Acute Effects of Citrulline Malate Supplementation on Nocturnal Blood Pressure Dipping After Exercise in Hypertensive Patients: A Randomized, Placebo-Controlled Trial.

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).

Effects of Acute Citrulline Malate Supplementation on CrossFit® Exercise Performance: A Randomized, Double-Blind, Placebo-Controlled, Cross-Over Study.

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.

Novel Chimeric Peptides Based on the Enolase Peptide Antigen (CEP-1) Bearing Three Post-Translational Modifications (Citrullination, Homocitrullination and Acetylation) for Determining the Diagnosis and Severity of Rheumatoid Arthritis.

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.

Hyperornithinemia-hyperammonemia-homocitrullinuria: a rare neurometabolic disorder in two siblings.

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.

Effects of 3 days of citrulline malate supplementation on short-duration repeated sprint running performance in male team sport athletes.

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 (Sdec: 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.

Comparative CKD risk prediction using homocitrulline and carbamylated albumin: two circulating markers of protein carbamylation.

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.

Acute and Chronic Citrulline Malate Supplementation on Muscle Contractile Properties and Fatigue Rate of the Quadriceps.

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.

A critical review of citrulline malate supplementation and exercise performance.

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.

Homocitrullination of lysine residues mediated by myeloid-derived suppressor cells in the tumor environment is a target for cancer immunotherapy.

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.

Acute Effect of Citrulline Malate on Repetition Performance During Strength Training: A Systematic Review and Meta-Analysis.

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.

GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N5-Carboxy-ornithine and N6-Carboxy-lysine.

Derivatization of amino acids by 2 M HCl/CH3OH (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, (d3Me)m-(PFP)n, from synthetic amino acids and 2 M HCl/CD3OD 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 N5-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 N6-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 C1-Carboxylic groups and on their activated Nureido groups. Procedure B also allows in situ preparation of (d3Me)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.

Acute citrulline-malate supplementation is ineffective during aerobic cycling and subsequent anaerobic performance in recreationally active males.

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.

Effect of citrulline on post-exercise rating of perceived exertion, muscle soreness, and blood lactate levels: A systematic review and meta-analysis.

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.

Functional interactions between complex I and complex II with nNOS in regulating cardiac mitochondrial activity in sham and hypertensive rat hearts.

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.

Quantification of the Effect of Citrulline and Homocitrulline Residues on the Collision-Induced Fragmentation of Peptides.

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.

Acute effects of Nitrosigine® and citrulline malate on vasodilation in young adults

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.

Studies on citrullinated LL-37: detection in human airways, antibacterial effects and biophysical properties.

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.

Antibodies and B cells recognising citrullinated proteins display a broad cross-reactivity towards other post-translational modifications.

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.

Effects of Citrulline Supplementation on Exercise Performance in Humans: A Review of the Current Literature.

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.