A Newly Developed Method-Based Xanthine Oxidoreductase Activities in Various Human Liver Diseases.

Studies evaluating xanthine oxidoreductase (XOR) activities in comprehensive liver diseases are scarce, and different etiologies have previously been combined in groups for comparison. To accurately evaluate XOR activities in liver diseases, the plasma XOR activities in etiology-based comprehensive liver diseases were measured using a novel, sensitive, and accurate assay that is a combination of liquid chromatography and triple quadrupole mass spectrometry to detect [13C2, 15N2]uric acid using [13C2, 15N2]xanthine as a substrate. We also mainly evaluated the association between the plasma XOR activities and parameters of liver tests, purine metabolism-associated markers, oxidative stress markers, and an inflammation marker. In total, 329 patients and 32 controls were enrolled in our study. Plasma XOR activities were generally increased in liver diseases, especially in the active phase, such as in patients with hepatitis C virus RNA positivity, those with abnormal alanine transaminase (ALT) levels in autoimmune liver diseases, and uncured hepatocellular carcinoma patients. Plasma XOR activities were numerically highest in patients with acute hepatitis B. Plasma XOR activities were closely correlated with parameters of liver tests, especially serum ALT levels, regardless of etiology and plasma xanthine levels. Our results indicated that plasma XOR activity might reflect the active phase in various liver diseases.

Systemic Endothelial Function, Plasma Xanthine Oxidoreductase Activity, and Blood Pressure Variability in Patients with Stable Coronary Artery Disease.

Background and Objectives: Although previous studies showed that an activity of xanthine oxidoreductase (XOR), a rate-limiting enzyme in purine metabolism, beyond the serum uric acid level, was associated with the development of coronary artery disease (CAD), the underlying mechanisms are unclear. Because endothelial dysfunction and a greater blood pressure (BP) variability may play a role, we investigated the relations among the endothelial function, XOR, and BP variability. Materials and Methods: This was a post-hoc study using pooled data of patients with a stable CAD from two prospective investigations, in which the systemic endothelial function was assessed with the reactive hyperemia index (RHI) and the XOR activity was measured. The BP variability was evaluated using BP measurements during the three- and four-day hospitalization. Results: A total of 106 patients with a stable CAD undergoing a percutaneous coronary intervention were included. Of the 106 patients, 46 (43.4%) had a systemic endothelial dysfunction (RHI < 1.67). The multivariable analysis identified a higher body mass index (BMI), female gender, and diabetes as factors associated with an endothelial dysfunction. A higher BMI was also related to an elevated XOR activity, in addition to current smoking. No significant correlation was observed between the RHI and XOR activity. Similarly, the in-hospital BP variability was associated with neither the endothelial function nor XOR. Conclusions: Among patients with a stable CAD, several factors were identified as being associated with a systemic endothelial dysfunction or an elevated XOR activity. However, no direct relations between the endothelial function, XOR, and BP variability were found.

Increased plasma XOR activity induced by NAFLD/NASH and its possible involvement in vascular neointimal proliferation.

Xanthine oxidoreductase (XOR) is an enzyme that catalyzes hypoxanthine to xanthine and xanthine to uric acid, respectively. However, the underlying mechanisms of increased plasma XOR and its pathological roles in systemic diseases, such as atherosclerosis, are not fully understood. In this study, we found that changes in plasma XOR activity after bariatric surgery closely associated with those in liver enzymes, but not with those in BMI. In a mouse model of nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH), plasma XOR activity markedly increased. Besides, purine catabolism was accelerated in the plasma per se of NASH mice and human patients with high XOR activity. In our NASH mice, we observed an increased vascular neointima formation consisting of dedifferentiated vascular smooth muscle cells (SMCs), which was significantly attenuated by topiroxostat, a selective XOR inhibitor. In vitro, human liver S9-derived XOR promoted proliferation of SMCs with phenotypic modulation and induced ROS production by catabolizing hypoxanthine released from human endothelial cells. Collectively, the results from human and mouse models suggest that increased plasma XOR activity, mainly explained by excess hepatic leakage, was involved in the pathogenesis of vascular injury, especially in NAFLD/NASH conditions.

Independent association of plasma xanthine oxidoreductase activity with hypertension in nondiabetic subjects not using medication.

Xanthine oxidoreductase (XOR), a rate-limiting and catalyzing enzyme of uric acid formation in purine metabolism, is involved in reactive oxygen species generation. Plasma XOR activity has been shown to be a novel metabolic biomarker related to obesity, liver dysfunction, hyperuricemia, dyslipidemia, and insulin resistance. However, the association between plasma XOR activity and hypertension has not been fully elucidated. We investigated the association of hypertension with plasma XOR activity in 271 nondiabetic subjects (male/female: 119/152) who had not taken any medications in the Tanno-Sobetsu Study, a population-based cohort. Males had higher plasma XOR activity than females. Plasma XOR activity was positively correlated with mean arterial pressure (r = 0.128, P = 0.036). When the subjects were divided by the presence and absence of hypertension into an HT group (male/female: 34/40) and a non-HT group (male/female: 85/112), plasma XOR activity in the HT group was significantly higher than that in the non-HT group (median: 39 vs. 28 pmol/h/mL, P = 0.028). There was no significant difference in uric acid levels between the two groups. Multivariable logistic regression analysis showed that plasma XOR activity (odds ratio: 1.091 [95% confidence interval: 1.023-1.177] per 10 pmol/h/mL, P = 0.007) was an independent determinant of the risk for hypertension after adjustment for age, sex, current smoking and alcohol consumption, estimated glomerular filtration rate, brain natriuretic peptide, and insulin resistance index. The interaction of sex with plasma XOR activity was not significant for the risk of hypertension. In conclusion, plasma XOR activity is independently associated with hypertension in nondiabetic individuals who are not taking any medications.

Greater coronary lipid core plaque assessed by near-infrared spectroscopy intravascular ultrasound in patients with elevated xanthine oxidoreductase: a mechanistic insight.

Elevated serum uric acid level was reportedly associated with greater coronary lipid plaque. Xanthine oxidoreductase (XOR) is a rate-limiting enzyme in purine metabolism and believed to play an important role in coronary atherosclerosis. However, the relation of XOR to coronary lipid plaque and its mechanism are unclear. Patients with stable coronary artery disease undergoing elective percutaneous coronary intervention under near-infrared spectroscopy intravascular ultrasound (NIRS-IVUS) guidance were prospectively enrolled. They were divided into three groups according to serum XOR activities: low, normal, and high. Coronary lipid core plaques in non-target vessels were evaluated by NIRS-IVUS with lipid core burden index (LCBI) and a maximum LCBI in 4 mm (maxLCBI4mm). Systemic endothelial function and inflammation were assessed with reactive hyperemia index (RHI) and high-sensitivity C-reactive protein, neutrophil-to-lymphocyte ratio, and platelet-to-lymphocyte ratio. Of 68 patients, 26, 31, and 11 were classified as low, normal, and high XOR activity groups. LCBI (474.4 ± 171.6 vs. 347.4 ± 181.6 vs. 294.0 ± 155.9, p = 0.04) and maxLCBI4mm (102.1 ± 56.5 vs. 65.6 ± 48.5 vs. 55.6 ± 37.8, p = 0.04) were significantly higher in high XOR group than in normal and low XOR groups. Although RHI was significantly correlated with body mass index, diabetes, current smoking, and high-density lipoprotein cholesterol, no relation was found between XOR activity and RHI. There were also no relations between XOR activity and C-reactive protein, neutrophil-to-lymphocyte ratio, or platelet-to-lymphocyte ratio. In conclusion, elevated XOR activity was associated with greater coronary lipid core plaque in patients with stable coronary artery disease, without significant relations to systemic endothelial function and inflammation.

Differential regulation of hypoxanthine and xanthine by obesity in a general population.

AIMS/INTRODUCTION: Uric acid is synthesized by oxidation of hypoxanthine and xanthine using a catalyzing enzyme, xanthine oxidoreductase (XOR), which can be a source of reactive oxygen species. Plasma XOR activity is a metabolic biomarker associated with obesity, hyperuricemia, liver dysfunction and insulin resistance. However, it has recently been reported that XOR activity in fat tissue is low in humans, unlike in rodents, and that hypoxanthine is secreted from human fat tissue. MATERIALS AND METHODS: The associations of obesity with hypoxanthine, xanthine and plasma XOR activity were investigated in 484 participants (men/women: 224/260) of the Tanno-Sobetsu Study. RESULTS: Levels of hypoxanthine, xanthine and plasma XOR activity were significantly higher in men than in women. In 59 participants with hyperuricemia, 11 (men/women: 11/0) participants were being treated with an XOR inhibitor and had a significantly higher level of xanthine, but not hypoxanthine, than that in participants without treatment. In all of the participants, hypoxanthine concentration in smokers was significantly higher than that in non-smokers. Stepwise and multivariate regression analyses showed that body mass index, smoking habit and xanthine were independent predictors of hypoxanthine after adjustment of age, sex and use of antihyperuricemic drugs. Whereas, alanine transaminase, hypoxanthine and plasma XOR activity were independent predictors for xanthine, and alanine transaminase, triglycerides and xanthine were independent predictors for plasma XOR activity. CONCLUSIONS: The concentration of hypoxanthine, but not that of xanthine, is independently associated with obesity and smoking habit, indicating differential regulation of hypoxanthine and xanthine in a general population.

Insulin Resistance Associated with Plasma Xanthine Oxidoreductase Activity Independent of Visceral Adiposity and Adiponectin Level: MedCity21 Health Examination Registry.

BACKGROUND: Higher levels of uric acid production have been reported in individuals with visceral fat obesity, and obesity is known to enhance xanthine oxidoreductase (XOR) activity, although the precise mechanism remains unclear. We investigated the associations of visceral fat area (VFA), serum adiponectin level, and insulin resistance with plasma XOR activity using our novel highly sensitive assay based on [13C2,15N2] xanthine and liquid chromatography/triple quadrupole mass spectrometry. METHODS: This cross-sectional study included 193 subjects (92 males and 101 females) registered in the MedCity21 health examination registry. Plasma XOR activity, serum adiponectin level, and VFA obtained by computed tomography were measured, and insulin resistance was determined based on the homeostasis model assessment (HOMA-IR) index. RESULTS: The mean values for VFA, log HOMA-IR, and log plasma XOR activity were 76.8 ± 45.8 cm2, 0.14 ± 0.30, and 1.50 ± 0.44 pmol/h/mL, respectively. Multiple regression analysis showed that HOMA-IR was significantly (p=0.020) associated with plasma XOR activity independent of other factors, including VFA and adiponectin level, as well as age, sex, alcohol drinking habit, smoking habit, alanine transaminase, HbA1c, and eGFR. The "sex∗HOMA - IR" interaction was not significant (p=0.020) associated with plasma XOR activity independent of other factors, including VFA and adiponectin level, as well as age, sex, alcohol drinking habit, smoking habit, alanine transaminase, HbA1c, and eGFR. The ". CONCLUSIONS: Our results indicate that insulin resistance is associated with plasma XOR activity and that relationship is independent of visceral adiposity and adiponectin level, suggesting that the development of insulin resistance resulting from increased visceral adiposity and/or reduced serum adiponectin contributes to increased uric acid production by stimulating XOR activity.

Independent links between plasma xanthine oxidoreductase activity and levels of adipokines.

AIMS/INTRODUCTION: Xanthine oxidoreductase (XOR) is a rate-limiting enzyme that catalyzes uric acid formation in the purine metabolism, is involved in an increase in reactive oxygen species. Plasma XOR activity has been shown to be associated with obesity, smoking, liver dysfunction, hyperuricemia, dyslipidemia and insulin resistance. MATERIALS AND METHODS: The association between plasma XOR activity, measured by using liquid chromatography and mass spectrometry, and levels of adipokines, including adiponectin, fatty acid-binding protein 4 (FABP4) and fibroblast growth factor 21 (FGF21), was investigated in 282 participants (male/female: 126/156) of the Tanno-Sobetsu Study who were not taking medication. RESULTS: Women had lower plasma XOR activity than did men. Smoking habit was associated with increased activity. Plasma XOR activity was positively correlated with concentrations of FABP4 (r = 0.192, P < 0.001) and FGF21 (r = 0.208, P < 0.001), homeostasis model assessment of insulin resistance as an index of insulin resistance and uric acid, and was negatively correlated with adiponectin level (r = -0.243, P = 0.001). Multivariate regression analyses showed that levels of adiponectin, FABP4 and FGF21 were independent determinants of plasma XOR activity after adjusting age, sex, uric acid and homeostasis model assessment of insulin resistance. With additional adjustment of smoking habit, the level of FABP4, but not that of adiponectin or FGF21, remained as an independent predictor of plasma XOR activity. CONCLUSIONS: Plasma XOR activity was independently associated with levels of adipokines in a general population of individuals not taking medication.

Relationship between Xanthine Oxidoreductase Redox and Oxidative Stress among Chronic Kidney Disease Patients.

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.

Plasma Xanthine Oxidoreductase Activity as a Novel Biomarker of Metabolic Disorders in a General Population.

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<0.001), waist circumference, blood pressure, and levels of liver enzymes including alanine transaminase (r=0.694, P<0.001), uric acid (r=0.249, P<0.001), triglycerides (r=0.312, P<0.001), hemoglobin A1c, fasting glucose, insulin and HOMA-R (r=0.238, P<0.001) as a marker of insulin resistance and was negatively correlated with high-density lipoprotein cholesterol level. On stepwise and multivariate regression analyses, BMI, smoking and levels of alanine transaminase, uric acid, triglycerides and HOMA-R were independent predictors of plasma XOR activity after adjustment for age and gender. CONCLUSIONS: Plasma XOR activity is a novel biomarker of metabolic disorders in a general population.

Quantitative analytical method for determining the levels of gastric inhibitory polypeptides GIP1-42 and GIP3-42 in human plasma using LC-MS/MS/MS.

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.

Discovery and pharmacological characterization of N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide hydrochloride (anagliptin hydrochloride salt) as a potent and selective DPP-IV inhibitor.

In the course of our program for discovery of novel DPP-IV inhibitors, a series of pyrazolo[1,5-a]pyrimidines were found to be novel DPP-IV inhibitors. We identified N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide hydrochloride (4a) and described its pharmacological profiles.