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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.

Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome is an extremely rare disorder of urea cycle, with few patients reported worldwide. Despite hyperammonemia control, the long-term outcome remains poor with progressive neurological deterioration. We report the clinical, biochemical, and molecular features of two Lebanese siblings diagnosed with this disorder and followed for 8 and 15 years, respectively. Variable clinical manifestations and neurological outcome were observed. The patient with earlier onset of symptoms had a severe neurological deterioration while the other developed a milder form of the disease at an older age. Diagnosis was challenging in the absence of the complete biochemical triad and the non-specific clinical presentations. Whole exome sequencing revealed a homozygous variant, p.Phe188del, in the SLC25A15 gene, a French- Canadian founder mutation previously unreported in Arab patients. Hyperammonemia was controlled in both patients but hyperonithinemia persisted. Frequent hyperalaninemia spikes and lactic acidosis occured concomitantly with the onset of seizures in one of the siblings. Variable neurological deterioration and outcome were observed within the same family. This is the first report from the Arab population of the long-term outcome of this devastating neurometabolic disorder., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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: Citrulline malate (CM), especially when used in conjunction with physical exercise, has demonstrated potential as a non-pharmacological adjunct in the management of hypertension. Nevertheless, its impact on nocturnal blood pressure dipping remains unexplored., Objective: Evaluate the impact of a single dose of CM on nocturnal blood pressure dipping after exercise in hypertensive individuals., Methods: In a double-blind, placebo-controlled, parallel-group clinical trial, twenty hypertensive adults (55 ± 16 years) were randomly assigned to either a CM (6 g) or placebo (6 g of corn starch) group (PLA). Resting blood pressure was measured after a 20-min period of comfortable seating in a calm environment. Both groups underwent 40 min of treadmill running/walking at an intensity of 60-70% of their reserve heart rate, 120 min after ingesting the substances. Ambulatory blood pressure monitoring was employed to measure blood pressure over 24 h., Results: No significant differences in systolic blood pressure values were observed between the CM and PLA groups at rest, during wakefulness, sleep, or over a 24-h period. However, CM exhibited a significant reduction in diastolic blood pressure in several metrics: delta 24 h (-14 mmHg vs -6 mmHg, p  = 0.047), delta wakefulness (-12 mmHg vs -4 mmHg, p  = 0.024), percent delta 24 h (-16% vs -6%, p  = 0.024), and percent delta wakefulness (-14% vs -4%, p  = 0.013). No significant differences were found between CM and PLA in terms of systolic and diastolic nocturnal absolute reductions (-13 mmHg vs -12 mmHg, p  = 0.808, and -13 mmHg vs -8 mmHg, p  = 0.273, respectively) or nocturnal percentage decrease (-9.9% vs -9.4%, p  = 0.844, and -15.3% vs -11.7%, p  = 0.399, respectively)., Conclusions: The study found no significant changes in the post-exercise nocturnal blood pressure dip following a single dose of CM supplementation. However, a notable reduction in diastolic blood pressure was observed during the waking period and over the average 24-h monitoring period., Clinical Trial Registry Number and Website: ClinicalTrials.gov platform (NCT03378596).

As a nitric oxide (NO) enhancer, citrulline malate (CM) has recently been touted as a potential ergogenic aid to both resistance and high-intensity exercise performance, as well as the recovery of muscular performance. The mechanism has been associated with enhanced blood flow to active musculature, however, it might be more far-reaching as either ammonia homeostasis could be improved, or ATP production could be increased via greater availability of malate. Moreover, CM might improve muscle recovery via increased nutrient delivery and/or removal of waste products. To date, a single acute 8 g dose of CM on either resistance exercise performance or cycling has been the most common approach, which has produced equivocal results. This makes the effectiveness of CM to improve exercise performance difficult to determine. Reasons for the disparity in conclusions seem to be due to methodological discrepancies such as the testing protocols and the associated test-retest reliability, dosing strategy (i.e., amount and timing), and the recent discovery of quality control issues with some manufacturers stated (i.e., citrulline:malate ratios). Further exploration of the optimal dose is therefore required including quantification of the bioavailability of NO, citrulline, and malate following ingestion of a range of CM doses. Similarly, further well-controlled studies using highly repeatable exercise protocols with a large aerobic component are required to assess the mechanisms associated with this supplement appropriately. Until such studies are completed, the efficacy of CM supplementation to improve exercise performance remains ambiguous., (© 2021. The Author(s).)

This study compared the acute and chronic impact of citrulline malate (CM) supplementation on muscle contractile properties and fatigue rate of the quadriceps. Eighteen recreationally trained males consumed both a placebo (PL) and CM treatment for two separate dosing periods. The first experimental testing session for each dosing period was considered the baseline day, the second session the acute day, and the third session the chronic day, which followed seven consecutive days of supplementation. All testing sessions included exercising on a cycle ergometer at 50%-60% of their max power output for 30 min followed by performing the Thorstensson test on an isokinetic dynamometer. A two-way (Supplement × Time) analysis of variance with repeated measures resulted in no significant interactions (p > .05) (PL: baseline day, acute day, chronic day vs. CM: baseline day, acute day, chronic day) for peak power (in watts) (469 ± 81, 490 ± 97, 502 ± 99 vs. 464 ± 85, 480 ± 103, 501 ± 81); peak torque (in newton meters) (150 ± 26, 157 ± 32, 161 ± 31 vs. 149 ± 27, 156 ± 33, 161 ± 26); fatigue rate (in percentage) (57 ± 9, 57 ± 10, 58 ± 9 vs. 57 ± 10, 56 ± 9, 58 ± 9); and heart rate (in beats per minute) (156 ± 17, 146 ± 13, 146 ± 9 vs. 155 ± 11, 146 ± 11, 146 ± 9). The results of this study suggest that neither acute nor chronic supplementation of CM had an effect on recovery or fatigue rate of the quadriceps.

Citrulline malate (CitMal) is a dietary supplement that is suggested to enhance strength training performance. However, there is conflicting evidence on this matter. Thus, the purpose of this meta-analysis was to determine whether supplementing with CitMal prior to strength training could increase the total number of repetitions performed before reaching voluntary muscular failure. A systematic search was conducted wherein the inclusion criteria were double-blind, placebo-controlled studies in healthy participants that examined the effect of CitMal on repetitions to failure during upper body and lower body resistance exercises. The Hedges's g standardized mean differences (SMD) between the placebo and CitMal trials were calculated and used in a random effect model. Two separate subanalyses were performed for upper body and lower body exercises. Eight studies, including 137 participants who consisted of strength-trained men (n = 101) and women (n = 26) in addition to untrained men (n = 9), fulfilled the inclusion criteria. Across the studies, 14 single-joint and multijoint exercises were performed with an average of 51 ± 23 total repetitions during 5 ± 3 sets per exercise at ∼70% of one-repetition maximum. Supplementing with 6-8 g of CitMal 40-60 min before exercise increased repetitions by 3 ± 5 (6.4 ± 7.9%) compared with placebo (p = .022) with a small SMD (0.196). The subanalysis for the lower body resulted in a tendency for an effect of the supplement (8.1 ± 8.4%, SMD: 0.27, p = .051) with no significant effect for the upper body (5.7 ± 8.4%, SMD: 0.16, p = .131). The current analysis observed a small ergogenic effect of CitMal compared with placebo. Acute CitMal supplementation may, therefore, delay fatigue and enhance muscle endurance during high-intensity strength training.

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.

Citrulline-malate (CM) purportedly increases exercise performance through increased nitric oxide production. The effects of CM on muscular strength performance are well-documented; however, the benefits of CM on aerobic and anaerobic biking performance are not well researched. Therefore, the present investigation examined the acute CM supplementation effects on aerobic and anaerobic cycling performance in recreationally active males. Methods : 28 recreationally active males (20.9 ± 2.8 years) completed randomized, double-blind, crossover trials consuming CM (12g dextrose + 8g CM) or a placebo (12g dextrose). Participants performed an aerobic cycling protocol (time-to-exhaustion [TTE]), followed by a subsequent 30-second Wingate cycling test, 60-minutes after supplement consumption. Results : Dependent t -tests showed no significant differences ( p  > 0.05) for TTE (PLA: 315.4 s ± 137.7 s; CM: 314.1 s ± 107.1 s) and Total Work Completed (TWC) (PLA: 74.7 ± 34.1 kilojoules (kJ); CM: 74.1 ± 26.4 kJ) during the aerobic cycling protocol. Dependent t -tests also showed no significant differences ( p  > 0.05) for mean watts (PLA: 586.1 ± 87.7 Watts (W); CM: 588.0 ± 93.0 W), peak watts (PLA: 773.0 ± 136.7 W; CM: 786.7 ± 133.0 W), and fatigue index (PLA: 12.9 ± 6.4 FI; CM: 14.3 ± 7.2 FI) during the Wingate protocol. Repeated-measures ANOVA results indicated a significant effect between each 5 s interval ( p  < 0.001), but no differences were observed between trials ( p  > 0.05). Conclusion : Acute CM supplementation in recreationally active males provides no ergogenic benefit in aerobic cycling performance followed by an anaerobic cycling test.

Nitric oxide (NO) affects mitochondrial activity through its interactions with complexes. Here, we investigated regulations of complex I (C-I) and complex II (C-II) by neuronal NO synthase (nNOS) in the presence of fatty acid supplementation and the impact on left ventricular (LV) mitochondrial activity from sham and angiotensin II (Ang-II)-induced hypertensive (HTN) rats. Our results showed that nNOS protein was expressed in sham and HTN LV mitochondrial enriched fraction. In sham, oxygen consumption rate (OCR) and intracellular ATP were increased by palmitic acid (PA) or palmitoyl-carnitine (PC). nNOS inhibitor, S-methyl-l-thiocitrulline (SMTC), did not affect OCR or cellular ATP increment by PA or PC. However, SMTC increased OCR with PA + malonate (a C-II inhibitor), but not with PA + rotenone (a C-I inhibitor), indicating that nNOS attenuates C-I with fatty acid supplementation. Indeed, SMTC increased C-I activity but not that of C-II. Conversely, nNOS-derived NO was increased by rotenone + PA in LV myocytes. In HTN, PC increased the activity of C-I but reduced that of C-II, consequently OCR was reduced. SMTC increased both C-I and C-II activities with PC, resulted in OCR enhancement in the mitochondria. Notably, SMTC increased OCR only with rotenone, suggesting that nNOS modulates C-II-mediated OCR in HTN. nNOS-derived NO was partially reduced by malonate + PA. Taken together, nNOS attenuates C-I-mediated mitochondrial OCR in the presence of fatty acid in sham and C-I modulates nNOS activity. In HTN, nNOS attenuates C-I and C-II activities whereas interactions between nNOS and C-II maintain mitochondrial activity.

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.)

Posttranslational modifications of proteins like citrullination and carbamylation are associated with several diseases. Detailed analytical characterization of citrullinated and carbamylated proteins or peptides could be difficult due to the low concentration of the analytes in complex biological samples. High structural similarity and chemical behavior of citrullinated and carbamylated residues also pose a challenge. We previously reported the "citrulline effect" phenomenon that is manifested in the generation of intense y type ions originating from Cit-Zzz amide bond scissions in collision-induced dissociation tandem mass spectra of citrullinated tryptic peptides. In this study, we created a rigorous tryptic-like model system of both citrulline and homocitrulline-containing peptides that included appropriate and well-defined controls and fragment analogues to quantify the citrulline effect and investigate whether there is an effect for homocitrulline residues as well. Our results show that citrulline residues significantly increased fragmentation at their C-terminus relatively independent of the identity of the following amino acid. In comparison, homocitrulline residues displayed inconclusive results at the same energies. However, the strength of effects was dependent on collision energy and the position of citrulline and homocitrulline in the sequences. As newer software algorithms tend to observe structure-intensity relationships during annotation, this finding increases reliable identification of modified proteins/peptides.

Gonzalez, AM and Trexler, ET. Effects of citrulline supplementation on exercise performance in humans: A review of the current literature. J Strength Cond Res 34(5): 1480-1495, 2020-L-citrulline, a nonessential amino acid found primarily in watermelon, has recently garnered much attention for its potential to augment L-arginine bioavailability, nitric oxide production, and exercise performance. Over the past decade, L-citrulline has received considerable scientific attention examining potentially ergogenic properties for both aerobic and anaerobic exercise performance. Thus, the purpose of this article is to summarize the theoretical rationale behind L-citrulline supplementation and to comprehensively review the available scientific evidence assessing the potential ergogenic value of L-citrulline supplementation on vascular function and exercise performance in humans. In addition, research that has investigated the potential synergistic effects of L-citrulline with other dietary ingredients (e.g., arginine, antioxidants, nitrates, and branched-chain amino acids) is reviewed. Oral L-citrulline and citrulline malate supplementation have shown to increase plasma citrulline and arginine concentrations, along with total nitrate and nitrite concentrations. Although blood flow enhancement is a proposed mechanism for the ergogenic potential of L-citrulline, evidence supporting acute improvements in vasodilation and skeletal muscle tissue perfusion after supplementation is scarce and inconsistent. Nevertheless, several studies have reported that L-citrulline supplementation can enhance exercise performance and recovery. Given the positive effects observed from some investigations, future studies should continue to investigate the effects of both acute and chronic supplementation with L-citrulline and citrulline malate on markers of blood flow and exercise performance and should seek to elucidate the mechanism underlying such effects.

Objective: Autoantibodies against antigens carrying distinct post-translational modifications (PTMs), such as citrulline, homocitrulline or acetyllysine, are hallmarks of rheumatoid arthritis (RA). The relation between these anti-modified protein antibody (AMPA)-classes is poorly understood as is the ability of different PTM-antigens to activate B-cell receptors (BCRs) directed against citrullinated proteins (CP). Insights into the nature of PTMs able to activate such B cells are pivotal to understand the 'evolution' of the autoimmune response conceivable underlying the disease. Here, we investigated the cross-reactivity of monoclonal AMPA and the ability of different types of PTM-antigens to activate CP-reactive BCRs., Methods: BCR sequences from B cells isolated using citrullinated or acetylated antigens were used to produce monoclonal antibodies (mAb) followed by a detailed analysis of their cross-reactivity towards PTM-antigens. Ramos B-cell transfectants expressing CP-reactive IgG BCRs were generated and their activation on stimulation with PTM-antigens investigated., Results: Most mAbs were highly cross-reactive towards multiple PTMs, while no reactivity was observed to the unmodified controls. B cells carrying CP-reactive BCRs showed activation on stimulation with various types of PTM-antigens., Conclusions: Our study illustrates that AMPA exhibit a high cross-reactivity towards at least two PTMs indicating that their recognition pattern is not confined to one type of modification. Furthermore, our data show that CP-reactive B cells are not only activated by citrullinated, but also by carbamylated and/or acetylated antigens. These data are vital for the understanding of the breach of B-cell tolerance against PTM-antigens and the possible contribution of these antigens to RA-pathogenesis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

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.

The ergogenic effects of citrulline malate (CitMal) and beetroot juice (BEET) have been widely studied, but their effects on physiological outcomes related to resistance exercise are not fully understood. The purpose of this randomized, double-blind, crossover study was to investigate the effects of CitMal (8 g) and BEET (400 mg nitrate) on blood pressure (BP), blood flow, and energy efficiency during submaximal leg extension. Recreationally active males ( n  = 27; age: 22 ± 4 yrs) completed familiarization, followed by three testing visits. Supine and standing BP were measured upon arrival, followed by supplement ingestion, a 2-h rest period, postsupplement BP measurement, and a bout of repeated submaximal isotonic leg extensions at 25% of maximal voluntary contraction torque. Diameter (aDIAM) and blood flow (aBF) of the superficial femoral artery, and cross-sectional area (CSA) and echo intensity (EI) of the vastus lateralis, were measured before and after exercise via ultrasonography. Muscle blood flow (mBF) and oxygen consumption (mVO 2 ), along with whole-body energy expenditure (EE) and respiratory exchange ratio (RER), were measured before and during exercise via indirect calorimetry and near-infrared spectroscopy. Baseline RER values differed among treatments ( p  = 0.01); BEET was higher than CitMal ( p  = 0.01) but not PLA ( p  = 0.58); CitMal and PLA were not significantly different ( p  = 0.12). No other measurements were significantly affected by treatment (all p  > 0.05). Results suggest that neither CitMal nor BEET significantly influence resting BP, blood flow, or metabolic efficiency during submaximal leg extension in recreationally active males.

Citrulline malate (CM) is purported to buffer lactic acid, enhance oxygen delivery, and attenuate muscle soreness. Anaerobic exercise trials with CM have produced conflicting results. The aim of the current investigation was to test the efficacy of CM on resistance training (RT) with the hypothesis that CM would improve performance. A double-blind, counter-balanced, randomized control trial was utilized to assess the effects of CM on RT. Nineteen participants (8 female) (25.7 ± 7.7 years), regularly engaged in RT, consumed either 8 g of CM (1.1:1 ratio) or a placebo (6 g citric acid). Participants attempted to perform a German Volume Training (GVT) protocol comprising 10 sets of 10 repetitions of barbell curls at 80% of their one repetition maximum. Repeated ANOVA suggested no effect of CM on RT performance (treatment × time × order p  = .217). There was no difference ( p  = .320) in the total number of reps over the 10 sets (CM median = 57, IQR 45-73; placebo median = 61, IQR 51-69). Blood lactate and creatine kinase did not differ between CM and placebo ( p  > .05). Finally, total muscle soreness was reduced significantly in CM compared to placebo (treatment × time × order p  = .004). These results require corroboration; an ergogenic benefit is yet to be established, and weight trainers should exercise caution when assessing the efficacy of CM. Future research should focus on the potential effects of loading doses of CM.

Trexler, ET, Keith, DS, Schwartz, TA, Ryan, ED, Stoner, L, Persky, AM, and Smith-Ryan, AE. Effects of citrulline malate and beetroot juice supplementation on blood flow, energy metabolism, and performance during maximum effort leg extension exercise. J Strength Cond Res 33(9): 2321-2329, 2019-Citrulline malate (CitMal) and beetroot juice (BEET) are increasingly popular ergogenic aids, but few studies have rigorously investigated their effects on resistance exercise performance and underlying mechanisms. The current randomized, double-blind, crossover study evaluated the effects of CitMal and BEET supplementation on blood flow, metabolic efficiency, and performance during maximal isokinetic leg extension exercise. After familiarization, 27 recreationally active men (age: 22 ± 4 years) completed 3 visits in which subjects ingested a treatment beverage (CitMal [8 g], BEET [400-mg nitrate], or placebo [PLA]), followed by a 2-hour rest period, warm-up, and 5 sets of 30 concentric leg extensions. Before and after exercise, ultrasound was used to measure diameter (aDIAM) and blood flow (aBF) of the superficial femoral artery, along with cross-sectional area and echo intensity of the vastus lateralis. Plasma analytes (lactate, nitrate/nitrite [NOx], and urea nitrogen [BUN]) were also assessed at these times, and indirect calorimetry was used to measure energy expenditure and respiratory exchange ratio before and during exercise. Resting NOx values were higher in BEET (233.2 ± 1.1 μmol·L) compared with CitMal (15.3 ± 1.1, p < 0.0001) and PLA (13.4 ± 1.1, p < 0.0001). Postexercise NOx values, adjusted for resting differences, were higher in BEET (86.3 ± 1.2 μmol·L) than CitMal (21.3 ± 1.1, p < 0.0001) and PLA (18.1 ± 1.1, p < 0.0001). No other variables were affected by treatment (all p > 0.05). While BEET increased NOx, neither treatment was found to enhance performance, blood flow, metabolic efficiency, nor the hormonal response to leg extension exercise.

Farney, TM, Bliss, MV, Hearon, CM, and Salazar, DA. The effect of citrulline malate supplementation on muscle fatigue among healthy participants. J Strength Cond Res 33(9): 2464-2470, 2019-The focus of the investigation was to examine the effects of citrulline malate (CM) on muscular fatigue in healthy, recreationally trained participants. Twelve participants (men = 6 and women = 6) (24.1 ± 3.9 years) visited the laboratory on 3 separate days, all separated by 1 week. Each visit consisted of consuming 1 of 3 treatments: placebo, CM (8 g), and control in which no drink mixture was consumed. For each day of testing, participants consumed assigned treatment and performed 1 high-intensity exercise trial consisting of squats, lunge jumps, squat jumps, and lateral jumps. Participants performed the exercises in the listed order, which was designated as 1 round. Each participant performed 3 rounds, with the work-to-rest ratio being 20 seconds of work and 30 seconds of rest. A 1-minute rest was given between rounds. A pre/post-exercise isokinetic leg extension test was performed to measure for peak power, peak torque, and rate of fatigue. In addition, blood lactate was obtained pre/post-exercise. There were no treatment or interaction effects (p > 0.05) for peak torque, peak power, rate of fatigue, or blood lactate accumulation. However, there was a statistical significant decrease from pre/post-exercise for peak torque (p = 0.003), peak power (p = 0.003), and rate of fatigue (p = 0.001). In addition, lactate accumulation did increase significantly from pre/post-exercise (p = 0.0001). Finally, neither total work nor final heart rate was statistically significant between the treatments (p > 0.05). Citrulline malate was not effective in improving performance or alleviating fatigue after a high-intensity exercise session.

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.

Manipulating phase separation structures of thermoresponsive polymers will enhance the usefulness of structure-controllable materials in fields such as drug delivery and tissue engineering. However, behaviors of upper critical solution temperature (UCST) have been less investigated so far, despite the importance of UCST. Here, we examined two citrulline-based polypeptides, poly(d-ornithine- co-d-citrulline) (PdOC) and poly(dl-ornithine- co-dl-citrulline) (PdlOC), to investigate how stereoregularity of the polypeptides influences UCST behavior, in addition to poly(l-ornithine- co-l-citrulline) (PlOC) previously studied. Homochiral PlOC and PdOC showed phase separation temperatures ( T p s) higher than that of racemic PdlOC. Moreover, PdlOC underwent liquid to coacervate phase separation at T p , whereas PlOC and PdOC underwent liquid to solid-like aggregation transitions. From a structural point of view, circular dichroism and small-angle X-ray scattering measurements revealed that homochiral PlOC and PdOC polypeptides formed α-helical structures and assembled into a regular hexagonal lattice upon phase separation. Interactions between the pendent ureido groups of homochiral POCs appear to play pivotal roles in helical folding and assembly into the hexagonal structure. In addition, T p change in response to biodegradation was confirmed for both PlOC and PdlOC. The biodegradability was considerably influenced by phase-separated structures. These findings of UCST-type POCs in this study would provide important insights into structure-controllable and thermoresponsive biomaterials.

Gonzalez, AM, Spitz, RW, Ghigiarelli, JJ, Sell, KM, and Mangine, GT. Acute effect of citrulline malate supplementation on upper-body resistance exercise performance in recreationally resistance-trained men. J Strength Cond Res 32(11): 3088-3094, 2018-To investigate the effect of citrulline malate (CM) supplementation on upper-body resistance exercise performance, 12 recreationally resistance-trained men (21.4 ± 1.6 years; 163.0 ± 46.2 cm; 85.0 ± 12.4 kg) underwent 2 testing sessions administered in a randomized, double-blind fashion. During each visit, participants were provided either 8 g of CM or a placebo (PL) 40 minutes before beginning a standardized warm-up and initiating a barbell bench press resistance exercise protocol consisting of 5 sets of 15 repetitions at 75% 1 repetition maximum with 2-minute rest intervals. Participants were instructed to complete as many repetitions as possible until either reaching 15 repetitions or muscular failure. Total number of repetitions performed and power output were recorded for each set. Subjective measures of energy, focus, fatigue, and perceived exertion, along with muscle thickness of the triceps brachii, were assessed before and after exercise. Significant (p ≤ 0.05) main effects for time were observed for all variables except for subjective feelings of energy (p = 0.085). A group × time interaction (F = 2.86, p = 0.034, n = 0.21) was observed for repetitions performed, where participants performed more (p = 0.015) repetitions on set 3 during PL (5.7 ± 1.2 repetitions) compared with CM (4.6 ± 1.2 repetitions). However, during set 4, participants tended (p = 0.089) to perform more repetitions during CM (4.8 ± 1.8 repetitions) compared with PL (4.3 ± 1.3 repetitions). No other differences were observed between trials. Supplementation with 8 g of CM 40 minutes before the barbell bench press resistance exercise protocol did not increase exercise performance, augment the muscle swelling response to training, or alter subjective measures of focus, energy, and fatigue in recreationally resistance-trained men.

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.

Antibodies to proteins/peptides containing citrulline are hallmarks of Rheumatoid Arthritis (RA). These antibodies are strongly associated with the expression of the Shared Epitope (SE). RA patients also generate antibodies to homocitrulline-containing proteins/peptides (also referred to as anti-carbamylated protein antibodies (Anti-CarP)). This study was undertaken to investigate the relationship between homocitrulline and citrulline immune responses using an established mouse model of RA: DR4-transgenic (DR4tg) mice that express the human SE. C57BL/6 (B6) and DR4tg (on a B6 background) mice were immunized subcutaneously with a homocitrullinated peptide (HomoCitJED). Splenic T cell proliferation was evaluated by 3 H-thymidine incorporation assay. Antibodies to homocitrullinated and citrullinated antigens were screened by enzyme-linked immunosorbent assay (ELISA). Antibody cross-reactivity was examined by inhibition with HomoCitJED and its citrullinated counterpart peptide, CitJED (the number of homocitrullines in HomoCitJED is equal to the number of citrullines in CitJED). HomoCitJED-immunized DR4tg mice developed early T and B cell responses to HomoCitJED and late responses to CitJED. These mice also developed anti-CCP2 antibodies. In some mice, antibodies to HomoCitJED were also reactive to CitJED. B6 mice immunized with HomoCitJED developed late T and B cell responses to HomoCitJED, but did not generate responses to citrullinated antigens. Unlike DR4tg mice, anti-HomoCitJED antibodies from B6 mice did not react to CitJED. In conclusion, DR4tg mice immunized with HomoCitJED developed immune responses to CitJED, indicating cross-reactivity. CitJED immune responses were dependent on the SE. HomoCitJED responses occurred in the absence of the SE (B6 mice); however, they developed earlier in DR4tg SE-expressing mice., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Spontaneous urea dissociation in water solution is a prominent source of protein carbamylation in our body. Protein carbamylation is a well-known phenomenon since early seventies. Some years ago, much interest in the diagnostic power of carbamylated protein arouse. Recently the target of the researches focused on its potential cardiovascular pathogenicity. Some authors claimed that this could be a reason for higher cardiovascular mortality in uremic patients. Nutritional therapy, amino acids supplementation and intensive dialysis regimen are some of the therapeutic tools tested to lower the carbamylation burst in this population., (Copyright by Società Italiana di Nefrologia SIN, Rome, Italy.)

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.)

Carbamylation corresponds to the non-enzymatic binding of isocyanic acid to protein amino groups and participates in protein molecular aging, characterized by the alteration of their structural and functional properties. Carbamylated proteins exert deleterious effects in vivo and are involved in the progression of various diseases, including atherosclerosis and chronic kidney disease. Therefore, there is a growing interest to evaluate the carbamylation rate of blood or tissue proteins, since carbamylation-derived products (CDPs) constitute valuable biomarkers in these contexts. Homocitrulline, formed by isocyanic acid covalently attaches to the ε-NH 2 group of lysine residue side chain, is the most characteristic CDP. Sensitive and specific quantification of homocitrulline requires mass spectrometry-based methods. This unit describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of homocitrulline, with special emphasis on pre-analytical steps that allow quantification of total or protein-bound homocitrulline in serum or tissue samples. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)

Objective: Antibodies that target citrullinated protein/peptide (ACPA) and homocitrullinated/carbamylated protein/peptide (AHCPA) are associated with rheumatoid arthritis (RA). The relationship between ACPA and AHCPA remains unclear. We examined the expression and cross-reactivity of these antibodies using citrulline- and homocitrulline-containing synthetic peptides, CitJED and HomoCitJED, respectively, which have equal numbers of citrulline or homocitrulline residues on the same peptide backbone., Methods: Serum from healthy subjects (n = 51) and patients with RA (n = 137), systemic lupus erythematosus (SLE; n = 37), and psoriatic arthritis (PsA; n = 37) were screened for IgG anti-CitJED and anti-HomoCitJED antibodies by ELISA. Cross-reactivity of these antibodies was examined by inhibition with various concentrations of CitJED and HomoCitJED., Results: Out of 137 patients with RA, antibodies to CitJED and HomoCitJED were detected in 69 (50%) and 78 (57%), respectively. Anti-CitJED and HomoCitJED antibodies were 77% concordant and their levels were strongly correlated [Spearman correlation coefficient (r s ) = 0.6676]. Sera from 25/27 patients (93%) with RA were inhibited by both CitJED and HomoCitJED with equal or higher affinity for the cognate (homologous) peptide., Conclusion: Antibodies to CitJED and HomoCitJED frequently occurred in RA, but were not found in SLE or PsA, suggesting that these antibodies are specific to RA. Cross-reactivity between anti-HomoCitJED and anti-CitJED antibodies suggests that ACPA and AHCPA are derived from the same B cell population and both may contribute to the pathogenesis of RA.

Reduced skeletal muscle density in older adults is associated with insulin resistance, decreased physical function, and an increased all-cause mortality risk. To elucidate mechanisms that may underlie the maintenance of skeletal muscle density, we conducted a secondary analysis of previously published muscle composition and serum metabolomic data in 73 older adults (average age, 78y). Multivariable-adjusted linear regression was used to examine associations between 321 metabolites with muscle composition, defined as the ratio between normal density (NDM) with low density (LDM) thigh muscle cross sectional area (NDM/LDM). Sixty metabolites were significantly (p≤0.05 and q<0.30) associated with NDM/LDM. Decreased renal function and the immune response have been previously linked with reduced muscle density, but the mechanisms underlying these connections are less clear. Metabolites that were significantly associated with muscle composition were then tested for their association with circulating markers of renal function (blood urea nitrogen, creatinine, uric acid), and with the immune response (neutrophils/lymphocytes) and activation (kynurenine/tryptophan). 43 significant NDM/LDM metabolites (including urea) were co-associated with at least 1 marker of renal function; 23 of these metabolites have been previously identified as uremic solutes. The neutrophil/lymphocyte ratio was significantly associated with NDM/LDM (β±SE: -0.3±0.1, p=0.01, q=0.04). 35 significant NDM/LDM metabolites were co-associated with immune activation. Carbamylation (defined as homocitrulline/lysine) was identified as a pathway that may link renal function and immune activation with muscle composition, as 29 significant NDM/LDM metabolites were co-associated with homocitrulline/lysine, with at least 2 markers of renal function, and with kynurenine/tryptophan. When considering that elevated urea and uremic metabolites have been linked with an increased systemic microbial burden, that antimicrobial defense can be reduced in the presence of carbamylation, and that adipocytes can promote host defense, we propose the novel hypothesis that the age-related increase in adipogenesis within muscle may be a compensatory antimicrobial response to protect against an elevated microbial burden., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: to determine the effectiveness of citrulline use for correcting endothelial dysfunction in children resi dents of radioactively contaminated areas., Materials and Methods: A group of children residents of radioactively contaminated areas with the presence of clinical and paraclinical signs of endothelial dysfunction was selected to assess the effectiveness of correcting endothelial dysfunction by the usage of NO potential donator - citrulline according to the data of selective screen ing. There were determined the biochemical parameters of the content of NO stable metabolites, L arginine, lipid peroxidation, antioxidant enzymes in the blood serum; the indices of cellular and humoral immunity; the instrumen tal indices of vascular endothelium dependent reaction on occlusion test, the lung ventilation capacity, the bioelec tric activity of the myocardium, the autonomic regulation of the cardiovascular system.Examined children were received a course of citrulline malate., Results and Conclusions: An increased content of serum L arginine, nitrite, and amounts of NO metabolites was established in children with endothelial dysfunction who were received a course of citrulline malate. Bronchospasm elimination was noted in the significant part of examined patients after the drug use. Decreased recovery period and increased period of hypercompensation for thermographic circulation index in the test with post occlusion reac tive hyperemia were detected by an evaluation of indicators for vascular endothelium dependent vasodilatation using thermographic method indicating an increased endothelial vasomotor capacity. There was tendency to improve the processes of autonomic regulation of the heart rhythm and repolarization of the heart muscle. The antioxidant effect of used citrulline malate course was determined as: decreased content of serum LPO end products that react with thiobarbituric acid under elevated activity of antioxidant - catalase. An increase in the percentage of T lymphocyte, normalization of their subpopulation composition was noted in dynamics of citrulline malate application., (Ye. I. Stepanova, I. Ye.Kolpakov, V. M. Zygalo, V. Yu. Vdovenko, V. H. Kondrashova, O. S. Leonovich, V. V. Kryzhanivska.)

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.

Objectives: Over 50% of patients with rheumatoid arthritis (RA) harbour a variety of anti-modified protein antibodies (AMPA) against different post-translationally modified (PTM) proteins, including anti-carbamylated protein (anti-CarP) antibodies. At present, it is unknown how AMPA are generated and how autoreactive B cell responses against PTM proteins are induced. Here we studied whether PTM foreign antigens can breach B cell tolerance towards PTM self-proteins., Methods: Serum reactivity towards five carbamylated proteins was determined for 160 patients with RA and 40 healthy individuals. Antibody cross-reactivity was studied by inhibition experiments. Mass spectrometry was performed to identify carbamylated self-proteins in human rheumatic joint tissue. Mice were immunised with carbamylated or non-modified (auto)antigens and analysed for autoantibody responses., Results: We show that anti-CarP antibodies in RA are highly cross-reactive towards multiple carbamylated proteins, including modified self-proteins and modified non-self-proteins. Studies in mice show that anti-CarP antibody responses recognising carbamylated self-proteins are induced by immunisation with carbamylated self-proteins and by immunisation with carbamylated proteins of non-self-origin. Similar to the data observed with sera from patients with RA, the murine anti-CarP antibody response was, both at the monoclonal level and the polyclonal level, highly cross-reactive towards multiple carbamylated proteins, including carbamylated self-proteins., Conclusions: Self-reactive AMPA responses can be induced by exposure to foreign proteins containing PTM. These data show how autoreactive B cell responses against PTM self-proteins can be induced by exposure to PTM foreign proteins and provide new insights on the breach of autoreactive B cell tolerance., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

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.)

Objectives: Smoking has been connected to citrullination of antigens and formation of anti-citrullinated peptide antibodies (ACPAs) in rheumatoid arthritis (RA). Since smoking can modify proteins by carbamylation (formation of homocitrulline), this study was conducted to investigate these effects on vimentin in animal models and RA., Methods: The efficiency of enzymatic carbamylation of vimentin was characterised. B-cell response was investigated after immunisation of rabbits with different vimentin isoforms. Effects of tobacco smoke exposure on carbamylation of vimentin and formation of autoantibodies were analysed in mice. The antibody responses against isoforms of vimentin were characterised with respect to disease duration and smoking status of patients with RA., Results: Enzymatic carbamylation of vimentin was efficiently achieved. Subsequent citrullination of vimentin was not disturbed by homocitrullination. Sera from rabbits immunised with carbamylated vimentin (carbVim), in addition to carbVim also recognised human IgG-Fc showing rheumatoid factor-like reactivity. Smoke-exposed mice contained detectable amounts of carbVim and developed a broad immune response against carbamylated antigens. Although the prevalence of anti-carbamylated antibodies in smokers and non-smokers was similar, the titres of carbamylated antibodies were significantly increased in sera of smoking compared with non-smoking RA. CarbVim antibodies were observed independently of ACPAs in early phases of disease and double-positive patients for anti-mutated citrullinated vimentin (MCV) and anti-carbVim antibodies showed an extended epitope recognition pattern towards MCV., Conclusions: Carbamylation of vimentin is inducible by cigarette smoke exposure. The polyclonal immune response against modified antigens in patients with RA is not exclusively citrulline-specific and carbamylation of antigens could be involved in the pathogenesis of disease., Trial Registration Number: ISRCTN36745608; EudraCT Number: 2006-003146-41., Competing Interests: Competing interests: HB is an employee of Orgentec Diagnostika GmbH, Mainz, Germany; test kits were kindly provided by Orgentec., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

In 2011 a novel autoantibody system, anti-carbamylated protein (anti-CarP) antibodies, was described in rheumatoid arthritis (RA) patients. Anti-CarP antibody positivity associates with a more severe disease course, is observed years before disease onset, and may predict the development of RA in arthralgia patients. Although many clinical observations have been carried out, information on the antigenic targets of anti-CarP antibodies is limited. Most studies on anti-CarP antibodies utilize an ELISA-based assay with carbamylated fetal calf serum (Ca-FCS) as antigen, a complex mixture of proteins. Therefore, we analysed the molecular identity of proteins within Ca-FCS that are recognized by anti-CarP antibodies. Ca-FCS was fractionated using ion exchange chromatography, selecting one of the fractions for further investigation. Using mass-spectrometry, carbamylated alpha-1-antitrypsin (Ca-A1AT) was identified as a potential antigenic target of anti-CarP antibodies in RA patients. A1AT contains several lysines on the protein surface that can readily be carbamylated. A large proportion of the RA patients harbour antibodies that bind human Ca-A1AT in ELISA, indicating that Ca-A1AT is indeed an autoantigen for anti-CarP antibodies. Next to the Ca-A1AT protein, several homocitrulline-containing peptides of A1AT were recognized by RA sera. Moreover, we identified a carbamylated peptide of A1AT in the synovial fluid of an RA patient using mass spectrometry. We conclude that Ca-A1AT is not only a target of anti-CarP antibodies but is also present in the synovial compartment, suggesting that Ca-A1AT recognized by anti-CarP antibodies in the joint may contribute to synovial inflammation in anti-CarP-positive RA., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Carbamylation is a non-enzymatic post-translational modification induced upon exposure of free amino groups to urea-derived cyanate leading to irreversible changes of protein charge, structure and function. Levels of carbamylated proteins increase significantly in chronic kidney disease and carbamylated albumin is considered as an important biomarker indicating mortality risk. High plasma concentrations and long half-life make fibrinogen a prime target for carbamylation. As aggregation and cross-linking of fibrin monomers rely on lysine residues, it is likely that carbamylation impacts fibrinogen processing. In this study we investigated carbamylation levels of fibrinogen from kidney disease patients as well as the impact of carbamylation on fibrinogen cleavage by thrombin, fibrin polymerisation and cross-linking in vitro. In conjunction, all these factors determine clot structure and stability and thus control biochemical and mechanical properties. LC-MS/MS analyses revealed significantly higher homocitrulline levels in patient fibrinogen than in fibrinogen isolated from control plasma. In our in vitro studies we found that although carbamylation does not affect thrombin cleavage per se, it alters fibrin polymerisation kinetics and impairs cross-linking and clot degradation. In addition, carbamylated fibrin clots had reduced fiber size and porosity associated with decreased mechanical stability. Using mass spectroscopy, we discovered that N-terminally carbamylated fibrinopeptide A was generated in this process and acted as a strong neutrophil chemoattractant potentially mediating recruitment of inflammatory cells to sites of fibrin(ogen) turnover. Taken together, carbamylation of fibrinogen seems to play a role in aberrant fibrin clot formation and might be involved in haemostatic disorders associated with chronic inflammatory diseases.

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.)

Purpose: Citrulline malate (CM) is a nonessential amino acid that increases exercise performance in males. However, based on physiological differences between genders, these results cannot be extrapolated to females. Therefore, the purpose of this investigation was to evaluate effects of acute CM supplementation on upper- and lower-body weightlifting performance in resistance-trained females., Methods: Fifteen females (23 ± 3 years) completed two randomized, double-blind trials consuming either CM (8 g dextrose + 8 g CM) or a placebo (8 g dextrose). One hour after supplement consumption, participants performed six sets each of upper- (i.e., bench press) and lower-body (i.e., leg press) exercises to failure at 80 % of previously established one-repetition maximum. Immediately after each set, repetitions completed, heart rate and rating of perceived exertion (RPE) were recorded., Results: Repeated-measures analysis of variance indicated that subjects completed significantly (p = .045) more repetitions throughout upper-body exercise when consuming CM versus placebo (34.1 ± 5.7 vs. 32.9 ± 6.0, respectively). When consuming CM, similar significant (p = .03) improvements in total repetitions completed were observed for lower-body exercise (66.7 ± 30.5 vs. 55.13 ± 20.64, respectively). Overall RPE score was significantly lower (p = .02) in upper-body exercise when subjects consumed CM versus placebo (7.9 ± 0.3 and 8.6 ± 0.2, respectively). The supplement consumed exhibited no significant effects on heart rate at any time point., Conclusions: Acute CM supplementation in females increased upper- and lower-body resistance exercise performance and decreased RPE during upper-body exercise. These data indicate that athletes competing in sports with muscular endurance-based requirements may potentially improve performance by acutely supplementing CM.