectrometry. Associations with midline shift (MLS) and the matrix metalloproteinase-9 (MMP-9) level that are further modified by glibenclamide treatment are compared with placebo. Hypoxanthine is the only measured metabolite that associates with MLS and MMP-9. In sensitivity analyses, greater hypoxanthine levels also associate with increased net water uptake (NWU), as measured on serial head computed tomography (CT) scans. Finally, we find that treatment with i.v. glibenclamide reduces plasma hypoxanthine levels across all post-treatment time points. Hypoxanthine, which has been previously linked to inflammation, is a biomarker of brain edema and a treatment response marker of i.v. glibenclamide treatment.

Hypoxanthine Reduces Radiation Damage in Vascular Endothelial Cells and Mouse Skin by Enhancing ATP Production via the Salvage Pathway.

An effective method that can protect radiation-damaged tissues from apoptosis and promote tissue repair has not been reported to date. Hypoxanthine (Hx) is an intermediate metabolite in the purine degradation system that serves as a substrate for ATP synthesis via the salvage pathway. In this study, we focused on the transient decrease in intracellular ATP concentration after radiation exposure and examined the protective effect of Hx against radiation-induced tissue damage. Human umbilical vein endothelial cells were X irradiated, and the cell viability and incidence of apoptosis and DNA double-strand breaks (DSBs) were evaluated at different Hx concentrations. We found that in the presence of 2-100 µM Hx, the percentages of DSBs and apoptotic cells after 2, 6 and 10 Gy dose of radiation significantly decreased, whereas cell viability increased in a concentration-dependent manner. Moreover, the addition of Hx increased the levels of AMP, ADP, and ATP in the cells at 2 h postirradiation, suggesting that Hx was used for adenine nucleotide synthesis through the salvage pathway. Administration of a xanthine oxidoreductase inhibitor to a mouse model of radiation dermatitis resulted in increased blood Hx levels that inhibited severe dermatitis and accelerated recovery. In conclusion, the findings provide evidence that increasing the levels of Hx to replenish ATP could be an effective strategy to reduce radiation-induced tissue damage and elucidating the detailed mechanisms underlying the protective effects of Hx could help develop new protective strategies against radiation.

Study on the levels of N-nitrosamine compounds and untargeted metabolomics in patients with colorectal cancer.

Plasma samples were collected from 34 patients with advanced CRC and 92 healthy persons (control group), and the levels of 9 VNAs were measured using GC-MS. Untargeted metabolomics analysis was performed using LC-MS/MS. Partial least squares discriminant analysis (PLS-DA) and hierarchical cluster analysis were used to determine differential metabolites between the 2 groups. Receiver operating characteristic (ROC) curve analysis and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed on the differential metabolites. It turned out that the detection rates of N-nitrosodimethylamine (NDMA) and N-nitrosopyrrolidine (NPYR) in patients with CRC were higher than in the control group (P < 0.05). N-nitrosomethylethylamine (NMEA) and N-nitrosodiphenylamine (NDPhA) were not detected in CRC patients. NDMA, N-nitrosodibutylamine (NDBA), N-nitrosopiperidine (NPIP), and NPYR were detected in male and female patients with CRC. There was no difference in VNAs exposure between the sexes of CRC patients. In the positive and negative ion mode, a total of 132 differential metabolites and 6 differential metabolic pathways were detected. Adenosine 5'-monophosphate, hypoxanthine, 11,12-epoxy-(5Z,8Z,11Z)-icosatrienoic acid, 16(R)-HETE, acetylcarnitine, and lysophosphatidic acid (LPA 20:5, LPA 20:4) were candidate biomarkers with higher predictive value. Hypoxanthine and xanthine metabolic pathways were associated with changes in VNAs in CRC patients. In summary, the effects of changes of VNAs in the plasma of CRC patients (especially NDMA and NPYR) on the progression of CRC should attract attention. Abnormalities of adenine and guanine and downstream hypoxanthine-xanthine metabolic pathways were closely related to changes of VNAs and metabolomics in CRC patients.

Proof of concept: hypoxanthine from stored red blood cells induces neutrophil activation.

BACKGROUND: Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated. MATERIALS AND METHODS: Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO2. Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex® assay. RESULTS: In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-α- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation. DISCUSSION: Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes.

Pharmacokinetic/Pharmacodynamic Modelling of Allopurinol, its Active Metabolite Oxypurinol, and Biomarkers Hypoxanthine, Xanthine and Uric Acid in Hypoxic-Ischemic Encephalopathy Neonates.

BACKGROUND: Allopurinol, an xanthine oxidase (XO) inhibitor, is a promising intervention that may provide neuroprotection for neonates with hypoxic-ischemic encephalopathy (HIE). Currently, a double-blind, placebo-controlled study (ALBINO, NCT03162653) is investigating the neuroprotective effect of allopurinol in HIE neonates. OBJECTIVE: The aim of the current study was to establish the pharmacokinetics (PK) of allopurinol and oxypurinol, and the pharmacodynamics (PD) of both compounds on hypoxanthine, xanthine, and uric acid in HIE neonates. The dosage used and the effect of allopurinol in this population, either or not undergoing therapeutic hypothermia (TH), were evaluated. METHODS: Forty-six neonates from the ALBINO study and two historical clinical studies were included. All doses were administered on the first day of life. In the ALBINO study (n = 20), neonates received a first dose of allopurinol 20 mg/kg, and, in the case of TH (n = 13), a second dose of allopurinol 10 mg/kg. In the historical cohorts (n = 26), neonates (all without TH) received two doses of allopurinol 20 mg/kg in total. Allopurinol and oxypurinol population PK, and their effects on inhibiting conversions of hypoxanthine and xanthine to uric acid, were assessed using nonlinear mixed-effects modelling. RESULTS: Allopurinol and oxypurinol PK were described by two sequential one-compartment models with an autoinhibition effect on allopurinol metabolism by oxypurinol. For allopurinol, clearance (CL) was 0.83 L/h (95% confidence interval [CI] 0.62-1.09) and volume of distribution (Vd) was 2.43 L (95% CI 2.25-2.63). For metabolite oxypurinol, CL and Vd relative to a formation fraction (fm) were 0.26 L/h (95% CI 0.23-0.3) and 11 L (95% CI 9.9-12.2), respectively. No difference in allopurinol and oxypurinol CL was found between TH and non-TH patients. The effect of allopurinol and oxypurinol on XO inhibition was described by a turnover model of hypoxanthine with sequential metabolites xanthine and uric acid. The combined allopurinol and oxypurinol concentration at the half-maximal XO inhibition was 0.36 mg/L (95% CI 0.31-0.42). CONCLUSION: The PK and PD of allopurinol, oxypurinol, hypoxanthine, xanthine, and uric acid in neonates with HIE were described. The dosing regimen applied in the ALBINO trial leads to the targeted XO inhibition in neonates treated with or without TH.

Astaxanthin attenuated hyperuricemia and kidney inflammation by inhibiting uric acid synthesis and the NF-κ B/NLRP3 signaling pathways in potassium oxonate and hypoxanthine-induced hyperuricemia mice.

Inflammation is an important pathological feature of hyperuricemia, which in turn aggravates hyperuricemia. Astaxanthin is a carotenoid with strong antioxidant capacity and possesses many biological activities. This study was aimed to evaluate the effect of astaxanthin (ASX) on hyperuricemia and kidney inflammation in potassium oxonate (PO) and hypoxanthine (HX)-induced hyperuricemic mice. Male ICR mice were administered intragastrically with PO and HX (250 mg/kg, respectively) for 14 days. ASX was given by gavage one hour after PO and HX administration. ASX treatment significantly reversed PO and HX-induced hyperuricemia and kidney inflammation in mice as evidenced by decreased serum levels of uric acid (UA), creatinine (Cr), blood urea nitrogen (BUN), and inflammatory factors (IL-1ß, IL-6, and TNF-α) and increased activities of antioxidant enzymes (CAT, SOD and GSH-Px). Furthermore, ASX administration effectively inhibited the activities of key enzymes related to UA synthesis (xanthine oxidase (XOD) and adenosine deaminase (ADA)) and modulated the protein expressions of NF-κ B p65, p-NF-κ B p65, Iκ Bα, p-Iκ Bα, NLRP3, ASC, Caspase-1, and cleavedCaspase-1 involved in inflammation pathways. Our results suggested that ASX improved hyperuricemia and kidney inflammation induced by PO and HX, probably by reducing UA synthesis and suppressing the NF-κ B and NLRP3 pathways simultaneously.

Sonneratia apetala seed oil attenuates potassium oxonate/hypoxanthine-induced hyperuricemia and renal injury in mice.

Sonneratia apetala seeds are considered as prospective nutraceuticals with a high content of unsaturated fatty acids (UFAs) which are mainly distributed in the oil. It is well-known that UFAs could exhibit urate-lowering potency and protect against renal injury, indicating that S. apetala seed oil (SSO) may possess hypouricemic and nephroprotective effects. Consequently, the present work attempted to probe into the effects and mechanisms of SSO on potassium oxonate/hypoxanthine-induced hyperuricemia and associated renal injury. The results indicated that SSO treatment prominently inhibited the increase of serum uric acid (UA), creatinine (CRE), and urea nitrogen (BUN) levels and hepatic xanthine oxidase (XOD) activity in hyperuricemia mice. Kidney indexes and histopathological lesions were also remarkably ameliorated. Additionally, SSO treatment improved the renal anti-oxidant status in hyperuricemia mice by significantly reversing the increase in ROS and MDA levels as well as the decline in SOD, CAT and GSH-Px activities. SSO dramatically downregulated the expression and secretion of pro-inflammatory factors involving MCP-1, IL-1ß, IL-6, IL-18 and TNF-α elicited by hyperuricemia. Furthermore, after SSO treatment, increased protein expressions of GLUT9, URAT1 and OAT1 in the hyperuricemia mice were obviously reversed. SSO treatment enormously restored Nrf2 activation and subsequent translation of related anti-oxidative enzymes in the kidneys. TXNIP/NLRP3 inflammasome activation was also obviously suppressed by SSO. In conclusion, SSO exerted favorable hypouricemic effects owing to its dual functions of downregulating the XOD activity and modulating the expressions of renal urate transport-associated proteins, and it also could alleviate hyperuricemia-induced renal injury by restoring the Keap1-Nrf2 pathway and blocking the TXNIP/NLRP3 inflammasome activation.

Berberrubine attenuates potassium oxonate- and hypoxanthine-induced hyperuricemia by regulating urate transporters and JAK2/STAT3 signaling pathway.

Phellodendri Chinensis Cortex (PC) is a traditional medicinal material used to treat gout and hyperuricemia (HUA) in China. Berberine (BBR), the main component of PC, possesses anti-hyperuricemic and anti-gout effects. However, BBR exhibits low bioavailability due to its extensive metabolism and limited absorption. Thus, the metabolites of BBR are believed to be the potential active forms responsible for its in vivo biological activities. Berberrubine (BRB), one of the major metabolites of BBR, exhibits appreciable biological activities even superior to BBR. In this work, the anti-hyperuricemic efficacy of BRB was investigated in HUA model mice induced by co-administration with intraperitoneal potassium oxonate (PO) and oral hypoxanthine (HX) for 7 days. Results showed that administration with BRB (6.25, 12.5, and 25.0 mg/kg) significantly decreased the serum levels of uric acid (UA) by 49.70%, 75.35%, and 75.96% respectively, when compared to the HUA group. In addition, BRB sharply decreased the levels of blood urea nitrogen (BUN) (by 19.62%, 28.98%, and 38.72%, respectively) and serum creatinine (CRE) (by 16.19%, 25.07%, and 52.08%, respectively) and reversed the PO/HX-induced renal histopathological damage dose-dependently. Additionally, BRB lowered the hepatic XOD activity, downregulated the expressions of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1), upregulated expressions of organic anion transporter 1/3 (OAT1/3) and ATP-binding cassette transporter subfamily G member 2 (ABCG2) at both protein and mRNA levels, and suppressed the activation of the JAK2/STAT3 signaling pathway. In addition, BRB significantly decreased the levels of inflammatory mediators (IL-1ß, IL-6, and TNF-α). In conclusion, our study indicated that BRB exerted anti-hyperuricemic effect, at least in part, via regulating the urate transporter expressions and suppressing the JAK2/STAT3 signaling pathway. BRB was believed to be promising for further development into a potential therapeutic agent for HUA treatment.