Bioavailability Of Arginine Research Articles

Arginine Deiminase of <i>Streptococcus pyogenes</i> M49-16 Disrupts the Confluence of the Monolayer and the Structure of the Actin Cytoskeleton of Endothelial Cells <i>In</i> <i>Vitro</i>

The actin cytoskeleton is involved in the regulation of the barrier function of the endothelium. The bioavailability of arginine is an important factor determining of actin cytoskeleton dynamics. Pathogenic microorganisms can use arginine-hydrolyzing enzymes to disrupt the confluences of the vascular endothelium for subsequent dissemination. In this study, the effect of streptococcal arginine deiminase on the human umbilical vein endothelial cells monolayer confluence and the actin cytoskeleton structure in vitro was studied. The original technique for obtaining supernatants by sonication destroyed streptococcal cells (SDSCs) of the original strain of Streptococcus pyogenes M49-16 and its isogenic mutant with the inactivated arginine deiminase gene S. pyogenes M49-16delArcA was used in this study. The changes in the L-arginine concentration were evaluated by the modified Sakaguchi colorimetric method. The structure of the actin cytoskeleton was analyzed after cells staining with fluorescent dye labeled phalloidin. The confluence of the endothelial cell monolayer was evaluated morphologically after staining the cells with crystal violet dye. It was found that in the presence of the parental strain-derived SDSC, a significant decrease in the arginine concentration in the endothelial cells culture medium caused dynamic changes in the actin cytoskeleton structure. After 48 hours, lamellae and stress fibers formed. After 72 hours, the content of F-actin decreased and the confluence of the monolayer of endothelial cells was disrupted. Such changes were not detected when cells were cultured under standard conditions and in the presence of mutant strain-derived SDSC. The results obtained show that pathogenic microbes can use arginine depletion to regulate endothelial barrier function and dissemination in the host organism.

Disulphide bonds and cross‐linked amino acids may affect amino acid utilization in feather meal fed to rainbow trout (Oncorhynchus mykiss)

A digestibility trial was carried to assess the apparent digestibility coefficient (ADC) of crude protein (CP) and amino acids (AA) in two feather meals (FeM) of different origin (FeM1 and FeM2) and in their enzymatically pre-treated ingredient stream (PTFeM1 and PTFeM2) in rainbow trout. The ADC of CP was 11%–17% higher (p < 0.05) in the PTFeMs than in their FeM counterparts. There was no difference (p > 0.05) of ADC of CP among FeMs and among PTFeMs. The FT-Raman analysis showed a reduction in concentration and conformational stability of disulphide bonds in the PTFeMs when compared to their FeM counterparts. Moreover, important discrepancies in disulphide concentration were observed between FeMs and between PTFeMs. These observations suggest that the ADC of CP in FeM is not only governed by disulphide bond content and other variables should be considered. Higher concentrations of the cross-linked amino acids (CLAA) lanthionine, lysinoalanine and β-aminoalanine were measured in FeM1 and PTFeM1 as compared to FeM2 and PTFeM2 respectively. Previous studies have suggested inverse relationships between CLAAs and the digestibility as well as bioavailability of AAs in feed ingredients. A previous study conducted in our laboratory showed a lower (p < 0.05) bioavailability of arginine in FeM1 and PTFeM1 than in FeM2 and PTFeM2, respectively, in rainbow trout. Our results suggest that (a) the digestibility of AAs in FeM may be affected by disulphide bond content and the presence of CLAAs, and (b) CLAA content may be indicator of bioavailability of AAs in FeM.

A novel enzymatic pre‐treatment improves amino acid utilization in feather meal fed to rainbow trout (Oncorhynchus mykiss)

A growth trial followed by a digestibility trial were conducted to assess the bioavailability of arginine (Arg) and amino acid digestibility of two feather meals (FeMs) and their pre-treated counterparts (PTFeMs). Two FeMs, identified as FeM1 and FeM2, were incubated in 200% distilled water (%FeM w/w), with 0.05% of Savinase® 16L (%FeM v/w) and 2% sodium sulphite (%FeM w/w) at 55ºC for 24 hr in a shaking incubator to produce PTFeM1 and PTFeM2 respectively. A basal diet, highly deficient in Arg (12.0 g/kg Arg) was formulated and 10 other diets were formulated to contain 13.5 or 15.0 g/kg Arg by adding increasing amounts of L-Arg, FeMs or PTFeMs. The experimental diets were fed for 8 weeks using a standard protocol. At 13.5 g/kg Arg, the PTFeMs supported 10.5% to 11.5% higher growth rates (p < 0.05) than their untreated counterparts. The diets formulated with PTFeMs presented 3.5%–4.5% and 2.7%–5.9% higher (p < 0.05) apparent digestibility coefficients of protein when compared to the diets containing the FeM counterparts at 13.5 and 15.0 g/kg Arg respectively. The results from this study demonstrate the potential of the pre-treatment to improve the nutritive value of FeMs in rainbow trout.

Endothelial Phenotype Evoked by Low Dose Carvedilol in Pulmonary Hypertension.

Background: The therapeutic benefits of β-blockers are well established in left heart failure. The Pulmonary Arterial Hypertension Treatment with Carvedilol for Heart Failure [PAHTCH] study showed safety and possible benefit of carvedilol in pulmonary arterial hypertension (PAH) associated right heart failure over 6 months. This study aims at evaluating the short-term cardiovascular effects and early mechanistic biomarkers of carvedilol therapy.Methods: Thirty patients with pulmonary hypertension (PH) received low dose carvedilol (3.125 mg twice daily) for 1 week prior to randomization to placebo, low-dose, or dose-escalating carvedilol therapy. Echocardiography was performed at baseline and 1 week. Exercise capacity was assessed by 6 min walk distance (6MWD). The L-arginine/nitric oxide pathway and other biological markers of endothelial function were measured.Results: All participants tolerated 1 week of carvedilol without adverse effects. After 1 week of carvedilol, 6MWD and heart rate at peak exercise did not vary (both p > 0.1). Heart rate at rest and 1 min post walk dropped significantly (both p < 0.05) with a trend for increase in heart rate recovery (p = 0.08). Right ventricular systolic pressure (RVSP) decreased by an average of 13 mmHg (p = 0.002). Patients who had a decrease in RVSP of more than 10 mm Hg were defined as responders (n = 17), and those with a lesser drop as non-responders (n = 13). Responders had a significant drop in pulmonary vascular resistance (PVR) after 1 week of carvedilol (p = 0.004). In addition, responders had a greater decrease in heart rate at rest and 1 min post walk compared to non-responders (both p < 0.05). Responders had higher plasma arginine and global bioavailability of arginine at baseline compared to non-responders (p = 0.03 and p = 0.05, respectively). After 1 week of carvedilol, responders had greater increase in urinary nitrate (p = 0.04). Responders treated with carvedilol had a sustained drop in RVSP and PVR after 6 months of carvedilol with no change in cardiac output.Conclusions: Low-dose carvedilol for 1 week can potentially identify a PH responder phenotype that may benefit from β-blockers that is associated with less endothelial dysfunction.Clinical Trial Registration: http://www.clinicaltrials.gov. identifier: NCT01586156.

Plasma metabolomic profiling of a ketamine and placebo crossover trial of major depressive disorder and healthy control subjects.

(R,S)-Ketamine produces rapid, robust, and sustained antidepressant effects in major depressive disorder. Specifically, its pharmacological efficacy in treatment refractory depression is considered a major breakthrough in the field. However, the mechanism of action of ketamine's rapid effect remains to be determined. In order to identify pathways that are responsible for ketamine's effect, a targeted metabolomic approach was carried out using a double-blind, placebo-controlled crossover design, with infusion order randomized with medication-free patients with treatment-resistant major depressive disorder (29 subjects) and healthy controls (25 subjects). The metabolomic profile of these subjects was characterized at multiple time points, and a comprehensive analysis was investigated between the following: MDD and healthy controls, treatment and placebo in both groups and the corresponding response to ketamine treatment. Ketamine treatment resulted in a general increase in circulating sphingomyelins, levels which were not correlated with response. Ketamine response resulted in more pronounced effects in the kynurenine pathway and the arginine pathway at 4h post-infusion, where a larger decrease in circulating kynurenine levels and a larger increase in the bioavailability of arginine were observed in responders to ketamine treatment, suggesting possible mechanisms for response to ketamine treatment.

Molecular docking studies of ephedrine, eugenol, and their derivatives as arginase inhibitors: Implications in asthma

Background: Arginine being a common substrate for arginase and nitric oxide synthase (NOS) an imbalance between enzymes could lead to a shift in airway responses. Reports suggest that increased arginase reduces the substrate availability to NOS and attributes to the airway hyperresponsiveness. Hence, inhibition of arginase might enhance the bioavailability of arginine to NOS and generates nitric oxide (NO) a bronchodilator. Molecules from Ephedra and Eugenia caryophyllus are documented for bronchodilator properties. However, the mechanism of action of these molecules for enhancing bronchodilation is not well characterized. The objective of the present study is to assess whether these molecules could inhibit the arginase by binding at its active site and helps in bronchodilation using in silico approach. Methods: The crystal structures of the arginase and NOS enzymes were selected from the protein database. The molecules from Ephedra and Eugenia caryophyllus were obtained from Pubchem. Drug likeliness and bioactivity of the molecules were assessed by Molinspiration. The successful molecules were docked with active sites of enzymes using docking software, and the docked complexes were analyzed using Accelrys Discovery Studio. Results: Molecules from Ephedra and Eugenia caryophyllus were able to interact to arginase at the active site whereas away from the active site in case of NOS. The molecules showed differential binding affinities, and some of them had higher binding affinity than substrate arginine. Conclusion: In silico study suggests that molecules of Ephedra and Eugenia were capable of blocking the active site of arginase. We speculate that if these molecules are used as therapeutics, they could inhibit the arginase activity and this might increase arginine availability to NOS to produce NO which acts as bronchodilator. Our study suggests that molecules which bind to active site of arginine and do not affect the active site of NOS might be the potential molecules for arginase associated asthma.

Development and evaluation of a host-targeted antiviral that abrogates herpes simplex virus replication through modulation of arginine-associated metabolic pathways

Since their inception five decades ago, most antivirals have been engineered to disrupt a single viral protein or process that is essential for viral replication. This approach has limited the overall therapeutic effectiveness and applicability of current antivirals due to restricted viral specificity, a propensity for development of drug resistance, and an inability to control deleterious host-mediated inflammation. As obligate intracellular parasites, viruses are reliant on host metabolism and macromolecular synthesis pathways. Of these biosynthetic processes, many viruses, including Herpes simplex viruses (HSV), are absolutely dependent on the bioavailability of arginine, a non-essential amino acid that is critical for many physiological and pathophysiological processes associated with either facilitating viral replication or progression of disease. To assess if targeting host arginine-associated metabolic pathways would inhibit HSV replication, a pegylated recombinant human Arginase I (peg-ArgI) was generated and its in vitro anti-herpetic activity was evaluated. Cells continuously treated with peg-ArgI for over 48 h exhibited no signs of cytotoxicity or loss of cell viability. The antiviral activity of peg-ArgI displayed a classical dose-response curve with IC50’s in the sub-nanomolar range. peg-ArgI potently inhibited HSV-1 and HSV-2 viral replication, infectious virus production, cell-to-cell spread/transmission and virus-mediated cytopathic effects. Not unexpectedly given its host-targeted mechanism of action, peg-ArgI showed similar effectiveness at controlling replication of single and multidrug resistant HSV-1 mutants. These findings illustrate that targeting host arginine-associated metabolic pathways is an effective means of controlling viral replicative processes. Further exploration into the breadth of viruses inhibited by peg-ArgI, as well as the ability of peg-ArgI to suppress arginine-associated virus-mediated pathophysiological disease processes is warranted.

Arginine Silicate Supplementation Decreases Markers of Cardiovascular, Renal and Metabolic Dysfunction and Increases Markers of Vasodilation and Cardiovascular Health in Healthy Adult Males

Arginine silicate (ASI; Nitrosigine®) is a new dietary ingredient with enhanced absorption and bioavailability of arginine and silicon. Human pharmacokinetic data show increased blood levels of the...

Bioavailability of arginine from Indian mustard protein concentrate and meal compared with that of a soy protein concentrate in rainbow trout (Oncorhynchys mykiss)

Relative bioavailability (RBV) of arginine (Arg) from Indian mustard protein concentrate (IMC, 62% crude protein) and Indian mustard meal (IMM, 42% crude protein), and a commercially available soy protein concentrate (SPC, 57% crude protein) was compared with that of crystalline L-arginine (L-Arg) in rainbow trout. A basal diet highly deficient in Arg (1.23%) was formulated. Eight other isoproteic and isoenergetic diets were formulated to contain ~1.35% and ~1.5% Arg by adding increasing amount of IMC, IMM, SPC and L-Arg. The experimental diets were fed for 16 weeks using a standard protocol. Growth rate, weight gain (g fish � 1 ) and protein (PD, g fish � 1 ) and lipid (LD, g fish � 1 ) deposition were increased linearly with increasing level of Arg from all ingredients. Arg availability from protein-bound sources were equal or higher than those from L-Arg. RBV of Arg from IMC, IMM and SPC were ranged from 100% and 123% than that from L-Arg (assumed as 100% bio-available). Among the ingredients, only the RBV of Arg from IMC was significantly higher than those from SPC (P < 0.05). The findings suggest that the RBV of Arg from IMC and IMM are very good and comparable to that of the commercial SPC used in this study.

Endotoxemia affects citrulline, arginine and glutamine bioavailability

Sepsis considerably alters the intestinal barrier functions, which in turn modify the absorption and bioavailability of nutrients. However, the effects of septic shock on aminoacid (AAs) bioavailability are poorly documented. The aim of this study was to compare the bioavailability of citrulline, arginine and glutamine during endotoxemia. Thirty-six rats were randomised into two groups: control and lipopolysaccharides (LPS). The LPS group received an intraperitoneal injection of endotoxins (7·5 mg/kg). After 12 h, each group was again randomised into three subgroups, each of which received an oral bolus of citrulline, arginine or glutamine (5·7 mmol/kg). Blood samples were collected at various times from 0 to 600 min after AA administration. The concentrations of citrulline, arginine, glutamine and their metabolites arginine and ornithine were measured to determine pharmacokinetic parameters Area Under Curve (AUC), C(max) and T(max). The AUC values of citrulline decreased in LPS rats [citrulline, control: 761 ± 67 and LPS: 508 ± 72 μmol min/mL (P = 0·02)]. Maximum concentrations of citrulline were also significantly decreased by endotoxemia (P = 0·01). The pharmacokinetic parameters of arginine and glutamine were not significantly modified by endotoxemia. The AUC value of arginine from citrulline conversion was diminished in endotoxemic rats. The other pharmacokinetic parameters of arginine were not significantly modified after arginine or citrulline supply in either group (control or LPS). Endotoxemia affects the bioavailability of AAs differently according to the amino acid considered. This feature may be important for nutritional strategy in ICU patients.

Genetic analysis of arginase 1 in Th2-dominated lung inflammation (163.12)

Abstract Arginase 1 (Arg1) expression in myeloid lineage cells, and especially alternatively-activated macrophages, increases dramatically in the lungs of mice with acute and chronic Th2-driven inflammation. Arg1 has been proposed to contribute to asthma pathogenesis by regulating at least four pathways: inhibiting the production of nitric oxide by substrate competition, regulating fibrosis and polyamine supply, regulating the bioavailability of arginine in the lung, and inhibiting T cell proliferation. Here we used mice lacking Arg1 in myeloid lineage cells, to investigate the contribution of Arg1 to asthma pathophysiology and lung inflammation. In six distinct model systems encompassing acute and chronic Th2-mediated inflammation, we observed neither a pathogenic nor protective role for myeloid-expressed Arg1 in any measured parameter of lung pathophysiology. Collectively, our results argue that (i) attempts to inhibit arginases in the lung by small molecule arginase inhibitors or RNAi should be examined with caution and (ii) whether macrophages use Arg1 to limit Th2 responses depends on the organ and type of infection or injury.

Development of a test diet for assessing the bioavailability of arginine in feather meal fed to rainbow trout ( Oncorhynchus mykiss)

A 12-week slope ratio growth assay was undertaken to estimate the bioavailability of arginine in feather meal fed to rainbow trout. Fish were fed to satiety with isonitrogenous and isoenergetic diets containing graded levels of arginine provided by either synthetic l-arginine or steam-hydrolysed feather meal. Weight gain of the fish responded in a significantly linear (P < 0.0125) fashion to increase dietary levels of synthetic l-arginine. Weight gain of the fish did not significantly improve with increasing arginine level provided by feather meal. This result was unexpected since all diets were formulated to contain digestible essential amino acid (EAA) levels that were significantly in excess of NRC (1993) requirements. However, significant improvement in the feed efficiency, as well as nitrogen and energy utilization efficiencies, of fish fed the diets with increasing levels of feather meal, suggests that digestible nutrients from feather meal were, on average, very efficiently utilized by the fish. In a follow up experiment, supplementation of a high feather meal diet from the first experiment with a mixture of synthetic EAA significantly (P < 0.05) improved weight gain of the fish. These results indicate that the diets formulated to contain increasing levels of feather meal in the first experiment may have become deficient in one or more essential amino acids. This study highlights the need to have more accurate information on EAA requirements of fish for the development of slope ratio assays aimed at assessing the bioavailability of amino acids in different ingredients. It also shows the importance of generating more information on the bioavailability of individual EAA in feather meals.

Despite a large first‐pass extraction for urea synthesis, the systemic bioavailability of meal arginine is high and dose‐dependent in men

Dietary Arginine is supposed to be largely extracted by the liver for ureagenesis but favourable effects of dietary Arginine are mainly ascribed to a systemic utilization for NO synthesis.Using [15N2‐(guanido)]‐Arginine, we traced the fate of L‐Arginine at two nutritional levels in a complete amino acid meal given to healthy men. We monitored the postprandial appearance of meal arginine (mArg) in the plasma, body and urinary urea derived from mArg, and plasma citrulline and urinary nitrate derived from mArg[FM1].The relative urea production from mArg was high but similar whatever the mArg level (49±2% of mArg) and completed early after ingestion. With the lower level of mArg, intact mArg accounted for 56±4% of the marked postprandial increase in plasma arginine. In contrast, plasma concentrations of mArg‐derived citrulline were very low. After the 2.7‐fold higher mArg intake, plasma mArg AUC was 3.2±0.2‐fold higher, and plasma mArg‐derived citrulline AUC and urinary mArg‐derived nitrate excretion were 1.8±0.3 and 2.2±0.3‐fold higher, respectively.Because its relative splanchnic extraction for urea synthesis stays steady, mArg exhibits a high incremental systemic bioavailability. However, our data suggests that the utilization of mArg for NO synthesis, although modulated by the systemic availability of mArg, stays very low in these healthy men.Supported by a grant of the French ministry of research[FM1] OU, pour gagner un peu de place," mArg‐derived body and urinary urea, and mArg‐derived plasma citrulline and urinary nitrate"

Nitric Oxide and Arginine Dysregulation: A Novel Pathway to Pulmonary Hypertension in Hemolytic Disorders

Secondary pulmonary hypertension (PH) is emerging as one of the leading causes of mortality and morbidity in patients with hemolytic anemias such as sickle cell disease (SCD) and thalassemia. Impaired nitric oxide (NO) bioavailability represents the central feature of endothelial dysfunction, and is a major factor in the pathophysiology of PH. Inactivation of NO correlates with hemolytic rate and is associated with the erythrocyte release of cell-free hemoglobin, which consumes NO directly, and the simultaneous release of the arginine-metabolizing enzyme arginase, which limits bioavailability of the NO synthase substrate arginine during the process of intravascular hemolysis. Rapid consumption of NO is accelerated by oxygen radicals that exists in both SCD and thalassemia. A dysregulation of arginine metabolism contributes to endothelial dysfunction and PH in SCD, and is strongly associated with prospective patient mortality. The central mechanism responsible for this metabolic disorder is enhanced arginine turnover, occurring secondary to enhanced plasma arginase activity. This is consistent with a growing appreciation of the role of excessive arginase activity in human diseases, including asthma and pulmonary arterial hypertension. New treatments aimed at improving arginine and NO bioavailability through arginase inhibition, suppression of hemolytic rate, oral arginine supplementation, or use of NO donors represent potential therapeutic strategies for this common pulmonary complication of hemolytic disorders.

Metabolic effects of a novel silicate inositol complex of the nitric oxide precursor arginine in the obese insulin-resistant JCR:LA- cp rat

Insulin resistance is a major contributor to macro- and microvascular complications, particularly in the presence of the metabolic syndrome, and is also associated with polycystic ovary syndrome. Impaired nitric oxide metabolism and endothelial function are important components of the vascular disease. Increasing the bioavailability of arginine, the precursor of nitric oxide, thus potentially offers protection against end-stage disease. We have recently demonstrated that dietary supplementation with a novel silicate inositol arginine complex reduces vasculopathy and glomerular sclerosis in the insulin-resistant JCR:LA- cp rat. The objective of this study was to address the absorption of, and the underlying metabolic alterations caused by, the arginine silicate inositol complex and arginine HCl (as a reference agent) in obese insulin-resistant male and female JCR:LA- cp rats. Male and female rats were treated with the preparations at 1.0 mg/(kg d) (expressed as arginine HCl) from 8 to 12 and 12 to 18 weeks of age, respectively. Obese female, but not male, rats treated with the arginine silicate inositol complex showed a reduced rate of weight gain without concomitant reduction in food intake. Plasma silicon levels were raised very significantly in arginine silicate–treated rats, consistent with significant absorption of the complex. In male rats, arginine levels were elevated by treatment with arginine silicate only; and female rats responded to both preparations. Plasma concentrations of oxides of nitrogen in rats treated with the silicate complex showed a dimorphism, decreasing in male and increasing in female rats. Fasting insulin levels were elevated in male rats treated with the arginine silicate complex, whereas fasting and postprandial insulin levels were decreased in female rats. Furthermore, female, but not male, rats treated with either of the arginine preparations showed significant reductions in cholesterol, triglyceride, and phospholipid concentrations. We conclude that the arginine silicate inositol complex is absorbed efficiently, raising plasma arginine levels, and is more biologically effective than the free amino acid hydrochloride. This has different beneficial metabolic effects in both sexes of an animal model of insulin resistance and cardiovascular disease, consistent with reduction in end-stage disease.

New Strategies for the Treatment of Pulmonary Hypertension in Sickle Cell Disease

Nitric oxide (NO) is inactivated in sickle cell disease (SCD), while bioavailability of arginine, the substrate for NO synthesis, is diminished. Impaired NO bioavailability represents the central feature of endothelial dysfunction, and is a key factor in the pathophysiology of SCD. Inactivation of NO correlates with the hemolytic rate and is associated with erythrocyte release of cell-free hemoglobin and arginase during hemolysis. Accelerated consumption of NO is enhanced further by the inflammatory environment of oxidative stress that exists in SCD. Based upon its critical role in mediating vasodilation and cell growth, decreased NO bioavailability has also been implicated in the pathogenesis of pulmonary arterial hypertension (PHT). Secondary PHT is a common life-threatening complication of SCD that also occurs in most hereditary and chronic hemolytic disorders. Aberrant arginine metabolism contributes to endothelial dysfunction and PHT in SCD, and is strongly associated with prospective patient mortality. The central mechanism responsible for this metabolic disorder is enhanced arginine turnover, occurring secondary to enhanced plasma arginase activity. This is consistent with a growing appreciation of the role of excessive arginase activity in human diseases, including asthma and PHT. Decompartmentalization of hemoglobin into plasma consumes endothelial NO and thus drives a metabolic requirement for arginine, whose bioavailability is further limited by arginase activity. New treatments aimed at maximizing both arginine and NO bioavailability through arginase inhibition, suppression of hemolytic rate, or oral arginine supplementation may represent novel therapeutic strategies.

Elevated Plasma Arginase Levels in Hemoglobinopathies.

The decreased bioavailability of arginine (Arg) and the resulting lower nitric oxide (NO) production has been shown to be an important factor in the pathology of sickle cell disease (SCD) and thalassemia. Vascular alterations leading to pulmonary hypertension are important factors of heart failure and death in these hemoglobinopathies. Red blood cell (RBC) hemolysis in these patients will release arginase into the circulation and contribute to the reduction of plasma Arg levels, change the Arg-to-ornithine ratio, and increase other downstream amino acid metabolites. Such compounds, including proline and polyamines, may contribute to vascular and airway remodeling. To study the contribution of arginase released from RBC, we measured the arginase activity and arginase protein concentration in the plasma and RBC lysates of normal controls, SCD patients, and thalassemia patients.Arginase activity was determined by the conversion of 14C-labeled guanidine-L-arginine to 14C-labeled urea, then to 14CO2 by urease and trapped as Na214CO3 for scintillation counting. Arginase and hemoglobin concentration were measured by ELISA (BioVendor, Candler, NC and Bethyl Labs Inc., Montgomery, TX respectively). Mean cellular hemoglobin concentration was measured with a Coulter Counter (Beckman Coulter, Fullerton, CA) or Technicon H3 analyzer (Tarrytown, NY). RBC were separated by density gradient centrifugation with OptiPrep (Axis Shield, Dundee, UK). Arginase specific activity (SA) was calculated by dividing the activity by the concentration to yield mole of Arg metabolized per g of arginase per hour. We found a higher level of arginase in the plasma and RBC of SCD and thalassemia patients as compared to normal controls. These levels correlated with cell-free hemoglobin content (plasma) or reticulocyte count (RBC). The light density fraction (<1.077 g/ml) of RBC, enriched in reticulocytes, showed higher arginase levels confirming increased arginase levels in young RBC. Interestingly, as the plasma arginase protein and activity increased with cell-free hemoglobin, the SA decreased. Similarly in RBC lysates, the SA decreases with hemoglobin concentration. This suggests that a factor exists within the RBC that negatively modulates arginase activity. Due to the high SA of arginase at low hemoglobin concentrations, low levels of intravascular hemolysis can generate relatively high levels of Arg breakdown.We conclude that low levels of intravascular hemolysis of RBC with increased arginase content in SCD and thalassemia, play a role in the altered Arg metabolism and decreased NO production. While the reason for high levels of arginase in the RBC as well as complete clinical impact of elevated arginase activity in plasma remain to be determined, dysregulated Arg metabolism and a shift toward Arg catabolic products, may be associated with the development of pulmonary hypertension and mortality in SCD and thalassemia.