Plasma Xanthine Oxidase Activity Research Articles

Release of hepatic xanthine oxidase (XO) to the circulation is protective in intravascular hemolytic crisis

Xanthine oxidase (XO) catalyzes the catabolism of hypoxanthine to xanthine and xanthine to uric acid, generating oxidants as a byproduct. Importantly, XO activity is elevated in numerous hemolytic conditions including sickle cell disease (SCD); however, the role of XO in this context has not been elucidated. Whereas long-standing dogma suggests elevated levels of XO in the vascular compartment contribute to vascular pathology via increased oxidant production, herein, we demonstrate, for the first time, that XO has an unexpected protective role during hemolysis. Using an established hemolysis model, we found that intravascular hemin challenge (40 μmol/kg) resulted in a significant increase in hemolysis and an immense (20-fold) elevation in plasma XO activity in Townes sickle cell phenotype (SS) sickle mice compared to controls. Repeating the hemin challenge model in hepatocyte-specific XO knockout mice transplanted with SS bone marrow confirmed the liver as the source of enhanced circulating XO as these mice demonstrated 100% lethality compared to 40% survival in controls. In addition, studies in murine hepatocytes (AML12) revealed hemin mediates upregulation and release of XO to the medium in a toll like receptor 4 (TLR4)-dependent manner. Furthermore, we demonstrate that XO degrades oxyhemoglobin and releases free hemin and iron in a hydrogen peroxide-dependent manner. Additional biochemical studies revealed purified XO binds free hemin to diminish the potential for deleterious hemin-related redox reactions as well as prevents platelet aggregation. In the aggregate, data herein reveals that intravascular hemin challenge induces XO release by hepatocytes through hemin-TLR4 signaling, resulting in an immense elevation of circulating XO. This increased XO activity in the vascular compartment mediates protection from intravascular hemin crisis by binding and potentially degrading hemin at the apical surface of the endothelium where XO is known to be bound and sequestered by endothelial glycosaminoglycans (GAGs).

PS-BPC12-3: PLASMA XANTHINE OXIDASE ACTIVITY AND ITS RELATED FACTORS IN ESSENTIAL HYPERTENSIVE PATIENTS

Objective: Xanthine oxidase (XO) activity has a key role in the development of oxidative stress and progression of cardiovascular diseases. In resistant hypertension, plasma XO activity is related to increased urinary sodium excretion and left ventricular hypertrophy. In this study, plasma XO activity and its related factors were investigated in essential hypertensive patients. Design and method: In 53 essential hypertensive patients (30 men, 64 ± 13 years) without taking anti-hyperuricemic agents, plasma XO activity was measured by using a high-performance liquid chromatography method. 38 healthy volunteers without any specific comorbidities were included as a control cohort. Associations between plasma XO activity and other clinical variables were also investigated in essential hypertensive patients. Results: Plasma XO activity was increased in essential hypertensive patients compared with the healthy cohort (log10XO 1.33 ± 0.47 nmol/L/hr vs 0.98 ± 0.37, p < 0.001). In essential hypertensive patients, plasma XO activity did not associate with age, blood pressure, estimated glomerular filtration and urinary protein excretion. There were weak correlations between plasma XO activity and the levels of serum uric acid and blood glucose (r = 0.28, 0.32, p < 0.05, respectively). Plasma XO activity strongly correlated with body mass index (BMI) and alanine aminotransferase (ALT) (r = 0.46, 082, p < 0.001, respectively), and in multiple regression analysis, BMI and ALT were independent determinants for plasma XO activity (R2 0.57, p < 0.001). Conclusions: In essential hypertension, plasma XO activity was increased accompanying with obesity and liver disfunction, irrespective of blood pressure elevation and renal dysfunction.

Zinc normalizes hepatic lipid handling via modulation of ADA/XO/UA pathway and caspase 3 signaling in highly active antiretroviral therapy-treated Wistar rats

BackgroundAlthough highly active antiretroviral therapy (HAART) is effective in the management of HIV, it has been reported to induce hepatic injury and non-alcoholic fatty liver (NAFLD). However, there is a lack of data on the roles of the adenosine deaminase (ADA)/xanthine oxidase (XO)/uric acid (UA) pathway and caspase 3 signaling in HAART-induced NAFLD. Also, whether or not zinc confers protection against HAART-induced NAFLD is not known. AimThis study evaluated the involvement of the ADA/XO/UA pathway and caspase 3 signaling in HAART-induced hepatic lipid accumulation. It also evaluated the possible protective effect of zinc in HAART-induced hepatic lipid accumulation and injury. MethodsThirty two male Wistar rats (n = 8/group) were assigned into four groups namely; vehicle-treated (p.o), zinc-treated (3 mg/kg/day of elemental zinc; p.o), HAART-treated (a cocktail of 52.9 mg/kg of Efavirenz, 26.48 mg/kg of Lamivudine, and 26.48 mg/kg of Tenofovir; p.o), and HAART + zinc-treated groups. The treatment lasted for 8 weeks. ResultsHAART administration led to increased body weight and hepatic weight, but unaltered hepatic organo-somatic index. HAART exposure also resulted in impaired glucose homeostasis, evidenced by increased fasting blood glucose, hyperinsulinemia, and insulin resistance (IR), increased plasma and hepatic cholesterol and triglycerides, and impaired hepatic function as depicted by elevated hepatic injury markers and reduced glycogen synthase activity and glycogen content. These findings were accompanied by increased plasma and hepatic ADA and XO activities, UA and malondialdehyde levels, inflammatory markers, and caspase 3 activities. However, HAART suppressed plasma and hepatic antioxidant defenses. Furthermore, HAART distorted hepatic histoarchitecture and reduced hepatic sinusoidal diameter. Co-administration of zinc with HAART normalized HAART-induced alterations. ConclusionsThese findings showed that downregulation of the ADA/XO/UA pathway and caspase 3 signalings may rescue the liver from HAART-induced lipid accumulation and injury.

THE ACTIVITY OF XANTHINE OXIDASE IN PLASMA IN PATIENTS WITH TYPE 2 DIABETES MELLITUS: THE RELATIONSHIP BETWEEN HYPERURICEMIA, INSULIN RESISTANCE, AND LIVER DYSFUNCTION

We aimed to investigate the clinical implication of plasma xanthine oxidase (XO) activity in patients with type 2 diabetes mellitus (DM). Materials and methods. 127 patients were examined for type 2 DM (72 female and 55 male). Serum biochemical variables include aspartate aminotransferase (AST), alanine aminotransferase (ALT), fasting (FPG) and postprandial (PPG) plasma glucose, glycohemoglobin A1c (HbA1c), total cholesterol, triglyceride, high‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol, creatinine (Cre) and serum urine acid (SUA) was measured with a conventional automated analyzer. The level of fasting insulin (IRI) was determined by the immuno-chemiluminescent method. Homeostasis model assessment of insulin resistance (IR) (HOMA-IR) index was calculated by the formula: HOMA-IR=fasting insulin (μmol/L)×FPG(mmol/L)/22.5. The plasma XO activity was determined by a photometric method. The rate of glomerular filtration (eGFR) was calculated according to CKD-EPI formulas. All statistical analyses were performed using STATISTICA software version 10.0. Results. The plasma lnXO activity was correlated with IRI (rs=0.52; P<0.001) and HOMA‐IR (rs=0.51; P<0.002). In addition, the plasma lnXO activity was correlated with BMI (rs=0.40; P<0.001). In contrast, plasma lnXO activity was not correlated with age (rs=-0.07; P=0.61), HbA1c (rs=-0.11; P=0.41), FPG (rs=-0,4; P=0.74) and PPG (rs=-0.09; P=0.57). lnXO activity and indices of liver dysfunction or parameters closely related to SUA level in patients with type 2 DM: lnALT (rs=0.73; P<0.001), lnAST (rs=0.69; P<0.001), ALT/AST (rs=0.85; P<0.001). The plasma lnXO activity was correlated with Cre (rs=-0.15; P=0.46) and eGFR (rs=0.13; P=0.34). In a series of multiple regression analyses, plasma lnXO activity was used as a dependent variable, and age, BMI, HbA1c, SUA, eGFR, lnAST, lnALT, IRI, and HOMA-IR as explanatory variables were carried out. When used as explanatory variables, the value of lnALT (Model 1) was an independent strong factor for determining plasma XO activity (R2=95.3%; P<0.00001). When used as explanatory variables, the value of lnALT (Model 2) was also an independent strong factor for determining the plasma XO activity (R2=97.2%; P<0.00001). We aimed to investigate the clinical implication of plasma xanthine oxidase (XO) activity in patients with type 2 diabetes mellitus (DM). Materials and methods. 127 patients were examined for type 2 DM (72 female and 55 male). Serum biochemical variables include aspartate aminotransferase (AST), alanine aminotransferase (ALT), fasting (FPG) and postprandial (PPG) plasma glucose, glycohemoglobin A1c (HbA1c), total cholesterol, triglyceride, high‐density lipoprotein cholesterol, low‐density lipoprotein cholesterol, creatinine (Cre) and serum urine acid (SUA) was measured with a conventional automated analyzer. The level of fasting insulin (IRI) was determined by the immuno-chemiluminescent method. Homeostasis model assessment of insulin resistance (IR) (HOMA-IR) index was calculated by the formula: HOMA-IR=fasting insulin (μmol/L)×FPG(mmol/L)/22.5. The plasma XO activity was determined by a photometric method. The rate of glomerular filtration (eGFR) was calculated according to CKD-EPI formulas. All statistical analyses were performed using STATISTICA software version 10.0. Results. The plasma lnXO activity was correlated with IRI (rs=0.52; P<0.001) and HOMA‐IR (rs=0.51; P<0.002). In addition, the plasma lnXO activity was correlated with BMI (rs=0.40; P<0.001). In contrast, plasma lnXO activity was not correlated with age (rs=-0.07; P=0.61), HbA1c (rs=-0.11; P=0.41), FPG (rs=-0,4; P=0.74) and PPG (rs=-0.09; P=0.57). lnXO activity and indices of liver dysfunction or parameters closely related to SUA level in patients with type 2 DM: lnALT (rs=0.73; P<0.001), lnAST (rs=0.69; P<0.001), ALT/AST (rs=0.85; P<0.001). The plasma lnXO activity was correlated with Cre (rs=-0.15; P=0.46) and eGFR (rs=0.13; P=0.34). In a series of multiple regression analyses, plasma lnXO activity was used as a dependent variable, and age, BMI, HbA1c, SUA, eGFR, lnAST, lnALT, IRI, and HOMA-IR as explanatory variables were carried out. When used as explanatory variables, the value of lnALT (Model 1) was an independent strong factor for determining plasma XO activity (R2=95.3%; P<0.00001). When used as explanatory variables, the value of lnALT (Model 2) was also an independent strong factor for determining the plasma XO activity (R2=97.2%; P<0.00001). When used as explanatory variables without parameters for liver functions, the value of HOMA‐IR was an independent factor for determining the plasma XO activity (Model 3) (R2=97.3%; P<0.00001). These data suggest that the values of ALT and HOMA-IR, a clinical marker for hepatic IR, might be independent factors for directly influencing the plasma XO activity in patients with type 2 DM. Conclusions. The value of plasma xanthine oxidase activity was significantly correlated with indices of insulin resistance and liver dysfunction in patients with type 2 diabetes mellitus. The values of alanine aminotransferase and homeostatic model assessment for insulin resistance (HOMA-IR), a clinical marker for hepatic insulin resistance, might be independent factors for directly influencing the plasma xanthine oxidase activity in patients with type 2 diabetes mellitus.

Racial Differences in XO (Xanthine Oxidase) and Mitochondrial DNA Damage-Associated Molecular Patterns in Resistant Hypertension.

We previously reported increased plasma XO (xanthine oxidase) activity in patients with resistant hypertension. Increased XO can cause mitochondrial DNA damage and promote release of fragments called mitochondrial DNA damage-associated molecular patterns (mtDNA DAMPs). Here, we report racial differences in XO activity and mtDNA DAMPs in Black and White adults with resistant hypertension. This retrospective study includes 91 resistant hypertension patients (44% Black, 47% female) with blood pressure >140/90 mm Hg on ≥4 medications and 37 normotensive controls (30% Black, 54% female) with plasma XO activity, mtDNA DAMPs, and magnetic resonance imaging of left ventricular morphology and function. Black-resistant hypertension patients were younger (mean age 52±10 versus 59±10 years; P=0.001), with higher XO activity and left ventricular wall thickness, and worse diastolic dysfunction than White resistant hypertension patients. Urinary sodium excretion (mg/24 hour per kg) was positively related to left ventricular end-diastolic volume (r=0.527, P=0.001) and left ventricular mass (r=0.394, P=0.02) among Black but not White resistant hypertension patients. Patients with resistant hypertension had increased mtDNA DAMPs versus controls (P<0.001), with Black mtDNA DAMPS greater than Whites (P<0.001). Transmission electron microscopy of skeletal muscle biopsies in resistant hypertension patients demonstrates mitochondria cristae lysis, myofibrillar loss, large lipid droplets, and glycogen accumulation. These data warrant a large study to examine the role of XO and mitochondrial mtDNA DAMPs in cardiac remodeling and heart failure in Black adults with resistant hypertension.

PLASMA XANTHINE OXIDASE ACTIVITY IS ASSOCIATED WITH LOW ESTIMATED GLOMERULAR FILTRATION RATE IN NEWLY DIAGNOSED HYPERTENSIVES

Abstract Objective: Elevated circulating uric acid level has emerged as an independent predictor of adverse outcomes in hypertension. Increased reactive oxygen species that could be caused by increased activity of xanthine oxidase (XO) during the production of uric acid has been attributed to hypertension. However, the predictive value of XO on impaired glomerular filtration rate in hypertension is unknown. The present study aims at evaluating the correlations between plasma uric acid concentration and XO activity, and estimated glomerular filtration rate (eGFR) in newly diagnosed non-diabetic hypertensive subjects. Design and method: Cross-sectional analyses were performed in treatment-naïve hypertensives with blood pressure equals to or more than 140/90 mmHg (n = 46) and normotensive with blood pressure equals to or less than 120/80 mmHg (n = 17), who were without history of diabetes mellitus, hyperlipidemia, smoking, thyroid disease and insulin or steroid use. Both groups were matched for age, sex and BMI. Using the modification of diet in renal disease equation, individuals with &lt; median value of 91 ml/min/1.73 m2 were considered to have impaired eGFR. Results: Our results showed that hypertensives had higher uric acid level than the normotensives. Activity of XO was comparable in both the hypertensives and normotensives. Interestingly, hypertensives or normotensives with impaired eGFR had higher (p &lt; 0.0001) XO activity than those with normal eGFR. Using multivariate regression analysis, results showed that XO activity (beta = 0.277; p &lt; 0.0001) was an independent determinant of impaired eGFR in hypertensives only. The receiver operating characteristic curve analysis also showed that plasma XO activity (0.699) was a better predictor of impaired eGFR in hypertensives than plasma uric acid. Conclusions: These results demonstrate that plasma XO activity is significantly associated with low eGFR in hypertensives, independently of plasma uric acid level. The findings could imply that elevated plasma activity of XO may be a stronger risk factor that would promote the development of hypertension-related renal dysfunction.

Local and Systemic Oxidative Stress in Balkan Endemic Nephropathy Is Not Associated with Xanthine Oxidase Activity.

Balkan endemic nephropathy (BEN) represents a chronic tubulointerstitial nephropathy which is followed by the progression of kidney fibrosis to end-stage kidney failure. The critical involvement of poisons in food (aristolochic acid (AA), ochratoxin, and heavy metals) and selenium deficiency are among nutritive factors which contribute to the pathogenesis of BEN, due to reactive oxygen species (ROS) liberation and/or decreased antioxidative defence system. The aim of the study is to distinguish a possible systemic and local origin of ROS through the measurement of xanthine oxidase (XO) activity in urine and plasma, along with the determination of the oxidative changes in lipids and proteins. The study included 50 patients with BEN and 38 control healthy subjects. We noted increased levels of both thiobarbituric acid-reactive substances (TBARS) and advanced oxidation protein products (AOPPs) in the plasma of patients with BEN, compared to the control group (p < 0.001). The urinary levels of AOPPs were higher in patients with BEN in comparison to the control (p < 0.001). The specific activity of XO was significantly lower in plasma and urine in BEN samples, compared to controls (p < 0.005). Based on these results, we hypothesize that XO might not be considered a direct systemic or local contributor to ROS production in BEN, most probably because of the diminished kidney functional tissue mass and/or AA-induced changes in purine nucleotide conformation. The increased AOPP and TBARS level in both plasma and urine in BEN may predict ROS systemic liberation with toxic local effects.

Xanthine Oxidase Activity in Patients with Sickle Cell Anaemia Presenting with Vaso-Occlusive Crises in University College Hospital, Ibadan

Recurrent bone pain or vaso-occlusive crisis (VOC) constitutes a major presentation in patients with sickle cell anaemia. This is partly attributable to vasospasm, oxidative stress and inflammation. Xanthine oxidase is released during VOC and it had been implicated in the pathogenesis of bone pain crisis in patients with sickle cell anaemia. This study was aimed at comparing the plasma levels of xanthine oxidase activity in adult patients with Sickle cell anaemia (SCA), during bone pain crisis (BPC) and steady state and correlating it with the severity of bone pain crisis. Thirty adult sickle cell anemia patients with BPC and 30 in steady state attending Haematology clinic at the University College Hospital, Ibadan, Nigeria and 30 normal haemoglobin A, age- and sex-matched controls were enrolled for this study. Severity of BPC was assessed with total summary pain score. Haematological parameters were determined by a 5-part autoanalyzer. Plasma xanthine oxidase activity was determined using enzyme linked immunosorbent assay (ELISA). Data obtained were analyzed using the Statistical Package for the Social Science (SPSS) version 20. Results were considered statistically significant if p<0.05. Biochemical parameters were correlated with the severity of bone pain crisis. Plasma xanthine oxidase activity was significantly elevated in the BPC group than in the steady state group (p=0.000) and HbA group (p=0.000). There steady state group plasma xanthine oxidase activity was also significantly higher than that of HbA group (p-0.032). Similarly, the plasma Plasma xanthine oxidase activity was significantly higher in all BPC severity group than steady state group with associated significantly low positive correlation of xanthine oxidase activity and bone pain severity. This study established that plasma xanthine oxidase activity was higher in patients with SCA presenting in bone pain crisis than steady state and HbA individual with associated positive correlation between plasma xanthine oxidase activity and bone pain severity.

Heme Overload Triggers Xanthine Oxidase Release and Mediates the Inflammatory Response

Heme crisis drives a number of hemolytic conditions including malaria, sepsis, blood transfusions, and cardiac bypass. Free heme, released from hemoglobin, causes endothelial damage via direct and iron (Fe)-mediated generation of reactive species, as well as activation of endothelial cells and macrophages leading to an inflammatory response. An enzyme that has been shown to have elevated activity in many hemolytic conditions is xanthine oxidase (XO). XO generates hydrogen peroxide (H2O2) as a byproduct of the oxidation of hypoxanthine and xanthine in the final steps of the purine degradation pathway. While XO activity is known to be increased in hemolytic diseases, its exact role has yet to be established. In order to study the role of XO in hemolytic disease, we developed a novel model of heme crisis in which we injected mice with two identical doses of hemin one hour apart and monitored the mice for 24 hours in order to deplete heme scavenging mechanisms before inducing heme crisis. Heme crisis induced damage was evaluated by hemopexin ELISA, plasma XO activity by HPLC, H&amp;E staining of liver, lung, and kidney tissue, qRT-PCR of inflammatory cytokines, and hematological analysis of circulating leukocytes, RBCs, and platelets. To isolate the role of XO, our heme crisis model was repeated; however, prior to hemin injection mice were pretreated with the FDA approved, XO inhibitor febuxostat (10 mg/kg/day) in drinking water. Liver, lung and kidney injury and inflammation was again evaluated with H&amp;E staining, qRT-PCR of inflammatory cytokines, and hematological analysis. In addition, the interaction between heme and XO was explored in vitro using evaluation of hemin degradation via spectrophotometry and computational modeling. We found that mice treated with two doses of 50 μmol/kg hemin had a 92.3% decrease in hemopexin, and a 20-fold increase in plasma XO activity compared to controls. H&amp;E staining showed severe liver hemorrhaging, increased cell infiltration in the lung, and cellular disorganization in the kidney. The pro-inflammatory cytokines, IL-6, TNFα, and IL-1β, were all significantly increased in the liver, lung, and kidney, with IL-6 having the greatest fold change in all three organs. Systemic inflammation was also suggested via significant increases in circulating monocytes and granulocytes. Additionally, hematological analysis showed decreased RBCs and platelets, indicating additional hemolysis and platelet activation. While these markers of injury and inflammation were observed with 50 μmol/kg hemin, lower doses of hemin showed no effect. Together, these results indicate that our heme crisis model mimics the pro-inflammatory state, and organ damage observed in patients during severe hemolysis. Interestingly, when mice were pre-treated with febuxostat, organ damage was observed at lower doses of hemin (25 μmol/kg) compared to untreated mice, as observed by H&amp;E staining. Inhibition of XO also had a significant impact on the inflammatory response. While circulating monocytes were decreased in mice pre-treated with febuxostat, the pro-inflammatory cytokines IL-6, TNFα, and IL-1β, were further exacerbated in the liver, lung, and kidney. This suggests that XO may play a role in mediating the inflammatory response induced by heme crisis. To explore how XO could mediate the inflammatory response we conducted in vitro enzymatic XO experiments with hemin. We found that XO was able to degrade hemin as observed by a decrease in absorbance at 618 nm. Additionally, based on a spectral shift observed when hemin and XO were incubated together, we hypothesized that XO may have the ability to bind hemin. This was further supported by computational modeling in which a potential heme binding site was discovered in the FAD domain of XO with a kd=128 nM. This suggests that XO may have the ability to bind hemin. Thus, during substrate oxidation, H2O2 is produced in the same XO domain of the potential heme binding site, allowing for increased chance of the H2O2 induced heme splitting reaction. We further hypothesize that the uric acid produced by xanthine oxidation may serve as an Fe chelator to scavenge free Fe released by a heme splitting reaction. By creating a microenvironment that can split heme and scavenge Fe, XO may be able to mediate the inflammatory response induced by heme crisis. Disclosures Straub: Bayer Pharmaceuticals: Research Funding.

Malaria inflammation by xanthine oxidase-produced reactive oxygen species.

Malaria is a highly inflammatory disease caused by Plasmodium infection of host erythrocytes. However, the parasite does not induce inflammatory cytokine responses in macrophages in vitro and the source of inflammation in patients remains unclear. Here, we identify oxidative stress, which is common in malaria, as an effective trigger of the inflammatory activation of macrophages. We observed that extracellular reactive oxygen species (ROS) produced by xanthine oxidase (XO), an enzyme upregulated during malaria, induce a strong inflammatory cytokine response in primary human monocyte‐derived macrophages. In malaria patients, elevated plasma XO activity correlates with high levels of inflammatory cytokines and with the development of cerebral malaria. We found that incubation of macrophages with plasma from these patients can induce a XO‐dependent inflammatory cytokine response, identifying a host factor as a trigger for inflammation in malaria. XO‐produced ROS also increase the synthesis of pro‐IL‐1β, while the parasite activates caspase‐1, providing the two necessary signals for the activation of the NLRP3 inflammasome. We propose that XO‐produced ROS are a key factor for the trigger of inflammation during malaria.

Plasma xanthine oxidase activity is related to increased sodium and left ventricular hypertrophy in resistant hypertension

BackgroundThe extra-renal effects of aldosterone on left ventricular (LV) structure and function are exacerbated by increased dietary sodium in persons with hypertension. Previous studies demonstrated endothelial dysfunction and increased oxidative stress with high salt diet in normotensive salt-resistant subjects. We hypothesized that increased xanthine oxidase (XO), a product of endothelial cells, is related to 24-h urinary sodium and to LV hypertrophy and function in patients with resistant hypertension (RHTN). MethodsThe study group included persons with RHTN (n = 91), defined as a blood pressure > 140/90 mmHg on ≥ 3 medications at pharmacologically effective doses. Plasma XO activity and 24-h urine were collected, and cardiac magnetic resonance imaging (MRI) was performed to assess LV function and morphology. Sixty-seven normotensive persons on no cardiovascular medications served as controls. A subset of RHTN (n = 19) received spironolactone without salt restriction for six months with follow-up XO activity measurements and MRI analyses. ResultsXO activity was increased two-fold in RHTN vs. normal and was positively correlated with LV mass, LV diastolic function, and 24-h urinary sodium. In RHTN patients receiving spironolactone without salt restriction, LV mass decreased, but LV diastolic function and XO activity did not improve. Baseline urinary sodium was positively associated with rate of change of LV mass to volume ratio and the LV E/A ratio. ConclusionsThese results demonstrate a potential role of endothelium-derived oxidative stress and excess dietary salt in the pathophysiology of LV hypertrophy and diastolic dysfunction in persons with RHTN unaffected by the addition of spironolactone.

56 - Excess circulating free heme triggers immense elevation in plasma xanthine oxidase activity

Heme, an iron-containing compound and critical prosthetic group for metalloproteins, is found most commonly as an element of hemoglobin. Excess circulating heme is a key driver of hemolytic diseases including thalassemia, malaria, sickle cell disease, sepsis as well as a side effect of transplantation and cardiac bypass. Severe hemolysis (heme crisis) saturates heme degradation pathways such as hemopexin leading to abundant circulating free heme that can directly generate reactive oxygen species (ROS) and/or stimulate elevation in enzymatic sources of ROS, ultimately leading to overt tissue damage. A key enzymatic source of ROS reported to be upregulated in sickle cell disease is xanthine oxidase (XO). As such, we investigated the relationship between circulating XO activity and heme crisis in mice. Both 24 hour survival and plasma XO activity were assessed in C57BL6/J mice injected (tail-vein) with hemin (0-100 μmol/kg). Hemin dose-dependently reduced survival (100% (25 μmol/kg), 87% (50 μmol/kg), 12% (70 μmol/kg) and 0% (100 μmol/kg)). Hematological analysis revealed an increase in monocyte recruitment and diminished platelet counts with increasing doses of hemin. Doses of 50 and 70 μmol/kg hemin elevated plasma XO activity 20-fold and 39-fold, respectively compared to controls and correlated with substantial loss of hepatic XO. Histological analysis (H&E staining) also indicated that liver tissue from mice injected with 50 μmol/kg hemin demonstrated significant alterations in tissue organization. In conclusion, heme crisis induces extensive hepatocellular release of XO into the circulation where it may actively participate in the pathologic process. To validate this assumption, we have generated a liver-specific XO knockout and are currently investigating mechanisms underpinning heme crisis-associated hepatocellular release of XO as well as the impact of circulating XO on cardiovascular function. These ongoing experiments may serve to identify novel clinical strategies to address heme crisis.

189 - Hepatic-specific ablation or whole-body inhibition of xanthine oxidase in mice corrects obesity-induced systemic hyperuricemia without improving metabolic abnormalities

Rationale: Systemic hyperuricemia (HyUA) in obesity/T2DM, facilitated by elevated xanthine oxidase (XO) activity, which is the sole source of UA in mammals, has been proposed to contribute to the pathogenesis of insulin resistance/dyslipidemia in obesity. However, this hypothesis has not been assessed directly. Objectives The effects of tissue-specific, genetic ablation of XO (a liver-specific knockout (LXO)) and whole-body pharmacologic inhibition of XO (febuxostat (FEBUX)) on obesity-induced insulin resistance/dyslipidemia were assessed. Findings Deletion of hepatic XO substantially lowered liver and plasma UA levels. When exposed to an obesogenic diet LXO and floxed (FLX, control) mice became equally obese. However, unlike obese floxed, liver and systemic HyUA in LXO mice was absent. Despite this, obese LXO mice became as insulin resistant and dyslipidemic as obese FLX mice. In obese C57BL/6J mice, FEBUX dramatically lowered plasma and tissue UA and XO activity compared to vehicle controls (CONT). However, obesity-associated insulin resistance/dyslipidemia were similar in FEBUX and CONT. Furthermore, neither pharmacologic inhibition nor hepatic ablation of XOR significantly altered indices of oxidant stress in the liver at 20 weeks of high-fat feeding compared with untreated controls. Conclusions These data demonstrate that liver XO expression is a critical determinant of blood UA homeostasis; that deletion of liver XO is sufficient to prevent the systemic HyUA of obesity, and that neither prevention nor correction of HyUA improve insulin resistance/dyslipidemia in obesity or diminish liver oxidant load. Thus, systemic HyUA, while clearly a biomarker of the metabolic abnormalities of obesity, does not appear to be a causative factor.

A4690 Sodium acetate and mineralocorticoid receptor blockade improve gestational androgen excess-induced glucose dysmetabolism in rats by suppressing adenosine deaminase/xanthine oxidase pathway

Objectives: The effects of sodium acetate and mineralocorticoid receptor (MR) on glucose dysmetabolism and poor fetal outcome induced by gestational androgen excess is not yet well elucidated. We tested the hypothesis that blockade of MR or treatment with sodium acetate will prevent glucose dysmetabolism induced by late gestational androgen excess through suppression of adenosine deaminase (ADA)/xanthine oxidase (XO) pathway. Methods: Thirty-six pregnant Wistar rats were treated (sc) with olive oil, testosterone propionate (0.5 mg/kg) singly or in combination with sodium acetate (200 mg/kg; po), androgen receptor (AR) blocker (flutamide; 7.5 mg/kg; po), non-selective MR blocker (spironolactone 0.25 mg/kg; po) or selective MR blocker (eplerenone; 0.5 mg/kg; po) between gestational days 14 and 19. Glucose dysmetabolism was accessed by elevated fasting blood glucose, impaired oral glucose tolerance test and homeostatic model assessment of insulin resistance. Results: The results showed that late gestational androgen excess led to glucose dysregulation, increased plasma testosterone, plasma and cardiac ADA and XO activities. Conversely, gestational androgen excess resulted in reduced plasma and cardiac anti-oxidant defenses (glutathione peroxidase, reduced glutathione/glutathione disulphide ratio, glucose-6-phosphate dehydrogenase and adenosine). However, all these effects were ameliorated by AR blockade, MR blockade or sodium acetate comparatively. Conclusion: The study demonstrates that improvement of glucose dysregulation induced by late gestational androgen excess by AR blockade, MR blockade or sodium acetate occurs by improvement of anti-ox

A4800 Dipeptidyl peptidase-4 inhibition prevents increased hepatic triglyceride content in insulin-resistant female rats

Objectives: Dipeptidyl peptidase-4 (DPP-4) inhibition has been show to exert beneficial effects against insulin resistance (IR) and fatty liver disease. Combined oral contraceptive (COC) treatment has been shown to be associated with glucose deregulation and increased hepatic triglyceride (TG) levels, but the mechanisms are elusive. We therefore, hypothesized that DPP-4 inhibition (DPP-4i) will ameliorate COC-induced glucose dysregulation and hepatic TG accumulation through adenosine deaminase (ADA) /xanthine oxidase (XO) /uric acid-dependent pathway. Methods: Female Wistar rats received (po) vehicle and COC (1.0 μg ethinylestradiol plus 5.0 μg levonorgestrel; po) with or without DPP-4 inhibitor (sitagliptin; 100 mg/kg; po) for 8 weeks (n = 6/group). Glucose dysmetabolism was accessed by elevated fasting blood glucose, impaired oral glucose tolerance test and homeostatic model assessment of insulin resistance. Results: Treatment with COC led to increased plasma fasting glucose, triglyceride-glucose index, 1-h postload glucose response, insulin, free fatty acid, IR and impaired glucose tolerance. COC treatment also resulted in increased plasma and hepatic triglycerides (TG), TG/HDL-cholesterol ratio, malondialdehyde, uric acid, lactate dehydrogenase, DPP-4, ADA and XO activities. On the other hand, COC led to reduction in nitric oxide level. However, DPP-4i significantly ameliorated these alterations induced by COC treatment except plasma and hepatic XO activity. Conclusion: This demonstrates that increased uric acid, DPP-4 and ADA activities are involved in glucose dysregulation and hepatic TG accumulation induced by COC treatment. Therefore, DPP-4i would impact positively on cardiometabolic disorders, at least in part, through ADA and uric acid suppression.

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&lt;b&gt;Are patients with higher plasma xanthine oxidase activity more likely to have cardiac diastolic dysfunction.&lt;/b&gt;

Inhibition of adenosine deaminase and xanthine oxidase by valproic acid abates hepatic triglyceride accumulation independent of corticosteroids in female rats treated with estrogen-progestin.

Elevated circulating uric acid has been postulated to play an important pathophysiological role in estrogen-progestin combined oral contraceptive (COC)-induced hypertension and endothelial dysfunction. We hypothesized that disruption of glucoregulation and liver triglyceride (TG) accumulation induced by COC use would be abated by valproic acid (VPA) treatment through suppression of adenosine deaminase (ADA) and xanthine oxidase (XO) activities. Female Wistar rats aged 9-10 weeks were treated with a combination of estrogen-progestin COC steroids (1.0 μg ethinylestradiol and 5.0 μg levonorgestrel; p.o.) with or without VPA (100.0 mg/kg; p.o.) daily for 6 weeks. The result shows that the disrupted glucoregulation and associated elevated hepatic ADA activity, plasma and hepatic XO activity, uric acid (UA), TG/HDL-cholesterol, total cholesterol, and malondialdehyde induced by COC treatment were attenuated by VPA treatment. However, VPA did not have any effect on plasma aldosterone, corticosterone, ADA, circulating and hepatic free fatty acid. Our results demonstrate that suppression of plasma and hepatic XO activities, along with hepatic ADA activity and UA by VPA treatment, protects against disrupted glucoregulation and increased liver TG by COC independent of elevated corticosteroids. The findings imply that VPA would provide protection against the development of cardiometabolic disorder via inhibition of the ADA/XO/UA-mediated pathway.

Activity of xanthine oxidase in plasma correlates with indices of insulin resistance and liver dysfunction in patients with type 2 diabetes mellitus and metabolic syndrome: A pilot exploratory study.

Aims/IntroductionThere is controversy as to whether hyperuricemia is an independent risk factor for cardiometabolic diseases. The serum level of uric acid is affected by a wide variety of factors involved in its production and excretion. In contrast, evidence has accumulated that locally‐ and systemically‐activated xanthine oxidase (XO), a rate‐limiting enzyme for production of uric acid, is linked to metabolic derangement in humans and rodents. We therefore explored the clinical implication of plasma XO activity in patients with type 2 diabetes mellitus and metabolic syndrome (MetS).Materials and MethodsWe enrolled 60 patients with type 2 diabetes mellitus and MetS. MetS was defined according to the 2005 International Diabetes Federation guidelines. Plasma XO activity was measured by highly‐sensitive fluorometric assay measuring the conversion of pterin to isoxanthopterin, and explored associations between the value of plasma XO activity and metabolic parameters.ResultsThe value of plasma XO activity was correlated with indices of insulin resistance and the level of circulating liver transaminases. In contrast, the level of serum uric acid was not correlated with indices of insulin resistance. The value of plasma XO activity was not correlated with the serum uric acid level.ConclusionsPlasma XO activity correlates with indices of insulin resistance and liver dysfunction in Japanese patients with type 2 diabetes mellitus and MetS. Through assessing the plasma XO activity, patients showing normal levels of serum uric acid with higher activity of XO can be screened, thereby possibly providing a clue to uncovering metabolic risks in type 2 diabetes mellitus and MetS patients.

An effort toward molecular neuroeconomics of food deprivation induced food hoarding in mice: focus on xanthine oxidoreductase gene expression and xanthine oxidase activity.

The crucial role of xanthine oxidoreductase (XOR) gene and its active isoform, xanthine oxidase (XO), in purine metabolism and cellular oxidative status led us to investigative their fluctuations in food deprivation induced food hoarding in mice. After, 10h food deprivation, mice that hoarded lesser than 5g were considered as 'low-hoarders' while mice that hoarded higher than 20g were considered as 'high-hoarders'. Mice who hoarded between 5 to 20g of food were excluded from study. An increase (1.133-fold) in encephalic XOR expression has been found in high-hoarders compared with low-hoarders without sex consideration. An increase (~ 50-fold) in encephalic XOR in female high-hoarders vs. female low-hoarders while a decrease (0.026-fold) in encephalic XOR in male high-hoarders vs. male low-hoarders demonstrated that food deprivation is associated with sex-dependent alteration in XOR expression. The encephalic and hepatic XO activities were not different in male high-hoarders vs. male low-hoarders while encephalic XO activity has been also increased significantly in female high-hoarders (~ 4 times) compared to female low-hoarders. The plasma and hepatic XO activities tended to be increased in female high-hoarders as compared to female low-hoarders, however the uric acid levels in plasma, liver and brain tissues were not altered in female high-hoarders as compared to female low-hoarders. In sum, this study generally proposed that different gene expression space is behind of hoarding behavior in a food-deprived mouse model. Specifically, this is the first study that examined the levels of encephalic XO activity and XOR expression in hoarding behavior, although additional studies are requested.

The Relationship between Xanthine Oxidoreductase and Xanthine Oxidase Activities in Plasma and Kidney Dysfunction

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.