Increased plasma arginase activity in human sepsis: association with increased circulating neutrophils.

BACKGROUND: The pathophysiology of sepsis is incompletely understood. Impaired bioavailability of L-arginine, the substrate for NO synthesis, is linked to sepsis severity, and plasma arginase has been linked to hypoargininemia in other disease states. Circulating neutrophils are increased in sepsis and constitutively express arginase. We investigated whether plasma arginase activity is increased in human sepsis and whether this is associated with neutrophil numbers and activation. METHODS: We used HPLC and a radiometric assay to evaluate plasma amino acid concentrations and plasma arginase activity. The relationships between plasma arginase activity, neutrophil count, neutrophil activity and plasma L-arginine and arginine metabolites were evaluated in 44 sepsis patients and 25 controls. RESULTS: Plasma arginase activity was increased in sepsis patients, correlated with neutrophil count (r=0.44; p=0.003), but was independent of sepsis severity (SOFA or APACHE II score). Plasma HNP1-3 correlated with neutrophil count (r=0.31; p=0.04), was elevated in shock (median 180 ng/mL vs. 83 ng/mL sepsis without shock, p=0.0006) and correlated with SOFA score. Sepsis patients with high neutrophil counts had significantly higher plasma HNP1-3 and arginase activity and lower plasma L-arginine concentrations than those with lower neutrophil counts and controls. CONCLUSIONS: Plasma arginase activity, potentially derived in part from neutrophil activation, is elevated in sepsis, and may contribute to impaired bioavailability of L-arginine in sepsis.

Neutrophils with myeloid derived suppressor function deplete arginine and constrain T cell function in septic shock patients.

INTRODUCTION: Impaired T cell function in sepsis is associated with poor outcome, but the mechanisms are unclear. In cancer, arginase-expressing myeloid derived suppressor cells (MDSCs) deplete arginine, impair T cell receptor CD3 zeta-chain expression and T cell function and are linked to poor clinical outcome, but their role during acute human infectious disease and in particular sepsis remains unknown. Hypoarginemia is prevalent in sepsis. This study aimed to determine whether neutrophils that co-purify with PBMC express arginase, and if arginine depletion constrains T cell CD3 zeta-chain expression and function in human sepsis. METHODS: Using flow cytometry, cell culture, HPLC, arginase activity and mRNA detection, our study examined whether neutrophils, with reduced buoyant density isolated in the Ficoll interface, metabolise L-arginine and suppress T cell proliferation in sepsis. A total of 35 sepsis patients (23 with septic shock) and 12 hospital controls in a tertiary referral hospital in tropical Australia were evaluated. RESULTS: Only sepsis patients had interphase neutrophils, neutrophils co-purifying with mononuclear cells (≤1.077 specific gravity). The percentage of interphase neutrophils in sepsis was proportional to sepsis severity and correlated with plasma IL-6 concentrations. Ex vivo, sepsis-derived interphase neutrophils expressed arginase, metabolised culture L-arginine and suppressed T cell proliferation and CD3 zeta-chain expression. In vivo, in septic shock there was a longitudinal inverse association between interphase neutrophil number and CD3 zeta-chain expression. Depletion or inhibition of interphase neutrophils in vitro restored zeta-chain expression and T cell function. CONCLUSIONS: For the first time during an acute human infection, interphase neutrophils that express arginase were found to circulate in sepsis, in proportion to disease severity. These neutrophil-MDSCs impair T cell CD3 zeta-chain expression and T cell function via L-arginine metabolism, and likely contribute to the T cell dysfunction seen in sepsis. Modulation of neutrophil-MDSC or their downstream effects warrant consideration as targets for novel adjunctive therapies in sepsis.

Increased asymmetric dimethylarginine in severe falciparum malaria: association with impaired nitric oxide bioavailability and fatal outcome.

Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is a predictor of mortality in critical illness. Severe malaria (SM) is associated with decreased NO bioavailability, but the contribution of ADMA to the pathogenesis of impaired NO bioavailability and adverse outcomes in malaria is unknown. In adults with and without falciparum malaria, we tested the hypotheses that plasma ADMA would be: 1) increased in proportion to disease severity, 2) associated with impaired vascular and pulmonary NO bioavailability and 3) independently associated with increased mortality. We assessed plasma dimethylarginines, exhaled NO concentrations and endothelial function in 49 patients with SM, 78 with moderately severe malaria (MSM) and 19 healthy controls (HC). Repeat ADMA and endothelial function measurements were performed in patients with SM. Multivariable regression was used to assess the effect of ADMA on mortality and NO bioavailability. Plasma ADMA was increased in SM patients (0.85 microM; 95% CI 0.74-0.96) compared to those with MSM (0.54 microM; 95%CI 0.5-0.56) and HCs (0.64 microM; 95%CI 0.58-0.70; p<0.001). ADMA was an independent predictor of mortality in SM patients with each micromolar elevation increasing the odds of death 18 fold (95% CI 2.0-181; p = 0.01). ADMA was independently associated with decreased exhaled NO (r(s) = -0.31) and endothelial function (r(s) = -0.32) in all malaria patients, and with reduced exhaled NO (r(s) = -0.72) in those with SM. ADMA is increased in SM and associated with decreased vascular and pulmonary NO bioavailability. Inhibition of NOS by ADMA may contribute to increased mortality in severe malaria.

Sepsis-associated microvascular dysfunction measured by peripheral arterial tonometry: an observational study.

INTRODUCTION: Sepsis has a high mortality despite advances in management. Microcirculatory and endothelial dysfunction contribute to organ failure, and better tools are needed to assess microcirculatory responses to adjunctive therapies. We hypothesised that peripheral arterial tonometry (PAT), a novel user-independent measure of endothelium-dependent microvascular reactivity, would be impaired in proportion to sepsis severity and related to endothelial activation and plasma arginine concentrations. METHODS: Observational cohort study in a 350-bed teaching hospital in tropical Australia. Bedside microvascular reactivity was measured in 85 adults with sepsis and 45 controls at baseline and 2-4 days later by peripheral arterial tonometry. Microvascular reactivity was related to measures of disease severity, plasma concentrations of L-arginine (the substrate for nitric oxide synthase), and biomarkers of endothelial activation. RESULTS: Baseline reactive hyperaemia index (RH-PAT index), measuring endothelium-dependent microvascular reactivity; (mean [95% CI]) was lowest in severe sepsis (1.57 [1.43-1.70]), intermediate in sepsis without organ failure (1.85 [1.67-2.03]) and highest in controls (2.05 [1.91-2.19]); P < 0.00001. Independent predictors of baseline RH-PAT index in sepsis were APACHE II score and mean arterial pressure, but not plasma L-arginine or markers of endothelial activation. Low baseline RH-PAT index was significantly correlated with an increase in SOFA score over the first 2-4 days (r = -0.37, P = 0.02). CONCLUSIONS: Endothelium-dependent microvascular reactivity is impaired in proportion to sepsis severity and suggests decreased endothelial nitric oxide bioavailability in sepsis. Peripheral arterial tonometry may have a role as a user-independent method of monitoring responses to novel adjunctive therapies targeting endothelial dysfunction in sepsis.

Ex-vivo changes in amino acid concentrations from blood stored at room temperature or on ice: implications for arginine and taurine measurements.

BACKGROUND: Determination of the plasma concentrations of arginine and other amino acids is important for understanding pathophysiology, immunopathology and nutritional supplementation in human disease. Delays in processing of blood samples cause a change in amino acid concentrations, but this has not been precisely quantified. We aimed to describe the concentration time profile of twenty-two amino acids in blood from healthy volunteers, stored at room temperature or on ice. METHODS: Venous blood was taken from six healthy volunteers and stored at room temperature or in an ice slurry. Plasma was separated at six time points over 24 hours and amino acid levels were determined by high-performance liquid chromatography. RESULTS: Median plasma arginine concentrations decreased rapidly at room temperature, with a 6% decrease at 30 minutes, 25% decrease at 2 hours and 43% decrease at 24 hours. Plasma ornithine increased exponentially over the same period. Plasma arginine was stable in blood stored on ice, with a < 10% change over 24 hours. Plasma taurine increased by 100% over 24 hours, and this change was not prevented by ice. Most other amino acids increased over time at room temperature but not on ice. CONCLUSION: Plasma arginine concentrations in stored blood fall rapidly at room temperature, but remain stable on ice for at least 24 hours. Blood samples taken for the determination of plasma amino acid concentrations either should be placed immediately on ice or processed within 30 minutes of collection.

Pharmacokinetics of L-arginine in adults with moderately severe malaria.

Severe malaria is associated with decreased nitric oxide (NO) production and low plasma concentrations of L-arginine, the substrate for NO synthase. Supplementation with L-arginine has the potential to improve NO bioavailability and outcomes. We developed a pharmacokinetic model for L-arginine in moderately severe malaria to explore the concentration-time profile and identify important covariates. In doses of 3, 6, or 12 g,L-arginine was infused over 30 min to 30 adults with moderately severe malaria, and plasma concentrations were measured at 8 to 11 time points. Patients who had not received L-arginine were also assessed and included in the model. The data were analyzed using a population approach with NONMEM software. A two-compartment linear model with first-order elimination best described the data, with a clearance of 44 liters/h (coefficient of variation [CV] = 52%) and a volume of distribution of 24 liters (CV = 19%). The natural time course of L-arginine recovery was described empirically by a second-order polynomial with a time to half recovery of 26 h. The half-life of exogenous L-arginine was reduced in patients with malaria compared with that for healthy adults. Weight and ethnicity were significant covariates for clearance. MATLAB simulations of dosing schedules for use in future studies predicted that 12 g given over 6, 8, or 12 h will provide concentrations above the K(m) of endothelial cell CAT-1 transporters in 90%, 75%, and 60% of patients, respectively.

Simultaneous determination of multiple amino acids in plasma in critical illness by high performance liquid chromatography with ultraviolet and fluorescence detection.

There is increasing recognition that the host response to critical illness includes derangement of multiple amino acid pathways, including amino acids (AAs) central to metabolism and immune, endothelial and neurological function. To characterise concentration changes of these plasma amino acid we report the development and validation of a method for the quantification of AAs in small volumes of plasma (50µL) using HPLC with simultaneous UV and fluorescence (FL) detection. Protein precipitation and pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is followed by reversed phase HPLC separation. Calibration curves were built with norleucine as an internal standard. Thirty-three (including the 20 proteinogenic) AAs, were selected as standards and their corresponding concentrations in the plasma of healthy human controls and patients with severe falciparum malaria were quantified. This method enables the detection of perturbations in arginine metabolism, aromatic amino acid pathways, methionine transsulfuration and transmethylation pathways and other metabolic pathways.

An observational cohort study of the kynurenine to tryptophan ratio in sepsis: association with impaired immune and microvascular function.

Both endothelial and immune dysfunction contribute to the high mortality rate in human sepsis, but the underlying mechanisms are unclear. In response to infection, interferon-γ activates indoleamine 2,3-dioxygenase (IDO) which metabolizes the essential amino acid tryptophan to the toxic metabolite kynurenine. IDO can be expressed in endothelial cells, hepatocytes and mononuclear leukocytes, all of which contribute to sepsis pathophysiology. Increased IDO activity (measured by the kynurenine to tryptophan [KT] ratio in plasma) causes T-cell apoptosis, vasodilation and nitric oxide synthase inhibition. We hypothesized that IDO activity in sepsis would be related to plasma interferon-γ, interleukin-10, T cell lymphopenia and impairment of microvascular reactivity, a measure of endothelial nitric oxide bioavailability. In an observational cohort study of 80 sepsis patients (50 severe and 30 non-severe) and 40 hospital controls, we determined the relationship between IDO activity (plasma KT ratio) and selected plasma cytokines, sepsis severity, nitric oxide-dependent microvascular reactivity and lymphocyte subsets in sepsis. Plasma amino acids were measured by high performance liquid chromatography and microvascular reactivity by peripheral arterial tonometry. The plasma KT ratio was increased in sepsis (median 141 [IQR 64-235]) compared to controls (36 [28-52]); p<0.0001), and correlated with plasma interferon-γ and interleukin-10, and inversely with total lymphocyte count, CD8+ and CD4+ T-lymphocytes, systolic blood pressure and microvascular reactivity. In response to treatment of severe sepsis, the median KT ratio decreased from 162 [IQR 100-286] on day 0 to 89 [65-139] by day 7; p = 0.0006) and this decrease in KT ratio correlated with a decrease in the Sequential Organ Failure Assessment score (p<0.0001). IDO-mediated tryptophan catabolism is associated with dysregulated immune responses and impaired microvascular reactivity in sepsis and may link these two fundamental processes in sepsis pathophysiology.

Asymmetric dimethylarginine, endothelial nitric oxide bioavailability and mortality in sepsis.

BACKGROUND: Plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are raised in patients with chronic vascular disease, causing increased cardiovascular risk and endothelial dysfunction, but the role of ADMA in acute inflammatory states is less well defined. METHODS AND RESULTS: In a prospective longitudinal study in 67 patients with acute sepsis and 31 controls, digital microvascular reactivity was measured by peripheral arterial tonometry and blood was collected at baseline and 2-4 days later. Plasma ADMA and L-arginine concentrations were determined by high performance liquid chromatography. Baseline plasma L-arginine: ADMA ratio was significantly lower in sepsis patients (median [IQR] 63 [45-103]) than in hospital controls (143 [123-166], p<0.0001) and correlated with microvascular reactivity (r = 0.34, R(2) = 0.12, p = 0.02). Baseline plasma ADMA was independently associated with 28-day mortality (Odds ratio [95% CI] for death in those in the highest quartile (≥ 0.66 µmol/L) = 20.8 [2.2-195.0], p = 0.008), and was independently correlated with severity of organ failure. Increase in ADMA over time correlated with increase in organ failure and decrease in microvascular reactivity. CONCLUSIONS: Impaired endothelial and microvascular function due to decreased endothelial NO bioavailability is a potential mechanism linking increased plasma ADMA with organ failure and death in sepsis.

HPLC analysis of asymmetric dimethylarginine, symmetric dimethylarginine, homoarginine and arginine in small plasma volumes using a Gemini-NX column at high pH.

There is increasing recognition of the clinical importance of endogenous nitric oxide synthase inhibitors in critical illness. This has highlighted the need for an accurate high performance liquid chromatography (HPLC) method for detection of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) in small volumes of blood. Here, the validation of an accurate, precise HPLC method for the determination of ADMA, SDMA, homoarginine and arginine concentrations in plasma is described. Solid phase extraction is followed by derivatisation with AccQ-Fluor and reversed phase separation on a Gemini-NX column at pH 9. Simultaneous detection by both UV-vis and fluorescence detectors affords extra validation. This solid phase extraction method gives absolute recoveries of more than 85% for ADMA and SDMA and relative recoveries of 102% for ADMA and 101% for SDMA. The intra-assay relative standard deviations are 2.1% and 2.3% for ADMA and SDMA, respectively, with inter-assay relative standard deviations of 2.7% and 3.1%, respectively. Advantages of this method include improved recovery of all analytes using isopropanol in the solid phase extraction; sharp, well-resolved chromatographic peaks using a high pH mobile phase; a non-endogenous internal standard, n-propyl L-arginine; and accurate and precise determination of methylated arginine concentrations from only 100microL of plasma.

Recovery of endothelial function in severe falciparum malaria: relationship with improvement in plasma L-arginine and blood lactate concentrations.

BACKGROUND: Severe malaria is characterized by microvascular obstruction, endothelial dysfunction, and reduced levels of L-arginine and nitric oxide (NO). L-Arginine infusion improves endothelial function in moderately severe malaria. Neither the longitudinal course of endothelial dysfunction nor factors associated with recovery have been characterized in severe malaria. METHODS: Endothelial function was measured longitudinally in adults with severe malaria (n = 49) or moderately severe malaria (n = 48) in Indonesia, using reactive hyperemia peripheral arterial tonometry (RH-PAT). In a mixed-effects model, changes in RH-PAT index values in patients with severe malaria were related to changes in parasitemia, lactate, acidosis, and plasma L-arginine concentrations. RESULTS: Among patients with severe malaria, the proportion with endothelial dysfunction fell from 94% (46/49 patients) to 14% (6/42 patients) before discharge or death (P < .001). In severe malaria, the median time to normal endothelial function was 49 h (interquartile range, 20-70 h) after the start of antimalarial therapy. The mean increase in L-arginine concentrations in patients with severe malaria was 11 micromol/L/24 h (95% confidence interval [CI], 9-13 micromol/L/24 h), from a baseline of 49 micromol/L (95% CI, 37-45 micromol/L). Improvement of endothelial function in patients with severe malaria correlated with increasing levels of L-arginine (r = 0.56; P = .008) and decreasing levels of lactate (r = -0.44; P = .001). CONCLUSIONS: Recovery of endothelial function in severe malaria is associated with recovery from hypoargininemia and lactic acidosis. Agents that can improve endothelial NO production and endothelial function, such as L-arginine, may have potential as adjunctive therapy early during the course of severe malaria.

Impaired nitric oxide bioavailability and L-arginine reversible endothelial dysfunction in adults with falciparum malaria.

Severe falciparum malaria (SM) is associated with tissue ischemia related to cytoadherence of parasitized erythrocytes to microvascular endothelium and reduced levels of NO and its precursor, l-arginine. Endothelial function has not been characterized in SM but can be improved by l-arginine in cardiovascular disease. In an observational study in Indonesia, we measured endothelial function using reactive hyperemia-peripheral arterial tonometry (RH-PAT) in 51 adults with SM, 48 patients with moderately severe falciparum malaria (MSM), and 48 controls. The mean RH-PAT index was lower in SM (1.41; 95% confidence interval [CI] = 1.33-1.47) than in MSM (1.82; 95% CI = 1.7-2.02) and controls (1.93; 95% CI = 1.8-2.06; P < 0.0001). Endothelial dysfunction was associated with elevated blood lactate and measures of hemolysis. Exhaled NO was also lower in SM relative to MSM and controls. In an ascending dose study of intravenous l-arginine in 30 more patients with MSM, l-arginine increased the RH-PAT index by 19% (95% CI = 6-34; P = 0.006) and exhaled NO by 55% (95% CI = 32-73; P < 0.0001) without important side effects. Hypoargininemia and hemolysis likely reduce NO bioavailability. Endothelial dysfunction in malaria is nearly universal in severe disease, is reversible with l-arginine, and likely contributes to its pathogenesis. Clinical trials in SM of adjunctive agents to improve endothelial NO bioavailability, including l-arginine, are warranted.