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Nitric oxide depletion secondary to arginase induced arginine deficiency has been shown to be important in the pathophysiology of vaso-occlusion in sickle cell pain crisis. Our objective of this study was to perform a comprehensive amino acid evaluation during sickle cell pain crisis. In a total of 58 subjects (29 in steady-state sickle cell disease and 29 with sickle cell pain crisis), the amino acids related to nitric oxide pathway was significantly decreased during sickle cell pain crisis compared to steady-state sickle cell disease: arginine (p = 0.001), citrulline (p = 0.012), and ornithine (p = 0.03). In addition, the amino acids related to energy metabolism was significantly decreased during a pain crisis: asparagine (p0.001), serine (p = 0.002), histidine (p = 0.017), alanine (p = 0.004), tyrosine (p = 0.012), methionine (p = 0.007), cystine (p = 0.016), isoleucine (p = 0.016) and lysine (p = 0.006). The amino acid related to oxidative stress were significantly higher during a sickle cell pain crisis (glutamic acid (p0.001). Furthermore, multivariate analysis with partial least squares-discriminant analysis (PLS-DA) showed that deficiencies of the amino acids arginine, asparagine, citrulline, methionine and alanine were the most important related to sickle cell pain crisis.

Aging is associated with a decline in physiological function and exercise performance. These effects are mediated, at least in part, by an age-related decrease in the bioavailability of nitric oxide (NO), a ubiquitous gasotransmitter and regulator of myriad physiological processes. The decrease in NO bioavailability with aging is especially apparent in sedentary individuals, whereas older, physically active individuals maintain higher levels of NO with advancing age. Strategies which enhance NO bioavailability (including nutritional supplementation) have been proposed as a potential means of reducing the age-related decrease in physiological function and enhancing exercise performance and may be of interest to a range of older individuals including those taking part in competitive sport. In this brief review we discuss the effects of aging on physiological function and endurance exercise performance, and the potential role of changes in NO bioavailability in these processes. We also provide a summary of current evidence for dietary supplementation with substrates for NO production - including inorganic nitrate and nitrite, l-arginine and l-citrulline - for improving exercise capacity/performance in older adults. Additionally, we discuss the (limited) evidence on the effects of (poly)phenols and other dietary antioxidants on NO bioavailability in older individuals. Finally, we provide suggestions for future research.

Purpose To determine if plasma levels of six arginine and citrulline-related metabolites (arginine, citrulline, asymmetric dimethylarginine [ADMA], ornithine, proline, and argininosuccinate) differ between patients with type 2 diabetes and diabetic retinopathy (DR) and type 2 diabetic controls or between patients with proliferative DR (PDR) and non-proliferative DR (NPDR). Design Cross-sectional study Methods Adults with type 2 diabetes were recruited from the Vanderbilt Eye Institute. Exclusion criteria included non-diabetic retinal disease. Plasma metabolite levels were quantified in 159 diabetic controls and 156 DR patients (92 NPDR, 64 PDR) using isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Metabolite levels were compared using Wilcoxon Rank Sum test and logistic regressions adjusting for age, sex, hemoglobin A1c, diabetes duration, statin use, and angiotensin-converting enzyme inhibitor or angiotensin II receptor blocker use. A secondary analysis that included creatinine in the regression model was performed for the subset of patients with available creatinine values (135 diabetic controls, 100 DR patients [58 NPDR, 42 PDR]). Results Multivariable logistic regression analyses determined arginine (OR = 1.20, [1.06-1.38], P = 0.0067) and citrulline (OR = 1.53, [1.20-1.98], P = 0.0025) were significantly elevated in DR patients compared to diabetic controls. While ADMA differed between NPDR and PDR patients in the primary analysis (OR = 1.56, [1.15-2.16], P = 0.0051), it was not significantly different when adjusting for creatinine (OR = 1.30, [0.90-1.91], P = 0.15). Conclusions Plasma arginine and citrulline were significantly elevated in type 2 diabetic patients with DR compared to diabetic controls. None of the tested metabolites significantly differed between NPDR and PDR patients in the adjusted analysis.

Diagnosing internal herniation (IH) in Roux-en-Y gastric bypass (RYGB) patients with acute abdominal pain poses a diagnostic challenge. Diagnostic laparoscopy is often required for a definitive diagnosis. We hypothesized that intestinal ischemia biomarkers would aid in the diagnosing of IH.To explore intestinal ischemia biomarkers in diagnosing IH.University Hospital, Sweden.Prospective inclusion of 46 RYGB patients admitted for acute abdominal pain between June 2015 and December 2017. Blood samples for analysis of citrulline, intestinal fatty acid-binding protein (I-FABP), and D-dimer were drawn72 hours from admission and compared between patients with IH (n = 8), small bowel obstruction (SBO) (n = 5), other specified diagnoses (n = 12), or unspecified abdominal pain (n = 21). Levels of white blood cell count (WBC), C-reactive protein (CRP), and lactate at admission were compared. A prospective pain questionnaire for time of pain onset and level of pain at onset and at admission was analyzed.None of the investigated biomarkers differed significantly between diagnosis categories. Most patients with IH had normal CRP, WBC, and D-dimer levels while their lactate levels were significantly lower (P = .029) compared with the rest of the cohort. Neither pain level nor pain duration differed between the groups.This study shows that citrulline, I-FABP, and D-dimer cannot be used to diagnose IH and indicates that CRP, D-dimer, and lactate are rarely elevated by an IH. Furthermore, pain intensity and duration cannot differentiate patients with IH. A diagnostic laparoscopy remains the gold standard to diagnose and rule out an IH.

The use of cyclosporine A (CsA) is associated with different adverse effects including hypertension and nephrotoxicity. The present study aimed to compare the inhibitory effects of l-argininel-citrulline on CsA-induced blood pressure and biochemical changes in the serum of rats.Thirty-six rats were divided into 6 groups received daily: (1) 1ml distilled water, (2) 200mg/kg l-citrulline IP, (3) 25mg/kg CsA SC, (4) CsA+l-citrulline with the same dose of the former groups, (5) 200mg/kg l-arginine IP and (6) l-arginie+CsA with the same doses of group 4 for 7 days.The changes in the blood pressure, heart rate, creatinine, BUN, glucose and C-reactive protein (CRP) of the serum were determined in the treated animals. Significant (p0.001) increase was shown in the blood pressure and heart rate of CsA treated rats compared to the control group. There were also a significant (p0.05) increase in the creatinine, BUN and glucose, but a decrease in the CRP value in the CsA-treated group. However, l-citrulline significantly (p0.001) inhibited the changes in the blood pressure and heart rate in CsA-treated as well as it was able to reduce blood pressure in non-treated group significantly (p0.01). l-citrulline also inhibited the increased levels of BUN and creatinine induced by CsA, while, l-arginine was able to prevent the increased blood pressure and creatinine occurs after administration of CsA.These findings suggest that the l-citrulline is more efficient than l-arginine against the adverse effects induced by cyclosporine.

Proximal urea cycle disorders (PUCDs) are not included in most newborn screening programs due to the lack of adequate markers to monitor. Failure to alter citrulline and glutamine levels, the prognostic markers commonly used, can results in high false negative. Therefore, new biomarkers, prognostic of PUCDs, are strongly desirable.We used tandem mass spectrometry to analyze blood spot from PUCDs patients during their follow up in our referral center focusing on glutamine to glutamate (Gln/Glu) ratio. We reanalyzed the same specimens of three patients after two months and the specimen of a new patient with suspicious of PUCD disorder.Specimens of our patients shown a significant elevation of the ratio Gln/Glu compared to that of a healthy population (p 0.05) as well as the specimens analyzed after two months, while the glutamine concentration dropped. New patient, showing high value of the ratio, was molecularly confirmed as PUCD patient. We further analyzed the blood spots from a neonatal population in order to fix a cut-off value and include it in a newborn screening panel.Our preliminary results suggest that the Gln/Glu ratio could be a very useful diagnostic marker, more stable over time than glutamine, which could improve the performance in early PUCDs identification.

Citrullination is a post-translational modification (PTM) that converts peptidyl-arginine into peptidyl-citrulline; citrullination is catalyzed by the protein arginine deiminases (PADs). This PTM is associated with several physiological processes, including the epigenetic regulation of gene expression, neutrophil extracellular trap formation, and DNA-damage induced apoptosis. Notably, aberrant protein citrullination is relevant to several autoimmune and neurodegenerative diseases and certain forms of cancer. As such, the PADs are promising therapeutic targets. In this review, we discuss recent advances in the development of PAD inhibitors and activity-based probes, the development and use of citrulline-specific probes in chemoproteomic applications, and methods to site-specifically incorporate citrulline into proteins.

Background Watermelon (Citrullus lanatus) represents the largest cultivated member of the Cucurbitaceae family, showing great relevance from the economic point of view and largely consumed around the world accounting for about 7% of the world area dedicated to the production of vegetables, with 67% of the total quantity produced in China. It is characterized by several bioactive compounds, showing different chemical structures, such as carotenoids, xanthophylls, phenolic compounds, citrulline, and unsaturated fatty acids. The good amount of total polyphenols, vitamin C, citrulline and the excellent supply of lycopene, about 40% compared to raw tomatoes, give a measure of the importance of this fruit respect to the other crops considering the beneficial effects of these compounds on human health. Scope and approach In the last decades, this great interest towards natural bioactive compounds led many researchers to study watermelon as natural source of bioactive compounds, mainly when considering some biological properties, including antioxidant, antimicrobial and anti-inflammatory activities, inhibition or induction of enzymes, inhibition of receptor activities, together with induction/inhibition of gene expression. However, the most of scientific literature on this topic was based on in vitro assays, thus limiting the comprehensive understanding of the real health-promoting outcomes. Key findings and conclusions Therefore, the aim of this review was to present the up-to-date research carried out on watermelon phytochemicals, showing the most important biological activities reported from both in vitro and in vivo trials. Besides, the potential exploitation of watermelon by-products in the green circular economy and food-waste valorization has been discussed.

Summary Background & aims Plasma citrulline, a non-protein amino acid, is a biochemical marker of small intestine enterocyte mass in humans. Indeed, citrulline is highly correlated with residual bowel length in patients with short bowel syndrome. It is known to be synthesised in epithelial cells of the small intestine from other amino acids (precursors). Citrulline is then released into systemic circulation and interconverted into arginine in kidneys. If plasma citrulline concentration depends on abundance of intestinal amino acid transporters is not known. The aim of the present study was to explore whether plasma citrulline concentration correlates with the expression of intestinal amino acid transporters. Furthermore, we assessed if arginine in urine correlates with plasma citrulline. Methods Duodenal samples, blood plasma and urine were collected from 43 subjects undergoing routine gastroduodenoscopy. mRNA expression of seven basolateral membrane amino acid transporters/transporter subunits were assessed by real-time PCR. Plasma and urine amino acid concentrations of citrulline, its precursors and other amino acids were analysed using High Performance Liquid Chromatography measurements. Amino acid transporter mRNA expression was correlated with blood plasma and urine levels of citrulline and its precursors using Spearman's rank correlation. Likewise, urine arginine was correlated with plasma citrulline. Results Plasma citrulline correlated with the mRNA expression of basolateral amino acid transporter LAT4 (Spearman's r = 0.467, p = 0.028) in small intestine. None of the other basolateral membrane transporters/transporter subunits assessed correlated with plasma citrulline. Plasma citrulline correlated with urinary arginine, (Spearman's r = 0.419, p = 0.017), but not with urinary citrulline or other proteinogenic amino acids in the urine. Conclusions In this study, we showed for the first time that small intestinal basolateral LAT4 expression correlates with plasma citrulline concentration. This finding indicates that LAT4 has an important function in mediating citrulline efflux from enterocytes. Furthermore, urine arginine correlated with plasma citrulline, indicating arginine in the urine as possible additional marker for small intestine enterocyte mass. Finally, basolateral LAT4 expression along the human small intestine was shown for the first time.

Despite the saturating concentrations of intracellular l -arginine, nitric oxide (NO) production in endothelial cells (EC) can be stimulated by exogenous arginine. This phenomenon, termed the “arginine paradox” led to the discovery of an arginine recycling pathway in which l -citrulline is recycled to l -arginine by utilizing two important urea cycle enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Prior work has shown that ASL is present in a NO synthetic complex containing hsp90 and endothelial NO synthase (eNOS). However, it is unclear whether hsp90 forms functional complexes with ASS and ASL and if it is involved regulating their activity. Thus, elucidating the role of hsp90 in the arginine recycling pathway was the goal of this study. Our data indicate that both ASS and ASL are chaperoned by hsp90. Inhibiting hsp90 activity with geldanamycin (GA), decreased the activity of both ASS and ASL and decreased cellular l -arginine levels in bovine aortic endothelial cells (BAEC). hsp90 inhibition led to a time-dependent decrease in ASS and ASL protein, despite no changes in mRNA levels. We further linked this protein loss to a proteasome dependent degradation of ASS and ASL via the E3 ubiquitin ligase, C-terminus of Hsc70-interacting protein (CHIP) and the heat shock protein, hsp70. Transient over-expression of CHIP was sufficient to stimulate ASS and ASL degradation while the over-expression of CHIP mutant proteins identified both TPR- and U-box-domain as essential for ASS and ASL degradation. This study provides a novel insight into the molecular regulation l -arginine recycling in EC and implicates the proteasome pathway as a possible therapeutic target to stimulate NO signaling.

Introduction Ornithine transcarbamylase deficiency (OTCD) is the most common urea cycle disorder. Late-onset OTCD manifests after the neonatal period; therefore, if pre-symptomatic diagnosis and treatment are performed, it can improve the prognosis by preventing hyperammonemia. However, pre-symptomatic diagnosis is unreliable as the specific screening marker of OTCD has not been established yet. This retrospective study aimed to evaluate the pre-symptomatic blood citrulline levels in patients with late-onset OTCD. Methods Patients with late-onset OTCD who were born after the newborn screening based on tandem mass spectrometry (MS/MS-NBS) was started and were referred to Hyogo College of Medicine Hospital between 2014 and 2018 were included. Pre-symptomatic blood citrulline levels measured by MS/MS-NBS were retrospectively evaluated. Results Four patients were included in this study. The pre-symptomatic blood citrulline levels were 2.02, 4.50, 4.97, and 3.75 µmol/l, respectively. Compared with the citrulline levels in all newborns in Hyogo prefecture, these values were significantly low. Conclusions These results suggest the possibility that hypocitrullinemia detected by the MS/MS-NBS can be used as a screening marker for some patients with late-onset OTCD. Further retrospective evaluation of pre-symptomatic citrulline levels in patients with late-onset OTCD, as well as prospective monitoring of hypocitrullinemia on the MS/MS-NBS should be conducted.

Background Recent data propose a diagnostic and prognostic capacity for citrullinated histone H3 (H3Cit), a marker of neutrophil extracellular traps (NETs), in pathologic conditions such as cancer and thrombosis. However, current research is hampered by lack of standardized assays. Objectives We aimed to develop an assay to reliably quantify nucleosomal H3Cit in human plasma. Methods We assessed the common practice of in vitro enzymatically modified histone H3 as calibration standards and the specificity of available intrapeptidyl citrulline antibodies. Based on our findings, we developed and validated a novel assay to quantify nucleosomal H3Cit in human plasma. Results We show that enzymatically citrullinated H3 proteins are compromised by high enzyme-dependent lot variability as well as instability in plasma. We furthermore demonstrate that the majority of commercially available antibodies against intrapeptidyl citrulline display poor specificity for their reported target when tested against a panel of semi-synthetic nucleosomes containing distinct histone H3 citrullinations. Finally, we present a novel assay utilizing highly specific monoclonal antibodies and semi-synthetic nucleosomes containing citrulline in place of arginine at histone H3, arginine residues 2, 8, and 17 (H3R2,8,17Cit) as calibration standards. Rigorous validation of this assay shows its capacity to accurately and reliably quantify nucleosomal H3Cit levels in human plasma with clear elevations in cancer patients compared to healthy individuals. Conclusions Our novel approach using defined nucleosome controls enables reliable quantification of H3Cit in human plasma. This assay will be broadly applicable to study the role of histone citrullination in disease and its utility as a biomarker.

Background Ornithine transcarbamylase deficiency (OTCD) is pleomorphic congenital hyperammonemia, in which the prognosis of the patient is determined both by genotype and environmental factors. This study investigated the clinical and biochemical characteristics of OTCD patients with different prognosis. Method Of 35 OTCD patients, six males deceased at the first disease-onset, 17 males survived and had controllable ammonia levels after treatment, and 12 females survived through the first disease-onset but had intractable hyperammonemia and high mortality. Fasting blood samples from patients collected at three disease stages were used for the analysis of amino acid (AA) profile, acylcarnitine profile, and micronutrients. Differences in profiles between patients and healthy controls and within patient groups were studied. Results All OTCD patients had accumulation of glutamine, homocitrulline, lysine, glutamate, cystathionine, and pipecolic acid, as well as deficiency of citrulline, tryptophan, threonine, and carnitine. For male non-survivors, most other AAs and long-chain acylcarnitines were elevated at disease onset, of which the levels of creatine, N-acetylaspartic acid, and homoarginine were remarkably high. Male survivors and female patients had most other AAs at low to normal levels. Compared with male survivors, female patients had much lower protein-intolerance, as indicated by significantly lower levels of protein consumption indicators, including essential AAs, 1-methylhistidine, acylcarnitines et al., but high levels of ammonia. Female patients still had significantly higher levels of citrulline, homocitrulline, and citrulline/arginine compared to male survivors. Conclusion Unique profiles were observed in each group of OTCD patients, indicating specific physiological changes that happened to them.

BACKGROUND & AIMS: Disease-associated factors influence parenteral support (PS) reduction in response to teduglutide in patients with intestinal failure associated-short bowel syndrome (SBS-IF). We sought to determine correlative relationships between plasma citrulline levels, small bowel length, and PS volume.METHODS: A post hoc analysis of plasma citrulline levels from patients in the STEPS 24-week study of teduglutide in patients with SBS-IF. Plasma citrulline was assessed in all patients; patients were stratified 3 times into subgroups based on bowel anatomy, cause of SBS-IF, and baseline PS volumes. Correlation analyses used simple linear regression models. Statistical comparisons between study groups were conducted using 2-sided t tests for 2 independent mean differences.RESULTS: Baseline plasma citrulline correlated with remnant small bowel length (r = 0.355, P = 0.002), but not with baseline PS volume (r = -0.167, P = 0.14), in the overall population. There was a robust correlation between the baseline and Week 24 citrulline (r = 0.705, P < 0.0001), and an inverse correlation between change from baseline in citrulline and PS volume from baseline to Week 24 (r = -0.359, P = 0.001). In all subgroups, patients treated with teduglutide showed numerically greater increases in plasma citrulline at Week 24 compared with placebo.CONCLUSION: Baseline plasma citrulline showed significant correlations with small bowel length in patients with ≥50% colon remaining/no stoma/colon-in-continuity, and patients with SBS-IF causes other than IBD/vascular disease. Citrulline levels may correlate with PS changes in response to teduglutide and more research may reveal a relationship between citrulline levels within the heterogeneous population of patients with SBS-IF. ClinicalTrials.gov NCT00798967, ClinicalTrialsRegister.eu 2008-006193-15.

Controlling metabolism of engineered microbes is important to modulate cell growth and production during a bioprocess. For example, external parameters such as light, chemical inducers, or temperature can act on metabolism of production strains by changing the abundance or activity of enzymes. Here, we created temperature-sensitive variants of an essential enzyme in arginine biosynthesis of Escherichia coli (argininosuccinate synthetase, ArgG) and used them to dynamically control citrulline overproduction and growth of E. coli. We show a method for high-throughput enrichment of temperature-sensitive ArgG variants with a fluorescent TIMER protein and flow cytometry. With 90 of the thus derived ArgG variants, we complemented an ArgG deletion strain showing that 90% of the strains exhibit temperature-sensitive growth and 69% of the strains are auxotrophic for arginine at 42 °C and prototrophic at 30 °C. The best temperature-sensitive ArgG variant enabled precise and tunable control of cell growth by temperature changes. Expressing this variant in a feedback-dysregulated E. coli strain allowed us to realize a two-stage bioprocess: a 33 °C growth-phase for biomass accumulation and a 39 °C stationary-phase for citrulline production. With this two-stage strategy, we produced 3 g/L citrulline during 45 h cultivation in a 1-L bioreactor. These results show that temperature-sensitive enzymes can be created en masse and that they may function as metabolic valves in engineered bacteria., Graphical abstract Image 1, Highlights • Method to enrich temperature-sensitive enzymes en masse. • Temperature-sensitive enzymes function as metabolic valve. • Temperature controlled two-stage production of citrulline.

Peptidylarginine deiminases (PADs) are phylogenetically conserved calcium-dependent enzymes which post-translationally convert arginine into citrulline in target proteins in an irreversible manner, leading to functional and structural changes in target proteins. Protein deimination can cause the generation of neo-epitopes, affect gene regulation and also allow for protein moonlighting and therefore facilitate multifaceted functions of the same protein. PADs are furthermore a key regulator of cellular release of extracellular vesicle (EVs), which are found in most body fluids and participate in cellular communication via transfer of cargo proteins and genetic material. In this study, post-translationally deiminated proteins and EVs were assessed in sera of two seal species, grey seal and harbour seal. We report a poly-dispersed population of serum-EVs, which were positive for phylogenetically conserved EV-specific markers and characterised by transmission electron microscopy. A number of deiminated proteins critical for immune and metabolic functions were identified in the seal sera and varied somewhat between the two species under study, while some targets were in common. EV profiles of the seal sera further revealed that key microRNAs for inflammation, immunity and hypoxia also vary between the two species. Protein deimination and EVs profiles may be useful biomarkers for assessing health status of sea mammals, which face environmental challenges, including opportunistic infection, pollution and shifting habitat due to global warming.

Postmenopausal status is associated with an increase in total and abdominal body fat as well as increased incidence of insulin resistance and cardiovascular disease. The purpose of this study was to determine if watermelon supplementation affects select systemic markers of atherosclerosis and measures of insulin resistance in overweight and obese postmenopausal women. We hypothesized that overweight and obese postmenopausal women consuming 100% watermelon puree daily for 6 weeks would have improved levels of select systemic markers connected with cardiovascular disease without changing markers of insulin resistance. To test this hypothesis, overweight and obese postmenopausal women were recruited to participate in this study. Participants were randomly assigned to either the control group (no intervention) or the watermelon puree group (WM) for 6 weeks. Plasma concentration of markers connected with atherosclerosis and glycemic control were measured pre- and poststudy. A significant 6% decrease in soluble vascular cell adhesion molecule-1 occurred pre- to poststudy in WM, P = .003. The pattern of change in fasting blood glucose (P = .633), insulin (P = .158), and homeostatic model assessment–estimated insulin resistance (P = .174) did not differ between groups. Pre- to poststudy increases were measured in the fasting plasma concentration of l -arginine (8%, P = .005), cis-lycopene (32%, P = .003), and trans-lycopene (42%, P = .003) in WM. We conclude that 6 weeks of watermelon supplementation improved soluble vascular cell adhesion molecule-1 levels, a marker connected to atherogenesis, independent of changes in body composition or glycemic control.

Post-translational modifications of extracellular matrix proteins such as fibrinogen may lead to tolerance loss and have been implicated in rheumatoid arthritis (RA) pathogenesis. The purpose of this study was to determine whether fibrinogen (FIB) modified with citrulline (CIT), malondialdehyde-acetaldehyde (MAA) or both leads to altered macrophage polarization, peptidyl arginine deiminase (PAD) expression, or production of citrullinated proteins.PMA-treated U-937 cells (M0 cells) were stimulated with MAA, CIT or MAA-CIT modified FIB. Macrophage (M1/M2) phenotypes were evaluated by flow cytometry, RT-PCR, and ELISA. PAD enzyme expression and protein citrullination was evaluated using RT-PCR and Western Blot.Flow cytometry revealed that M0 macrophages stimulated with FIB-MAA-CIT resulted in mixed M1/M2 phenotypes as demonstrated by cell surface expression and mRNA levels of CD14, CD192, CD163, and CD206 (p 0.001 vs. others), and the release of IL-18, IP-10, CCL22, and IL-13 (p 0.001 vs. others). While FIB-MAA treated M0 cells demonstrated a mixed M1/M2 phenotype, cytokine and cell surface markers differed from FIB-MAA-CIT. Finally, M0 cells treated with FIB-CIT demonstrated markers and cytokines consistent with only the M1-like phenotype. Exposure of M0 cells to FIB-MAA-CIT (at 48 h) and FIB-MAA (at 24 h) led to increased mRNA expression and protein expression of PAD2 (p 0.001) with increased protein citrullination.These findings suggest that MAA-modification and citrullination of FIB, in isolation or combination, yield specific effects on macrophage polarization, PAD expression and citrullination that ultimately may induce inflammatory and fibrotic responses associated with RA.

BACKGROUND Arginine:glycine amidinotransferase, necessary for the conversion of arginine (Arg) to guanidinoacetic acid (GAA), is expressed mainly in kidney and pancreas. The methylation of GAA to creatine (Cre) primarily occurs in the liver. The role of the gut in Cre homeostasis has not been characterized. OBJECTIVE We aimed to quantify the contribution of kidney, pancreas, and gut as sources of GAA for Cre synthesis. METHODS Sow-reared, feed-deprived Yucatan miniature piglets (17-21 d old) were randomly assigned to acute intravenous treatments (expressed in μmol/kg/min) of: 1) Arg (4.8) + methionine (1.4) (Arg/Met), 2) Cre (0.6) with Arg/Met (Cre/Arg/Met), 3) citrulline (4.8) + methionine (1.4) (Cit/Met), or 4) alanine (6.2) (Ala). Suckling piglets were also studied. RESULTS Renal GAA release was higher during Cit/Met compared with all other treatments (53-360% higher; P

Supplementing the diet with functional ingredients is a key strategy to improve fish performance and health in aquaculture. The amino acids of the urea and nitric oxide (NO) cycles - arginine, ornithine and citrulline - perform crucial roles in the immune response through the generation of NO and the synthesis of polyamine used for tissue repair. We previously found that citrulline supplementation improves and maintains circulating free arginine levels in rainbow trout more effectively than arginine supplementation. Here, to test whether supplementation of urea cycle amino acids modulates the immune response in rainbow trout (Oncorhynchus mykiss), we supplemented a commercial diet with high levels (2% of total diet) of either arginine, ornithine or citrulline during a 7-week feeding trial, before challenging fish with the bacterium Aeromonas salmonicida. We carried out two separate experiments to investigate fish survival and 24 h post-infection to investigate the immediate response of free amino acid levels, and transcriptional changes in genes encoding urea cycle, NO cycle and polyamine synthesis enzymes. There were no differences in percentage fish mortality between diets, however there were numerous highly significant changes in free amino acid levels and gene expression to both dietary supplementation and infection. Out of 26 amino acids detected in blood plasma, 8 were significantly changed by infection and 9 by dietary supplementation of either arginine, ornithine or citrulline. Taurine, glycine and aspartic acid displayed the largest decreases in circulating levels in infected fish, while ornithine and isoleucine were the only amino acids that increased in concentration. We investigated transcriptional responses of the enzymes involved in arginine metabolism in liver and head kidney; transcripts for polyamine synthesis enzymes showed highly significant increases in both tissues across all diets following infection. The paralogous arginase-encoding genes, Arg1a, Arg1b, Arg2a and Arg2b, displayed complex responses across tissues and also due to diet and infection. Overall, these findings improve our understanding of amino acid metabolism following infection and suggests new potential amino acid targets for improving the immune response in salmonids.

International audience; BACKGROUND & AIMS: As citrulline is produced by small intestine, plasma citrulline concentration is decreased and may become essential in patients with short bowel syndrome (SBS). In a rat model of SBS, citrulline supplementation enhanced muscle protein synthesis. The aim of the study was to determine whether citrulline impacts whole body protein metabolism in patients with SBS. METHODS: Nine adults with non-malignant SBS (residual small bowel 90 ± 48 cm; mean ± SD) who were in near-normal nutritional status without any artificial nutrition, were recruited long after surgery. They received 7-day oral supplementation with citrulline (0.18 g/kg/day), or an iso-nitrogenous placebo in a randomized, double-blind, cross-over design with a 13-day wash-out between regimens, and an intravenous 5-h infusion of L-[1-13C]-leucine in the postabsorptive state to assess protein metabolism after each regimen. RESULTS: Plasma citrulline concentration rose 17-fold (25 ± 9 vs. 384 ± 95 μmol/L) and plasma arginine 3-fold after oral citrulline supplementation (both p < 4 × 10-6). Supplementation did not alter leucine appearance rate (97 ± 5 vs. 97 ± 5 μmol kg-1.h-1; p = 0.88), leucine oxidation (14 ± 1 vs. 12 ± 1 μmol kg-1.h-1; p = 0.22), or non-oxidative leucine disposal (NOLD), an index of whole-body protein synthesis (83 ± 4 vs. 85 ± 5 μmol kg-1.h-1; p = 0.36), nor insulin or IGF-1 plasma concentrations. In each of the 3 patients with baseline citrulline

Nitric oxide synthase (NOS) catalyzes the transformation of l -arginine, molecular oxygen (O2), and NADPH-derived electrons to nitric oxide (NO) and l -citrulline. Under some conditions, however, NOS catalyzes the reduction of O2 to superoxide (O2−) instead, a phenomenon that is generally referred to as uncoupling. In principle, both the heme in the oxygenase domain and the flavins in the reductase domain could catalyze O2− formation. In the former case the oxyferrous (Fe(II)O2) complex that is formed as an intermediate during catalysis would dissociate to heme and O2−; in the latter case the reduced flavins would reduce O2 to O2−. The NOS cofactor tetrahydrobiopterin (BH4) is indispensable for coupled catalysis. In the case of uncoupling at the heme this is explained by the essential role of BH4 as an electron donor to the oxyferrous complex; in the case of uncoupling at the flavins it is assumed that the absence of BH4 results in NOS monomerization, with the monomers incapable to sustain NO synthesis but still able to support uncoupled catalysis. In spite of little supporting evidence, uncoupling at the reductase after NOS monomerization appears to be the predominant hypothesis at present. To set the record straight we extended prior studies by determining under which conditions uncoupling of the neuronal and endothelial isoforms (nNOS and eNOS) occurred and if a correlation exists between uncoupling and the monomer/dimer equilibrium. We determined the rates of coupled/uncoupled catalysis by measuring NADPH oxidation spectrophotometrically at 340 nm and citrulline synthesis as the formation of [3H]-citrulline from [3H]-Arg. The monomer/dimer equilibrium was determined by FPLC and, for comparison, by low-temperature polyacrylamide gel electrophoresis. Uncoupling occurred in the absence of Arg and/or BH4, but not in the absence of Ca2+ or calmodulin (CaM). Since omission of Ca2+/CaM will completely block heme reduction while still allowing substantial FMN reduction, this argues against uncoupling by the reductase domain. In the presence of heme-directed NOS inhibitors uncoupling occurred to the extent that these compound allowed heme reduction, again arguing in favor of uncoupling at the heme. The monomer/dimer equilibrium showed no correlation with uncoupling. We conclude that uncoupling by BH4 deficiency takes place exclusively at the heme, with virtually no contribution from the flavins and no role for NOS monomerization.

Citrulline, a non-protein amino acid, is present in large amounts in watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai Cucurbitaceae) fruits. Amino acid profiling of various tissues of cv. Charleston Gray during plant development confirmed progressive accumulation of citrulline only in the fruit flesh and rind tissues. Citrulline content was positively correlated with precursor (ornithine) and by-product (arginine) amino acids during fruit ripening. Genetic variation in the partitioning of citrulline and related amino acids in the flesh and rind tissues was confirmed in a sub-set of watermelon cultivars. No correlation was established between morphological fruit traits (size and rind properties) and citrulline content. To understand the regulation of citrulline accumulation, we investigated the expression of genes associated with its biosynthesis and catabolism in flesh and rind tissues during fruit development. The expression of ornithine carbamoyltransferase (OTC) involved in the ultimate step of citrulline synthesis remained steady in both tissues. The expression of N-acetylornithine aminotransferase (N-AOA) involved in the production of N-acetylornithine and N-acetylornithine deacetylase (AOD-3) involved in ornithine synthesis coincided with increasing accumulation of citrulline in flesh and rind tissues during fruit development. Down-regulation N-acetylornithine-glutamate acetyltransferase (N-AOGA) suggests the subordinate role of the non-cyclic pathway in citrulline synthesis. Eccentricity between citrulline accumulation and expression of carbamoyl phosphate synthases (CPS-1, CPS-2) during fruit development suggest that the localized synthesis of carbamoyl phosphates may not be required for citrulline synthesis. Most genes involved in citrulline break-down (Argininosuccinate synthases - ASS-1, ASS-2, and ASS-3, Argininosuccinate lyases - ASL-1, Ornithine decarboxylase - ODC, Arginine decarboxylase - ADC) were consistently down-regulated during fruit development.

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of arginine residues to citrulline residues. Aberrant levels of PAD activity are associated with various diseases, such as rheumatoid arthritis, Alzheimer's disease, and multiple sclerosis, so there is a need for simple and convenient high-throughput screening systems to discover PAD inhibitors as candidate therapeutic agents. Here, we report a highly sensitive off/on-type fluorescence probe for PAD activity based on the donor-excited photoinduced electron transfer (d-PeT) mechanism, utilizing the specific cycloaddition reaction between the benzil group of the probe and the ureido group of the PAD product, citrulline, under acidic conditions. We synthesized and functionally evaluated a series of probes bearing substituents on the benzil phenyl group, and found that 4MEBz-FluME could successfully detect citrulline with higher sensitivity and broader dynamic range than our previously reported fluorescence probe, FGME. Moreover, we succeeded in establishing multiple assay systems for PAD subtypes activities, including PAD2 and PAD4, with 4MeBz-FluME thanks to its high sensitivity. We expect that our fluorescence probes will become a powerful tool for discovering PAD inhibitors of several subtypes. Thus, it should be suitable for high-throughput screening of chemical libraries for inhibitors of PADs.

BACKGROUND: Citrulline (CIT), is not extracted by the splanchnic area, can stimulate muscle protein synthesis and could potentially find clinical applications in conditions involving low amino acid (AA) intake, such as in malnourished older subjects. OBJECTIVE: Our purpose was to research the effects of CIT supplementation on protein metabolism in particular on non-oxidative leucine disposal (NOLD, primary endpoint), and splanchnic extraction of amino acids in malnourished older patients. DESIGN: This prospective randomized multicenter study determined whole-body and liver protein synthesis, splanchnic protein metabolism and appendicular skeletal muscle mass (ASMM) in 24 malnourished older patients [80-92 years; 18 women and 6 men] in inpatient rehabilitation units. All received an oral dose of 10 g of CIT or an equimolar mixture of six non-essential amino acids (NEAAs), as isonitrogenous placebo, for 3 weeks. RESULTS: NOLD and albumin fractional synthesis rates were not different between the NEAA and CIT groups. Splanchnic extraction of dietary amino acid tended to decrease (p = 0.09) in the CIT group (45.2%) compared with the NEAA group (60.3%). Total differences in AA and NEAA area under the curves between fed-state and postabsorptive-state were significantly higher in the CIT than in the NEAA group. There were no significant differences for body mass index, fat mass (FM), lean mass (LM) or ASMM in the whole population except for a tendential decrease in FM for the citrulline group (p = 0.089). Compared with Day 1, lean mass and ASMM significantly increased (respectively p = 0.016 and p = 0.018) at Day 20 in CIT-treated women (mean respective increase of 1.7 kg and 1.1 kg), and fat mass significantly decreased (p = 0.001) at Day 20 in CIT-group women (mean decrease of 1.3 kg). CONCLUSIONS: Our results demonstrate that CIT supplementation has no effect on whole-body protein synthesis or liver protein synthesis in malnourished older subjects. However, CIT supplementation was associated with a higher systemic AA availability. In the subgroup of women, CIT supplementation increased LM and ASMM, and decreased FM.

Peptidyl arginine deiminase (PAD) which mediates citrullination catalyzes the conversion of arginine residues of protein peptide chains to citrulline residues. Citrullination can be involved in the process of apoptosis, embryo development, regulation of myelin sheath function and other physiological processes. Besides, it can regulate the process of cell death, affect the formation of neutrophil extracellular traps (NETs) and produce anti-citrullinated protein antibody (ACPA) to participate in autoimmune diseases. In this manuscript, the regulatory effects of citrullination in normal physiology and autoimmune diseases are reviewed, and the effects of citrullination on immune cells in autoimmune diseases are discussed in detail.

As global pollution, microplastics pollution has aroused growing concerns. In our experiment, the effect of microplastics acute exposure on the liver of swordtail fish was investigated by using LC-MS metabolomics. Fishes treated with high concentration polystyrene microspheres (1 μm) for 72 h were divided into three concentration groups: (A) no microplastics, (B): 1 × 10

Peptidylarginine deiminase 4 (PAD4), a Ca

Meteorin-like (Metrnl) is a novel adipokine that is highly expressed in white adipose tissues. Whether Metrnl plays a role in rheumatoid arthritis (RA) remains unclear. In this study, sera from 159 RA patients, 28 osteoarthritis (OA) patients, and 50 healthy individuals were included. The serum levels of Metrnl were measured using an enzyme-linked immunosorbent assay. Clinical parameters, including disease activity score 28 (DAS28), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), rheumatoid factor (RF), antibodies to cyclic citrulline peptide (anti-CCP), inflammatory cytokines, and blood biochemical indices were collected.Metrnl levels were higher in RA patients compared to OA patients and controls. In the RA group, serum Metrnl levels were positively correlated with DAS28, RF, and CRP levels. However, in the RA group, serum Metrnl levels were not correlated with ESR, anti-CCP, immunoglobulins, and blood biochemical indices.This study showed that Metrnl is involved in the pathogenesis of RA. Increase in serum Metrnl levels is closely related to RA activity.

Although major adverse effects associated with nonsteroidal anti-inflammatory drugs (NSAIDs) are gastric injury, assessment of NSAIDs-induced gastrointestinal adverse effects is mostly dependent on endoscopy due to the lack of plasma biomarkers. Several amino acids associated with collagenase activity and gastric mucosal mass have been suggested as plasma biomarker candidates for gastric injury. Therefore, this study aimed to develop a liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the plasma biomarker candidates, i.e., acetylcarnitine, proline, hydroxyproline, citrulline, and arginine and evaluate their potential as a biomarker for NSAIDs-induced gastric injury. The method utilized simple protein precipitation with methanol and D4-citrulline as an internal standard (IS). The assay resulted in the lower limit of quantification (LLOQ) of 0.1 μg/mL for acetylcarnitine and 1 μg/mL for proline, hydroxyproline, citrulline, and arginine in the surrogate blank plasma. The intra- and inter-day accuracy ranged 82.5–111.2% for acetylcarnitine, 95.4–103.3% for proline, 98.9–106.4% for hydroxyproline, 99.5–103.5% for citrulline, and 87.4–105.3% for arginine. The precision was within 6.17%, 3.63%, 6.20%, 6.31%, and 6.17% for acetylcarnitine, proline, hydroxyproline, citrulline, and arginine, respectively. The developed assay was successfully applied to monitor the changes of the plasma levels of the five amino acids in rats and Beagle dogs following repeated oral administrations of aceclofenac. In rats, plasma concentrations of proline, hydroxyproline, and citrulline were significantly reduced after 4 days of aceclofenac administration compared to the control group. In dogs, plasma concentrations of proline and citrulline were significantly decreased after 7 days of aceclofenac administration compared to those obtained after the first aceclofenac administration. These data indicate that plasma levels of proline, hydroxyproline, and citrulline may be used as quantitative biomarkers of NSAIDs-induced gastric damage. The present assay could also be utilized to monitor the changes of these amino acids as potential indicators for various physiological and pathophysiological conditions.

Background Although fruit and vegetable-rich diets have beneficial effects on cardiovascular diseases, we have little knowledge of the impact of fruits and their constituents, iridoids and anthocyanins, on the l -arginine-ADMA-DDAH pathway. Our previous study demonstrated the modulation of those factors by the oral administration of the cornelian cherry fruit. Hypothesis/purpose We have assessed the effects of the oral administration of two main constituents isolated from the cornelian cherry fruit, iridoid loganic acid and anthocyanins, on l -arginine, its derivatives (ADMA, SDMA), metabolites (DMA, l -citrulline), and the hepatic DDAH activity and its isoform expression in rabbits fed a high-cholesterol diet. We have also analyzed eNOS expression in the thoracic aorta as well as the redox status in blood. Study Design In the present study, we used an animal model of diet induced atherosclerosis. For 60 days, white New Zealand rabbits were fed a standard diet, a 1% cholesterol enriched diet, or concomitantly with the investigated substances. l -arginine, ADMA, SDMA, DMA, and l -citrulline were assessed using the LC-MS/MS method. DDAH activity and redox parameters were analyzed spectrophotometrically. DDAH1 and DDAH2 isoform expressions were assessed by western blotting, mRNA expression of eNOS was quantified by real-time PCR. Results We demonstrated that the administration of loganic acid (20 mg/kg b.w.), and to a lesser extent of anthocyanins (10 mg/kg b.w.), caused an increase in the l -arginine level and the l -arginine/ADMA ratio. Also, both substances decreased ADMA, DMA, and l -citrulline, but not SDMA levels. Anthocyanins, but not loganic acid, enhanced the activity of DDAH in the liver. Anthocyanins also significantly enhanced both DDAH1 and DDAH2 expression, while loganic acid to a lesser extent enhanced DDAH1 but not DDAH2 expression. Both loganic acid and anthocyanins pronouncedly increased mRNA expression of eNOS in thoracic aortas. Both loganic acid and anthocyanins reversed the blood glutathione level depleted by dietary cholesterol. Cholesterol feeding decreased the blood GPx level, and the change was not reversed by anthocyanins or loganic acid. We did not observe any significant differences in the blood levels of MDA or SOD among the groups. Conclusion Iridoids and anthocyanins may modulate the l -arginine-ADMA pathway in subjects fed a high-cholesterol diet.

Peptidylarginine deiminase-4 (PAD4) is a calcium-dependent enzyme that catalyzes the conversion of arginine into citrulline of macromolecules in the body. It governs several processes including apoptosis, innate immunity (Netosis), and pluripotency. Dysregulated PAD4 plays a vital role in the occurrence and development of Rheumatoid arthritis (RA). Therefore, PAD4 is considered a promising target for diagnosing and treating RA. Over the last few years research has been carried out on PAD4 inhibitors. When administered it circulates to the entire body and inhibits PAD4 causing immunosuppression which may lead to infection. A growing number of studies have demonstrated infiltration and differentiation of monocytes and macrophages into the inflamed synovium, inducing overexpression of PAD4 levels in the inflamed joints. To overcome the above-mentioned critical issues, the targeted drug delivery systems inhibit PAD4 at the inflamed site. This review provides an update on the PAD4 inhibitors and emerging advanced drug delivery systems for the treatment of RA. Finally, we concluded that active targeting of PAD4 inhibitors to inflamed joints via hybrid nanocarriers provided an improved therapeutic efficacy, minimized extra synovial toxicity, and prevent the occurrence of inflammation in RA.

White striping (WS) is a major problem affecting the broiler industry. Fillets affected by this myopathy present pathologies that compromise the quality of the meat, and most importantly, make the fillets more prone to rejection by the consumer. The exact etiology is still unknown, which is why a metabolomics analysis was performed on breast samples of broilers. The overall objective was to identify biological pathways involved in the pathogenesis of WS. The analysis was performed on a total of 51 muscle samples and distinction was made between normal (n = 19), moderately affected (n = 24) and severely affected (n = 8) breast fillets. Samples were analyzed using gas chromatographic mass spectral analysis and liquid chromatography quadrupole time-of-flight mass spectrometry. Data were subsequently standardized, normalized and analyzed using various multivariate statistical procedures. Metabolomics allowed for the identification of several pathways that were altered in white striped breast fillets. The tricarboxylic acid cycle exhibited opposing directionalities. This is described in literature as the backflux and enables the TCA cycle to produce high-energy phosphates through matrix-level phosphorylation and, therefore, produce energy under conditions of hypoxia. Mitochondrial fatty acid oxidation was limited due to disturbances in especially cis-5–14:1 carnitine (log2 FC of 2, P < 0.01). Because of this, accumulation of harmful fatty acids took place, especially long-chain ones, which damages cell structures. Conversion of arginine to citrulline increased presumably to produce nitric oxide, which enhances blood flow under conditions of hypoxia. Nitric oxide however also increases oxidative damage. Increases in taurine (log2 FC of 1.2, P < 0.05) suggests stabilization of the sarcolemma under hypoxic conditions. Lastly, organic osmolytes (sorbitol, taurine, and alanine) increased (P < 0.05) in severely affected birds; likely this disrupts cell volume maintenance. Based on the results of this study, hypoxia was the most likely cause/initiator of WS in broilers. We speculate that birds suffering from WS have a vascular support system in muscle that is borderline adequate to support growth, but triggers like activity results in local hypoxia that damages tissue.

NG-Hydroxy- l -arginine (NOHA) is the intermediate product of the conversion of l -arginine to l -citrulline and nitric oxide (NO) by nitric oxide synthase (NOS). NO is further oxidized to nitrite and nitrate which circulate in the blood and are excreted in the urine. Nitrite and nitrate may therefore serve as surrogates of NO synthesis. NOHA has been reported to occur in various cells and in blood of animals and humans. The concentration of nitrite in the circulation is comparable to the concentration of NOHA in plasma and serum of humans and laboratory animals. NOHA is a relatively unstable compound and the interaction of its NG-hydroxy group with redox active species or during sample treatment such as derivatization in the heat may yield N-containing compounds including nitrite and nitrate. In theory, NOHA may interfere with the analysis of nitrite and nitrate. In the present study, we investigated a possible interference of synthetic NOHA (0–400 μM) with the gas chromatography-negative ion chemical ionization–mass spectrometry (GC-NICI-MS) method of analysis of circulating and urinary nitrite and nitrate involving derivatization with pentafluorobenzyl (PFB) bromide in aqueous acetone at 50 °C for 5 min (nitrite) or for 60 min (nitrite and nitrate). Our results show that NOHA does not interfere with the measurement of nitrite and nitrate in human plasma and urine by this method at concentrations up to 400 μM.

The arginine deiminase (ADI) pathway catalyses the degradation of l -arginine via the intermediate l -citrulline into l -ornithine, ammonia, and CO2. This constitutes an important metabolic feature of some lactic acid bacteria (LAB) in their response to acid stress. We investigated the capacity of four thermophilic starters to degrade arginine via the ADI pathway and studied their influence on the ripening and quality of experimental Gruyere cheeses. One of the starters could not degrade arginine, while another had an impaired ADI pathway and formed mainly citrulline. In cheeses produced with the two starters exhibiting a fully functional ADI pathway, ornithine content, pH-value, and CO2 and eye formation were significantly increased, indicating that the quality and ripening of the cheeses were affected by ADI metabolism. This is the first study investigating thermophilic starter cultures that contain Lactobacillus delbrueckii subsp. lactis with a functional ADI pathway converting arginine into ornithine and CO2 in cheese.

Watermelon (Citrullus lanatus) is rich in l-citrulline, an l-arginine precursor that may reduce cardiovascular disease risk. The purpose of this study was to compare the effects of watermelon powder and l-arginine on lipid profiles, antioxidant capacity, and inflammation in rats fed an atherogenic diet. We hypothesized that watermelon and l-arginine would increase antioxidant capacity and reduce blood lipids and inflammation by modulating hepatic gene expression. Male Sprague-Dawley rats aged 21 days (N = 32) were assigned to 3 groups and fed diets containing watermelon powder (0.5% wt/wt), l-arginine (0.3% as 0.36% l-arginine HCl wt/wt), or a control diet for 9 weeks. Watermelon and l-arginine supplementation improved lipid profiles by lowering serum concentrations of triglycerides, total cholesterol, and low-density lipoprotein cholesterol (P .050). Serum concentrations of C-reactive protein were significantly lower (P .050) in the watermelon and l-arginine groups. Rats in the watermelon and l-arginine groups showed reduced oxidative stress, increased total antioxidant capacity, and higher concentrations of superoxide dismutase and glutathione S-transferase (P .050). Concentrations of aspartate aminotransferase, alkaline phosphatase, and lactate dehydrogenase were lower (P .050) in the watermelon and l-arginine groups. Watermelon and l-arginine consumption upregulated hepatic gene expression of endothelial nitric oxide synthase and downregulated expression of fatty acid synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, sterol regulatory element-binding protein 1, sterol regulatory element-binding protein 2, cyclooxygenase-2, and nuclear factor-κB p65 (P .050). The results support the hypothesis that watermelon and arginine improve cardiovascular disease risk factors including lipid profile, antioxidant capacity, and inflammation by altering relevant gene expression.

Plasma citrulline is decreased in cases of severe intestinal injury with apparent villus and cellular atrophy. However, the fluctuation of plasma citrulline in slight intestinal injury remains to be investigated. To clarify this, irinotecan at 30 mg/kg or 60 mg/kg was administered intravenously to rats. Irinotecan reduced plasma citrulline concentrations compared to those in the pair-fed control, being concurrent with slight single cell necrosis and mucosal epithelium regeneration in the small intestine without apparent villus and cellular atrophy. Gene expression of enzymes converting glutamine to citrulline was decreased in the small intestine of the injury model. Moreover, citrulline and arginine levels in the ileum were decreased without alterations to glutamine and glutamate levels, indicating that citrulline synthesis from glutamine was impaired. Metabolome analysis revealed that plasma citrulline and arginine levels were decreased, while there were no marked alterations in other amino acids, metabolites of glycolysis, ketone bodies, or fatty acids. These results suggested that a decreased plasma citrulline level was unlikely to result from amino acid catabolism in response to malnutrition. In conclusion, plasma citrulline concentration reflects slight intestinal injury without apparent villus and cellular atrophy, and thus, it would be a sensitive biomarker for the small intestinal injury.

Publisher's version (útgefin grein), Pentraxins arefluid phase pattern recognition molecules that form an important part of the innate immunedefence and are conserved betweenfish and human. In Atlantic cod (Gadus morhuaL.), two pentraxin-likeproteins have been described, CRP-I and CRP-II. Here we show for thefirst time that these two CRP forms arepost-translationally deiminated (an irreversible conversion of arginine to citrulline) and differ with respect totissue specific localisation in cod ontogeny from 3 to 84 days post hatching. While both forms are expressed inliver, albeit at temporally differing levels, CRP-I shows a strong association with nervous tissue while CRP-II isstrongly associated to mucosal tissues of gut and skin. This indicates differing roles for the two pentraxin types inimmune responses and tissue remodelling, also elucidating novel roles for CRP-I in the nervous system. Thepresence of deimination positive bands for cod CRPs varied somewhat between mucus and serum, possiblyfacilitating CRP protein moonlighting, allowing the same protein to exhibit a range of biological functions andthus meeting different functional requirements in different tissues. The presentedfindings may further currentunderstanding of the diverse roles of pentraxins in teleost immune defences and tissue remodelling, as well as invarious human pathologies, including autoimmune diseases, amyloidosis and cancer., Thanks are due to Matthías Oddgeirsson, Agnar Steinarsson and other staff the staff at the Marine Institute's Mariculture Laboratory, Staður Grindavík, Iceland for providing sampling facilities and the fish. The authors also thank Margrét Jónsdóttir, Keldur, Institute for Experimental Pathology University of Iceland, for cod larvae sample preparation. This work was partly supported by The Icelandic Research Council (RANNIS), EC grant Fishaid QLK2-CT-2000-01076 and a University of Westminster start-up grant to SL. The authors declare no competing interest.

Introduction COMP-4 is a natural compound-based dietary supplement consisting of the combination of ginger, Paullinia cupana, muira puama and l -citrulline, which when given long-term has been shown in the aged rat to a) upregulate iNOS in the penile smooth muscle cells (SMC), b) reverse the corporal SMC apoptosis and fibrosis associated with corporal veno-occlusive dysfunction (CVOD), and c) improve resulting erectile function. To elucidate the mechanism of how COMP-4 and its individual components modulate the iNOS-cGMP pathway, an in vitro study was conducted using a rat corporal primary SMC culture to determine its effect on NOS, soluble guanylate cyclase (sGC), cGMP and the phosphodiesterase 5 enzyme (PDE5). Materials and methods Primary SMC cultures using the explant technique were initiated by cutting small pieces of corporal tissue from 8 week old Sprague-Dawley rats. The SMC were grown in Dulbecco media with 20% fetal calf serum. The SMC were then incubated with or without COMP-4 (0.69 mg/ml) or its ingredients alone (ginger: 0.225 mg/ml; muira puama, Paullinia cupana and l -citrulline each at 0.9 mg/ml) for up to 24 h mRNA and protein were extracted and used for the determination of NOS, sGC and PDE5 content. cGMP content was determined by ELISA. L-NIL (4 μM) was used as an inhibitor of iNOS activity. Results Compared to the control values, COMP-4 upregulated expression of cGMP by 85%, induced a 42 fold increase in sGC as well as a 15 fold increase in both iNOS protein and mRNA content while it decreased both PDE5 mRNA and protein content each by about 50%. L-NIL completely inhibited the effect of COMP-4 on cGMP production. When compared with each of the individual four components of COMP-4, it appears that COMP-4 itself had the most profound effect in modulating each one the specific steps within the iNOS-cGMP pathway. Conclusions This in vitro study demonstrates that COMP-4 is capable of activating the endogenous cellular iNOS-cGMP pathway within the CSM cells, which is theorized to be responsible for reducing the fibrosis and apoptosis as well as the CVOD observed in the aging rat penis. Further studies will be necessary in order to determine whether supplementation of COMP-4 on a daily basis may be beneficial in halting or reversing this aging related erectile dysfunction in the clinical setting.

Current newborn screening (NBS) for urea cycle disorders (UCD) is incomplete as only distal UCDs are included in most NBS programs by measuring elevated amino acid concentrations. NBS for the proximal UCDs involves the detection in NBS spots of low citrulline values, a finding which is often overlooked because it is considered to be inadequate. We retrospectively analyzed NBS blood spots from known UCD patients comparing the utility of the Region 4 Stork (R4S) interpretive tools to conventional cutoff based interpretation. This study shows the utility of R4S tools in detecting all UCDs, and provides evidence to support the nomination to add proximal UCDs to the recommended uniform screening panel.

The urea cycle disorders are a group of inherited biochemical diseases caused by a complete or partial deficiency of any one of the enzymes or transport proteins required to convert toxic ammonia into urea and to produce arginine and citrulline. The clinical manifestations of these disorders are mostly the result of acute or chronic hyperammonemia, which affects the central nervous system. Affected individuals can also develop hepatic dysfunction. These disorders can present at any age from the immediate newborn to later in life. Early diagnosis and treatment are key to improving outcomes.

Objectives: L-Glutamine is FDA-approved for sickle cell disease (SCD), yet the mechanism(s)-of-action are poorly understood. We performed a pharmacokinetics (pK) study to determine the metabolic fate of glutamine supplementation on plasma and erythrocyte amino acids in patients with SCD. Design: A pK study was performed where patients with SCD fasting for > 8 h received oral L-glutamine (10 g). Blood was analyzed at baseline, 30/60/90 min/2/3/4/8 hrs. A standardized diet was administered to all participants at 3 established time-points (after 2/5/7hrs). A subset of patients also had pK studies performed without glutamine supplementation to follow normal diurnal fluctuations in amino acids. Setting: Comprehensive SCD Center in Oakland, California Results: Five patients with SCD were included, three of whom performed pK studies both with and without glutamine supplementation. Average age was 50.6 ± 5.6 years, 60% were female, 40% SS, 60% SC. Plasma glutamine levels increased significantly after oral glutamine supplementation, compared to minimal fluctuations with diet. Plasma glutamine concentration peaked within 30-min of ingestion (p = 0.01) before decreasing to a plateau by 2-h that remained higher than baseline by 8 h. Oral glutamine also increased plasma arginine concentration, which peaked by 4-h (p = 0.03) and remained elevated through 8-h. Erythrocyte glutamine levels began to increase by 8-h, while erythrocyte arginine concentration peaked at 4-h. Conclusions: Oral glutamine supplementation acutely improved glutamine and arginine bioavailability in both plasma and erythrocytes. This is the first study to demonstrate that glutamine therapy increases arginine bioavailability and may provide insight into shared mechanisms-of-action between these conditionally-essential amino acids.

Augmented aortic systolic blood pressure (SBP) and wave reflection via sympathetic-mediated vasoconstriction elevates the risk for adverse cardiovascular events in older adults. L-citrulline (L-CIT) supplementation has shown to reduce aortic SBP and pulse pressure (PP) responses to cold pressor test (CPT) induced sympathoactivation in young men. The aim of this study was to elucidate the efficacy of L-CIT supplementation to attenuate aortic hemodynamic responses to CPT in older adults.Sixteen older adults were randomly assigned to placebo or L-CIT (6 g/day) for 14-days in a crossover, double-blind, placebo-controlled design. Brachial SBP and aortic SBP, PP, augmented pressure (AP), augmentation index standardized at 75 bpm (AIx@75), and pressure of the forward (Pf) and reflected (Pb) waves were evaluated at rest and during CPT pre- and post-intervention. Although no hemodynamic changes at rest, brachial SBP (Δ-12 ± 18 vs. Δ4 ± 14 mmHg; P = 0.008) and aortic SBP (Δ-10 ± 14 vs. Δ4 ± 12 mmHg; P = 0.005), PP (Δ-10 ± 12 vs. Δ4 ± 11 mmHg; P = 0.002), AP (Δ-4 ± 4 vs. Δ2 ± 7 mmHg; P = 0.004), AIx@75 (Δ-3.2 ± 7.2 vs. Δ2.2 ± 6.9%; P = 0.038), Pf (Δ-6 ± 10 vs. Δ3 ± 9 mmHg; P = 0.019), and Pb (Δ-4 ± 6 vs. Δ2 ± 6 mmHg; P = 0.008) responses to the CPT were significantly attenuated following L-CIT supplementation vs. placebo.L-CIT supplementation attenuated aortic pulsatile pressure and pressure wave reflection responses to CPT in older adults, providing possible cardioprotection during cold-induced sympathoactivation in older adults.

Citrulline can be converted into argininosuccinate by argininosuccinate synthetase (ASS1) in the urea cycle and the citrulline-nitric oxide cycle. However, the regulation and biological function of citrulline metabolism remain obscure in the immune system. Unexpectedly, we found that macrophage citrulline declines rapidly after interferon gamma (IFN-γ) and/or lipopolysaccharide (LPS) stimulation, which is required for efficient proinflammatory signaling activation. Mechanistically, IFN-γ and/or LPS stimulation promotes signal transducers and activators of transcription 1 (STAT1)-mediated ASS1 transcription and Janus kinase2 (JAK2)-mediated phosphorylation of ASS1 at tyrosine 87, thereby leading to citrulline depletion. Reciprocally, increased citrulline directly binds to JAK2 and inhibits JAK2-STAT1 signaling. Blockage of ASS1-mediated citrulline depletion suppresses the host defense against bacterial infection in vivo. We therefore define a central role for ASS1 in controlling inflammatory macrophage activation and antibacterial defense through depletion of cellular citrulline and, further, identify citrulline as an innate immune-signaling metabolite that engages a metabolic checkpoint for proinflammatory responses.

Increased presence of IL-22+ cells in the skin is a characteristic finding in skin barrier defects, such as psoriasis and atopic dermatitis. However, mechanistic insight into effects of IL-22 on epidermal functioning is yet to be elucidated. One crucial step during epidermal differentiation is deimination or citrullination. Here, we show reduced levels of peptidylarginine deiminase 1, an enzyme that converts peptidylarginine into citrulline in lesional psoriatic skin. IL-22 signaling through the IL-22 receptor complex was found to suppress expression of peptidylarginine deiminase 1 in epidermal keratinocytes. Subsequently, total peptidylarginine deiminase activity and extent of protein deimination in keratinocytes treated with IL-22 were reduced together with a significant decrease in deimination of keratin 1 and FLG, both important for epidermal differentiation. Vitamin D and acitretin partly restored the peptidylarginine deiminase 1 defect caused by IL-22. Collectively, we show that IL-22 downregulates deimination, thus identifying a potential target for treatment of skin barrier defects.

Inflammatory bowel disease (IBD) is a group of chronic inflammatory gastrointestinal diseases that causes worldwide suffering. Lactobacillus helveticus is a probiotic that can enhance intestinal barrier function via alleviation of excessive inflammatory response. Citrulline, a functional amino acid, has been reported to stimulate muscle synthesis and to function with a prebiotic-like action with certain Lactobacillus strains. The aim of this study was to investigate the potential synergistic effect of combining L. helveticus and citrulline on protection against damage induced by dextran sulfate sodium (DSS) in a mouse model. Six-week-old male C57BL/6J mice were fed with DSS water and randomly divided for administering with different milk treatments: 1) plain milk (control or DSS control), 2) 1% (w/v) citrulline enriched milk (Cit_milk), 3) milk fermented with L. helveticus (LHFM) and 4) DSS+milk fermented with L. helveticus with 1% (w/v) citrulline (Cit_LHFM). The treatment effects on the survival and macroscopic and microscopic signs were examined. All treatments presented different degrees of protective effects on attenuating the damages induced by DSS. All treatments reduced the body weight loss, disease activity index (DAI), histological scores, pro-inflammatory cytokine expression (IL-6, TNF-α and IFN-γ) and production (IL-4) (all P

Introduction Sleep apnea syndrome is characterized by intermittent hypoxia (IH), leading to an increase in blood pressure (BP), infarct size (IS), endothelial dysfunction, insulin resistance, and dyslipidemia. Insulin resistance and the metabolic syndrome associated with IH are also accompanied by an alteration in the metabolism of Arginine (Arg) [and its precursor Citrulline (Cit)]. As Arg availability directly impacts nitric oxide (NO) production, we speculate that myocardial Arg deficiency during IH may contribute to cardiovascular alterations induced by IH. Objective Knowing that Cit is a much better precursor of Arg than Arg itself, we propose to use Cit during IH to protect against IH-induced increase in BP and IS. Methods Four groups of rats (n = 8), were submitted to normoxia (N) or IH [14 days (d), 8 h per d, 30 s-O2 21%/30 s-O2 5%] and supplemented or not with Cit (1 g.kg−1.d−1). One normoxic per-feed (NPf) supplementary group was added to assess the impact of anorexia associated with IH. BW and food intake were daily measured. After 14 d, BP was measured by carotid catheterization. Hearts were perfused by the Langendorff method and submitted to global and total ischemia (30 min)-reperfusion (120 min) before measuring IS related to left ventricle area (I/V) by colorimetry and planimetry. Results Irrespective of diet, IH increases both systolic BP (141.3 ± 1.1 vs. 170.6 ± 5.5 mmHg in N and IH respectively, n = 4–8, P = 0.05) and I/V (27.2 ± 4.7% vs. 43.6 ± 4.5% in N and IH, respectively, n = 5–8, P Conclusion Under IH, Cit seems to increase the BP but decrease I/V. After completing groups, further biochemical and histological analyses will be performed to enlighten the mechanisms (NOx, NO synthase, oxidative stress…) potentially involved in the impact of Cit chronic supplementation.

Peptidyl arginine deiminases (PADs) catalyze the post-translational deimination of peptidyl arginine residues to form citrulline residues. Aberrant citrullination of histones by one of the PAD isozymes, PAD4, is associated with various diseases, including rheumatoid arthritis, so high-throughput screening systems are needed to identify PAD4 inhibitors as chemical tools to investigate the role of PAD4, and as candidate therapeutic agents. Here, we utilized the addition-cyclization reaction between phenylglyoxal and citrulline under acidic conditions to design turn-on fluorescent probes for citrulline based on the donor-excited photoinduced electron transfer (d-PeT) mechanism. Among several derivatives of phenylglyoxal bearing a fluorescent moiety, we found that FGME enabled detection of citrulline without a neutralization process, and we used it to establish a simple methodology for turn-on fluorescence detection of citrulline.

The HLA-DRB1 locus is strongly associated with rheumatoid arthritis (RA) susceptibility, whereupon citrullinated self-peptides bind to HLA-DR molecules bearing the shared epitope (SE) amino acid motif. However, the differing propensity for citrullinated/non-citrullinated self-peptides to bind given HLA-DR allomorphs remains unclear. Here, we used a fluorescence polarization assay to determine a hierarchy of binding affinities of 34 self-peptides implicated in RA against three HLA-DRB1 allomorphs (HLA-DRB1*04:01/*04:04/*04:05) each possessing the SE motif. For all three HLA-DRB1 allomorphs, we observed a strong correlation between binding affinity and citrullination at P4 of the bound peptide ligand. A differing hierarchy of peptide-binding affinities across the three HLA-DRB1 allomorphs was attributable to the β-chain polymorphisms that resided outside the SE motif and were consistent with sequences of naturally presented peptide ligands. Structural determination of eight HLA–DR4–self-epitope complexes revealed strict conformational convergence of the P4-Cit and surrounding HLA β-chain residues. Polymorphic residues that form part of the P1 and P9 pockets of the HLA-DR molecules provided a structural basis for the preferential binding of the citrullinated self-peptides to the HLA-DR4 allomorphs. Collectively, we provide a molecular basis for the interplay between citrullination of self-antigens and HLA polymorphisms that shape peptide–HLA-DR4 binding affinities in RA.