Gary Cunningham

l-Citrulline ameliorates chronic hypoxia-induced pulmonary hypertension in newborn piglets

Newborn piglets develop pulmonary hypertension and have diminished pulmonary vascular nitric oxide (NO) production when exposed to chronic hypoxia. NO is produced by endothelial NO synthase (eNOS) in the pulmonary vascular endothelium using l-arginine as a substrate and producing l-citrulline as a byproduct. l-Citrulline is metabolized to l-arginine by two enzymes that are colocated with eNOS in pulmonary vascular endothelial cells. The purpose of this study was to determine whether oral supplementation with l-citrulline during exposure of newborn piglets to 10 days of chronic hypoxia would prevent the development of pulmonary hypertension and increase pulmonary NO production. A total of 17 hypoxic and 17 normoxic control piglets were studied. Six of the 17 hypoxic piglets were supplemented with oral l-citrulline starting on the first day of hypoxia. l-Citrulline supplementation was provided orally twice a day. After 10 days of hypoxia or normoxia, the animals were anesthetized, hemodynamic measurements were performed, and the lungs were perfused in situ. Pulmonary arterial pressure and pulmonary vascular resistance were significantly lower in hypoxic animals treated with l-citrulline compared with untreated hypoxic animals (P < 0.001). In vivo exhaled NO production (P = 0.03) and nitrite/nitrate accumulation in the perfusate of isolated lungs (P = 0.04) were significantly higher in l-citrulline-treated hypoxic animals compared with untreated hypoxic animals. l-Citrulline supplementation ameliorated the development of pulmonary hypertension and increased NO production in piglets exposed to chronic hypoxia. We speculate that l-citrulline may benefit neonates exposed to prolonged periods of hypoxia from cardiac or pulmonary causes.

A study of aspirin and clopidogrel in idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is characterised by in situ thrombosis and increased thromboxane (Tx) A2 synthesis; however, there are no studies of antiplatelet therapy in IPAH. The aim of the current study was to determine the biochemical effects of aspirin (ASA) and clopidogrel on platelet function and eicosanoid metabolism in patients with IPAH. A randomised, double-blind, placebo-controlled crossover study of ASA 81 mg once daily and clopidogrel 75 mg once daily was performed. Plasma P-selectin levels and aggregometry were measured after exposure to adenosine diphosphate, arachidonic acid and collagen. Serum levels of TxB2 and urinary metabolites of TxA2 and prostaglandin I2 (Tx-M and PGI-M, respectively) were assessed. A total of 19 IPAH patients were enrolled, of whom nine were being treated with continuous intravenous epoprostenol. ASA and clopidogrel significantly reduced platelet aggregation to arachidonic acid and adenosine diphosphate, respectively. ASA significantly decreased serum TxB2, urinary Tx-M levels and the Tx-M/PGI-M ratio, whereas clopidogrel had no effect on eicosanoid levels. Neither drug significantly lowered plasma P-selectin levels. Epoprostenol use did not affect the results. In conclusion, aspirin and clopidogrel inhibited platelet aggregation, and aspirin reduced thromboxane metabolite production without affecting prostaglandin I2 metabolite synthesis. Further clinical trials of aspirin in patients with idiopathic pulmonary arterial hypertension should be performed.

Low plasma citrulline levels are associated with acute respiratory distress syndrome in patients with severe sepsis

IntroductionThe role of nitric oxide synthase (NOS) in the pathophysiology of acute respiratory distress syndrome (ARDS) is not well understood. Inducible NOS is upregulated during physiologic stress; however, if NOS substrate is insufficient then NOS can uncouple and switch from NO generation to production of damaging peroxynitrites. We hypothesized that NOS substrate levels are low in patients with severe sepsis and that low levels of the NOS substrate citrulline would be associated with end organ damage including ARDS in severe sepsis.MethodsPlasma citrulline, arginine and ornithine levels and nitrate/nitrite were measured at baseline in 135 patients with severe sepsis. ARDS was diagnosed by consensus definitions.ResultsPlasma citrulline levels were below normal in all patients (median 9.2 uM, IQR 5.2 - 14.4) and were significantly lower in ARDS compared to the no ARDS group (6.0 (3.3 - 10.4) vs. 10.1 (6.2 - 16.6), P = 0.002). The rate of ARDS was 50% in the lowest citrulline quartile compared to 15% in the highest citrulline quartile (P = 0.002). In multivariable analyses, citrulline levels were associated with ARDS even after adjustment for covariates including severity of illness.ConclusionsIn severe sepsis, levels of the NOS substrate citrulline are low and are associated with ARDS. Low NOS substrate levels have been shown in other disease states to lead to NOS uncoupling and oxidative injury suggesting a potential mechanism for the association between low citrulline and ARDS. Further studies are needed to determine whether citrulline supplementation could prevent the development of ARDS in patients with severe sepsis and to determine its role in NOS coupling and function.

Rescue Treatment with L-Citrulline Inhibits Hypoxia-Induced Pulmonary Hypertension in Newborn Pigs

Infants with cardiopulmonary disorders associated with hypoxia develop pulmonary hypertension. We previously showed that initiation of oral L-citrulline before and continued throughout hypoxic exposure improves nitric oxide (NO) production and ameliorates pulmonary hypertension in newborn piglets. Rescue treatments, initiated after the onset of pulmonary hypertension, better approximate clinical strategies. Mechanisms by which L-citrulline improves NO production merit elucidation. The objective of this study was to determine whether starting L-citrulline after the onset of pulmonary hypertension inhibits disease progression and improves NO production by recoupling endothelial NO synthase (eNOS). Hypoxic and normoxic (control) piglets were studied. Some hypoxic piglets received oral L-citrulline starting on Day 3 of hypoxia and continuing throughout the remaining 7 days of hypoxic exposure. Catheters were placed for hemodynamic measurements, and pulmonary arteries were dissected to assess NO production and eNOS dimer-to-monomer ratios (a measure of eNOS coupling). Pulmonary vascular resistance was lower in L-citrulline-treated hypoxic piglets than in untreated hypoxic piglets but was higher than in normoxic controls. NO production and eNOS dimer-to-monomer ratios were greater in pulmonary arteries from L-citrulline-treated than from untreated hypoxic animals but were lower than in normoxic controls. When started after disease onset, oral L-citrulline treatment improves NO production by recoupling eNOS and inhibits the further development of chronic hypoxia-induced pulmonary hypertension in newborn piglets. Oral L-citrulline may be a novel strategy to halt or reverse pulmonary hypertension in infants suffering from cardiopulmonary conditions associated with hypoxia.

Prolonged hypoxia augments l-citrulline transport by System A in the newborn piglet pulmonary circulation

AIMS Pulmonary arterial endothelial cells (PAECs) express the enzymes needed for generation of l-arginine from intracellular l-citrulline but do not express the enzymes needed for de novo l-citrulline synthesis. Hence, l-citrulline levels in PAECs are dependent on l-citrulline transport. Once generated, l-arginine can be converted to l-citrulline and nitric oxide (NO) by the enzyme NO synthase. We sought to determine whether hypoxia, a condition aetiologically linked to pulmonary hypertension, alters the transport of l-citrulline and the expression of the sodium-coupled neutral amino acid transporters (SNATs) in PAECs from newborn piglets. METHODS AND RESULTS PAECs isolated from newborn piglets were cultured under normoxic and hypoxic conditions and used to measure SNAT1, 2, 3, and 5 protein expression and (14)C-l-citrulline uptake. SNAT1 protein expression was increased, while SNAT2, SNAT3, and SNAT5 expression was unaltered in hypoxic PAECs. (14)C-l-citrulline uptake was increased in hypoxic PAECs. Studies with inhibitors of System A (SNAT1/2) and System N (SNAT3/5) revealed that the increased (14)C-l-citrulline uptake was largely due to System A-mediated transport. Additional studies were performed to evaluate SNAT protein expression and l-citrulline levels in lungs of piglets with chronic hypoxia-induced pulmonary hypertension and comparable age controls. Lungs from piglets raised in chronic hypoxia exhibited greater SNAT1 expression and higher l-citrulline levels than lungs from controls. CONCLUSION Increased SNAT1 expression and the concomitant enhanced ability to transport l-citrulline in PAECs could represent an important regulatory mechanism to counteract NO signalling impairments known to occur during the development of chronic hypoxia-induced pulmonary hypertension in newborns.

γ-Glutamylcysteine ameliorates oxidative injury in neurons and astrocytes in vitro and increases brain glutathione in vivo.

γ-Glutamylcysteine (γ-GC) is an intermediate molecule of the glutathione (GSH) synthesis pathway. In the present study, we tested the hypothesis that γ-GC pretreatment in cultured astrocytes and neurons protects against hydrogen peroxide (H(2)O(2))-induced oxidative injury. We demonstrate that pretreatment with γ-GC increases the ratio of reduced:oxidized GSH levels in both neurons and astrocytes and increases total GSH levels in neurons. In addition, γ-GC pretreatment decreases isoprostane formation both in neurons and astrocytes, as well as nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation in astrocytes in response to H(2)O(2)-induced oxidative stress. Furthermore, GSH and isoprostane levels significantly correlate with increased neuron and astrocyte viability in cells pretreated with γ-GC. Finally, we demonstrate that administration of a single intravenous injection of γ-GC to mice significantly increases GSH levels in the brain, heart, lungs, liver, and in muscle tissues in vivo. These results support a potential therapeutic role for γ-GC in the reduction of oxidant stress-induced damage in tissues including the brain.

An ethnic-specific polymorphism in the catalytic subunit of glutamate-cysteine ligase impairs the production of glutathione intermediates in vitro.

Glutathione plays a crucial role in free radical scavenging, oxidative injury, and cellular homeostasis. Previously, we identified a non-synonymous polymorphism (P462S) in the gene encoding the catalytic subunit of glutamate-cysteine ligase (GCLC), the rate-limiting enzyme in glutathione biosynthesis. This polymorphism is present only in individuals of African descent. Presently, we report that this ethnic-specific polymorphism (462S) encodes an enzyme with significantly decreased in vitro activity when expressed by either a bacterial or mammalian cell expression system. In addition, overexpression of the 462P wild-type GCLC enzyme results in higher intracellular glutathione concentrations than overexpression of the 462S isoform. We also demonstrate that apoptotically stimulated mammalian cells overexpressing the 462S enzyme have increased caspase activation and increased DNA laddering compared to cells overexpressing the wild-type 462P enzyme. Finally, we genotyped several African and African-descent populations and demonstrate that the 462S polymorphism is in Hardy-Weinberg disequilibrium, with no individuals homozygous for the 462S polymorphism identified. These findings describe a glutathione production pathway polymorphism present in individuals of African descent with significantly decreased in vitro activity.

Simple and inexpensive quantification of ammonia in whole blood.

Quantification of ammonia in whole blood has applications in the diagnosis and management of many hepatic diseases, including cirrhosis and rare urea cycle disorders, amounting to more than 5 million patients in the United States. Current techniques for ammonia measurement suffer from limited range, poor resolution, false positives or large, complex sensor set-ups. Here we demonstrate a technique utilizing inexpensive reagents and simple methods for quantifying ammonia in 100 μL of whole blood. The sensor comprises a modified form of the indophenol reaction, which resists sources of destructive interference in blood, in conjunction with a cation-exchange membrane. The presented sensing scheme is selective against other amine containing molecules such as amino acids and has a shelf life of at least 50 days. Additionally, the resulting system has high sensitivity and allows for the accurate reliable quantification of ammonia in whole human blood samples at a minimum range of 25 to 500 μM, which is clinically for rare hyperammonemic disorders and liver disease. Furthermore, concentrations of 50 and 100 μM ammonia could be reliably discerned with p = 0.0001.

A phase 1 dose-finding study of intravenous L-citrulline in sickle cell disease: a potential novel therapy for sickle cell pain crisis

Acute pain from vaso-occlusion (VOC) in sickle cell disease (SCD) is the most frequent cause of emergency room visits and hospital admissions, contributing to the high burden of health care costs (Lanzkron et al, 2010). While major advances in the care of patients with SCD have occurred over the last 30 years, very little progress has been made in the actual treatment of VOC. Nitric oxide (NO) is a powerful vasodilator that plays a fundamental role in VOC (Morris, 2008). NO is produced from L-citrulline and L-arginine, amino acids generated through the urea cycle from the NO synthase (NOS) family of enzymes (Moncada & Higgs, 1993). There are three NOS isoforms: neuronal (nNOS) found in neuronal tissue, inducible NOS (iNOS) found in cells and tissues, and endothelial NOS (eNOS) found in vascular endothelial cells. Shen et al (2005) showed that citrulline was the major supply for intracellular L-arginine and endothelial NO production in murine endothelial cells. Furthermore, Wijnands et al (2012) showed that L-citrulline supplementation restored intracellular NO production, which was related to the degree of eNOS phosphorylation. Moreover, enhanced arginase-induced arginine consumption is believed to play an integral role in the pathogenesis of sickle cell complications. In a more recent study, L-citrulline supplementation increased NO production and improved microcirculatory flow during conditions with arginase-induced arginine deficiency (Wijnands et al, 2015). However, intravenous citrulline has never been evaluated in human SCD. Hence, this study aimed to characterize the pharmacokinetic (PK) and safety profile of intravenous (IV) citrulline in this unique patient population. A single centre open label phase 1 trial of IV citrulline was performed in participants with SCD following approval from the University of Mississippi Medical Center (UMMC) institutional review board. The study was registered at ClinicalTrials.gov (NCT02314689; NCT02697240), where the inclusion and exclusion criteria are described. Th phase 1 study was performed in two steps. Step 1 included a dose escalation bolus infusion of IV citrulline in steady-state SCD to determine the PK and safety profile with a peak goal plasma citrulline concentration of 80–100 lmol/l (Barr et al, 2007). Step 2 of the study was performed to evaluate safety and PK during a vaso-occlusive crisis. The study drug, L-citrulline, was administered as open label vials of 50 mg/ml (5%) isotonic solution. Plasma sampling for PK studies were collected at specific time points. Briefly, for amino acid analysis, deproteinated plasma samples were subjected to cation exchange chromatography using a 4-component pH and ionic strength graded lithium citrate buffer system on a Beckmann 7300 amino acid analyser (Beckmann, Palo Alto, CA). Data obtained for each patient was fitted to a single-compartment PK model. The appearance of citrulline in plasma was described by a zeroorder process (rate of citrulline appearance, Rapp) to account for endogenous production, whereas the removal of citrulline was determined by a first-order process (constant of citrulline removal, krem). It was assumed that the values of all parameters remained constant for each patient during the course of plasma sampling. Scientist v2.0 (Micromath Scientific Software, St. Louis, MO) was used to fit the plasma citrulline concentration to the PK model by a weighted, least squares procedure to obtain values for Rapp, krem, and the volume of distribution (Vd). Clearance was calculated as the product of krem and Vd. For safety assessments, the Investigator determined the intensity of any adverse event (AE) according to the National Cancer Institute Common Terminology Criteria for Adverse Events Version 4.03 (https://evs.nci.nih.gov/ftp1/CTCAE/ CTCAE_4.03_2010-06-14_QuickReference_5x7.pdf) and their causal relationship. A Data Safety Monitoring Board, comprising 3 physicians who were not related to the study, reviewed every third subject. A total of 8 subjects with SCD were enrolled in this phase 1 study of IV citrulline: four participants were enrolled in Step 1 and another four participants in Step 2. Patient demographics, genotype and baseline blood counts are shown in Table SI. In the first cohort of four participants, the IV bolus infusion of 20 mg/kg of L-citrulline yielded a mean peak level of 259 lmol/l and trough level in the range of 20–40 lmol/l at 4 h after infusion (Fig 1). Citrulline PK parameters for Step 1 with bolus infusion are shown in Table SII. Pharmacokinetic model simulations indicated a 20 mg/kg bolus dose of IV citrulline followed by 7 mg/kg per hour continuous infusion was needed to maintain the target citrulline plasma concentration of 100 lmol/l. Subsequently, four subjects with VOC were enrolled to receive IV citrulline bolus and continuous infusion. Individuals with VOC showed significantly lower baseline citrulline levels compared to steady-state (mean SD: 9 37 1 43 vs. correspondence