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Objectives Reactive oxygen species generated by xanthine oxidoreductase (XOR) are associated with the progression of atherosclerosis. However, changes in plasma XOR (pXOR) activity after percutaneous coronary intervention (PCI) for stable coronary artery disease (CAD) remains unknown. Methods Herein, we compared the change in the pXOR activity in patients undergoing PCI with that in patients undergoing coronary angiography (CAG) and further evaluated the relation between changes in pXOR activity and in-hospital and long-term outcomes of patients undergoing PCI. The pXOR activity of 80 consecutive patients who underwent PCI and 25 patients who underwent CAG during the hospitalization was analyzed daily. The percentage changes from baseline regulated time interval was evaluated. Results We found that although pXOR activity decreased after PCI, and remained low until discharge, no significant changes were observed in patients undergoing CAG. Furthermore, among the patients undergoing PCI, those who experienced in-hospital adverse events, had a higher percentage of pXOR reduction 3 days after PCI. There was no association between these changes and long-term events. Conclusions A significant change in pXOR activity was observed in patients undergoing PCI than in patients undergoing CAG, and there seems to be a correlation between the in-hospital outcomes and the percentage reduction from baseline in pXOR activity.

Xanthine oxidoreductase (XOR) catalyzes the oxidation of hypoxanthine to xanthine, and of xanthine to uric acid. XOR also enhances the production of reactive oxygen species and causes endothelial dysfunction. In this study, we evaluated the association of XOR and its substrate with the vascular complications in 94 Japanese inpatients with type 2 diabetes (T2DM). The plasma XOR activity and plasma xanthine levels were positively correlated with the body mass index, aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-GTP, fasting plasma insulin, and the homeostasis model of assessment of insulin resistance (HOMA-IR), and negatively correlated with the high density lipoprotein cholesterol. The plasma XOR activity also showed a positive correlation with the serum triglyceride. Multivariate analyses identified AST, ALT, fasting plasma insulin and HOMA-IR as being independently associated with the plasma XOR activity. The plasma XOR activity negatively correlated with the duration of diabetes, and positively correlated with the coefficient of variation of the R-R interval and sensory nerve conduction velocity. Furthermore, the plasma XOR activity was significantly decreased in patients with coronary artery disease. Thus, the plasma XOR activity might be a surrogate marker for the development of vascular complications, as well as liver dysfunction and insulin resistance, in T2DM.Trial registration: This study is registered at the UMIN Clinical Trials Registry (UMIN000029970; https://www.umin.ac.jp/ctr/index-j.htm). The study was conducted from Nov 15, 2017.

Endothelial dysfunction represents a predominant early feature of diabetes, rendering patients with diabetes prone to renal complications, e.g., proteinuria. Recent studies have indicated a possible role for xanthine oxidase (XO) in the pathogenesis of vascular dysfunctions associated with diabetes. In the present study, we investigated the contribution of XO activation on the progression of diabetic nephropathy in a mouse model using selective XO inhibitors. Male Ins2Akita heterozygous mice were used with wild-type mice as controls. Akita mice were treated with topiroxostat (Topi) or vehicle for 4 wk. Serum uric acid levels were significantly reduced in Akita + Topi mice compared with Akita + vehicle mice. The Akita + Topi group had a significant reduction in urinary albumin excretion compared with the Akita + vehicle group. Mesangial expansion, glomerular collagen type IV deposition, and glomerular endothelial injury (assessed by lectin staining and transmission electron microscopy) were considerably reduced in the Akita + topi group compared with the Akita + vehicle group. Furthermore, glomerular permeability was significantly higher in the Akita + vehicle group compared with the wild-type group. These changes were reduced with the administration of Topi. We conclude that XO inhibitors preserve glomerular endothelial functions and rescue compromised glomerular permeability, suggesting that XO activation plays a vital role in the pathogenesis of diabetic nephropathy.

Background Xanthine oxidoreductase (XOR) is a rate-limiting enzyme that catalyzes the oxidation of hypoxanthine to xanthine, and of xanthine to uric acid. XOR also enhances the production of reactive oxygen species (ROS) and causes endothelial dysfunction. To explore the association of XOR and its substrate with the vascular complications in patients with type 2 diabetes mellitus (hereafter simply, diabetes), we measured the plasma XOR activity and plasma levels of xanthine and hypoxanthine in patients with type 2 diabetes. Materials and Methods Plasma XOR activity and the plasma levels of xanthine and hypoxanthine were measured, and their associations with the clinical parameters and vascular complications were investigated in 94 Japanese inpatients with type 2 diabetes. Results Both the plasma XOR activity and plasma xanthine levels were correlated with the serum uric acid level. The plasma XOR activity was correlated with the plasma xanthine level, but not the plasma hypoxanthine level. The plasma XOR activity and plasma xanthine levels were positively correlated with the body mass index (BMI), serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), serum γ-GTP, fasting plasma insulin, and the homeostasis model of assessment of insulin resistance (HOMA-IR), and negatively correlated with the serum high density lipoprotein cholesterol levels (HDL-C). The plasma XOR activity also showed a positive correlation with the serum triglyceride (TG) levels. Plasma xanthine was positively correlated with the Fib4-index. Multivariate analyses identified AST, ALT, fasting plasma insulin and HOMA-IR as being independently associated with the plasma XOR activity. In addition, a negative correlation was observed between the plasma XOR activity and the duration of diabetes, and positive correlations were observed between the plasma XOR activity and the coefficient of variation of the R-R interval (CVR-R) and sensory nerve conduction velocity (SCV). Furthermore, the plasma XOR activity was found to be significantly decreased in patients with coronary artery disease. Conclusions The plasma XOR activity might be a surrogate marker for the development of vascular complications, as well as liver dysfunction and insulin resistance, in patients with type 2 diabetes

Xanthine oxidoreductase (XOR) inhibitor administration reduces uric acid and reactive oxygen species (ROS) production, and also lowers blood pressure (BP). However, the associations of plasma XOR activity, uric acid level, and oxidative stress levels with BP remain unclear. This cross-sectional study included 156 subjects (68 males, 88 females) registered in the MedCity21 health examination registry without anti-hypertensive or anti-hyperuricemic agent administration. Plasma XOR activity was measured using our highly sensitive novel assay, which is unaffected by uric acid in the sample. BP was also determined, and serum uric acid and derivative of reactive oxygen metabolites (d-ROMs) levels were simultaneously measured. Median plasma XOR activity, serum uric acid, d-ROMs, and mean arterial pressure (MAP) values were 25.7 pmol/h/mL, 5.4 mg/dL, 305 Carr U, and 89.0 mmHg, respectively. Multiple regression analysis showed that plasma XOR activity (β = 0.211, p = 0.019), but not serum uric acid (β = 0.072, p = 0.502), was significantly associated with MAP. In subjects with lower but not higher d-ROMs level, an independent association of plasma XOR activity with MAP was observed (β = 0.428, p = 0.001 and β = 0.019, p = 0.891, respectively; p for interaction = 0.046). XOR may contribute to the pathophysiology of higher BP through ROS but not uric acid production, especially in patients with lower oxidative stress.

The aim of present study was to investigate the association between plasma xanthine oxidoreductase activity, which has gained attention as a novel preventive target of cardiovascular disease, and various physiological parameters and was to determine the effects of habitual exercise on plasma xanthine oxidoreductase activity in middle-aged and older women. In the cross-sectional study, we investigated the association between plasma xanthine oxidoreductase activity and various physiological parameters in 94 middle-aged and older women. In the interventional study, subjects (n = 22) were divided into two groups: exercise (n = 12) or the control group (n = 10), whereby we examined the effect of 12-week aerobic exercise training on plasma xanthine oxidoreductase activity in middle-aged and older women. The cross-sectional study demonstrated that plasma xanthine oxidoreductase activity was significantly associated with various physiological parameters, including visceral fat and daily step counts. In the interventional study, the plasma xanthine oxidoreductase activity significantly decreased after the 12-week aerobic exercise training, its changes were inversely associated with the changes in daily step counts. Our results revealed that the plasma xanthine oxidoreductase activity was associated with visceral fat accumulation and lack of exercise, and it was decreased by the aerobic exercise training.

The aim of this study was to confirm the renoprotective effect of xanthine oxidoreductase (XOR) inhibitor, topiroxostat, compared with another XOR inhibitor, febuxostat, under decreased angiotensin II type 1a (AT1a) receptor expression in the model of renal injury caused by adenine. To evaluate the degree of tubular damage using urinary liver-type fatty acid-binding protein (L-FABP) under decreased AT1a expression, we used AT1a receptor knockdown hetero and human L-FABP chromosomal transgenic (Tg) mice (AT1a+/-L-FABP+/-). Male AT1a+/-L-FABP+/- mice were divided into two groups: the adenine diet group (n = 40) was given a diet containing only 0.2% w/w adenine, and the normal diet group (n = 5) was given a normal diet. When renal dysfunction was confirmed in the adenine diet group 4 weeks after starting the diet, the adenine diet group was further divided into five groups. The adenine diet group (n = 8) was continuously given only the adenine diet. Each group receiving high-dose (3mg/kg) or low-dose (1mg/kg) topiroxostat (Topiroxostat-H group, n = 8, Topiroxostat-L group, n = 8) or febuxostat (Febuxostat-H group, n = 8, Febuxostat-L group, n = 8) was given the adenine diet including the drug for another 4 weeks. The levels of renal XOR, renal dysfunction, urinary L-FABP, tubulointerstitial damage, hypoxia, and oxidative stress were decreased or attenuated after treatment with topiroxostat or febuxostat compared with the adenine diet group. Furthermore, antioxidant capacity was maintained owing to these treatments. In conclusion, topiroxostat and febuxostat attenuated renal damage under decreased AT1a expression in the adenine-induced renal injury model.

Oxidative stress due to purine degradation is associated with the development of chronic heart failure (CHF). Xanthine oxidoreductase (XOR) is a rate-limiting enzyme of purine degradation that plays a key role in uric acid (UA) production with a resultant increase in reactive oxygen species. However, the relationship between plasma XOR activity and CHF severity and clinical outcome remains unclear.We measured XOR activity in 440 patients with CHF and 44 control subjects. Abnormally high and low XOR activities were identified based on the results for 95% of the control subjects (high and low XOR activities ≥120 and33pmol/100μL/h, respectively). The prevalence rates of high and low XOR activities increased with advancing New York Heart Association functional class. There were 158 cardiac events during a median follow-up period of 1034days. Multivariate Cox proportional hazard regression analysis showed that both high and low XOR activities were significantly associated with cardiac events in patients with CHF after adjustment for confounding risk factors including serum UA and loop diuretic use. Kaplan-Meier analysis revealed that the cardiac event rate was significantly higher in patients with either high or low XOR activity. The net reclassification index was significantly improved by adding XOR activity to the basic risk factors.We provide the first evidence of an association of plasma XOR activity with CHF severity and clinical outcome. Plasma XOR activity could be used to identify high-risk CHF patients and could be a therapeutic target for XOR inhibitors.

Associations between elevated plasma xanthine oxidoreductase (XOR) activity and various pathologies have been widely reported. However, it has been difficult to accurately measure human plasma XOR activity because the XOR activity of humans is lower than that of animals such as mouse. We developed a highly sensitive assay for XOR activity utilizing a combination of [13C2,15N2] xanthine and liquid chromatography/triple quadrupole mass spectrometry. In the present study, we established and validated a novel human plasma XOR activity assay utilizing this technique. The calibration curve of [13C2,15N2]uric acid showed linearity over the range of 4-4000nM (r2>0.995) with a lower limit of quantitation of 4nM which corresponds to an XOR activity of 6.67pmol/h/mL plasma. Intra- and inter-assay coefficients of variation of pooled human plasma XOR activity were 6.5% and 9.1%, respectively. Plasma XOR activities of 20 healthy volunteers ranged from 32.8 to 227pmol/h/mL (mean±SD=89.1±55.1, n=20), which correlated with alanine transaminase (r=0.827), aspartate transaminase (r=0.487), and uric acid (r=0.502). The established assay is expected to be useful for investigating the function of XOR and the effect of its inhibitors in various diseases.

Xanthine oxidoreductase (XOR), which is mainly produced by the liver, has been reported to be associated with insulin resistance in addition to disorder of uric acid metabolism, which is common in obese people. Moreover, nonalcoholic fatty liver disease (NAFLD) is known to trigger insulin resistance, but the mechanism has not been sufficiently elucidated. In this study, we used FibroScan equipped with controlled attenuation parameter (CAP) estimation to measure intrahepatic fat accumulation and to evaluate the association of insulin resistance with intrahepatic fat accumulation and XOR activity by adjusting for the effects of visceral fat. Of the 200 individuals who had undergone health checkups at our medical center, 142 individuals (59 men) were included in the study after excluding 58 individuals because of incomplete data, habitual alcohol consumption, or ongoing treatment with hypoglycemic and antihyperuricemic agents. NAFLD was defined as a CAP value ≥236 dB/m. Plasma XOR activity was measured by liquid chromatography-triple quadrupole mass spectrometry using [13C2, 15N2] xanthine as a stable isotope substrate developed by Sanwa Kagaku Kenkyusho Co., Ltd. The association of insulin resistance (HOMA-IR ≥2.5) with NAFLD was analyzed by multivariate logistic analysis while adjusting the effects of other factors. NAFLD, as well as visceral fat area, were found to be an independent explanatory factor for insulin resistance. However, when logarithmic values of XOR activity levels (ln-XOR) were added to the analysis model, ln-XOR, but not NAFLD, was a significant explanatory factor for insulin resistance (odds ratio [95% confidence interval]: 4.36 [2.21-8.60]). This finding suggested that XOR of the liver was involved in the association between NAFLD and insulin resistance. In conclusion, NAFLD and insulin resistance are associated via XOR Activity. Disclosure S. Fukumoto: Research Support; Self; Astellas Pharma Inc., Chugai Pharmaceutical Co., Ltd., Kowa Pharmaceutical Europe Co. Ltd., Mitsubishi Tanabe Pharma Corporation, Taisho Toyama Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Novo Nordisk Inc., Sanofi K.K. M. Kurajoh: None. T. Nakamura: None. T. Murase: None. Y. Kakutani: None. Y. Yamazaki: None. A. Ochi: None. K. Motoyama: None. T. Morioka: Research Support; Self; Eli Lilly and Company, Merck & Co., Inc., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Sanofi K.K., Takeda Pharmaceutical Company Limited. Speaker's Bureau; Self; Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk Inc., Roche Diagnostics K.K., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited. Other Relationship; Self; Ono Pharmaceutical Co., Ltd. T. Shoji: Research Support; Self; Bayer Yakuhin, Ltd., Chugai Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd. Speaker's Bureau; Self; Kissei Pharmaceutical Co., Ltd., Kowa, Kyowa Hakko Kirin Co., Ltd. M. Emoto: Research Support; Self; Ono Pharmaceutical Co., Ltd., Sumitomo Dainippon Pharma Co., Ltd. Speaker's Bureau; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Eli Lilly and Company, Kowa Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Taisho Toyama Pharmaceutical CO., Ltd., Takeda Pharmaceutical Company Limited. M. Inaba: None. Funding Japan Society for the Promotion of Science

Background Long-acting glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are widely used for treatment of type 2 diabetes (T2DM) in the United States, the European Union, and Japan. In our previous work, we designed and characterized a novel GLP-1 RA, SKL-18287. This RA consists of only natural L-amino acids, and is believed to exist in an oligomer form in systemic circulation. This unique feature may allow high biological stability and a long-lasting glucose lowering effect in T2DM treatment. In the present study, we investigated the pharmacokinetic properties of SKL-18287 in rats, monkeys, and mini-pigs. Tissue distributions of radioactivity were also studied in rats after subcutaneous administration of [3H]-SKL-18287. Methods Plasma concentrations of SKL-18287 were measured by LC-MS/MS after intravenous and subcutaneous administration of SKL-18287 in rats, monkeys, and mini-pigs. Pharmacokinetic parameters were then calculated and compared among these animal species. Tissue concentrations of radioactivity were determined by liquid scintillation counting following sample combustion, after subcutaneous administration of [3H]-SKL-18287 to rats. Results SKL-18287 showed an extended half-life of over 5 h, with good subcutaneous bioavailability, in all animal species. Prediction of the pharmacokinetic profiles of SKL-18287 in humans using an animal scale-up approach revealed an SKL-18287 half-life of 14.8 h. The radioactivity concentration in the pancreas, a target tissue of GLP-1RA, was relatively higher than in other tissues, until 12 h after [3H]-SKL-18287 administration. Conclusion SKL-18287 might be sufficient to maintain an effective concentration for a once-daily treatment for T2DM, and is a unique GLP-1 RA with a pancreas-selective feature.

The aim of the present study was to reveal the effect of a xanthine oxidoreductase (XOR) inhibitor, topiroxostat (Top), compared with another inhibitor, febuxostat (Feb), in an adenine-induced renal injury model. We used human liver-type fatty acid-binding protein (L-FABP) chromosomal transgenic mice, and urinary L-FABP, a biomarker of tubulointerstitial damage, was used to evaluate tubulointerstitial damage. Male transgenic mice ( n = 24) were fed a 0.2% (wt/wt) adenine-containing diet. Two weeks after the start of this diet, renal dysfunction was confirmed, and the mice were divided into the following four groups: the adenine group was given only the diet containing adenine, and the Feb, high-dose Top (Top-H), and low-dose Top (Top-L) groups were given diets containing Feb (3 mg/kg), Top-H (3 mg/kg), and Top-L (1 mg/kg) in addition to adenine for another 2 wk. After withdrawal of the adenine diet, each medication was continued for 2 wk. Serum creatinine levels, the degree of macrophage infiltration, tubulointerstitial damage, renal fibrosis, urinary 15-F2t-isoprostane levels, and renal XOR activity were significantly attenuated in the kidneys of the Feb, Top-L, and Top-H groups compared with the adenine group. Serum creatinine levels in the Top-L and Top-H groups as well as renal XOR in the Top-H group were significantly lower than those in the Feb group. Urinary excretion of L-FABP in both the Top-H and Top-L groups was significantly lower than in the adenine and Feb groups. In conclusion, Top attenuated renal damage in an adenine-induced renal injury model.

Topiroxostat, a xanthine oxidoreductase (XOR) inhibitor, has been shown to decrease the urinary albumin-to-creatinine ratio compared with placebo in hyperuricemic patients with stage 3 chronic kidney disease. Thus, we aimed to ascertain the albuminuria-lowering effect of topiroxostat in diabetic mouse. Db/db mice were fed standard diets with or without topiroxostat (0.1, 0.3, 1, and 3mg/kg/day) and febuxostat (0.1, 0.3, and 1mg/kg/day) for four weeks. Urinary albumin and purine bodies levels, XOR activities, and drug concentrations in the liver, kidney, and plasma were measured. Moreover, the XOR inhibitory activity of each XOR inhibitor was evaluated with or without an exogenous protein in vitro. Topiroxostat decreased dose-dependently the urinary albumin excretion, but febuxostat did not show such a tendency. Treatment with topiroxostat inhibited plasma XOR activity with dose-dependent increase in plasma purine levels, which was not observed by febuxostat. Pharmacokinetic/pharmacodynamic analysis revealed that topiroxostat and febuxostat concentration in each tissue showed a good correlation with both the hypouricemic effect and plasma drug concentration, whereas the change in albuminuria correlated neither with the change in uric acid nor with drug concentration in plasma. However, the change in urinary albumin and plasma XOR activity showed good correlation in topiroxostat group. The 50% inhibitory concentration (IC50 value) of febuxostat against plasma XOR in vitro was 12-fold higher than that of topiroxostat, and increased by approximately 13-fold by interfering with an exogenous protein. Topiroxostat caused reduced urinary albumin excretion, in which potent inhibition of the plasma XOR activity might be involved.

In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [(13) C2 ,(15) N2 ]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [(13) C2 ,(15) N2 ]xanthine as a substrate, [(13) C2 ,(15) N2 ]UA as an analytical standard, and [(13) C3 ,(15) N3 ]UA as an internal standard. The [(13) C2 ,(15) N2 ]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R(2) = 0.998, weighting of 1/x(2) ) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial-diluted mouse plasma was measured. Thereby, the XOR activity of the 1024-fold-diluted mouse plasma was 4.49 ± 0.44 pmol/100 μL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high-sensitivity measurements required for XOR activity analysis on various organs or human plasma.

Studies of pathological mechanisms and XOR inhibitor characterization, such as allopurinol, febuxostat, and topiroxostat, require accurate and sensitive measurements of XOR activity. However, the established assays have some disadvantages such as susceptibility to endogenous substances such as uric acid (UA), xanthine, or hypoxanthine. Here, we aimed to develop a novel XOR activity assay utilizing a combination of high-performance liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) for tissues such as the liver, kidney, and plasma. Stable isotope-labeled [(15)N2]-xanthine was utilized as substrate and the production of [(15)N2]-uric acid was determined. [(15)N2]-UA production by XOR was dependent on the amounts of [(15)N2]-xanthine and enzyme and the time of reaction. Because high concentrations of endogenous xanthine and hypoxanthine affect XOR activities, we employed a multi-component analysis using LC/HRMS to improve the accuracy of XOR activity assay. Quantification of [(15)N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [(15)N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.1±0.7, 158±5, 928±25pmol/min/mg of protein, respectively (mean±SD, n=5). Furthermore, we measured the XOR activities in the same samples using the LC/ultraviolet and LC/fluorescence (FL) methods. The level of [(15)N2]-xanthine oxidation by XOR was equal to that of xanthine oxidation and approximately 7.9-8.9 times higher than that of pterin oxidation. We found a good correlation between XOR activities examined using LC/MS assay with [(15)N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [(15)N2]-xanthine and the LC/FL assay with pterin were useful, the former provided information regarding XOR activities that more directly reflected the physiological condition than the latter.

Hypouricemia is a high-risk factor of exercise-induced acute kidney injury (EIAKI) probably through a lack of an antioxidant effect of uric acid. Xanthine oxidoreductase (XOR) is an enzyme that catalyzes the formation of uric acid from hypoxanthine and xanthine, leading to an increase in superoxide and reactive oxygen species. Activation of XOR has been proposed to promote oxidative stress-related tissue injury. We measured plasma XOR activity by a sensitive and accurate assay using a combination of liquid chromatography and triple quadrupole mass spectrometry in subjects with relatively low levels of uric acid (≤4.0 mg/dL) who were recruited from 627 subjects (male/female: 292/335) in the Tanno-Sobetsu Study, a population-based cohort. The numbers of subjects with uric acid ≤4.0 mg/dL, ≤3.0 mg/dL and ≤2.0 mg/dL were 72 (11.5%, male/female: 5/67), 13 (2.1%, all females) and 2 (0.3%, both females), respectively. Plasma XOR activities in 5 male subjects were below the median value of the 292 male subjects. In 12 (17.9%) of the 67 female subjects with uric acid ≤4.0 mg/dL, plasma XOR activities were above the upper quartile value of the 335 female subjects. Eleven of the 12 female subjects with high plasma XOR activity and a low uric acid level had liver dysfunction and/or insulin resistance. In conclusion, unexpected high plasma XOR activities were found in some female subjects with relatively low levels of uric acid. Measurement of plasma XOR activity may help to identify hypouricemic patients with a high risk for EIAKI.

BACKGROUND Xanthine oxidoreductase (XOR) is an enzyme that catalyzes the formation of uric acid from hypoxanthine and xanthine, leading to an increase in superoxide and reactive oxygen species. Activation of XOR promotes oxidative stress-related tissue injury. We investigated the associations between metabolic parameters and plasma XOR activity measured by a sensitive and accurate assay using a combination of liquid chromatography and triple quadrupole mass spectrometry to detect [13C2,15N2]-uric acid using [13C2,15N2]-xanthine as a substrate.Methods and Results:A total of 627 Japanese subjects (M/F, 292/335) from the Tanno-Sobetsu Study, a population-based cohort, were recruited. Plasma XOR activity was significantly higher in males than in females, and habitual smoking was associated with elevation of activity. Plasma XOR activity was positively correlated with body mass index (BMI; r=0.323, P

Increased reactive oxygen species (ROS) contributes to the development of endothelial dysfunction, which is involved in coronary artery spasm (CAS). Xanthine oxidoreductase (XOR) plays a pivotal role in producing both uric acid and ROS. However, the association between plasma XOR activity and CAS has not been elucidated. The aim of this study was to investigate whether plasma XOR activity is associated with CAS. We measured XOR activity in 104 patients suspected for CAS, who presented without significant coronary artery stenosis and underwent intracoronary acetylcholine provocation tests. CAS was provoked in 44 patients and they had significantly higher XOR activity as compared with those without CAS. The patients were divided into three groups based on the XOR activity. The prevalence rate of CAS was increased with increasing XOR activity. A multivariate logistic regression analysis showed that the 3rd tertile group exhibited a higher incidence of CAS as compared with the 1st tertile group [odds ratio (OR) 6.9, P = 0.001) and the 2nd tertile group (OR 3.2, P = 0.033) after adjustment for conventional CAS risk factors, respectively. The C index was significantly improved by the addition of XOR activity to the baseline model based on CAS risk factors. Furthermore, the 3rd tertile group had the highest incidence of severe spasm defined as total obstruction, flow-limiting stenosis, diffuse spasm, multivessel spasm, and/or lethal arrhythmia. This is a first report to elucidate the association of plasma XOR activity with CAS. Increased plasma XOR activity is significantly associated with CAS.

Xanthine oxidase (XO), an isoform of xanthine oxidoreductase (XOR), is thought to increase the cardiovascular burden among chronic kidney disease (CKD) patients via oxidative radical production. Plasma XOR redox, which is characterized by the ratio of XO to total XOR, changes under different oxidative conditions associated with kidney dysfunction. However, the relationship between plasma XOR redox and oxidative stress (OS) is unclear. Thus, we aimed to clarify whether OS is related to XOR redox. We used the redox state of human serum albumin (HSA) as a marker to investigate the status of OS in CKD patients. HSA is composed of human mercaptoalbumin (HMA), which possesses not oxidized cysteine residues, reversibly oxidized human nonmercaptoalbumin-1 (HNA-1), and strongly oxidized human nonmercaptoalbumin-2 (HNA-2). The subjects included 13 nondialysis patients (7 males and 6 females) with varying degrees of kidney function. We found that ƒ(HMA) was negatively (R=−0.692, P=0.0071) and ƒ(HNA-1) was positively (R=0.703, P=0.0058) correlated with plasma XO/XOR. ƒ(HNA-2) showed no correlation with XO/XOR (R=0.146, P=0.6412), indicating that plasma XOR redox is not related to the irreversible oxidation of HSA. In conclusion, plasma XOR redox is closely related to HSA redox, particularly reversible oxidation of HSA.

Aim Combination therapy with sofosbuvir and ribavirin (SOF/RBV) has been recently available for chronic hepatitis C patients with genotype 2 (CHG2) in Japan. The domestic phase III clinical trial showed a high antiviral effect with a relatively safe adverse event (AE) profile. Our aim was to report an important AE detected during treatment. Methods A prospective multi-institutional study of 12-week combination therapy with SOF/RBV for CHG2 was carried out to evaluate efficacy and safety. Results The eligible subjects included 142 patients. Out of 50 assessable patients, 16% of the patients were diagnosed with hyperuricemia. The proportions of subjects with grade 1, grade 3, and grade 4 hyperuricemia were 12, 2, and 2%, respectively. Serum uric acid (UA) levels at week 1 of the therapy (W1) were numerically the highest during therapy in patients with hyperuricemia, and the ratio of W1/baseline serum UA levels was significantly higher than that of post-treatment week 4 or 8/baseline serum UA levels in assessable patients. Serum UA levels at W1 were significantly correlated with body mass index. The difference between serum UA levels at W1 and baseline serum UA levels was significantly correlated with the difference between serum creatinine levels at W1 and baseline serum creatinine levels. Conclusions Elevated serum UA level was a notable AE associated with SOF/RBV therapy for CHG2. However, because of the small number of subjects, the exact frequency of AEs should be re-evaluated with larger cohorts. We need to remember that elevated serum UA level might develop during the therapy, especially at W1.

Xanthine oxidoreductase activity (XOR-a) plays an important role as a pivotal source of reactive oxygen species. In the present study, we investigated factors associated with plasma XOR-a in 163 hemodialysis patients (age 67.3 ± 10.9 years; 89 males and 74 females), using a newly established, highly-sensitive assay based on [13C2,15N2] xanthine and liquid chromatography/triple quadrupole mass spectrometry. Plasma glucose and serum uric acid levels correlated significantly and positively with plasma XOR-a. In multiple regression analyses, the presence of type 2 diabetes mellitus (T2DM) and plasma glucose were associated significantly, independently, and positively with plasma XOR-a. While serum uric acid correlated significantly and positively with plasma XOR-a in hemodialysis patients without T2DM, plasma glucose and serum glycated albumin, a new marker of glycemic control in diabetic hemodialysis patients, correlated significantly and positively with plasma XOR-a in those with T2DM. Multivariate analyses in those with T2DM revealed that plasma glucose and serum glycated albumin were associated significantly and independently with plasma XOR-a, and that serum uric acid was associated significantly and independently with XOR-a in those without T2DM. Our results suggested that glycemic control in hemodialysis patients may be important in regard to a decrease in ROS induced by XOR.

Background and purpose Xanthine oxidoreductase (XOR), which catalyzes purine catabolism, has two interconvertible forms, xanthine dehydrogenase and xanthine oxidase, the latter of which produces superoxide during uric acid (UA) synthesis. An association between plasma XOR activity and cardiovascular and renal outcomes has been previously suggested. We investigated the potential association between cardiac parameters and plasma XOR activity among cardiology patients. Methods and results Plasma XOR activity was measured by [13C2,15N2]xanthine coupled with liquid chromatography/triplequadrupole mass spectrometry. Among 270 patients who were not taking UA-lowering drugs, XOR activity was associated with body mass index (BMI), alanine aminotransferase (ALT), HbA1c and renal function. Although XOR activity was not associated with serum UA overall, patients with chronic kidney disease (CKD), those with higher XOR activity had higher serum UA among patients without CKD. Compared with patients with the lowest XOR activity quartile, those with higher three XOR activity quartiles more frequently had left ventricular hypertrophy. In addition, plasma XOR activity showed a U-shaped association with low left ventricular ejection fraction (LVEF) and increased plasma B-type natriuretic peptide (BNP) levels, and these associations were independent of age, gender, BMI, ALT, HbA1C, serum UA, and CKD stages. Conclusions Among cardiac patients, left ventricular hypertrophy, low LVEF, and increased BNP were significantly associated with plasma XOR activity independent of various confounding factors. Whether pharmaceutical modification of plasma XOR activity might inhibit cardiac remodeling and improve cardiovascular outcome should be investigated in future studies.

The aim of this study was to confirm the renoprotective effect of xanthine oxidoreductase (XOR) inhibitor, topiroxostat, compared with another XOR inhibitor, febuxostat, under decreased angiotensin II type 1

Background: Oxidative stress (OS) is thought to play a role in detrimental events among patients with chronic kidney disease (CKD). Although the mechanism of OS increases in CKD patients is unclear, it has been suggested that increased activity of xanthine oxidase (XO), the superoxide-producing form of xanthine oxidoreductase (XOR), plays a large role in enhancing OS. Therefore, we measured the activities of plasma XOR and XO among CKD patients. Methods: Thirteen CKD patients were enrolled in this study. Their plasma XOR and XO activities were measured using a liquid chromatography-triple quadrupole mass spectroscopy (LC-TQMS) method, and the ratio of XO to XOR (XO/XOR) was calculated. Results: The XOR and XO activities (pmol/h/mL plasma) were 8.30–58.2 and 9.15–40.8, and XO/XOR value was 0.698–1.240. Both XOR and XO showed positive correlation with estimated glomerular filtration rate (eGFR) (R=0.751, P=0.0020 and R=0.773, P=0.0031, respectively). On the other hand, XO/XOR showed negative correlation with eGFR (R=-0.614, P=0.0236). Conclusion: XOR and XO activities are decreased, and XO/XOR ratio is increased, in accordance with renal dysfunction. High XO/XOR ratio in advanced CKD patients might contribute to the elevation of OS.

Gastric inhibitory polypeptide (GIP), an incretin, is an important subject in endocrinology. Some LC-MS assays have been proposed; however, their sensitivities are insufficient for the study of endogenous human incretin. Here, we describe a nanoflow LC hybrid triple quadrupole/linear ion trap MS assay for the simultaneous quantification of GIP1-42 and GIP3-42 from human plasma. We selected the surrogate peptide to avoid oxidative modification, and the endoproteinase Asp-N was selected for the proteolysis of GIP1-42 and GIP3-42. The phenylalanine residue at position 6 in both GIP1-42 and GIP3-42 was substituted with (13)C9,(15)N-labeled phenylalanine, and these substituted GIPs were used as the internal standards. This facilitated accurate and precise quantification because large corrections are possible at all steps of sample pretreatment and ionization efficiency. The lower limit of quantification was 1 pM for GIP1-42 and 10 pM for GIP3-42 by using 200 μL of plasma. Quantification of GIP1-42 and GIP3-42 in plasma from patients with type 2 diabetes was possible using this method, which included protein precipitation, Asp-N proteolysis, solid-phase extraction, nanoflow LC, and positive-ion multiple reaction monitoring cubed (MRM(3)) for GIP1-8, and MRM for GIP3-8 to achieve accurate, precise, and quantitative analysis that can be validated to support large clinical trials.

In this study, we developed a highly sensitive assay for xanthine oxidoreductase (XOR) activity utilizing a combination of [(13) C2 ,(15) N2 ]xanthine and liquid chromatography (LC)/triple quadrupole mass spectrometry (TQMS). In this assay, the amount of [(13) C2 ,(15) N2 ]uric acid (UA) produced by XOR was determined by using LC/TQMS. For this assay, we synthesized [(13) C2 ,(15) N2 ]xanthine as a substrate, [(13) C2 ,(15) N2 ]UA as an analytical standard, and [(13) C3 ,(15) N3 ]UA as an internal standard. The [(13) C2 ,(15) N2 ]UA calibration curve obtained using LC/TQMS under the selected reaction monitoring mode was evaluated, and the results indicated good linearity (R(2) = 0.998, weighting of 1/x(2) ) in the range of 20 to 4000 nM. As a model reaction of less active samples, the XOR activity of serial-diluted mouse plasma was measured. Thereby, the XOR activity of the 1024-fold-diluted mouse plasma was 4.49 ± 0.44 pmol/100 μL/h (mean ± standard deviation, n = 3). This value is comparable to the predicted XOR activity value of healthy human plasma. Hence, this combination method may be used to obtain high-sensitivity measurements required for XOR activity analysis on various organs or human plasma.

Focused structure-activity relationships of isoindoline class DPP-IV inhibitors have led to the discovery of 4b as a highly selective, potent inhibitor of DPP-IV. In vivo studies in Wistar/ST rats showed that 4b was converted into the strongly active metabolite 4l in high yield, resulting in good in vivo efficacy for antihyperglycemic activity.