Measuring Arginase Activity Research Articles

Synthesis of Mn2O3 Nanoparticles and Determination of Its Inhibition Effect On Sera of Iraqi Patients with Diabetes Mellitus Type-2 and Diabetes Nephropathy

Manganese is essential for the synthesis of antioxidant enzymes and metabolic issues in Diabetes type 2 (DMT2), which is a worldwide disease, Chronic metabolic disorders cause insulin resistance, hyperglycemia, and complications like diabetic nephropathy. Arginase converts arginine to ornithine and urea. Increased arginase activity in DMT2 and diabetes nephropathy (DN)which has been linked to kidney damage, and arginase inhibitors can increase NO which is essential to vascular function. However, the molecular mechanisms of arginased is regulationare in DMT2 and DN are still unclear. This study examined the effect of manganese oxide nanoparticles (Mn2O3NPs) on arginase activity inhibition inserum samples from DMT2 and DN patients. We hypothesized that Mn2O3NPs alter cell redox status and signaling pathways, affecting DMT2 and DN arginase activity. We used a colorimetric assay to measure arginase activity in 80 serum samples from DMT2 and DN patients treated with different MnO2 NP concentrations to test our hypothesis. The current study characterized nanoparticles using various techniques such as IR, SEM, AFM, XRD, and EDX, which found it within nanoscale nature. Our findings are that Mn2O3NPs modulate arginase activity specificity in DM2 samples. SuggestionsMn2O3NPs could be used to develop new treatments for these conditions.

Assays to Evaluate Toxoplasma-Macrophage Interactions.

The obligate intracellular protozoan parasite Toxoplasma gondii can infect any nucleated cell from a warm-blooded host. However, its interaction with host macrophages plays a critical role in shaping the immune response during infection. Therefore, assessing Toxoplasma-macrophage interactions at a cellular level is important. In this chapter, we describe assays that can be used to characterize Toxoplasma-macrophage interactions. These assays can also be used to evaluate other host-pathogen interactions. We describe multiplex approaches for measuring arginase activity, indoleamine 2,3 dioxygenase activity, cell death, and parasite growth during Toxoplasma-macrophage interactions. These assays can be used to compare how different Toxoplasma strains differ in their interaction with macrophages, and we describe how to properly assess Toxoplasma strain differences in Toxoplasma-macrophage interactions.

New approaches for biomarker stability determination in regulated bioanalysis: trending, bridging and incurred samples.

Aim: Determining the stability of biomarkers continues to present challenges. Disease states, complex matrices and differences between recombinant and endogenous analytes require new approaches to maintain stability and measure it. In this report, we determine stability for two assays using trending and statistical analysis. Methodology & results: Monitoring trends helps identify out of specification measurements and determine whether concerns are due to the stability of the analyte. We also describe challenges presented when measuring arginase activity in human sputum, a complex matrix, for respiratory diseases. We controlled preanalytical protease activity and collection heterogeneity and monitored incurred sample stability to improve stability of arginine. Conclusion: These new approaches to achieving and determining biomarker stability may provide solutions for increasingly complex biomarker measurements.

Tobacco and Antiretrovirals Modulate Transporter, Metabolic Enzyme, and Antioxidant Enzyme Expression and Function in Polarized Macrophages.

Background: Cigarette smoking increases systemic oxidative stress, inflammation, and viral replication in individuals with HIV. Macrophages are infected during HIV infection and serve as an important reservoir throughout the process. Macrophages exist in two phenotypes, the classically acti-vated M1 macrophage and alternatively activated M2 macrophage. The expression of drug efflux transporters and metabolic enzymes, which have direct effects on intracellular drug concentrations, differ between the pro-inflammatory M1 macrophage and the anti-inflammatory M2 macrophage.Objective: To further explain the role of tobacco use in worsened outcomes in the HIV + population receiving antiretroviral therapy.Methods: Western blotting was used to examine macrophage polarization and expression of drug efflux transporters, CYP enzymes, and antioxidant enzymes. The arginase assay was used to measure arginase activity. Cytokine production was measured using the human multiplex inflammatory cytokine assay kit. The 8-OHdG DNA Damage Quantification Direct Kit was used to quantify DNA damage. Viral replication under the influence of tobacco and antiretroviral drug use was measured by p24 Elisa.Results: We observed phenotypic shifts from M1 to M2 with both individual and combination treatments with cigarette smoke condensate and the protease inhibitor antiretroviral drug lopinavir. These shifts lead to changes in cytokine production, the expression of CYP enzymes, anti-oxidant enzymes, and drug efflux transporters, as well as changes in viral replication.Conclusion: This data suggest a mechanism by which tobacco use impairs HIV antiretroviral therapy to increase intracellular drug concentrations in this important cellular reservoir.

Disturbance of Arginase Activity and Nitric Oxide Levels in Iraqi Type 2 Diabetes Mellitus

This study is an attempt to find whether arginine metabolism dysregulation by arginase activity is related to hyperglycemia, followed by changes in nitric oxide (NO) generation in type 2 diabetic patients. This study includes 42 control subjects (Group I), and 92 Iraqi patients with type 2 diabetes mellitus (T2DM). The patient group was subdivided into two groups: Group II (54) with T2DM only and Group III (38) with T2DM and dyslipidemia (who were treating with atorvastatin along with diabetes treatment). The samples were obtained to measure arginase activity and NO levels. Serum arginase activity increased significantly in patients(groupII and groupIII) compared to control group. While serum NO level was significantly lower in diabetic patients as compared to control group, three significant correlations appeared in this study between glucose and arginase activity, glucose and NO levels, and between arginase activity and NO levels. The results also show that treatment with atorvastatin affects arginase activity and NO levels. Increasing in levels of arginase activity can be considered as an indicator of diabetic status. Endothelial dysfunctions accompanied with diabetes mellitus reverses correlation between arginase and NO in diabetic

Disturbance of Arginase Activity and Nitric Oxide Levels in Iraqi Type 2 Diabetes Mellitus

This study is an attempt to find whether arginine metabolism dysregulation by arginase activity is related to hyperglycemia, followed by changes in nitric oxide (NO) generation in type 2 diabetic patients. This study includes 42 control subjects (Group I), and 92 Iraqi patients with type 2 diabetes mellitus (T2DM). The patient group was subdivided into two groups: Group II (54) with T2DM only and Group III (38) with T2DM and dyslipidemia (who were treating with atorvastatin along with diabetes treatment). The samples were obtained to measure arginase activity and NO levels. Serum arginase activity increased significantly in patients(groupII and groupIII) compared to control group. While serum NO level was significantly lower in diabetic patients as compared to control group, three significant correlations appeared in this study between glucose and arginase activity, glucose and NO levels, and between arginase activity and NO levels. The results also show that treatment with atorvastatin affects arginase activity and NO levels. Increasing in levels of arginase activity can be considered as an indicator of diabetic status. Endothelial dysfunctions accompanied with diabetes mellitus reverses correlation between arginase and NO in diabetic

Determination of optimal conditions for activity of arginase in kidney of rabbit

Optimum conditions for arginase in kidney tissue may differ from those for arginase of liver. Because optimum conditions for measuring arginase activity in rabbit kidney tissue were not determined, it was aimed to optimize arginase of rabbit kidney tissue and compare it with arginase of liver. In the present study, the kidneys of six New Zealand rabbits aged 1 year old were used. It was found to be preincubation temperature 53 °C, preincubation period is 15 minutes, incubation period is 10 minutes and optimum pH is 10.1 in NaHCO3-NaOH buffer for arginase in rabbit kidney tissue. Enzyme achieved its highest activity at 1 mM MnCl2concentration. Km of arginase in rabbit kidney tissue for L-arginine was measured as 12.5 mM. It was determined that Mn+2 ions and preincubation at 53 °C were essential for the activation of the enzyme. As a result, optimal conditions for the kidney arginase were found to be different from those for the liver arginase. Our study indicates that arginases encoded by separate gene loci may have different optimal conditions

Identifying Factors of Microparticles Modified with Arginine Derivatives That Induce Phenotypic Shifts in Macrophages.

Macrophages are key players in the progression of many diseases, ranging from rheumatoid arthritis to cancer. Drug delivery systems have the potential not only to transport payloads to diseased tissue but also to influence cell behavior. Here, poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) microparticles were modified with 14 different arginine derivatives. These particles were then incubated with interleukin-4 or lipopolysaccharide-stimulated macrophages or naïve macrophages (RAW 264.7). The phenotypic state of the macrophages was assessed by measuring arginase activity, tumor necrosis factor-α (TNF-α) secretion, and nitrite production. Partial least-squares analysis revealed material properties and descriptors that shifted the macrophage phenotype for the three cell conditions in this study. Material descriptors relating to secondary bonding were suggested to play a role in shifting phenotypes in all three macrophage culture conditions. These findings suggest that macrophage responses could be altered through drug delivery vehicles, and this method could be employed to assist in screening potential candidates.

Lung arginase expression and activity is increased in cystic fibrosis mouse models.

The activity of arginase is increased in airway secretions of patients with cystic fibrosis (CF). Downstream products of arginase activity may contribute to CF lung disease. We hypothesized that pulmonary arginase expression and activity would be increased in mouse models of CF and disproportionally increased in CF mice with Pseudomonas aeruginosa pneumonia. Expression of arginase isoforms in lung tissue was quantified with reverse transcriptase-PCR in naive cystic fibrosis transmembrane conductance regulator (Cftr)-deficient mice and β-epithelial sodium channel-overexpressing [β-ENaC-transgenic (Tg)] mice. An isolated lung stable isotope perfusion model was used to measure arginase activity in Cftr-deficient mice before and after intratracheal instillation of Pseudomonas aeruginosa. The expression of arginase-2 in lung was increased in adult Cftr-deficient animals and in newborn β-ENaC-Tg. Arginase-1 lung expression was normal in Cftr-deficient and in newborn β-ENaC-Tg mice, but was increased in β-ENaC-Tg mice at age 1, 3, and 6 wk. Arginase activity was significantly higher in lung (5.0 ± 0.7 vs. 3.2 ± 0.3 nmol·(-1)·h(-1), P = 0.016) and airways (204.6 ± 49.8 vs. 79.3 ± 17.2 nmol·(-1)·h(-1), P = 0.045) of naive Cftr-deficient mice compared with sex-matched wild-type littermate controls. Infection with Pseudomonas aeruginosa resulted in a far greater increase in lung arginase activity in Cftr-deficient mice (10-fold) than in wild-type controls (6-fold) (P = 0.01). This is the first ex vivo characterization of arginase expression and activity in CF mouse lung and airways. Our data show that pulmonary arginase expression and activity is increased in CF mice, especially with Pseudomonas aeruginosa infections.

Quantitative Assessment of Macrophage Functions in Repair and Fibrosis

Macrophages play key roles in wound repair and fibrosis by regulating extracellular matrix turnover. Macrophages can process matrix components themselves, but also recruit and alter the functions of other cell types that directly build or degrade extracellular matrix. Classically activated macrophages (CAM, also called M1) tend to promote tissue injury while alternatively activated macrophages (AAM, also called M2) are often linked with the mechanisms of wound repair and fibrosis. However, rather than promoting collagen deposition, recent studies suggest that arginase-1-expressing AAM suppress chronic inflammation and fibrosis by inhibiting antigen-specific T cell responses. This unit describes methods to measure arginase activity in macrophages and whole tissues as well as assays to quantify the T cell suppressive activity of AAMs. Modified hydroxyproline and soluble collagen assays that can be used to quantify collagen levels in tissues and brochoalveolar lavage fluid are also described. The protocols in this unit should provide the investigator with all the necessary information required to measure arginase activity and to correlate the observed activity with the progression and resolution of fibrosis.

The evaluation of arginase activity during acute vasoocclusive sickle cell crisis

Study objectives: Nitric oxide is an endothelium-derived vasodilator that plays a significant role in the pathophysiology of sickle cell acute vaso-occlusive crisis (VOC) and may play an important role in new therapeutic strategies. Nitric oxide is formed from the conversion of the substrate l-arginine to citrulline by the enzyme endothelial nitric oxide synthase (eNOS). Our previous studies have demonstrated low nitric oxide and, more recently, low l-arginine levels during adult VOC. Although it seems intuitive that low substrate levels would explain low product, the low l-arginine levels in our study were still 10-fold higher than the k_m (3 μM) of eNOS and were more than sufficient to saturate eNOS, yet nitric oxide production was diminished, thereby suggesting a derangement in the normal l-arg-nitric oxide pathway. L-arginine supplementation, in other conditions, even in the face of this low but enzyme-saturating concentration, has been shown to enhance nitric oxide production. This phenomenon has been called the "arginine paradox." One explanation for this paradox involves the enzyme arginase. Arginase, a ubiquitous intra- and extracellular enzyme, catalyzes l-arginine to ornithine. Arginase activity has been found to be enhanced in certain pathologic states and may thus compete for available l-arginine, lessening l-arginine available to NOS. A pediatric study previously demonstrated enhanced arginase activity, as demonstrated by increased ornithine levels during VOC, which has not been examined in adult VOC. We hypothesize that arginase activity is enhanced during adult VOC and may thus play an important role in the l-arginine-nitric oxide pathway during adult VOC. The specific aim of this study is to measure arginase activity by l-arginine and ornithine measurement in VOC. Methods: Included were patients older than 18 years and with a chief complaint of typical VOC pain (defined as patient-described pain consistent in duration, severity, quality, and distribution with previous episodes of VOC and no evidence of coexistent illness), sickle cell patients not in VOC, and healthy controls. Subjects with sickle cell SS, SC, and β-thalassemia disease as documented by previous hemoglobin electrophoresis were eligible. Five milliliters of blood was obtained by standard venipuncture for l-arginine and ornithine analysis before standard analgesic therapy. Blood was immediately centrifuged at 10,000 RPM at 4°C for 10 minutes. The serum was separated and stored at –20°C. L-arginine and ornithine levels were measured by Molecular Structure Facility (University of California, Davis, CA) for an α equal to .05 and power of 0.9, and based on an l-arginine level expected SD of 3.0 μM/L. Results: The study included 42 sickle cell patients in typical VOC, 14 sickle cell controls not in crisis, and 8 healthy controls. As in our previous study, l-arginine was significantly low in VOC (median 23.160 μM/L, <i>P</i><.05) versus healthy sickle cell controls (median 38.560 μM/L) and healthy controls (median 73.010 μM/L). There was no significant difference between citrulline measurements in any of the groups. When ornithine was measured, a statistically significant difference (<i>P</i><.5) was found between sickle cell patients in VOC (median 22.610) and sickle cell healthy controls (36.780). No statistical difference was found between either sickle cell group and healthy controls (median 26.910). Conclusion: We found low ornithine levels in healthy sickle cell controls and VOC subjects. This finding does not support increased arginase activity in sickle cell disease. Interestingly, ornithine levels are significantly lower in VOC than in the healthy sickle cell controls, which may simply be explained by the low l-arginine levels already found in VOC. The significance of this, however, is unknown.