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Background and Objectives : Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (HHH; OMIM 238970) is one of the rare urea cycle disorders. Ornithine carrier 1 deficiency causes HHH syndrome, characterized by failure of mitochondrial ornithine uptake, hyperammonemia, and accumulation of ornithine and lysine in the cytoplasm. The initial presentation and time of diagnosis in HHH highly varies. Genetic analysis is critical for diagnosis. Materials and Methods : This study encompassed retrospective and prospective analyses of four unrelated Vietnamese children diagnosed with HHH syndrome. Results : The age of diagnosis ranged from 10 days to 46 months. All four cases demonstrated hyperornithinemia and prolonged prothrombin time. Three out of four cases presented with hyperammonemia, elevated transaminases, and uraciluria. No homocitrulline was detected in the urine. Only one case depicted oroticaciduria. Genetic analyses revealed three pathogenic variants in the SLC25A15 gene, with the c.535C>T (p.Arg179*) variant common in Vietnamese patients. The c.562_564del (p.Phe188del) and c.408del (p.Met137Cysfs*10) variants were detected in one case. The latter variant has yet to be reported in the literature on HHH patients. After intervention with a protein-restricted diet, ammonia-reducing therapy, and L-carnitine supplementation, hyperammonemia was not observed, and liver enzyme levels returned to normal. Conclusions : Our results highlighted the clinical and biochemical heterogeneity of HHH syndrome and posed that HHH syndrome should be considered when individuals have hyperammonemia, elevated transaminase, and decreased prothrombin time.

With the aim of improving the uncertainties associated with the correct diagnosis of seronegative rheumatoid arthritis (RA) and identifying those at risk of developing interstitial lung disease (ILD), we have designed new peptide antigens bearing three post-translational modifications (PTMs) (citrulline, homocitrulline and acetyl-lysine) related to RA that could complement existing tests based on anti-citrullinated peptide/protein antibodies (ACPAs). Several chimeric peptides were synthesized and comparatively tested as antigens in ELISAs with two cohorts of sera: 178 RAs and 110 healthy blood donors. The results indicated that although chimeric peptides containing all three PTMs and vimentin and enolase domains do not significantly outperform existing ACPA tests in terms of sensitivity and specificity, they show potential to complement current assays, especially when detecting antibodies in some seronegative patients. Furthermore, the presence of these autoantibodies significantly identified patients with RA and ILD. We can conclude that the identification of specific autoantibody profiles using synthetic antigens containing peptide domains derived from proteins present in the human joint could help in the early detection of the risk of ILD in patients with RA and be useful for adapting follow-up strategies and guiding decisions during treatment.

Given the increasing popularity of CrossFit ® as a high-intensity functional training program and the potential benefits of citrulline malate (CM) in enhancing exercise performance through its role as a precursor to L-arginine and nitric oxide production, this study aimed to investigate the acute effects of CM supplementation on CrossFit ® performance and cardiovascular function. Using a randomized, double-blind, placebo-controlled, cross-over design, 21 recreationally active participants (mean age 22.2 ± 2.6 years, mean body weight 75.9 ± 10.4 kg) with CrossFit ® experience completed the "Cindy" workout under CM and placebo conditions. Participants consumed 4.4 g of CM or a placebo 60 min before the workout, and the performance was measured by the number of rounds completed. Secondary outcomes included heart rate response, time spent in different heart rate intensity zones, and post-exercise recovery time. The results indicated no significant difference in the number of rounds completed between the CM and placebo conditions (13.5 ± 5.2 vs. 13.8 ± 6.7 rounds, respectively; p = 0.587). However, the time spent in zone 4 (80-90% of HR max) was significantly increased in the CM condition (527 ± 395 s vs. 453 ± 334 s; p = 0.017), suggesting a potential benefit for aerobic capacity and anaerobic threshold. No significant differences in post-exercise recovery time were observed (6.6 ± 4.7 h vs. 6.9 ± 4.7 h; p = 0.475). This study highlights the need for further research with larger sample sizes, both genders, and different CM dosages to clarify these findings and better understand CM's role in enhancing athletic performance.

Citrulline malate (CM) is purported to be an ergogenic aid during various types of exercise performance. However, the effects of CM on repeated sprint performance (RSP) are under-explored. In a placebo-controlled, double-blind, counterbalanced cross-over design, male university-level team sport athletes (n = 13) performed two familiarization trials, after which CM or placebo (PLA) (8 × 1 g tablets each day) were taken on the 2 days prior to, and with breakfast on the morning of, each main experimental trial. The main experimental trials employed a RSP protocol consisting of 10 repetitions of 40 m maximal shuttle run test (MST) with a 30 s interval between the start of each sprint. Sprint times and heart rate were recorded throughout the MST, and blood lactate concentrations were measured before, immediately after, and 5 min after completing the MST. CM resulted in better RSP compared to PLA, as indicated by a lower sprint performance decrement (S dec : CM, 4.68% ± 1.82% vs. PLA, 6.10% ± 1.83%; p = 0.03; ES = 0.77), which was possibly influenced by the fastest sprint time being faster in CM (CM, 8.16 ± 0.34 s vs. PLA, 8.29 ± 0.39 s; p = 0.011; ES = 0.34). There were no differences between CM and PLA in average sprint time (p = 0.54), slowest sprint time (p = 0.48), blood lactate concentrations (p = 0.73) or heart rate (p = 0.18), nor was there a condition × time interaction effect across the 10 sprints (p = 0.166). Three days of CM supplementation (8 g daily) attenuated the sprint performance decrement during short-duration high-intensity exercise in the form of running RSP in male university-level team sport athletes., (© 2024 The Authors. European Journal of Sport Science published by Wiley‐VCH GmbH on behalf of European College of Sport Science.)

Background: Protein carbamylation, a post-translational protein modification primarily driven by urea, independently associates with adverse clinical outcomes in patients with CKD. Biomarkers used to quantify carbamylation burden have mainly included carbamylated albumin (C-Alb) and homocitrulline (HCit, carbamylated lysine). In this study, we aimed to compare the prognostic utility of these two markers in order to facilitate comparisons of existing studies employing either marker alone, and to inform future carbamylation studies., Methods: Both serum C-Alb and free HCit levels were assayed from the same timepoint in 1632 individuals with CKD stages 2-4 enrolled in the prospective Chronic Renal Insufficiency Cohort (CRIC) study. Adjusted Cox proportional hazard models were used to assess risks for the outcomes of death (primary) and end stage kidney disease (ESKD) using each marker. C-statistics, net reclassification improvement, and integrated discrimination improvement were used to compare the prognostic value of each marker., Results: Participant demographics included mean (SD) age 59 (11) years; 702 (43%) females; 700 (43%) white. C-Alb and HCit levels were positively correlated with one another (Pearson correlation coefficient 0.64). Higher C-Alb and HCit levels showed similar increased risk of death (e.g., the adjusted hazard ratio [HR] for death in the 4th carbamylation quartile compared to the 1st was 1.90 (95% confidence interval [CI] 1.35-2.66) for C-Alb, and 1.89 [1.27-2.81] for HCit; and on a continuous scale, the adjusted HR for death using C-Alb was 1.24 [1.11 to 1.39] per standard deviation increase, and 1.27 [1.10-1.46] using HCit). Both biomarkers also had similar HRs for ESKD. The C-statistics were similar when adding each carbamylation biomarker to base models (e.g., for mortality models, the C-statistic was 0.725 [0.707-0.743] with C-Alb and 0.725 [0.707-0.743] with HCit, both compared to a base model 0.723). Similarities were also observed for the net reclassification improvement and integrated discrimination improvement metrics., Conclusions: C-Alb and HCit had similar performance across multiple prognostic assessments. The markers appear readily comparable in CKD epidemiological studies., (© 2024. The Author(s).)

Background: Homocitrullination is the post-translational modification of lysine that is recognized by T cells., Methods: This study identified homocitrullinated peptides from aldolase, enolase, cytokeratin and binding immunoglobulin protein and used human leukocyte antigen (HLA) transgenic mice to assess immunogenicity by enzyme-linked immunosorbent spot assay. Vaccine efficacy was assessed in tumor therapy studies using HLA-matched B16 melanoma expressing constitutive or interferon γ (IFNγ)-inducible major histocompatibility complex class II (MHC-II) as represented by most human tumors. To determine the mechanism behind the therapy, immune cell infiltrates were analyzed using flow cytometry and therapy studies in the presence of myeloperoxidase (MPO) inhibitor and T-cell depletion performed. We assessed the T-cell repertoire to homocitrullinated peptides in patients with cancer and healthy donors using flow cytometry., Results: Homocitrulline (Hcit) peptide vaccination stimulated strong CD4 T-cell responses and induced significant antitumor therapy in an established tumor model. The antitumor response was dependent on CD4 T cells and the effect was driven mainly via direct tumor recognition, as responses were only observed if the tumors were induced to express MHC-II. In vitro proliferation assays show that healthy donors and patients with cancer have an oligoclonal CD4 T-cell repertoire recognizing homocitrullinated peptides. Inhibition of cyanate generation, which mediates homocitrullination, by MPO inhibition reduced tumor therapy by the vaccine induced T cells (p = 0.0018). Analysis of the tumor microenvironment (TME) suggested that myeloid-derived suppressor cells (MDSCs) were a potential source of MPO. The selected B16 melanoma model showed MDSC infiltration and was appropriate to see if the Hcit vaccine could overcome the immunosuppression associated with MDSCs. The vaccine was very effective (90% survival) as the induced CD4 T cells directly targeted the homocitrullinated tumor and likely reversed the immunosuppressive environment., Conclusion: We propose that MPO, potentially produced by MDSCs, catalyzes the buildup of cyanate in the TME which diffuses into tumor cells causing homocitrullination of cytoplasmic proteins which are degraded and, in the presence of IFNγ, presented by MHC-II for direct CD4 T-cell recognition. Homocitrullinated proteins are a new target for cancer vaccines and may be particularly effective against tumors containing high levels of MPO expressing MDSCs., Competing Interests: Competing interests: KWC, WX, VAB and LGD have ownership interest in the patent. LGD is a director and shareholder in Scancell Ltd. All authors are employees of Scancell Ltd. except DB, CC and AKM., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Derivatization of amino acids by 2 M HCl/CH 3 OH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantitative measurement in biological samples by gas chromatography-mass spectrometry (GC-MS) as methyl ester pentafluoropropionic (PFP) derivatives, (Me) m -(PFP) n . This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d 3 Me) m -(PFP) n , from synthetic amino acids and 2 M HCl/CD 3 OD for use as internal standards. However, procedure A converts citrulline (Cit) to ornithine (Orn) and homocitrulline (hCit) to lysine (Lys) due to the instability of their carbamide groups under the acidic conditions of the esterification step. In the present study, we investigated whether reversing the order of the two-step derivatization may allow discrimination and simultaneous analysis of these amino acids. Pentafluoropropionylation (30 min, 65 °C) and subsequent methyl esterification (30 min, 80 °C), i.e., procedure B, of Cit resulted in the formation of six open and cyclic reaction products. The most abundant product is likely to be N 5 -Carboxy-Orn. The second most abundant product was confirmed to be Orn. The most abundant reaction product of hCit was confirmed to be Lys, with the minor reaction product likely being N 6 -Carboxy-Lys. Mechanisms are proposed for the formation of the reaction products of Cit and hCit via procedure B. It is assumed that at the first derivatization step, amino acids form ( N , O )-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP) 4 and hCit-(PFP) 4 are esterified on their C 1 -Carboxylic groups and on their activated N ureido groups. Procedure B also allows in situ preparation of (d 3 Me) m -(PFP) n from synthetic amino acids for use as internal standards. It is demonstrated that the derivatization procedure B enables discrimination between Cit and Orn, and between hCit and Lys. The utility of procedure B to measure simultaneously these amino acids in biological samples such as plasma and urine remains to be demonstrated. Further work is required to optimize the derivatization conditions of procedure B for biological amino acids.

Background: Citrulline is one of the non-essential amino acids that is thought to improve exercise performance and reduce post-exercise muscle soreness. We conducted a systematic review and meta-analysis to determine the effect of citrulline supplements on the post-exercise rating of perceived exertion (RPE), muscle soreness, and blood lactate levels., Methods: A random effects model was used to calculate the effect sizes due to the high variability in the study design and study populations of the articles included. A systematic search of PubMed, Web of Science, and ClinicalTrials.gov was performed. Eligibility for study inclusion was limited to studies that were randomized controlled trials involving healthy individuals and that investigated the acute effect of citrulline supplements on RPE, muscle soreness, and blood lactate levels. The supplementation time frame was limited to 2 h before exercise. The types and number of participants, types of exercise tests performed, supplementation protocols for L-citrulline or citrulline malate, and primary (RPE and muscle soreness) and secondary (blood lactate level) study outcomes were extracted from the identified studies., Results: The analysis included 13 eligible articles including a total of 206 participants. The most frequent dosage used in the studies was 8 g of citrulline malate. Citrulline supplementation significantly reduced RPE (n = 7, p = 0.03) and muscle soreness 24-h and 48-h after post-exercise (n = 7, p = 0.04; n = 6, p = 0.25, respectively). However, citrulline supplementation did not significantly reduce muscle soreness 72-h post-exercise (n = 4, p = 0.62) or lower blood lactate levels (n = 8, p = 0.17)., Conclusion: Citrulline supplements significantly reduced post-exercise RPE and muscle soreness without affecting blood lactate levels., (Copyright © 2020. Production and hosting by Elsevier B.V.)

Background: Athletes are increasingly exploring ways to enhance their physical performance. Increasing blood flow to the working tissues through endothelium-dependent vasodilation is one factor athletes use to realize these results. Sports supplements such as pre-workouts tout this benefit; however, many have not been tested under laboratory conditions to examine the effects of commonly used supplements on vasodilation. Two popular supplements are Nitrosigine® and citrulline malate (CM). Thus, the purpose of this experiment was to determine the effects of Nitrosigine and CM on vasodilation using ultrasound and flow mediated dilation (FMD)., Methods: Healthy, normotensive, and physically active male (n = 16) and female (n = 8) young adults participated in the present investigation. We utilized a randomized, double-blind, within-subjects design where participants reported for three trials, each preceded by a 7-day washout period. Baseline FMD measurement was obtained for each visit, followed by consumption of one clinical dose CM (8 g), Nitrosigine (1.5 g), or dextrose placebo (8 g). Following a 60-min digestion period, FMD was repeated. Supplementation order was randomized controlling for potential order effects., Results: Repeated measures ANOVA yielded a significant supplement (3) x time (2) effect (p < .001), such that Nitrosigine and CM yielded a greater improvement in FMD response than placebo. After supplementation, Nitrosigine and CM increased FMD by 31 and 34%, respectively, compared to a decrease of 2% during the placebo trial. After allometric scaling of the FMD values, supplement x time effect remained significant (p = .001) and changes were similar to non-scaled results. Nitrosigine (23%) and CM (25%) generated significantly greater allometric scaled FMD values when compared to the placebo trial (0.60%)., Discussion: Both Nitrisigine and CM increased endothelial-dependent vasodilation as measured by a change in FMD. Increased vasodilation leads to an increase in skeletal muscle blood flow resulting in potential improvements in exercise performance.

Arginine residues of the antimicrobial peptide LL-37 can be citrullinated by peptidyl arginine deiminases, which reduce the positive charge of the peptide. Notably, citrullinated LL-37 has not yet been detected in human samples. In addition, functional and biophysical properties of citrullinated LL-37 are not fully explored. The aim of this study was to detect citrullinated LL-37 in human bronchoalveolar lavage (BAL) fluid and to determine antibacterial and biophysical properties of citrullinated LL-37. BAL fluid was obtained from healthy human volunteers after intra-bronchial exposure to lipopolysaccharide. Synthetic peptides were used for bacterial killing assays, transmission electron microscopy, isothermal titration calorimetry, mass-spectrometry and circular dichroism. Using targeted proteomics, we were able to detect both native and citrullinated LL-37 in BAL fluid. The citrullinated peptide did not kill Escherichia coli nor lysed human red blood cells. Both peptides had similar α-helical secondary structures but citrullinated LL-37 was more stable at higher temperatures, as shown by circular dichroism. In conclusion, citrullinated LL-37 is present in the human airways and citrullination impaired bacterial killing, indicating that a net positive charge is important for antibacterial and membrane lysing effects. It is possible that citrullination serves as a homeostatic regulator of AMP-function by alteration of key functions.

Background: Studies have persuasively demonstrated that citrulline has a key role in the arginine-nitric oxide system, increasing nitric oxide bioavailability, an important mediator of peripheral vasodilation., Objective: To analyze the inter-individual post-exercise hypotension responsiveness following acute citrulline supplementation in hypertensives., Methods: Forty hypertensives were randomly assigned to one of the four experimental groups (control-placebo, control-citrulline, exercise-placebo, and exercise-citrulline). They ingested placebo or citrulline malate [CM] (6 grams). During the exercise session, individuals performed 40 minutes of walking/running on a treadmill at 60-70% of HR reserve. For the control session, the individuals remained seated at rest for 40 minutes. Office blood pressure (BP) was taken every 10 minutes until completing 60 minutes after the experimental session. The ambulatory BP device was programmed to take the readings every 20 minutes (awake time) and every 30 minutes (sleep time) over the course of 24 hours of monitoring. Statistical significance was defined as p < 0.05., Results: Unlike the other experimental groups, there were no "non-responders" in the exercise/citrulline (EC) for "awake" (systolic and diastolic BP) and "24 hours" (diastolic BP). The effect sizes were more consistent in the EC for systolic and diastolic ambulatorial BP response. The effects were "large" (> 0.8) for "awake", "asleep", and "24 hours" only in the EC for diastolic BP., Conclusion: CM supplementation can increase the post-exercise hypotensive effects in hypertensives. In addition, the prevalence of non-responders is lower when associated with aerobic exercise and CM supplementation.

Background: Use of supplements to aid performance is common practice amongst recreationally active individuals, including those without a sufficient evidence base. This investigation sought to assess whether acute supplementation with 8 g of citrulline malate (CM) (1.11: 1 ratio) would improve anaerobic performance., Methods: A randomised double blind placebo control trial was employed, using a counterbalanced design. We recruited recreationally active men and women to take part in an isokinetic chair protocol, based on German Volume Training (GVT) whereby participants attempted to perform 10 sets of 10 repetitions against a force representing 70% of their peak concentric force., Results: The number of repetitions achieved over the course of the GVT was 94.0 ± 7.9 and 90.9 ± 13.9 for placebo and CM respectively. There was no significant difference between the placebo and CM treatment for number of repetitions (P = 0.33), isometric (P = 0.60), concentric (P = 0.38), or eccentric (P = 0.65) peak force following the GVT. Total muscle soreness was significantly higher in the CM compared to the placebo treatment following the GVT protocol over 72 h (P = 0.01); although this was not accompanied by a greater workload/number of repetitions in the CM group., Conclusions: We conclude that an acute dose of CM does not significantly affect anaerobic performance using an isokinetic chair in recreational active participants. Practical implications include precaution in recommending CM supplementation. Coaches and athletes should be aware of the disparity between the chemical analyses of the products reviewed in the present investigation versus the manufacturers' claims.

Background: Supplementation of combined glutathione (GSH) with L-citrulline in response to a single bout of resistance exercise has been shown to increase plasma nitric oxide metabolites, nitrite and nitrate and cyclic guanosine monophosphate (cGMP), which may play a role in muscle protein synthesis. As a result, in response to resistance training (RT) these responses may establish a role for GSH + L-citrulline to increase muscle mass. This study attempted to determine the effects of an 8-week RT program in conjunction with GSH (Setria®) + L-citrulline, L-citrulline-malate, or placebo supplementation on lean mass and its association with muscle strength. The secondary purpose was to assess the safety of such supplementation protocol by assessing clinical chemistry markers., Methods: In a randomized, double-blind, placebo-controlled design, 75 resistance-trained males were randomly assigned to ingest GSH + L-citrulline (GSH + CIT), L-citrulline-malate, or cellulose placebo daily while also participating in 8 weeks of RT. The full dose of each supplement was delivered in capsules that were identical in weight, size, shape, and color. Participants completed testing sessions for body composition and muscle strength before and after 4 and 8 weeks of RT and supplementation. Venous blood samples were obtained before and after 8 weeks., Results: Leg press was increased with RT but was not significantly different between groups (p > 0.05); however, bench press strength was not increased with RT (p > 0.05). There were no significant changes in total body mass, fat mass, or total body water during 8 weeks of RT and supplementation. Lean mass increased in both GSH + CIT when compared to PLC; however, the increase was significant only after 4 weeks. Lean mass and strength were positively correlated (p < 0.05) in GSH + CIT, but not CIT-malate or PLC. Neither RT nor supplementation had any significant effects on blood clinical chemistry variables (p > 0.05)., Conclusion: Compared to PLC, supplementation of GSH + CIT during resistance training increased lean mass after 4 weeks of RT and was positively associated with muscle strength. However, after 8 weeks of RT there were no significant differences in any of the measured variables.

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background and Objectives: Causes of CKD differ in prognosis and treatment. Metabolomic indicators of CKD cause may provide clues regarding the different physiologic processes underlying CKD development and progression., Design, Setting, Participants & Measurements: Metabolites were quantified from serum samples of participants in the Modification of Diet in Renal Disease (MDRD) Study, a randomized controlled trial of dietary protein restriction and BP control, using untargeted reverse phase ultraperformance liquid chromatography tandem mass spectrometry quantification. Known, nondrug metabolites ( n =687) were log-transformed and analyzed to discover associations with CKD cause (polycystic kidney disease, glomerular disease, and other cause). Discovery was performed in Study B, a substudy of MDRD with low GFR ( n =166), and replication was performed in Study A, a substudy of MDRD with higher GFR ( n =423)., Results: Overall in MDRD, average participant age was 51 years and 61% were men. In the discovery study (Study B), 29% of participants had polycystic kidney disease, 28% had glomerular disease, and 43% had CKD of another cause; in the replication study (Study A), the percentages were 28%, 24%, and 48%, respectively. In the discovery analysis, adjusted for demographics, randomization group, body mass index, hypertensive medications, measured GFR, log-transformed proteinuria, and estimated protein intake, seven metabolites (16-hydroxypalmitate, kynurenate, homovanillate sulfate, N2,N2-dimethylguanosine, hippurate, homocitrulline, and 1,5-anhydroglucitol) were associated with CKD cause after correction for multiple comparisons ( P <0.0008). Five of these metabolite associations (16-hydroxypalmitate, kynurenate, homovanillate sulfate, N2,N2-dimethylguanosine, and hippurate) were replicated in Study A ( P <0.007), with all replicated metabolites exhibiting higher levels in polycystic kidney disease and lower levels in glomerular disease compared with CKD of other causes., Conclusions: Metabolomic profiling identified several metabolites strongly associated with cause of CKD., (Copyright © 2017 by the American Society of Nephrology.)

Objectives: The majority of rheumatoid arthritis (RA) patients express anti-citrullinated protein antibodies (ACPA). Unaffected first-degree relatives of RA patients (FDR) also express ACPA, commonly of the IgA isotype. IgG anti-homocitrullinated/carbamylated protein antibodies (AHCPA) have been detected in both RA and FDR. It is unknown whether other isotypes are expressed. We aim to investigate the AHCPA isotype profile in unaffected FDR of RA patients., Methods: The enrolled subjects were examined by a rheumatologist. FDR and healthy controls (HC) were excluded if they had swollen joints. Serum AHCPA targeting homocitrullinated fibrinogen was determined using enzyme linked immunoabsorbant assay (ELISA). FDR were genotyped for HLA-DR4 alleles encoding the shared epitope (SE)., Results: 125 RA (35 probands), 61 FDR and 40 HC were included. 20% of FDR expressed IgG AHCPA, compared to 30% in RA patients and 5% in HC (p=0.0010 for RA vs. HC). Levels of IgG AHCPA in FDR were similar to RA. FDR rarely expressed IgM (8%) and did not express IgA AHCPA. 20% of RA and 13% of HC subjects expressed IgM, but very few expressed IgA AHCPA (<7% in both groups). AHCPA expression in FDR was not significantly associated with joint symptoms, smoking or SE., Conclusions: IgG AHCPA is the most commonly expressed isotype in RA and FDR. The significance of IgG AHCPA in FDR is unclear as it was not associated with joint symptoms or other risk factors for RA. Longitudinal studies are needed to determine whether AHCPA is meaningful in populations at risk for RA.

Carbamylation is a non-enzymatic post-translational modification that physiologically occurs during aging and is a risk factor for various diseases. The most common product of carbamylation is homocitrulline (HCit), where a lysine (Lys) amino group has reacted with urea-derived cyanate. HCit has recently been detected in collagen; however, given that 15%-90% of total Lys in collagen is hydroxylated, it is unclear how hydroxylation affects collagen carbamylation. Here, we identified a collagen-specific carbamylation product, hydroxyhomocitrulline (HHCit), and showed that high levels of HHCit are correlated with age in rat tissue collagen and in vivo carbamylation in mice, as well as with the decline of kidney function in the serum of dialysis patients. Proteomic analysis of the carbamylated collagens identified α2(I) Lys 933 , a major cross-linking site, as a preferential HHCit site. Furthermore, our results suggest that hydroxylysine carbamylation affects the mechanical properties of connective tissue by competitively inhibiting collagen cross-link formation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The effects of citrulline malate (CM) on muscle recovery from resistance exercise remains unknown. We aimed to determine if citrulline malate supplementation improves muscle recovery after a single session of high-intensity resistance exercise (RE) in untrained young adult men. Nine young adult men (24.0 ± 3.3 years) participated in a double-blind crossover study in which they received 6 g of CM and placebo (PL) on two occasions, separated by a seven-day washout period. Each occasion consisted of a single session of high-intensity RE (0 h) and three subsequent fatigue tests sessions (at 24, 48, and 72 h) to assess the time course of muscle recovery. During the tests sessions, we assessed the following variables: number of maximum repetitions, electromyographic signal (i.e., root mean square (RMS) and median frequency (MF)), muscle soreness and perceived exertion, as well as blood levels of creatine kinase (CK), lactate, insulin, and testosterone:cortisol ratio. CK levels increased at 24 h post-exercise and remained elevate at 48 and 72 h, with no difference between CM and PL conditions. Muscle soreness increased at 24 h post-exercise, which progressively returned to baseline at 72 h in both conditions. Lactate levels increased immediately post-exercise and remained elevated at 24, 48, and 72 h in both conditions. No significant treatment × time interaction was found for all dependents variables (maximum repetitions, perceived exertion, CK, lactate, RMS, MF, and testosterone:cortisol ratio) during the recovery period. In conclusion, our data indicate that CM supplementation (single 6 g dose pre-workout) does not improve the muscle recovery process following a high-intensity RE session in untrained young adult men., Competing Interests: The authors declare no conflict of interest.

Objective: Anti-carbamylated protein (anti-CarP) antibodies are associated with the risk and severity of rheumatoid arthritis (RA) and are primarily directed against fibrinogen. The lack of understanding of anti-CarP antibody reactivity has limited analysis of the immunopathogenic associations in RA. To address this shortcoming, we mapped anti-CarP antibody epitope reactivity in RA patient sera., Methods: Immunoblotting identified a patient serum sample with specific reactivity to the carbamylated human fibrinogen β-chain. Liquid chromatography mass spectrometry (LC-MS) identified sites of homocitrullines (carbamylated lysines) present in the human fibrinogen β-chain. The reactivity of an anti-CarP antibody-positive cohort to specific peptides containing carbamylated lysines was determined by enzyme-linked immunosorbent assay, through direct binding (n = 63 sera) and by competition assays (n = 40 sera)., Results: Serum with specific reactivity to carbamylated, but not citrullinated, fibrinogen β-chain was identified in a specimen obtained from an RA patient. LC-MS identified carbamylation of 9 of 34 lysines in the human fibrinogen β-chain. Mapping of immunoreactivity to tryptic peptide fragments demonstrated several candidate carbamylated epitopes that were confirmed by competition experiments. Peptides containing a homocitrulline at position 83 appeared to be an immunodominant epitope in some RA patient sera, with additional reactivity to peptides containing homocitrullines at positions 52, 264, 351, 367, and 374., Conclusion: Anti-CarP antibodies appear to preferentially target specific regions of the human fibrinogen β-chain that contain homocitrullines. Interestingly, humoral immunoreactivity appears to be relatively restricted in some patients, which may enable detection of specific relationships with disease phenotype., (© 2017, American College of Rheumatology.)

NO is physiologically generated by endothelial and neuronal NO synthase (nNOS) isoforms. Although nNOS was first identified in brain, it is expressed in other tissues, including perivascular nerves, cardiac and skeletal muscle. Increasing experimental evidence suggests that nNOS has important effects on cardiovascular function, but its composite effects on systemic hemodynamics in humans are unknown. We undertook the first human study to assess the physiological effects of systemic nNOS inhibition on basal hemodynamics. Seventeen healthy normotensive men aged 24±4 years received acute intravenous infusions of an nNOS-selective inhibitor, S-methyl-l-thiocitrulline, and placebo on separate occasions. An initial dose-escalation study showed that S-methyl-l-thiocitrulline (0.1-3.0 µmol/kg) induced dose-dependent changes in systemic hemodynamics. The highest dose of S-methyl-l-thiocitrulline (3.0 µmol/kg over 10 minutes) significantly increased systemic vascular resistance (+42±6%) and diastolic blood pressure (67±1 to 77±3 mm Hg) when compared with placebo (both P <0.01). There were significant decreases in heart rate (60±4 to 51±3 bpm; P <0.01) and left ventricular stroke volume (59±6 to 51±6 mL; P <0.01) but ejection fraction was unaltered. S-methyl-l-thiocitrulline had no effect on radial artery flow-mediated dilatation, an index of endothelial NOS activity. These results suggest that nNOS-derived NO has an important role in the physiological regulation of basal systemic vascular resistance and blood pressure in healthy humans., (© 2017 The Authors.)

Background: Cyanate has recently gained attention for its role in the pathogenesis of vascular injury. Nonetheless, the effect of cyanate on angiogenesis remains unclear., Methods and Results: In this study, we demonstrated that oral administration of cyanate impaired blood perfusion recovery in a mouse hind-limb ischemia model. A reduction in blood perfusion recovery at day 21 was observed in the ischemic tissue of cyanate-treated mice. Likewise, there were fewer capillaries in the ischemic hind-limb tissue of cyanate-exposed mice. Our in vitro study showed that cyanate, together with its carbamylated products, inhibited the migration, proliferation, and tube-formation abilities of endothelial cells. Further research revealed that cyanate regulated angiogenesis partly by interrupting the vascular endothelial growth factor receptor 2/phosphatidylinositol 3-kinase/Akt pathway. The serum concentrations of homocitrulline, a marker of cyanate exposure, were determined in 117 patients with stable angina and chronic total occlusion. Consistent with the antiangiogenic role of cyanate, homocitrulline levels were increased in patients with poor coronary collateralization (n=58) compared with those with high collateralization (n=59; 21.09±13.08 versus 15.54±9.02 ng/mL, P=0.009). In addition, elevated homocitrulline concentration was a strong predictor of poor coronary collateral growth., Conclusions: Impaired angiogenesis induced by cyanate might contribute to poor coronary collateral growth., (© 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Background: Seropositive rheumatoid arthritis (RA) is characterized by autoantibodies binding to citrullinated and homocitrullinated proteins. We wanted to study the expression patterns of these disease-associated protein forms and if the rheumatoid nodule and synovial tissue itself contain biologically active levels of citrullinating peptidyl arginine deiminases 2, 3 and 4 and homocitrullination-facilitating neutrophil enzyme myeloperoxidase., Method: Total of 195 synovial samples from metatarsal joints from five ACPA/RF-positive RA patients (n = 77), synovial samples from knees of eight seropositive RA (n = 60), seven seronegative RA (n = 33) and five osteoarthritis (n = 25) patients were analyzed for citrulline and homocitrulline contents using HPLC. The location of citrulline- and homocitrulline-containing proteins, PAD 2, 3, 4 and myeloperoxidase were shown by immunostaining. Myeloperoxidase and citrulline- or homocitrulline-containing proteins were stained on Western blot., Results: Overall, necrosis was frequent in metatarsals of seropositive RA and absent in seronegative RA and osteoarthritis patients. In histological analysis, there was a significant local patterning and variation in the citrulline and homocitrulline content and it was highest in metatarsal synovial tissues of seropositive RA patients. We found peptidyl arginine deiminase 2, 3 and 4 in the lining and sublining layers of intact synovial tissue. Myeloperoxidase was found locally around necrotic areas. The tissues with necrosis contained the highest levels of citrulline and homocitrulline., Conclusions: Rheumatoid nodules and synovia contain significant amount of PAD2, 3 and 4 and myeloperoxidase enzymes. These enzymes could explain the levels of citrulline and homocitrulline in seropositive RA synovial and rheumatoid nodule tissues especially around necrotic tissue.

The biochemical mechanisms through which eosinophils contribute to asthma pathogenesis are unclear. Here we show eosinophil peroxidase (EPO), an abundant granule protein released by activated eosinophils, contributes to characteristic asthma-related phenotypes through oxidative posttranslational modification (PTM) of proteins in asthmatic airways through a process called carbamylation. Using a combination of studies we now show EPO uses plasma levels of the pseudohalide thiocyanate (SCN - ) as substrate to catalyze protein carbamylation, as monitored by PTM of protein lysine residues into N ϵ -carbamyllysine (homocitrulline), and contributes to the pathophysiological sequelae of eosinophil activation. Studies using EPO-deficient mice confirm EPO serves as a major enzymatic source for protein carbamylation during eosinophilic inflammatory models, including aeroallergen challenge. Clinical studies similarly revealed significant enrichment in carbamylation of airway proteins recovered from atopic asthmatics versus healthy controls in response to segmental allergen challenge. Protein-bound homocitrulline is shown to be co-localized with EPO within human asthmatic airways. Moreover, pathophysiologically relevant levels of carbamylated protein either incubated with cultured human airway epithelial cells in vitro, or provided as an aerosolized exposure in non-sensitized mice, induced multiple asthma-associated phenotypes including induction of mucin, Th2 cytokines, IFNγ, TGFβ, and epithelial cell apoptosis. Studies with scavenger receptor-A1 null mice reveal reduced IL-13 generation following exposure to aerosolized carbamylated protein, but no changes in other asthma-related phenotypes. In summary, EPO-mediated protein carbamylation is promoted during allergen-induced asthma exacerbation, and can both modulate immune responses and trigger a cascade of many of the inflammatory signals present in asthma., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: To evaluate the effect of neuronal nitric oxide synthase on the striated urethral sphincter and the urinary bladder., Materials and Methods: A coaxial catheter was implanted in the proximal urethra and another one in the bladder of female rats, which were anesthetized with subcutaneous injection of urethane. The urethral pressure with saline continuous infusion and bladder isovolumetric pressure were simultaneously recorded. Two groups of rats were formed. In group I, an intrathecal catheter was implanted on the day of the experiment at the L6-S1 level of the spinal cord; in group II, an intracerebroventricular cannula was placed 5-6 days before the experiment., Results: It was verified that the group treated with S-methyl-L-thio-citrulline, via intrathecal pathway, showed complete or partial inhibition of the urethral sphincter relaxation and total inhibition of the micturition reflexes. The urethral sphincter and the detrusor functions were recovered after L-Arginine administration. When S-methyl-Lthio-citrulline was administered via intracerebroventricular injection, there was a significant increase of urethral sphincter tonus while preserving the sphincter relaxation and the detrusor contractions, at similar levels as before the use of the drugs. Nevertheless there was normalization of the urethral tonus when L-Arginine was applied., Conclusions: The results indicate that, in female rats anaesthetized with urethane, the nNOS inhibitor administrated through the intrathecal route inhibits urethral sphincter relaxation, while intracerebroventricular injection increases the sphincter tonus, without changing bladder function. These changes were reverted by L-Arginine administration. These findings suggest that the urethral sphincter and detrusor muscle function is modulated by nitric oxide., Competing Interests: Conflicts of Interest: None declared., (Copyright® by the International Brazilian Journal of Urology.)

Objective: Rheumatoid arthritis (RA) is characterized by the presence of autoantibodies, including seropositivity for rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs). In addition, antibodies to carbamylated proteins (anti-CarP) are present in patients with RA and are associated with joint damage. This study was undertaken to assess the presence of anti-CarP antibodies in indigenous North Americans (First Nations [FN] populations) with RA compared to their at-risk first-degree relatives (FDRs) and healthy controls., Methods: Anti-CarP IgG and ACPAs (specifically, anti-cyclic citrullinated peptide [anti-CCP] antibodies) were measured by enzyme-linked immunosorbent assay in the sera of FN patients with RA (n = 95), their unaffected FDRs (n = 109), and healthy FN controls (n = 85). Antibodies to additional citrullinated peptides were measured using a multiplex ACPA array, and the number of peptides recognized was reported as an ACPA score. Groups were compared using the chi-square test and Mann-Whitney U test. Associations between RA and seropositivity for RF, ACPAs, and anti-CarP antibodies were determined by logistic regression., Results: Anti-CarP antibodies were more frequent in FN patients with RA (44.3%) compared to FDRs (18.3%) and FN controls (4.7%) (both P < 0.0001 versus RA). Moreover, anti-CarP antibodies were more frequent in FDRs than in FN controls (P = 0.008). The ACPA score was higher in anti-CCP-positive FN patients with RA than in anti-CCP-positive FN FDRs (median score 7 [interquartile range (IQR) 7] versus median score 1 [IQR 4]; P = 0.04). The association with RA was strongest when all 3 autoantibodies (RF, anti-CCP, and anti-CarP) were present in the patients' serum (odds ratio 194, 95% confidence interval 23-1,609, P < 0.0001)., Conclusion: Anti-CarP antibodies are prevalent in FN patients with RA and also more common in their at-risk FDRs compared to healthy controls. The results indicate an association of RF, ACPAs, and anti-CarP with RA that is strongest when all 3 autoantibodies are present. These findings may provide new insights into the evolution of autoimmunity in preclinical RA., (© 2016 The Authors. Arthritis & Rheumatology published by Wiley Periodicals, Inc. on behalf of the American College of Rheumatology.)

We showed previously that Bacillus anthracis edema toxin (ET), comprised of protective antigen (PA) and edema factor (EF), inhibits phenylephrine (PE)-induced contraction in rat aortic rings and these effects are diminished in endothelial-denuded rings. Therefore, employing rat aortic ring and in vivo models, we tested the hypothesis that nitric oxide (NO) contributes to ET's arterial effects. Compared with rings challenged with PA alone, ET (PA + EF) reduced PE-stimulated maximal contractile force (MCF) and increased the PE concentration producing 50% MCF (EC50) (P < 0.0001). Compared with placebo, l-nitro-arginine methyl-ester (l-NAME), an NO synthase (NOS) inhibitor, reduced ET's effects on MCF and EC50 in patterns that approached or were significant (P = 0.06 and 0.03, respectively). In animals challenged with 24-h ET infusions, l-NAME (0.5 or 1.0 mg·kg(-1)·h(-1)) coadministration increased survival to 17 of 28 animals (60.7%) compared with 4 of 27 (14.8%) given placebo (P = 0.01). Animals receiving l-NAME but no ET all survived. Compared with PBS challenge, ET increased NO levels at 24 h and l-NAME decreased these increases (P < 0.0001). ET infusion decreased mean arterial blood pressure (MAP) in placebo and l-NAME-treated animals (P < 0.0001) but l-NAME reduced decreases in MAP with ET from 9 to 24 h (P = 0.03 for the time interaction). S-methyl-l-thiocitrulline, a selective neuronal NOS inhibitor, had effects in rings and, at a high dose in vivo models, comparable to l-NAME, whereas N'-[3-(aminomethyl)benzyl]-acetimidamide, a selective inducible NOS inhibitor, did not. NO production contributes to ET's arterial relaxant, hypotensive, and lethal effects in the rat.

The aryl hydrocarbon receptor (AhR), a regulator of xenobiotic toxicity, is a member of the eukaryotic Per-Arnt-Sim domain protein family of transcription factors. Recent evidence identified a novel AhR DNA recognition sequence called the nonconsensus xenobiotic response element (NC-XRE). AhR binding to the NC-XRE in response to activation by the canonical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in concomitant recruitment of carbamoyl phosphate synthase 1 (CPS1) to the NC-XRE. Studies presented here demonstrate that CPS1 is a bona fide nuclear protein involved in homocitrullination (hcit), including a key lysine residue on histone H1 (H1K34hcit). H1K34hcit represents a hitherto unknown epigenetic mark implicated in enhanced gene expression of the peptidylarginine deiminase 2 gene, itself a chromatin-modifying protein. Collectively, our data suggest that AhR activation promotes CPS1 recruitment to DNA enhancer sites in the genome, resulting in a specific enzyme-independent post-translational modification of the linker histone H1 protein (H1K34hcit), pivotal in altering local chromatin structure and transcriptional activation., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

When exposed to a hypoxic environment, the body's first response is a reflex increase in ventilation, termed the hypoxic ventilatory response (HVR). With chronic sustained hypoxia (CSH), such as during acclimatization to high altitude, an additional time-dependent increase in ventilation occurs, which increases the HVR and is termed ventilatory acclimatization to hypoxia (VAH). This secondary increase persists after exposure to CSH and involves plasticity within the circuits in the central nervous system that control breathing. The mechanisms of HVR plasticity are currently poorly understood. We hypothesized that changes in neuronal nitric oxide synthase (nNOS) activity or expression in the nucleus tractus solitarius contribute to this plasticity and underlie VAH in rats. To test this, we treated rats held in normoxia or 10% O2 (CSH, PIO2 = 70 Torr) for 7-9 days and measured ventilation in conscious, unrestrained animals before and after microinjecting the general NOS antagonist L-NG-Nitroarginine methyl ester into the nucleus tractus solitarius (NTS) or systemically injecting the nNOS-specific antagonist S-methyl-l-thiocitrulline. Localization of injection sites in the NTS was confirmed by histology following the experiment. We found that 1) neither NTS-specific nor systemic nNOS antagonism had any effect on hypoxia-mediated changes in breathing or metabolism (P > 0.05), but 2) nNOS protein expression was increased in the middle and caudal NTS by CSH. A persistent HVR after nNOS blockade in the NTS contrasts with results in awake mice, and our findings do not support the hypotheses that nNOS in the NTS contribute to the HVR or VAH in awake rats., (Copyright © 2015 the American Physiological Society.)

Introduction: Smoking is a well-established risk factor for rheumatoid arthritis (RA), and it has been proposed that smoking-induced citrullination renders autoantigens immunogenic. To investigate this mechanism, we examined human lung tissue from 40 subjects with defined smoking status, with or without chronic obstructive pulmonary disease (COPD), and control tissues from other organs for citrullinated proteins and the deiminating enzymes peptidylarginine deiminase type-2 (PAD2) and -4 (PAD4)., Methods: Lung tissue samples, dissected from lobectomy specimens from 10 never smokers, 10 smokers without airflow limitation, 13 COPD smokers and eight COPD ex-smokers, and control tissue samples (spleen, skeletal muscle, liver, ovary, lymph node, kidney and heart), were analysed for citrullinated proteins, PAD2 and PAD4 by immunoblotting. Citrulline and homocitrulline residues in enolase and vimentin were analysed by partial purification by gel electrophoresis followed by mass spectrometry in 12 of the lung samples and one from each control tissues. Band intensities were scored semi-quantitatively and analysed by two-tailed Mann-Whitney T-test., Results: Within the lung tissue samples, citrullinated proteins, PAD2 and PAD4 were found in all samples, with an increase in citrullination in COPD (P = 0.039), but minimal difference between smokers and non-smokers (P = 0.77). Citrullination was also detected at lower levels in the tissues from other organs, principally in lymph node, kidney and skeletal muscle. Mass spectrometry of the lung samples showed that vimentin was citrullinated at positions 71, 304, 346, 410 and 450 in non-smokers and smokers both with and without COPD. A homocitrulline at position 104 was found in four out of six COPD samples and one out of six non-COPD. Citrulline-450 was also found in three of the control tissues. There were no citrulline or homocitrulline residues demonstrated in α-enolase., Conclusions: We have shown evidence of citrullination of vimentin, a major autoantigen in RA, in both non-smokers and smokers. The increase in citrullinated proteins in COPD suggests that citrullination in the lungs of smokers is mainly due to inflammation. The ubiquity of citrullination of vimentin in the lungs and other tissues suggests that the relationship between smoking and autoimmunity in RA may be more complex than previously thought.

Introduction: Our objective was to find out if there are antibodies binding to homocitrulline-containing type I and II collagen carboxyterminal telopeptides in sera of patients with rheumatoid arthritis (RA), and if these antibodies cross-react with citrulline and homocitrulline in the same peptide sequence., Methods: A total of 72 RA and 72 control sera were analyzed for binding using enzyme-linked immunosorbent assay to citrulline- or homocitrulline-containing type I and II collagen carboxyterminal telopeptides, as well as to cyclic citrullinated peptide (CCP) and to mutated citrullinated vimentin (MCV). Specificities of the antibodies were tested using inhibition-ELISA., Results: Of the RA sera, 39 (54%) and 41 (57%) were positive for binding to CCP and MCV, respectively. Further, 34 (47%) and 30 (42%) of the patients had specific antibodies binding to and being inhibited by citrulline-containing type I collagen telopeptides and by citrulline-containing type II collagen carboxyterminal telopeptides, respectively. The corresponding figures regarding homocitrulline-containing type I and homocitrulline-containing type II collagen telopeptides were 16 (22%) and 14 (19%). Most of the patients, who were seropositive for citrullinated peptides, showed binding in multiple assays. A total of 10 (14%) RA patients were positive for all the tested peptide pairs, while 28 (39%) of them had antibodies that contained overlapping specifities between citrulline and homocitrulline in the same peptide sequence., Conclusions: Antibodies to both citrulline and homocitrulline containing type I and II collagen telopeptides can be found in sera of RA patients. These antibodies are not constant from one RA patient to another, but contain separate or overlapping specificities within the same peptide sequence varying between individuals. Our results suggest some relationship between citrulline and homocitrulline-recognizing antibodies, since homocitrulline antibodies exist mainly in individuals seropositive to anti-CCP and anti-MCV.

The dramatic cardiovascular mortality of patients with chronic kidney disease is attributable in a significant proportion to endothelial dysfunction. Cyanate, a reactive species in equilibrium with urea, is formed in excess in chronic kidney disease. Cyanate is thought to have a causal role in promoting cardiovascular disease, but the underlying mechanisms remain unclear. Immunohistochemical analysis performed in the present study revealed that carbamylated epitopes associate mainly with endothelial cells in human atherosclerotic lesions. Cyanate treatment of human coronary artery endothelial cells reduced expression of endothelial nitric oxide synthase, and increased tissue factor and plasminogen activator inhibitor-1 expression. In mice, administration of cyanate, promoting protein carbamylation at levels observed in uremic patients, attenuated arterial vasorelaxation of aortic rings in response to acetylcholine without affecting the sodium nitroprusside-induced relaxation. Total endothelial nitric oxide synthase and nitric oxide production were significantly reduced in aortic tissue of cyanate-treated mice. This coincided with a marked increase of tissue factor and plasminogen activator inhibitor-1 protein levels in aortas of cyanate-treated mice. Thus, cyanate compromises endothelial functionality in vitro and in vivo. This may contribute to the dramatic cardiovascular risk of patients suffering from chronic kidney disease.

Background and Purpose: Citrulline is an amino acid which is produced by the urea cycle and also a precursor for NO, that is, a vasodilator for normal function of pulmonary vasculature. Thereby, enhancing l-citrulline malate in patients with idiopathic pulmonary arterial hypertension and those with congenital heart disease identified as Eisenmenger Syndrome results in reduction of pulmonary hypertension., Methods and Subjects: In this clinical trial before and after study, we assigned 25 patients with arterial pulmonary hypertension (idiopathic or Eisenmenger Syndrome) to receive l-citrulline malate 1g three times daily for two weeks. The primary measurement was the change in exercise capacity, as considered as a result of the total distance walked in six minutes, from baseline to week 2. We also assessed mean pulmonary artery pressure, the change in the quality of life, and the change in pro-brain natriuretic peptide (BNP) level. The study was not powered to evaluate mortality., Results: The mean walking distance in six minutes was significantly increased by about 44m (p=0.005) after receiving l-citrulline malate. Mean pulmonary artery pressure significantly reduced from 83.34mmHg before receiving l-citrulline malate to 79.1mmHg after that (p=0.01). All dimensions of the quality of life had statistical differences after receiving l-citrulline malate except limit due to physical health, limit due to emotional health and social functioning (p>0.05). Finally, pro-BNP difference was not statistically significant (p=0.9)., Conclusion: l-Citrulline malate improves the distance walk in six minutes and also the quality of life of patients with idiopathic arterial pulmonary hypertension and Eisenmenger Syndrome and also reduced mean arterial pulmonary hypertension., (Copyright © 2014 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Renal afferent arterioles (AFF) regulate glomerular capillary pressure through two main mechanisms: the myogenic response (MYO) and tubuloglomerular feedback (TGF). Because Rho-kinase and nitric oxide synthase (NOS) are established factors that modulate vascular tone, we examined the role of these factors in pressure-induced AFF tone in Wistar-Kyoto rats and in spontaneously hypertensive rats (SHR) using an intravital CCD camera. Elevated renal perfusion pressure elicited marked AFF constriction that was partially inhibited by gadolinium, furosemide and fasudil, which inhibit MYO, TGF and Rho-kinase, respectively; however, this AFF constriction was completely blocked by combined treatment with fasudil+gadolinium or fasudil+furosemide. S-methyl-L-thiocitrulline (SMTC) partially reversed the fasudil-induced inhibition of TGF-mediated, but not that of MYO-mediated, AFF constriction. In SHR, the pressure-induced AFF response was enhanced, and MYO- and TGF-induced constriction were exaggerated. In the presence of gadolinium, SMTC partially mitigated the fasudil-induced inhibition of TGF-mediated AFF constriction. Immunoblot analyses demonstrated that both Rho-kinase activity and neuronal NOS were augmented in SHR kidneys. In conclusion, Rho-kinase contributes to MYO- and TGF-mediated AFF responses, and these responses are enhanced in SHR. Furthermore, neuronal NOS-induced nitric oxide modulates the TGF mechanism. This mechanism constitutes a target for Rho-kinase in TGF-mediated AFF constriction.

Background: Antibodies binding to citrullinated proteins are a frequent finding in rheumatoid arthritis patients and may precede the onset of clinical symptoms several years. The antibodies are a predisposing factor for bone erosions but their origin is unknown. In this study we analyze in detail the levels of protein bound citrulline and homocitrulline in several tissue samples of a single erosive arthritic surgery patient., Methods: Serum antibodies binding to CCP, MCV and citrulline- or homocitrulline-containing type I and II collagen carboxytelopeptides were measured. Tissue samples of a single RA patient, taken in two separate operations performed with two-year time span were hydrolyzed and analyzed for citrulline and homocitrulline content by HPLC., Results: Protein-bound citrulline and homocitrulline were found in several joint tissues of a RA patient with ACPA-positive erosive disease. The amount of homocitrulline stayed relatively constant between the different tissues. The amount of citrulline in erosive tissue was 3-times higher than in non-erosive tissue in the first operation. In the samples of the second operation 3-4-times higher mean amounts of citrulline were found in two out of the six tissues investigated., Conclusions: Homocitrulline is present in rheumatoid nodule together with citrulline. There is more variation in the amount of citrulline than in the amount of homocitrulline between the tissues. The tissue sample containing the most citrulline was the most erosive.

Isoform-specific nitric oxide (NO) synthase (NOS) contributions to NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle are incompletely understood. The purpose of the present study was to investigate the role of neuronal NOS (nNOS) in the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of healthy rats. We hypothesized that acute pharmacological inhibition of nNOS would augment sympathetic vasoconstriction in resting and contracting skeletal muscle, demonstrating that nNOS is primarily responsible for NO-mediated inhibition of sympathetic vasoconstriction. Sprague-Dawley rats (n = 13) were anesthetized and instrumented with an indwelling brachial artery catheter, femoral artery flow probe, and lumbar sympathetic chain stimulating electrodes. Triceps surae muscles were stimulated to contract rhythmically at 60% of maximal contractile force. In series 1 (n = 9), the percent change in femoral vascular conductance (%FVC) in response to sympathetic stimulations delivered at 2 and 5 Hz was determined at rest and during muscle contraction before and after selective nNOS blockade with S-methyl-l-thiocitrulline (SMTC, 0.6 mg/kg iv) and subsequent nonselective NOS blockade with N(ω)-nitro-l-arginine methyl ester (l-NAME, 5 mg/kg iv). In series 2 (n = 4), l-NAME was injected first, and then SMTC was injected to determine if the effect of l-NAME on constrictor responses was influenced by selective nNOS inhibition. Sympathetic stimulation decreased FVC at rest (-25 ± 7 and -44 ± 8%FVC at 2 and 5 Hz, respectively) and during contraction (-7 ± 3 and -19 ± 5%FVC at 2 and 5 Hz, respectively). The decrease in FVC in response to sympathetic stimulation was greater in the presence of SMTC at rest (-32 ± 6 and -49 ± 8%FVC at 2 and 5 Hz, respectively) and during contraction (-21 ± 4 and -28 ± 4%FVC at 2 and 5 Hz, respectively). l-NAME further increased (P < 0.05) the sympathetic vasoconstrictor response at rest (-47 ± 4 and -60 ± 6%FVC at 2 and 5 Hz, respectively) and during muscle contraction (-33 ± 3 and -40 ± 6%FVC at 2 and 5 Hz, respectively). The effect of l-NAME was not altered by the order of nNOS inhibition. These data demonstrate that NO derived from nNOS and endothelial NOS contribute to the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle.

Nitric oxide (NO) release from endothelial NO synthase (eNOS) and/or neuronal NO synthase (nNOS) could be modulated by sympathetic nerve activity and contribute to increased blood flow after exercise. We examined the effects of brachial-arterial infusion of the nNOS selective inhibitor S-methyl-l-thiocitrulline (SMTC) and the nonselective NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA) on forearm arm blood flow at rest, during sympathetic activation by lower body negative pressure, and during lower body negative pressure immediately after handgrip exercise. Reduction in forearm blood flow by lower body negative pressure during infusion of SMTC was not significantly different from that during vehicle (-28.5 ± 4.02 vs. -34.1 ± 2.96%, respectively; P = 0.32; n = 8). However, l-NMMA augmented the reduction in forearm blood flow by lower body negative pressure (-44.2 ± 3.53 vs. -23.4 ± 5.71%; n = 8; P < 0.01). When lower body negative pressure was continued after handgrip exercise, there was no significant effect of either l-NMMA or SMTC on forearm blood flow immediately after low-intensity exercise (P = 0.91 and P = 0.44 for l-NMMA vs. saline and SMTC vs. saline, respectively; each n = 10) or high-intensity exercise (P = 0.46 and P = 0.68 for l-NMMA vs. saline and SMTC vs. saline, respectively; each n = 10). These results suggest that sympathetic activation increases NO release from eNOS, attenuating vasoconstriction. Dysfunction of eNOS could augment vasoconstrictor and blood pressure responses to sympathetic activation. However, neither eNOS nor nNOS plays an essential role in postexercise hyperaemia, even in the presence of increased sympathetic activation.

Endothelial nitric oxide synthase (eNOS) was assumed to be the only source of nitric oxide (NO) involved in the regulation of human coronary blood flow (CBF). However, our recent first-in-human study using the neuronal NOS (nNOS)-selective inhibitor S-methyl-L-thiocitrulline (SMTC) showed that nNOS-derived NO also plays a role. In this study, we investigated the relative contribution of nNOS and eNOS to the CBF response to a pacing-induced increase in cardiac workload. Incremental right atrial pacing was undertaken in patients with angiographically normal coronary arteries during intracoronary infusion of saline vehicle and then either SMTC or N(G)-monomethyl-l-arginine (l-NMMA; which inhibits both eNOS and nNOS). Intracoronary SMTC (0.625 μmol/min) and l-NMMA (25 μmol/min) reduced basal CBF to a similar extent (-19.2 ± 3.2% and 25.0 ± 2.7%, respectively; n = 10 per group). Pacing-induced increases in CBF were significantly blunted by l-NMMA (maximum CBF: 83.5 ± 14.2 ml/min during saline vs. 61.6 ± 9.5 ml/min during l-NMMA; P < 0.01). By contrast, intracoronary SMTC had no effect on the maximum CBF during pacing (98.5 ± 12.9 ml/min during saline vs. 102.1 ± 16.6 ml/min during SMTC; P = not significant). l-NMMA also blunted the pacing-induced increase in coronary artery diameter (P < 0.001 vs. saline), whereas SMTC had no effect. Our results confirm a role of nNOS in the regulation of basal CBF in humans but show that coronary vasodilation in response to a pacing-induced increase in cardiac workload is exclusively mediated by eNOS-derived NO.

Traditional risk factors fail to explain the increased risk for cardiovascular morbidity and mortality in ESRD. Cyanate, a reactive electrophilic species in equilibrium with urea, posttranslationally modifies proteins through a process called carbamylation, which promotes atherosclerosis. The plasma level of protein-bound homocitrulline (PBHCit), which results from carbamylation, predicts major adverse cardiac events in patients with normal renal function, but whether this relationship is similar in ESRD is unknown. We quantified serum PBHCit in a cohort of 347 patients undergoing maintenance hemodialysis with 5 years of follow-up. Kaplan-Meier analyses revealed a significant association between elevated PBHCit and death (log-rank P<0.01). After adjustment for patient characteristics, laboratory values, and comorbid conditions, the risk for death among patients with PBHCit values in the highest tertile was more than double the risk among patients with values in the middle tertile (adjusted hazard ratio [HR], 2.4; 95% confidence interval [CI], 1.5-3.9) or the lowest tertile (adjusted HR, 2.3; 95% CI, 1.5-3.7). Including PBHCit significantly improved the multivariable model, with a net reclassification index of 14% (P<0.01). In summary, serum PBHCit, a footprint of protein carbamylation, predicts increased cardiovascular risk in patients with ESRD, supporting a mechanistic link among uremia, inflammation, and atherosclerosis.

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β(3)-adrenergic receptor (β(3)-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β(3)-AR signaling and found that the β(3)-AR-mediated reduction in cell shortening and [Ca(2+)](i) transient amplitude was abolished both in eNOS(-/-) and nNOS(-/-) left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-L-thiocitrulline. LV superoxide (O(2)(·-)) production was increased in nNOS(-/-) mice and reduced by L-N(ω)-nitroarginine methyl ester (L-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS(-/-) myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O(2)(·-), only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca(2+)](i) transient response to β(3)-AR stimulation in nNOS(-/-) mice. In summary, our data show that increased O(2)(·-) production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β(3)-AR stimulation in nNOS(-/-) myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS(-/-) mice result from disruption of eNOS signaling.

Rheumatoid arthritis (RA) is a common autoimmune disease affecting around 1% of the population. Although major advances have been made in the treatment of RA, still relatively little is known on disease pathogenesis and aetiology. From treatment studies it has become clear that treating patients early in their disease course will provide the best results. However, especially in the early phase of arthritis, in particular when the patients do not yet fulfil the criteria for RA, it is difficult to decide which patients would benefit most from an early and aggressive intervention. Good biomarkers are important to guide decisions in the clinical management of RA. Next to the well-known rheumatoid factor (RF) and the anti-citrullinated protein antibodies (ACPA), several new markers are now likely to become available with interesting potential. Besides antibody responses directed against citrullinated proteins, also antibodies against carbamylated proteins (anti-CarP) have recently been shown to be present in RA. Interestingly these anti-CarP antibodies are also present in around 20% of the ACPA-negative RA patients and are associated with more severe joint damage in this group. Apart from the antibodies that help in establishing the diagnosis and prognosis, also novel biomarkers that reflect clinical disease activity scores are being discovered. The development of biomarker-based disease activity scores might allow easy and frequent monitoring of patients to rapidly adjust treatment.

Advanced age is associated with derangements in skeletal muscle microvascular function during the transition from rest to contractions. We tested the hypothesis that, contrary to what was reported previously in young rats, selective neuronal nitric oxide (NO) synthase (nNOS) inhibition would result in attenuated or absent alterations in skeletal muscle microvascular oxygenation (Po(2)(mv)), which reflects the matching between muscle O(2) delivery and utilization, following the onset of contractions in old rats. Spinotrapezius muscle blood flow (radiolabeled microspheres), Po(2)(mv) (phosphorescence quenching), O(2) utilization (Vo(2); Fick calculation), and submaximal force production were measured at rest and following the onset of contractions in anesthetized old male Fischer 344 × Brown Norway rats (27 to 28 mo) pre- and postselective nNOS inhibition (2.1 μmol/kg S-methyl-l-thiocitrulline; SMTC). At rest, SMTC had no effects on muscle blood flow (P > 0.05) but reduced Vo(2) by ∼23% (P < 0.05), which elevated basal Po(2)(mv) by ∼18% (P < 0.05). During contractions, steady-state muscle blood flow, Vo(2), Po(2)(mv), and force production were not altered after SMTC (P > 0.05 for all). The overall Po(2)(mv) dynamics following onset of contractions was also unaffected by SMTC (mean response time: pre, 19.7 ± 1.5; and post, 20.0 ± 2.0 s; P > 0.05). These results indicate that the locus of nNOS-derived NO control in skeletal muscle depends on age and metabolic rate (i.e., rest vs. contractions). Alterations in nNOS-mediated regulation of contracting skeletal muscle microvascular function with aging may contribute to poor exercise capacity in this population.

Introduction: Nitric oxide (NO) vasodilation critically modulates renal hemodynamics in the neonate compared with the adult. Based on the postnatal expression pattern of renal neuronal nitric oxide synthase (nNOS), the hypothesis was that nNOS is the major NOS isoform regulating renal hemodynamics in the immature, but not mature, kidney., Results: NOS inhibitors did not alter mean arterial pressure (MAP) in either group. Intrarenal S-methyl-L-thiocitrulline (L-SMTC) in newborns significantly reduced renal blood flow (RBF) 38 ± 4%, glomerular filtration rate (GFR) 42 ± 6%, and increased renal vascular resistance (RVR) 37 ± 7%, whereas intrarenal L-nitro-arginine methyl ester (L-NAME) affected RBF, GFR, and RVR equivalent to L-SMTC treatment. When L-NAME was administered after L-SMTC treatment, newborn renal hemodynamic changes were not further altered from what was observed when L-SMTC was administered alone. In contrast, in the adult, only intrarenal L-NAME, and not L-SMTC, affected renal hemodynamic responses., Discussion: In conclusion, these studies demonstrate that nNOS is an important regulator of renal hemodynamics in the newborn kidney, but not in the adult., Methods: Experiments compared renal hemodynamic responses with intrarenal infusion of L-NAME, an inhibitor of all NOS isoforms, with the selective nNOS inhibitor L-SMTC in the newborn piglet and the adult pig.

Homocitrulline (HCit), an amino acid formed by the carbamylation of ε-amino groups of lysine residues, is considered a promising biomarker for monitoring diseases such as chronic renal failure and atherosclerosis. This paper describes a tandem mass spectrometric method for total, protein-bound and free HCit measurement in plasma samples. HCit was separated from other plasma components by hydrophilic interaction liquid chromatography. Detection was achieved by monitoring transitions of 190.1 > 127.1 and 190.1 > 173.1 for HCit, and 183.1 > 120.2 for d(7)-citrulline used as internal standard. This method allowed HCit quantification within 5.2 min and was precise (inter-assay CV < 5.85%), accurate (mean recoveries ranging from 97% to 106%), and exhibited a good linearity from 10 nmol/L to 1.6 μmol/L. Plasma samples from control and uremic mice (n = 10) were analyzed. In control mice, mean total plasma HCit concentration was 0.78 ± 0.12 μmol/mol amino acids, whereas it was increased 2.7-fold in uremic mice plasma, reaching 2.10 ± 0.50 μmol/mol amino acids (p < 0.001). In conclusion, this method exhibits good analytical performances and meets the criteria of sensitivity suitable for HCit concentration assessment in plasma samples.

Unlabelled: The diagnostic and prognostic value of the serial measurement of antibodies, such as rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP), and anti-citrullinated vimentin (anti-Sa) antibodies, has not been defined in early rheumatoid arthritis (ERA)., Objectives: To prospectively assess the presence of RF, anti-CCP, and anti-Sa in ERA patients., Patients and Methods: Forty ERA (less than 12 months) patients of the Brasília cohort were evaluated and followed up for three years. Both clinical and demographic data were recorded, in addition to the results (ELISA) of RF (IgM, IgG, and IgA), anti-CCP (CCP2, CCP3, and CCP3.1), and anti-Sa at the baseline assessment and after 3, 6, 12, 18, 24 and 36 months of follow-up. The results were compared by use of Student t test and paired t test., Results: The patients' mean age was 45 years, and 90% of them were female. At the time of diagnosis, RF was identified in 50% of the patients (RF IgA, 42%; RF IgG, 30%; and RF IgM, 50%), anti-CCP in 52.5% (no difference between CCP2, CCP3, and CCP3.1), and anti-Sa in 10%. After three years, no difference was observed in RF and anti-CCP prevalence, but anti-Sa increased to 17.5% (P = 0.001)., Conclusion: Repeated RF and anti-CCP measurement, including different isotypes, during three years of follow-up showed no significant changes. The third generation of anti-CCP assays did not increase the diagnostic value of the second-generation assays.

Autoimmune responses against posttranslationally modified antigens are a hallmark of several autoimmune diseases. For example, antibodies against citrullinated protein antigens (ACPA) have shown their relevance for the prognosis and diagnosis of rheumatoid arthritis (RA), and have been implicated in disease pathogenesis. It is conceivable that other autoantibody systems, recognizing other posttranslationally modified proteins, are also present in RA. Here, we describe the presence of an autoantibody system that discriminates between citrulline- and homocitrulline-containing antigens in the sera of RA-patients. IgG antibodies recognizing carbamylated (homocitrulline-containing) antigens were present in sera of over 45% of RA-patients. Likewise, anticarbamylated protein (anti-CarP) IgA antibodies were observed in 43% of RA-sera. ACPA and anti-CarP antibodies are distinct autoantibodies because, in selected double-positive patients, the anti-CarP antibody binding to carbamylated antigens could be inhibited by carbamylated antigens, but not by control or citrullinated antigens. Similarly, ACPA-binding to citrullinated antigens could only be inhibited by citrullinated antigens. In line with this observation, 16% of ACPA-negative RA-patients, as measured by a standard ACPA assay, harbored IgG anti-CarP antibodies, whereas 30% of these patients tested positive for IgA anti-CarP antibodies. The presence of anti-CarP antibodies was predictive for a more severe disease course in ACPA-negative patients as measured by radiological progression. Taken together, these data show the presence of a unique autoantibody system recognizing carbamylated, but not citrullinated, protein antigens. These antibodies are predictive for a more severe clinical course in ACPA-negative RA-patients, indicating that anti-CarP antibodies are a unique and relevant serological marker for ACPA-negative RA.

Inhibitors of human dimethylarginine dimethylaminohydrolase-1 (DDAH-1) are of therapeutic interest for controlling pathological nitric oxide production. Only a limited number of biologically useful inhibitors have been identified, so structurally diverse lead compounds are desired. In contrast with previous assays that do not possess adequate sensitivity for optimal screening, herein is reported a high-throughput assay that uses an alternative thiol-releasing substrate, S-methyl-L-thiocitrulline, and a thiol-reactive fluorophore, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, to enable continuous detection of product formation by DDAH-1. The assay is applied to query two commercial libraries totaling 4446 compounds, and two representative hits are described, including a known DDAH-1 inhibitor. This is the most sensitive DDAH-1 assay reported to date and enables screening of compound libraries using [S] = K (M) conditions while displaying Z' factors from 0.6 to 0.8. Therefore, this strategy now makes possible high-throughput screening for human DDAH-1 inhibitors in pursuit of molecular probes and drugs to control excessive nitric oxide production.

Objective: To clarify the possible roles of protein-bound homocitrulline in causing an antibody response to citrulline and as a confounding factor in citrulline detection assays., Methods: Native, carbamoylated, and citrullinated albumins were used for testing the specificity of commercial antibodies against modified citrulline by Western blot. Rabbits were immunized with human albumin and/or bone type I collagen, both of which were treated with cyanate to produce homocitrulline, or with citrullinated synthetic telopeptide of type I collagen. These antisera were tested for binding to cyclic citrullinated peptide 2 (CCP-2), mutated citrullinated vimentin, and type I or II collagen telopeptides containing either citrulline or homocitrulline., Results: Commercial antibodies that had been considered to be specific for chemically modified citrulline were found to recognize both homocitrulline- and citrulline-containing albumins. The rabbits immunized with proteins containing homocitrulline produced high-affinity antibodies against CCP-2 and, to a lesser extent, against mutated citrullinated vimentin. The antisera also bound homocitrulline-containing collagen telopeptides and, less strongly, citrulline-containing telopeptides., Conclusion: Our findings demonstrate that homocitrulline, which is a structural analog of citrulline (longer by 1 carbon, and readily formed in the body), can be involved in raising citrulline-recognizing antibodies in experimental animals and can cause false-positive reactions in assays for citrulline., (Copyright © 2010 by the American College of Rheumatology.)