Hypoxanthine In Serum Research Articles

Quantitative analysis of 20 purine and pyrimidine metabolites by HILIC-MS/MS in the serum and hippocampus of depressed mice

Purine and pyrimidine metabolism are vital metabolic pathways in the development, proliferation or repairment of cells or tissues associated with various diseases. Here, a simple, all-in-one injection hydrophilic interaction liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of 20 metabolites: adenine, adenosine, deoxyadenosine, adenosine 5′-monophosphate, cyclic adenosine monophosphate, hypoxanthine, xanthine, inosine, deoxyinosine, xanthosine, xanthosine 5′-monophosphate and uric acid, which are products of purine metabolism; uridine, deoxyuridine, uridine 5′-monophosphate and uracil, are products of pyrimidine metabolism; and corticosterone, methionine, acetylcholine and serotonin. To minimize interference of endogenous molecules in sample matrixes, a combination of activated carbon adsorption and a serum substitute matrix (5% bovine serum albumin in phosphate buffered saline) was utilized and jointly applied. The sensitivity, linearity, stability, precision, accuracy and extraction recovery were evaluated, and the method was demonstrated to be accurate, sensitive and reliable. An analytical strategy was successfully applied to quantitatively determine 20 metabolite levels in the serum and hippocampus of mice with chronic social defeat stress-induced depression. The results showed greatly perturbed purine metabolism in the depressed mice, which was primarily characterized by dramatic increases in hypoxanthine, xanthine and inosine in serum and reduced levels of adenine, adenosine and adenosine 5′-monophosphate in the hippocampus. These findings suggest that this novel strategy can facilitate the quantitative analysis of adenine and other purine and pyrimidine metabolites in tissue and serum and exhibits great potential in the exploration of metabolism-related mechanisms of relevant diseases.

Serum Metabolite Biomarkers Predictive of Response to PD-1 Blockade Therapy in Non-Small Cell Lung Cancer.

Background: Despite remarkable success of immunotherapies with checkpoint blockade antibodies targeting programmed cell death protein 1 (PD-1), the majority of patients with non-small-cell lung cancer (NSCLC) have yet to receive durable benefits. We used the metabolomic profiling of early on-treatment serum to explore predictors of clinical outcomes of anti-PD-1 treatment in patients with advanced NSCLC. Methods: We recruited 74 Chinese patients who had stage IIIB/IV NSCLC-proven tumor progression and were treated with PD-1 inhibitor. The study was comprised of a discovery cohort of patients treated with nivolumab and two validation cohorts of patients receiving tislelizumab or nivolumab. Serum samples were collected 2–3 weeks after the first infusion of PD-1 inhibitor. Metabolomic profiling of serum was performed using ultrahigh performance lipid chromatograph-mass spectrometry. The serum metabolite biomarkers were identified using an integral workflow of nontargeted metabolomic data analysis. Results: A serum metabolite panel consisting of hypoxanthine and histidine was identified and validated as a predictor of response to PD-1 blockade treatment in patients with advanced NSCLC. High levels of both hypoxanthine and histidine in early on-treatment serum were associated with improved progression-free survival [hazard ratio (HR) = 0.078, 95% confidence interval (CI), 0.027–0.221, p < 0.001] and overall survival (HR = 0.124, 95% CI, 0.039–0.397, p < 0.001) in the discovery cohort. The serum metabolite panel showed a high sensitivity and specificity in distinguishing responders and non-responders in the validation cohorts 1 and 2, with an area under the receiver-operating characteristic curve of 0.933 and 1.000, respectively. High levels of serum hypoxanthine and histidine were correlated with improved progression-free survival in the validation cohort 1 (HR = 0.137, 95% CI, 0.040–0.467, p = 0.001) and in the validation cohort 2 (HR = 0.084, 95% CI, 0.009–0.762, p = 0.028). Conclusion: Our results revealed that hypoxanthine and histidine in early on-treatment serum are predictive biomarkers of response to PD-1 blockade therapy in patients with advanced NSCLC. The serum biomarker panel would enable early identification of NSCLC patients who may benefit from PD-1 blockade therapy.

The effect of dietary protein restriction in a case of molybdenum cofactor deficiency with MOCS1 mutation

Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism that results from mutations in genes involved in molybdenum cofactor (Moco) biosynthesis. MoCD is characterized clinically by intractable seizures and severe, rapidly progressing neurodegeneration leading to death in early childhood in the majority of known cases. We report on a patient with an unusual late disease onset and mild phenotype, characterized by delayed development and a decline triggered by a febrile illness and a subsequent dystonic movement disorder. Magnetic resonance imaging showed abnormal signal intensities of the bilateral basal ganglia. Blood and urine chemistry tests demonstrated remarkably low serum and urinary uric acid levels. A urine sulfite test was positive. Specific diagnostic workup showed elevated levels of xanthine and hypoxanthine in serum with increased urinary sulfocysteine (SSC) levels. Genetic analysis revealed a homozygous missense mutation at c.1510C > T (p.504R > W) in exon 10 of the MOCS1 in isoform 7 (rs1387934803). At age 1 year 4 months, the patient was placed on a low protein diet to reduce cysteine load and accumulation of sulfite and SCC in tissues. At 3 months after introduction of protein restriction, the urine sulfite test became negative and the urine SCC level was decreased. After starting the protein restriction diet, dystonic movement improved, and the patient's course progressed without regression and seizures. Electroencephalogram findings were remarkably improved. This finding demonstrates that the dietary protein restriction suppresses disease progression in mild cases of MoCD and suggests the effectiveness of dietary therapy in MoCD.

Metabolic disorder of amino acids, fatty acids and purines reflects the decreases in oocyte quality and potential in sows

Oocyte quality is closely related to female fertility. Nevertheless, core nutritional metabolites influencing oocyte quality are unclear. Herein, comprehensive metabolomics analysis of follicular fluid, serum, and urine from low reproductive performance (LRP) and normal reproductive performance (NRP) sows was conducted. Twenty-seven, fourteen and sixteen metabolites (involved in metabolism of amino acids, fatty acids, purine and pyrimidine) were altered in follicular fluid, serum and urine, respectively, in LRP compared with NRP sows, and could decrease oocyte quality and developmental potential, ultimately leading to low fertility. Deoxyinosine, guanidine acetate, thymidine, 5,6-epoxy-eicosatrienoic acid, carnosine, docosahexaenoic acid and carbamoyl phosphate in follicular fluid, cysteine, carnitine, serotonin, hypoxanthine, valine and arginine in serum, as well as carnitine, phenyl glycine, N-acetyl glutamine, propionyl carnitine and choline in urine could be selected as diagnostic markers to indicate oocyte quality. Consistent with metabolomics data, we confirmed changes in concentrations of fatty acids and amino acids in follicular fluid. Targeting purine metabolism, elevating levels of deoxyinosine in in-vitro maturation medium of porcine oocyte significantly promoted the blastocyst rate. Collectively, this study provided new information of potential targets for predicting oocyte quality and developmental potential, and may help with strategies for early diagnosis or therapeutic/dietary intervention in improving reproductive outcomes.

Liquid Chromatography-Mass Spectrometry-Based Metabolomics of Nonhuman Primates after 4 Gy Total Body Radiation Exposure: Global Effects and Targeted Panels.

Rapid assessment of radiation signatures in noninvasive biofluids may aid in assigning proper medical treatments for acute radiation syndrome (ARS) and delegating limited resources after a nuclear disaster. Metabolomic platforms allow for rapid screening of biofluid signatures and show promise in differentiating radiation quality and time postexposure. Here, we use global metabolomics to differentiate temporal effects (1-60 d) found in nonhuman primate (NHP) urine and serum small molecule signatures after a 4 Gy total body irradiation. Random Forests analysis differentially classifies biofluid signatures according to days post 4 Gy exposure. Eight compounds involved in protein metabolism, fatty acid β oxidation, DNA base deamination, and general energy metabolism were identified in each urine and serum sample and validated through tandem MS. The greatest perturbations were seen at 1 d in urine and 1-21 d in serum. Furthermore, we developed a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM) method to quantify a six compound panel (hypoxanthine, carnitine, acetylcarnitine, proline, taurine, and citrulline) identified in a previous training cohort at 7 d after a 4 Gy exposure. The highest sensitivity and specificity for classifying exposure at 7 d after a 4 Gy exposure included carnitine and acetylcarnitine in urine and taurine, carnitine, and hypoxanthine in serum. Receiver operator characteristic (ROC) curve analysis using combined compounds show excellent sensitivity and specificity in urine (area under the curve [AUC] = 0.99) and serum (AUC = 0.95). These results highlight the utility of MS platforms to differentiate time postexposure and acquire reliable quantitative biomarker panels for classifying exposed individuals.

Serum and brain purine levels in an experimental systemic infection of mice by Cryptococcus neoformans: Purinergic immunomodulatory effects

The aim of this study was to evaluate the purine levels in serum and brains of mice experimentally infected by Cryptococcus neoformans. Twenty-four mice were divided into the following groups: a control group (n = 12; Group A) and an infection group with animals that were infected (n = 12; Group B) with a 0.3-mL intraperitoneal injection containing 1.7 × 107C. neoformans cells. Blood and brains were collected on days 20 (n = 6 per group) and 50 (n = 6 per group) post-infection (PI). Histopathology and lung and brain cultures were performed to confirm fungal infection and tissue injuries. The levels of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine (ADO), inosine (INO), hypoxanthine (HYPO), xanthine (XAN) and uric acid (UA) in brains and serum were measured by high-performance liquid chromatography (HPLC) analysis. At both time points, histopathology analysis revealed inflammatory infiltrates in the brains and lungs of infected mice; clinical signs, such as piloerection and clinical respiratory distress, were also observed. ATP levels were significantly increased on days 20 and 50 PI (P < 0.01) in brains and serum, while brain ADO levels were increased on day 20 PI; brain and serum ADO levels were decreased on day 50 PI. Levels of ADP and AMP did not differ between groups (P > 0.05). Serum levels of INO of infected mice increased only on day 50 PI (P < 0.05). HYPO levels were reduced in the brains of infected animals at both experimental time points and were decreased in serum at day 50 PI (P < 0.05). XAN levels increased in infected mice only in serum on day 50 PI (P < 0.05). The endogenous anti-oxidant uric acid was significantly increased in brain (days 20 and 50 PI) and decreased in serum. It is possible that C. neoformans infection in mice leads to a high ATP/ADO ratio that may improve the brain pro-inflammatory response during both periods, while high ATP levels in serum act as a systemic signal to improve the immune response. Moreover, the anti-oxidant uric acid may increase in the brain to protect inflamed tissue from oxidative stress.

Real-time monitoring of plasmon-induced proton transfer of hypoxanthine in serum.

Intramolecular proton transfer of hypoxanthine, induced by application of a laser on the surface of a bare noble nanomaterial, was monitored in real time using surface-enhanced Raman spectroscopy (SERS). This monitoring demonstrated the dependence of the reaction on the identity of the nanomaterial and on the laser power density. The results pave the way for monitoring the proton transfer reaction in various relevant fields. In addition, we observed the presence of the proton transfer phenomenon of hypoxanthine in serum, providing a way to avoid the effect of proton transfer and hence achieve more reliable spectra of sera for clinical diagnosis.

Simultaneous Determination of Uric Acid, Xanthine and Hypoxanthine in Human Plasma and Serum by HPLC–UV: Uric Acid Metabolism Tracking

A simple reversed phase HPLC method with UV detection in isocratic conditions was developed and validated for the simultaneous determination of hypoxanthine, xanthine and uric acid levels in human plasma and serum. One analysis run takes 6.5 min including a short organic mobile phase gradient for column regeneration. Concentrations of uric acid, xanthine and hypoxanthine in plasma and serum samples were highly comparable. However, hypoxanthine levels were increased in serum compared to plasma samples due to a prolonged time between serum and blood elements separation. The method was validated for linearity, precision, accuracy, sensitivity and robustness in a similar manner to that for pharmacokinetic data and it is appropriate for physiological and pathophysiological levels of all analytes. The stability of stock standard solutions was verified using spectrophotometric analysis in different conditions. The method is simple and robust with a good precision for the measurement of hypoxanthine, xanthine and uric acid in human plasma and serum.

Hypercrosslinked strong cation-exchange polymers for selective extraction of serum purine metabolites associated with gout

In this study, hypercrosslinked strong cation-exchange polymer resins (HXLPP-SCX) were synthesized and employed as selective sorbents for the solid-phase extraction (SPE) of basic purine metabolites associated with gout. The HXLPP-SCX material was prepared based on hypercrosslinking reactions and sulfonated with concentrated H2SO4. This synthetic procedure is facile and efficient without using highly toxic reagent. The resulting resins were characterized in the form of monodisperse microspheres (mean diameters of 3‒5μm) with narrow pore size (2.1nm) and relatively high specific surface areas (801m2/g). The polymers also possess high ion-exchange capacity (IEC, 2.22mmol/g) and good adsorption and selectivity performances for basic compounds. The resins used as SPE sorbents permit the selective enrichment of three pivotal purine metabolites (hypoxanthine, xanthine and inosine) in human serum followed by HPLC analysis. Method validation including linearity range, sensitivity, accuracy and reproducibility were evaluated. This method was exemplarily applied in the analysis of serum purines in gout patients and healthy controls. The present results demonstrate a promising potential of this HXLPP-SCX material for the clinical sample pretreatment.

Simultaneous determination of uric acid, xanthine, hypoxanthine and caffeine in human blood serum and urine samples using electrochemically reduced graphene oxide modified electrode

This paper describes the fabrication of graphene on glassy carbon electrode (GCE) by electrochemical reduction of graphene oxide (GO) attached through 1,6-hexadiamine on GCE and the simultaneous determination of structurally similar four purine derivatives using the resultant electrochemically reduced GO (ERGO) modified electrode. The electrocatalytic activity of ERGO was investigated toward the oxidation of four important purine derivatives, uric acid (UA), xanthine (XN), hypoxanthine (HXN) and caffeine (CAF) at physiological pH. The modified electrode not only enhanced the oxidation currents of the four purine derivatives but also shifted their oxidation potentials toward less positive potentials in contrast to bare GCE. Further, it successfully separates the voltammetric signals of the four purine derivatives in a mixture and hence used for the simultaneous determination of them. Selective determination of one purine derivative in the presence of low concentrations other three purine derivatives was also realized at the present modified electrode. Using differential pulse voltammetry, detection limits of 8.8×10−8M, 1.1×10−7M, 3.2×10−7M and 4.3×10−7M were obtained for UA, XN, HXN and CAF, respectively. The practical application of the modified electrode was demonstrated by simultaneously determining the concentrations of UA, XN, HXN and CAF in human blood plasma and urine samples.

Simultaneous LC–UV–MS–MS Analysis of Nine Pivotal Metabolites in Human Serum: Application to Studies of Impaired Glucose Tolerance

A rapid, sensitive, and selective LC–UV–MS–MS method for simultaneous quantification of uric acid, creatinine, xanthine, creatine, hypoxanthine, adenosine, inosine, thymidine, and uridine in serum from patients with impaired glucose tolerance (IGT) has been developed and validated. After precipitation of protein in the serum with methanol samples were evaporated with a stream of nitrogen then reconstituted with aqueous ammonia (0.002 mol L−1). Chromatographic separation was performed on a C18 column with methanol–buffer (8 mmol L−1 ammonium acetate adjusted to pH 6.1 with glacial acetic acid) as mobile phase at a flow rate of 0.8 mL min−1. UV and MS–MS detection were used to quantify the metabolites on the basis of different concentrations and detector response. Linearity was excellent, with r 2 no 85, <10, and <10%, respectively. This reliable bioanalytical method enables evaluation of the levels of purines and pyrimidines in serum for IGT studies and diagnosis.

Cell-free plasma DNA and purine nucleotide degradation markers following weightlifting exercise

The present study aimed to investigate the acute effects of a single bout of high-intensive strength training on the production of cell-free plasma DNA (cf-DNA), as well as on the degradation of purine nucleotides as assessed by the concentration of xanthine (XA) and hypoxanthine (HX) in urine and serum. Twelve trained weightlifters performed six sets of six lifting exercises with 90-95% of the one repetition maximum. Blood samples and urine were obtained 1 h before training, immediately after finishing the exercise session and following 2 h of recovery. Cf-DNA, HX, and XA (in serum) significantly increased (P < 0.05-P < 0.001) immediately after heavy lifting exercise when compared with baseline levels, and significantly decreased (P < 0.05-P < 0.001) after 2 h of recovery. These results indicate that, cf-DNA and oxypurines might be relevant biomarkers for cellular damage, mechanical, energetic, and/or ischemic stress in context with exercise.

Quantification of uric acid, xanthine and hypoxanthine in human serum by HPLC for pharmacodynamic studies

A simple HPLC method was developed and validated for the determination of uric acid (UA), xanthine (X) and hypoxanthine (HX) concentrations in human serum to support pharmacodynamic (PD) studies of a novel xanthine oxidase inhibitor during its clinical development. Serum proteins were removed by ultrafiltration. The hydrophilic analytes and the I.S. were eluted by 100% aqueous phosphate buffer mobile phase. The hydrophobic matrix components (late peaks) were eluted with a step gradient of a higher organic mobile phase. Validation on linearity, sensitivity, precision, accuracy, stability, and robustness of the method for PD biomarkers (UA, X, and HX) was carried out in a similar manner to that for pharmacokinetic (PK) data where applicable. Issues of selectivity for endogenous biomarker analytes and individual concentration variations were addressed during method validation. Standards were prepared in analyte-free phosphate buffer. Quality control samples were prepared in control serum from individuals not dosed with the xanthine oxidase inhibitor. The method was simple and robust with good accuracy and precision for the measurement of serum UA, X, and HX concentrations.

A high-performance liquid chromatographic method for the determination of hypoxanthine, xanthine, uric acid and allantoin in serum.

A method was developed for the simultaneous determination of hypoxanthine, xanthine, uric acid and allantoin based on isocratic reversed-phase chromatography. This HPLC-method additionally allows the direct determination with UV-detection of inosine-5'-phosphate, uridine, thymine, orotic acid, allopurinol and oxipurinol, besides hypoxanthine, xanthine and uric acid in the same chromatographic run. Allantoin elutes in this system near the void volume and a fraction is collected covering the retention time range for this substance. After hydrolysis allantoin is converted to glyoxylate-2,4-dinitrophenylhydrazone, rechromatographed and detected at 360 nm. The coefficient of variation for this method does not exceed 5.0% for a serum concentration of 0.3 mumol/l hypoxanthine and is not greater than 5.3% for a xanthine concentration of 0.3 mumol/l serum. Recoveries were 90-110% for both hypoxanthine and xanthine. The determination of uric acid had an imprecision and inaccuracy not exceeding 1.45% in the concentration range of 103-568 mumol/l. Due to the more complex procedure required for the determination of allantoin, the coefficient of variation between days was 13.6% for a sample containing 0.8 mumol/l allantoin and the recoveries for this analyte were in the range of 86-93%. Reference ranges (mean +/- SD) determined on 171 serum samples from healthy adults were 12.7 +/- 6.6 mumol/l for hypoxanthine, 3.3 +/- 1.4 mumol/l for xanthine, and 15.7 +/- 7.9 mumol/l for allantoin. No significant age or sex dependence was observed. Uric acid concentrations were 320 +/- 55 mumol/l serum for men and 206 +/- 55 mumol/l for women.

Hyperamylasemia, specific pancreatic enzymes, and hypoxanthine during recovery from diabetic ketoacidosis.

The contribution of the exocrine pancreas to hyperamylasemia in diabetic ketoacidosis was investigated by measuring total amylase, salivary and pancreatic isoamylases, cathodic trypsin-like immunoreactivity, and pancreatic lipase in 12 consecutive patients recovering from diabetic ketoacidosis. Hyperamylasemia was present in six of the patients [50%; expected incidence: 21.1-78.9% (95% confidence limits)]--in five with simultaneously increased activities of all three specific pancreatic enzymes, and in one with only increased salivary isoamylase. The serum concentration of hypoxanthine--an indicator of the cellular energy state--was above normal in all patients at admission. We found no differences in concentrations of hypoxanthine in serum of patients with or without hyperamylasemia or in patients with or without increases in the specific pancreatic enzymes. In none of the patients was the clinical course or the time--concentration curves of the pancreatic enzymes consistent with acute pancreatitis. The pathogenic mechanism leading to hyperamylasemia in diabetic ketoacidosis remains uncertain.

Quantitation of Purines and Pyrimidines in Human Serum by High-Performance Liquid Chromatography

Abstract A high-performance liquid chromatography assay was developed for the purpose of examining xanthine, hypoxanthine, uridine, thymidine, allopurinol, and oxypurinol in serum of patients with advanced carcinoma treated with methorexate and 5-fluorouracil. After the addition of an internal standard, serum samples were extracted of purines and pyrimidines with trichloroacetic acid and neutralized with tert-amine. Aliquots of the extracted serum were injected onto a C18 reverse-phase column and purines and pyrimidines were eluted with a gradient of MeOH/H2O and KH2PO4 solutions. Absorbance was detected with a variable-wavelength UV spectrophotometer at 254 nm and 280 nm. This assay can be readily applied to quantitate baseline and treatment-induced variations in serum purine and pyrimidine levels which may correlate with clinical response and/or toxicity in patients with cancer.

Specific simultaneous assay of hypoxanthine and xanthine in serum by reversed-phase high-performance liquid chromatography using an immobilized xanthine oxidase reactor

Specific simultaneous assay of hypoxanthine and xanthine in serum by reversed-phase high-performance liquid chromatography using an immobilized xanthine oxidase reactor

An isotachophoretic method for the determination of hypoxanthine in serum

Analytical isotachophoresis was used for the determination of hypoxanthine in serum. 5-Fluorouracil was used as the internal standard. The inclusion of spacers in the system greatly improved the separation and quantification. The method can readily be adapted to microscale and thus can be used in pediatric practice. The method is applicable to hypoxanthine concentrations above 10 nmol/liter.

Simultaneous liquid chromatography of 5-fluorouracil, uridine, hypoxanthine, xanthine, uric acid, allopurinol, and oxipurinol in plasma.

A reversed-phase "high-pressure" liquid-chromatographic method is described for simultaneous analysis for 5-fluorouracil, uridine, hypoxanthine, xanthine, uric acid, allopurinol, and oxipurinol. Separation was optimal with phosphate buffer (50 mmol/L, pH 4.60) as eluent. A simple acid extraction procedure yielded quantitative recoveries and permitted adequate separation for interfering peaks. Compounds were identified by their retention times, absorbance ratios, co-elution with standards, and enzymatic shifts. With a computerized integrator we quantitated these compounds in widely varying concentrations with a single injection. The limit of sensitivity was 0.1 mumol/L for the compounds studied. This method was applied to determine mean values for those compounds in normal human plasma. They are (in mumol/L): uric acid 276 (SD 55), hypoxanthine 0.46 (SD 0.21), xanthine 0.40 (SD 0.27), and uridine 4.50 (SD 1.70). Erythrocytes and platelets can continue to release hypoxanthine and xanthine into plasma or serum after a blood specimen has been drawn. We believe this explains the higher values previously reported for hypoxanthine and xanthine in serum.

Quantitative estimation of uric acid, xanthine, and hypoxanthine in plasma using thin-layer chromatography

A rapid method for the quantitative separation and estimation of uric acid, xanthine, and hypoxanthine in plasma or serum using thin-layer chromatography and spectrophotometry is described. The compounds are first separated on 20 × 20 cm layers of ECTEOLA cellulosc 275 μ thick using 0.15 M NaCl as a solvent. Development is accomplished in 60 to 90 min and the R f values are 0.27, 0.34, and 0.46 for uric acid, xanthine, and hypoxanthine, respectively. The resolved compounds are then removed from the plate, eluted in 0.05 M borax and analyzed using the Beckman model DB spectrophotometer with UV equipment. Precision of the method is above ±5% for uric acid and ±1% for xanthine and hypoxanthine in the normal range. Because of the over-all simplicity of the procedure, it is possible to perform about twelve determinations in about two working days.