Protein Citrullination by Peptidyl Arginine Deiminase/Arginine Deiminase Homologs in Members of the Human Microbiota and Its Recognition by Anti-Citrullinated Protein Antibodies.

The human microbiome exists throughout the body, and it is essential for maintaining various physiological processes, including immunity, and dysbiotic events, which are associated with autoimmunity. Peptidylarginine deiminase (PAD) enzymes can citrullinate self-proteins related to rheumatoid arthritis (RA) that induce the production of anti-citrullinated protein antibodies (ACPAs) and lead to inflammation and joint damage. The present investigation was carried out to demonstrate the expression of homologs of PADs or arginine deiminases (ADs) and citrullinated proteins in members of the human microbiota. To achieve the objective, we used 17 microbial strains and specific polyclonal antibodies (pAbs) of the synthetic peptide derived from residues 100-200 of human PAD2 (anti-PAD2 pAb), and the recombinant fragment of amino acids 326 and 611 of human PAD4 (anti-PAD4 pAb), a human anti-citrulline pAb, and affinity ACPAs of an RA patient. Western blot (WB), enzyme-linked immunosorbent assay (ELISA), elution, and a test with Griess reagent were used. This is a cross-sectional case-control study on patients diagnosed with RA and control subjects. Inferential statistics were applied using the non-parametric Kruskal-Wallis test and Mann-Whitney U test generated in the SPSS program. Some members of phyla Firmicutes and Proteobacteria harbor homologs of PADs/ADs and citrullinated antigens that are reactive to the ACPAs of RA patients. Microbial citrullinome and homolog enzymes of PADs/ADs are extensive in the human microbiome and are involved in the production of ACPAs. Our findings suggest a molecular link between microorganisms of a dysbiotic microbiota and RA pathogenesis.

Exploring the effects of saccharides on the solvation behaviour of L-Citrulline in aqueous medium: Volumetric, transition state theory, transfer parameters and spectroscopic approach.

This study examines the volumetric, viscometric and UV-vis characteristics of L-Citrulline in water and aqueous saccharides at atmospheric pressure across the whole concentration range and the absorber operating temperature range of 293.15 K -313.15 K. Density, partial molar volume, apparent molar isobaric expansion, Hepler's constant and hydration number were among the examined volumetric parameters, and viscosity coefficients, viscosity deviation and free energy for viscous flow activation were among the examined viscometric parameters. Stronger interactions between L-Citrulline and L-Arabinose were indicated by the increase in the transfer characteristics in the following order: L-Arabinose > D-xylose. A comparison of the taste behaviours of L-Citrulline in water and in an aqueous solution of saccharides has also been attempted. L-Citrulline interacts with all solvents in a significant way, as evidenced by the UV-visible spectra suggested by the shift in UV-visible absorption maxima that correspond with a rise in L-Citrulline content in the solvent systems chosen.

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.

Effects of supplemental citrulline on thermal and intestinal morphology parameters during heat stress and feed restriction in growing pigs.

Study objectives were to characterize the effects of citrulline (CIT) on physiological and intestinal morphology metrics during heat stress (HS) and feed restriction. Forty crossbred gilts (30 ±â€…2 kg body weight [BW]) were assigned to one of five treatments: (1) thermoneutral (TN) fed ad libitum (AL) with control (CON) supplement (TNAL; n = 8), (2) TN pair-fed (PF) with CON (PF-CON; n = 8), (3) TN PF with CIT (PF-CIT; n = 8), (4) HS AL with CON (HS-CON; n = 8), and (5) HS AL with CIT (HS-CIT; n = 8). During the period (P) 1 (7 d), pigs were in TN conditions (23.6 °C) and fed AL their respective supplemental treatments. During P2 (2.5 d), HS-CON and HS-CIT pigs were fed AL and exposed to cyclical HS (33.6 to 38.3 °C), while TNAL, PF-CON, and PF-CIT remained in TN and were fed either AL or PF to their HS counterparts. Citrulline (0.13 g/kg BW) was orally administered twice daily during P1 and P2. HS increased rectal temperature (Tr), skin temperature (Ts), and respiration rate (RR) relative to TN pigs (0.8 °C, 4.7 °C, and 47 breaths/min, respectively; P < 0.01). However, HS-CIT had decreased RR (7 breaths/min, P = 0.04) and a tendency for decreased Tr (0.1 °C, P = 0.07) relative to HS-CON pigs. During P2, HS pigs had decreased feed intake (22%; P < 0.01) and a tendency for decreased average daily gain (P = 0.08) relative to TNAL pigs, and by experimental design, PF pigs followed this same pattern. Circulating lipopolysaccharide-binding protein tended to be decreased (29%; P = 0.08) in PF relative to TNAL pigs and was increased (41%; P = 0.03) in HS compared to PF pigs. Jejunum villus height was decreased in PF relative to TNAL pigs (15%; P = 0.03); however, CIT supplementation improved this metric during feed restriction (16%; P = 0.10). Jejunum mucosal surface area decreased in PF (16%; P = 0.02) and tended to decrease in HS (11%; P = 0.10) compared to TNAL pigs. Ileum villus height and mucosal surface area decreased in HS compared to TNAL pigs (10 and 14%, respectively; P ≤ 0.04), but both parameters were rescued by CIT supplementation (P ≤ 0.08). Intestinal myeloperoxidase and goblet cell area remained similar among treatments and intestinal segments (P > 0.24). In summary, CIT supplementation slightly improved RR and Tr during HS. Feed restriction and HS differentially affected jejunum and ileum morphology and while CIT ameliorated some of these effects, the benefit appeared dependent on intestinal section and stressor type.

Heat stress (HS) negatively affects animal health and production efficiency and is a significant economic burden to global animal agriculture. Although the mechanisms responsible for reduced animal productivity during HS are complex and multifaceted, increasing evidence points to decreased intestinal barrier function as an important mediator of this response. Furthermore, HS causes a voluntary reduction in feed intake, and feed restriction independently induces gastrointestinal hyperpermeability. Loss of intestinal barrier integrity facilitates bacteria translocation across the epithelium into local and systemic circulation, thus initiating an immune response. Dietary citrulline has been shown to support gut health by improving intestinal barrier integrity and modulating intestinal inflammation. Therefore, the current study investigated the effects of citrulline supplementation on physiological and intestinal morphology parameters in heat-stressed and feed-restricted growing pigs. Herein, citrulline supplementation reduced respiration rate and rectal temperature in pigs exposed to the thermal load. Heat stress and feed restriction compromised small intestinal morphology, and while supplementing citrulline improved some of these parameters, the effects depended on the intestinal region and stressor type. Additional research is needed to evaluate the potential effects of citrulline supplementation on gut health during HS or nutrient restriction.

An energy-conserving reaction in amino acid metabolism catalyzed by arginine synthetase.

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon Thermococcus kodakarensis, we identified an enzyme which we designate "arginine synthetase". In arginine synthesis, the enzyme converts citrulline, ATP, and free ammonia to arginine, adenosine 5'-diphosphate (ADP), and phosphate. In the reverse direction, arginine synthetase conserves the energy of arginine deimination and generates ATP from ADP and phosphate while releasing ammonia. The equilibrium constant of this reaction at pH 7.0 is [Cit][ATP][NH3]/[Arg][ADP][Pi] = 10.1 ± 0.7 at 80 °C, corresponding to a ΔG°' of -6.8 ± 0.2 kJ mol-1. Growth of the gene disruption strain was compared to the host strain in medium composed of amino acids. The results suggested that arginine synthetase is necessary in providing ornithine, the precursor for proline biosynthesis, as well as in generating ATP. Growth in medium supplemented with citrulline indicated that arginine synthetase can function in the direction of arginine synthesis. The enzyme is widespread in nature, including bacteria and eukaryotes, and catalyzes a long-overlooked energy-conserving reaction in microbial amino acid metabolism. Along with ornithine transcarbamoylase and carbamate kinase, the pathway identified here is designated the arginine synthetase pathway.

[Newborn screening, clinical features and genetic analysis for Citrin deficiency in Henan province].

OBJECTIVE: To explore the prevalence, clinical features, genetic characteristics and prognosis of Citrin deficiency in Henan province of China. METHODS: A total of 986 565 neonates screened by tandem mass spectrometry at the Third Affiliated Hospital of Zhengzhou University from January 2013 to December 2021 were retrospectively analyzed. Analysis of SLC25A13 gene variants and parental verification were carried out for neonates suspected for Citrin deficiency by next-generation sequencing. The clinical, biochemical and genetic characteristics of Citrin deficiency patients were integrated to guide the diet treatment and follow up the growth and development. Paired-t test was used to compare the amino acid levels in the peripheral blood samples before and after the treatment. RESULTS: Nine cases of Citrin deficiency were diagnosed among the 986 565 neonates. Specific elevation of citrulline was observed in all of the 9 cases. Six variants were detected by genetic sequencing, among which c.852_855delTATG, c.615+5G>A, c.550C>T and IVS16ins3kb were known pathogenic variants, whilst c.1111_1112delAT and c.837T>A were unreported previously. The detection rate for c. 852_855delTATG was the highest (61.6%, 11/18), followed by IVS16ins3kb (16.7%, 3/18). The clinical symptoms of all patients were relieved after the treatment, and the blood amino acid profile and biochemical parameters were significantly improved by gradually falling within the normal range. By June 2022, all patients had shown a good prognosis. CONCLUSION: The prevalence of Citrin deficiency among neonates from Henan Province by tandem mass spectrometry is 1/109 618, and the carrier rate for the pathogenic variants of the SLC25A13 gene was 1/166. The c.852_855delTATG may be a hot spot variant among the patients. Discovery of the novel variants has enriched the mutational spectrum of the SLC25A13 gene. Above results have provided a basis for the early diagnosis, treatment, prognosis and genetic counseling for the affected families.

The monocyte cell surface is a unique site of autoantigen generation in rheumatoid arthritis.

Although anti-citrullinated protein autoantibodies (ACPAs) are a hallmark serological feature of rheumatoid arthritis (RA), the mechanisms and cellular sources behind the generation of the RA citrullinome remain incompletely defined. Peptidylarginine deiminase IV (PAD4), one of the key enzymatic drivers of citrullination in the RA joint, is expressed by granulocytes and monocytes; however, the subcellular localization and contribution of monocyte-derived PAD4 to the generation of citrullinated autoantigens remain underexplored. In this study, we demonstrate that PAD4 displays a widespread cellular distribution in monocytes, including expression on the cell surface. Surface PAD4 was enzymatically active and capable of citrullinating extracellular fibrinogen and endogenous surface proteins in a calcium dose-dependent manner. Fibrinogen citrullinated by monocyte-surface PAD4 could be specifically recognized over native fibrinogen by a panel of eight human monoclonal ACPAs. Several unique PAD4 substrates were identified on the monocyte surface via mass spectrometry, with citrullination of the CD11b and CD18 components of the Mac-1 integrin complex being the most abundant. Citrullinated Mac-1 was found to be a target of ACPAs in 25% of RA patients, and Mac-1 ACPAs were significantly associated with HLA-DRB1 shared epitope alleles, higher C-reactive protein and IL-6 levels, and more erosive joint damage. Our findings implicate the monocyte cell surface as a unique and consequential site of extracellular and cell surface autoantigen generation in RA.

The Role of Citrullination Modification in CD4+ T Cells in the Pathogenesis of Immune-Related Diseases.

The post-translational modifications (PTMs) of proteins play a crucial role in increasing the functional diversity of proteins and are associated with the pathogenesis of various diseases. This review focuses on a less explored PTM called citrullination, which involves the conversion of arginine to citrulline. This process is catalyzed by peptidyl arginine deiminases (PADs). Different members of the PAD family have distinct tissue distribution patterns and functions. Citrullination is a post-translational modification of native proteins that can alter their structure and convert them into autoantigens; thus, it mediates the occurrence of autoimmune diseases. CD4+ T cells, including Th1, Th2, and Th17 cells, are important immune cells involved in mediating autoimmune diseases, allergic reactions, and tumor immunity. PADs can induce citrullination in CD4+ T cells, suggesting a role for citrullination in CD4+ T cell subset differentiation and function. Understanding the role of citrullination in CD4+ T cells may provide insights into immune-related diseases and inflammatory processes.

Mass Spectrometry Reveals Molecular Effects of Citrulline Supplementation during Bone Fracture Healing in a Rat Model.

Bone fracture healing is a complex process in which specific molecular knowledge is still lacking. The citrulline-arginine-nitric oxide metabolism is one of the involved pathways, and its enrichment via citrulline supplementation can enhance fracture healing. This study investigated the molecular effects of citrulline supplementation during the different fracture healing phases in a rat model. Microcomputed tomography (µCT) was applied for the analysis of the fracture callus formation. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid-chromatography tandem mass spectrometry (LC-MS/MS) were used for lipid and protein analyses, respectively. µCT analysis showed no significant differences in the fracture callus volume and volume fraction between the citrulline supplementation and control group. The observed lipid profiles for the citrulline supplementation and control group were distinct for the different fracture healing stages. The main contributing lipid classes were phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs). The changing effect of citrulline supplementation throughout fracture healing was indicated by changes in the differentially expressed proteins between the groups. Pathway analysis showed an enhancement of fracture healing in the citrulline supplementation group in comparison to the control group via improved angiogenesis and earlier formation of the soft and hard callus. This study showed the molecular effects on lipids, proteins, and pathways associated with citrulline supplementation during bone fracture healing, even though no effect was visible with µCT.

Investigating genetic links between blood metabolites and preeclampsia.

BACKGROUND: Observational studies have revealed that metabolic disorders are closely related to the development of preeclampsia (PE). However, there is still a research gap on the causal role of metabolites in promoting or preventing PE. We aimed to systematically explore the causal association between circulating metabolites and PE. METHODS: Single nucleotide polymorphisms (SNPs) from genome-wide association study (GWAS) of 486 blood metabolites (7,824 participants) were extracted as instrumental variables (P < 1 × 10- 5), GWAS summary statistics for PE were obtained from FinnGen consortium (7,212 cases and 194,266 controls) as outcome, and a two-sample Mendelian randomization (MR) analysis was conducted. Inverse variance weighted (IVW) was set as the primary method, with MR-Egger and weighted median as auxiliary methods; the instrumental variable strength and confounding factors were also assessed. Sensitivity analyses including MR-Egger, Cochran's Q test, MR-PRESSO and leave-one-out analysis were performed to test the robustness of the MR results. For significant associations, repeated MR and meta-analysis were performed by another metabolite GWAS (8,299 participants). Furthermore, significantly associated metabolites were subjected to a metabolic pathway analysis. RESULTS: The instrumental variables for the metabolites ranged from 3 to 493. Primary analysis revealed a total of 12 known (e.g., phenol sulfate, citrulline, lactate and gamma-glutamylglutamine) and 11 unknown metabolites were associated with PE. Heterogeneity and pleiotropy tests verified the robustness of the MR results. Validation with another metabolite GWAS dataset revealed consistency trends in 6 of the known metabolites with preliminary analysis, particularly the finding that genetic susceptibility to low levels of arachidonate (20:4n6) and citrulline were risk factors for PE. The pathway analysis revealed glycolysis/gluconeogenesis and arginine biosynthesis involved in the pathogenesis of PE. CONCLUSIONS: This study identifies a causal relationship between some circulating metabolites and PE. Our study presented new perspectives on the pathogenesis of PE by integrating metabolomics with genomics, which opens up avenues for more accurate understanding and management of the disease, providing new potential candidate metabolic molecular markers for the prevention, diagnosis and treatment of PE. Considering the limitations of MR studies, further research is needed to confirm the causality and underlying mechanisms of these findings.

Effect of Natural Osmolytes on Recombinant Tau Monomer: Propensity of Oligomerization and Aggregation.

The pathological misfolding and aggregation of the microtubule associated protein tau (MAPT), a full length Tau2N4R with 441aa, is considered the principal disease relevant constituent in tauopathies including Alzheimer's disease (AD) with an imbalanced ratio in 3R/4R isoforms. The exact cellular fluid composition, properties, and changes that coincide with tau misfolding, seed formation, and propagation events remain obscure. The proteostasis network, along with the associated osmolytes, is responsible for maintaining the presence of tau in its native structure or dealing with misfolding. In this study, for the first time, the roles of natural brain osmolytes are being investigated for their potential effects on regulating the conformational stability of the tau monomer (tauM) and its propensity to aggregate or disaggregate. Herein, the effects of physiological osmolytes myo-inositol, taurine, trimethyl amine oxide (TMAO), betaine, sorbitol, glycerophosphocholine (GPC), and citrulline on tau's aggregation state were investigated. The overall results indicate the ability of sorbitol and GPC to maintain the monomeric form and prevent aggregation of tau, whereas myo-inositol, taurine, TMAO, betaine, and citrulline promote tau aggregation to different degrees, as revealed by protein morphology in atomic force microscopy images. Biochemical and biophysical methods also revealed that tau proteins adopt different conformations under the influence of these osmolytes. TauM in the presence of all osmolytes expressed no toxicity when tested by a lactate dehydrogenase assay. Investigating the conformational stability of tau in the presence of osmolytes may provide a better understanding of the complex nature of tau aggregation in AD and the protective and/or chaotropic nature of osmolytes.

GnRH Induces Citrullination of the Cytoskeleton in Murine Gonadotrope Cells.

Peptidylarginine deiminases (PADs or PADIs) catalyze the conversion of positively charged arginine to neutral citrulline, which alters target protein structure and function. Our previous work established that gonadotropin-releasing hormone agonist (GnRHa) stimulates PAD2-catalyzed histone citrullination to epigenetically regulate gonadotropin gene expression in the gonadotrope-derived LßT2 cell line. However, PADs are also found in the cytoplasm. Given this, we used mass spectrometry (MS) to identify additional non-histone proteins that are citrullinated following GnRHa stimulation and characterized the temporal dynamics of this modification. Our results show that actin and tubulin are citrullinated, which led us to hypothesize that GnRHa might induce their citrullination to modulate cytoskeletal dynamics and architecture. The data show that 10 nM GnRHa induces the citrullination of ß-actin, with elevated levels occurring at 10 min. The level of ß-actin citrullination is reduced in the presence of the pan-PAD inhibitor biphenyl-benzimidazole-Cl-amidine (BB-ClA), which also prevents GnRHa-induced actin reorganization in dispersed murine gonadotrope cells. GnRHa induces the citrullination of ß-tubulin, with elevated levels occurring at 30 min, and this response is attenuated in the presence of PAD inhibition. To examine the functional consequence of ß-tubulin citrullination, we utilized fluorescently tagged end binding protein 1 (EB1-GFP) to track the growing plus end of microtubules (MT) in real time in transfected LßT2 cells. Time-lapse confocal microscopy of EB1-GFP reveals that the MT average lifetime increases following 30 min of GnRHa treatment, but this increase is attenuated by PAD inhibition. Taken together, our data suggest that GnRHa-induced citrullination alters actin reorganization and MT lifetime in gonadotrope cells.

L-Citrulline Ameliorates Iron Metabolism and Mitochondrial Quality Control via Activating AMPK Pathway in Intestine and Improves Microbiota in Mice with Iron Overload.

SCOPE: Oxidative stress caused by iron overload tends to result in intestinal mucosal barrier dysfunction and intestinal microbiota imbalance. As a neutral and nonprotein amino acid, L-Citrulline (L-cit) has been implicated in antioxidant and mitochondrial amelioration properties. This study investigates whether L-cit can alleviate iron overload-induced intestinal injury and explores the underlying mechanisms. METHODS AND RESULTS: C57BL/6J mice are intraperitoneally injected with iron dextran, then gavaged with different dose of L-cit for 2 weeks. L-cit treatment significantly alleviates intestine pathological injury, oxidative stress, ATP level, and mitochondrial respiratory chain complex activities, accompanied by ameliorating mitochondrial quality control. L-cit-mediated protection is associated with the upregulation of Glutathione Peroxidase 4 (GPX4) expression, inhibition Nuclear Receptor Coactivator 4 (NCOA4)-mediated ferritinophagy and ferroptosis, and improvement of gut microbiota. To investigate the underlying molecular mechanisms, Intestinal Porcine Epithelial Cell line-J2 (IPEC-J2) cells are treated with L-cit or AMP-activated Protein Kinase (AMPK) inhibitor. AMPK signaling has been activated by L-cit. Notably, Compound C abolishes L-cit's protection on intestinal barrier, mitochondrial function, and antioxidative capacity in IPEC-J2 cells. CONCLUSION: L-cit may restrain ferritinophagy and ferroptosis to regulate iron metabolism, and induce AMPK pathway activation, which contributes to exert antioxidation, ameliorate iron metabolism and mitochondrial quality control, and improve intestinal microbiota. L-cit is a promising therapeutic strategy for iron overload-induced intestinal injury.

Measurements of I-FABP and citrulline in the postoperative period of non-cardiac surgeries with gastrointestinal complications: A prospective cohort observational study.

BACKGROUND: Acute Gastrointestinal Injury (AGI) is associated with adverse clinical outcomes, including increased mortality. We aimed to investigate the potential of citrulline and intestinal fatty acid binding protein (I-FABP) as biomarkers for early AGI diagnosis and predicting outcomes in surgical patients. METHODS: Prospective cohort study involving patients who underwent non-cardiac surgeries and were admitted to Intensive Care Units. AGI diagnosis was based on specific criteria, and severity was categorised following established guidelines. Statistical analyses were performed to assess the diagnostic accuracy of the biomarkers and their association with outcomes, P significant when <0.05. RESULTS: AGI was identified in 40.3% of patients with varying severity. Mortality rates were significantly higher in the AGI group in the ICU (19.4% vs. 0%, p = 0.001) and hospital (22.6% vs. 2.17%, p = 0.003). Urinary I-FABP levels on days 3 and 7 showed reasonable and good accuracy for AGI diagnosis (AUC 0.732 and 0.813, respectively). Urinary I-FABP levels on days 2 and 3 accurately predict sepsis. Urinary citrulline levels on day one predicted mortality (AUC 0.87) furthermore urinary I-FABP levels on day 2 showed reasonable accuracy (sensitivity 83.3%, specificity 92.4%). CONCLUSION: Urinary I-FABP and citrulline levels are promising diagnostic and prognostic markers in ICU patients following non-cardiac surgeries.

Impact of supplementation with L-citrulline/arginine after liver transplantation in individuals with Urea Cycle Disorders.

OBJECTIVE: Liver transplantation (LTx) is an intervention when medical management is not sufficiently preventing individuals with urea cycle disorders (UCDs) from the occurrence of hyperammonemic events. Supplementation with L-citrulline/arginine is regularly performed prior to LTx to support ureagenesis and is often continued after the intervention. However, systematic studies assessing the impact of long-term L-citrulline/arginine supplementation in individuals who have undergone LTx is lacking to date. METHODS: Using longitudinal data collected systematically, a comparative analysis was carried out by studying the effects of long-term L-citrulline/arginine supplementation vs. no supplementation on health-related outcome parameters (i.e., anthropometric, neurological, and cognitive outcomes) in individuals with UCDs who have undergone LTx. Altogether, 52 individuals with male ornithine transcarbamylase deficiency, citrullinemia type 1 and argininosuccinic aciduria and a pre-transplant "severe" disease course who have undergone LTx were investigated by using recently established and validated genotype-specific in vitro enzyme activities. RESULTS: Long-term supplementation of individuals with L-citrulline/arginine who have undergone LTx (n = 16) does neither appear to alter anthropometric nor neurocognitive endpoints when compared to their severity-adjusted counterparts that were not supplemented (n = 36) after LTx with mean observation periods between four to five years. Moreover, supplementation with L-citrulline/arginine was not associated with an increase of disease-specific plasma arithmetic mean values for the respective amino acids when compared to the non-supplemented control cohort. CONCLUSION: Although supplementation with L-citrulline/arginine is often continued after LTx, this pilot study does neither identify altered long-term anthropometric or neurocognitive health-related outcomes nor does it find an adequate biochemical response as reflected by the unaltered plasma arithmetic mean values for L-citrulline or L-arginine. Further prospective analyses in larger samples and even longer observation periods will provide more insight into the usefulness of long-term supplementation with L-citrulline/arginine for individuals with UCDs who have undergone LTx.

Impact of citrulline substitution on clinical outcome after liver transplantation in carbamoyl phosphate synthetase 1 and ornithine transcarbamylase deficiency.

Carbamoyl phosphate synthetase 1 (CPS1) and ornithine transcarbamylase (OTC) deficiencies are rare urea cycle disorders, which can lead to life-threatening hyperammonemia. Liver transplantation (LT) provides a cure and offers an alternative to medical treatment and life-long dietary restrictions with permanent impending risk of hyperammonemia. Nevertheless, in most patients, metabolic aberrations persist after LT, especially low plasma citrulline levels, with questionable clinical impact. So far, little is known about these alterations and there is no consensus, whether l-citrulline substitution after LT improves patients' symptoms and outcomes. In this multicentre, retrospective, observational study of 24 patients who underwent LT for CPS1 (n = 11) or OTC (n = 13) deficiency, 25% did not receive l-citrulline or arginine substitution. Correlation analysis revealed no correlation between substitution dosage and citrulline levels (CPS1, p = 0.8 and OTC, p = 1). Arginine levels after liver transplantation were normal after LT independent of citrulline substitution. Native liver survival had no impact on mental impairment (p = 0.67). Regression analysis showed no correlation between l-citrulline substitution and failure to thrive (p = 0.611) or neurological outcome (p = 0.701). Peak ammonia had a significant effect on mental impairment (p = 0.017). Peak plasma ammonia levels correlate with mental impairment after LT in CPS1 and OTC deficiency. Growth and intellectual impairment after LT are not significantly associated with l-citrulline substitution.

Identification of novel serum metabolic signatures to predict chronic kidney disease among Chinese elders using UPLC-Orbitrap-MS.

BACKGROUND: Chronic kidney disease (CKD) is a major public health concern. However, validated and broadly applicable biomarkers for early CKD diagnosis are currently not available. We aimed to identify serum metabolic signatures at an early stage of CKD to provide a reference for future investigations into the early diagnostic biomarkers. METHODS: Serum metabolites were extracted from 65 renal dysfunction (RD) patients and 121 healthy controls (discovery cohort: 12 RD patients and 55 health participants; validation cohort: 53 RD patients and 66 health participants). Metabolite extracts were analyzed by ultraperformance liquid chromatography coupled with quadrupole-electrostatic field orbital trap mass spectrometry (UPLC-QE-Orbitrap MS) for untargeted metabolomics. Orthogonal partial least squares-discriminant analysis (OPLS-DA) was performed to detect different compounds between groups. Receiver operating characteristic (ROC) curve analysis was carried out to determine the diagnostic value of the validated differential metabolites between groups. We referred to the Kyoto Encyclopedia of Gene and Genomes (KEGG) to elucidate the metabolic pathways of the validated differential metabolites. RESULTS: A total of 22 and 23 metabolites had significantly different abundances in the discovery and validation cohort, respectively. Six of them (creatinine, L-proline, citrulline, butyrylcarnitine, 1-methylhistidine, and valerylcarnitine) in the RD group was more abundant than that of the health group in both cohorts. The combination of the six validated differential metabolites were able to accurately detect RD (AUC 0.86). Three of the six metabolites are involved in the metabolism of arginine and proline. CONCLUSIONS: The present study highlights that creatinine, L-proline, citrulline, butyrylcarnitine, 1-methylhistidine, and valerylcarnitine are metabolite indicators with potential predictive value for CKD.

Impact of a short-term nitrate and citrulline co-supplementation on sport performance in elite rowers: a randomized, double-blind, placebo-controlled crossover trial.

PURPOSE: Citrulline (CIT) and beetroot extract (BR) have separately shown benefits in rowing performance-related outcomes. However, effects of combined supplementation remain to be elucidated. The main purpose of this research was to study the effects of 1 week of daily co-supplementation of 3.5 g BR (500 mg NO3-) plus 6 g CIT on aerobic performance, maximal strength, and high-intensity power and peak stroke in elite male rowers compared to a placebo and to a BR supplementation. METHODS: 20 elite rowers participated in this randomized, double-blind, placebo-controlled crossover trial completing 1 week of supplementation in each group of study: Placebo group (PLAG); BR group (BRG); and BR + CIT group (BR-CITG). 3 main physical tests were performed: aerobic performance, Wingate test and CMJ jump, and metabolic biomarkers and physiological outcomes were collected. RESULTS: The Wingate all-out test showed no between-condition differences in peak power, mean power, relative power, or fatigue index (P > 0.05), but clearance of lactate was better in BR-CITG (P < 0.05). In the performance test, peak power differed only between PLAG and BR-CITG (P = 0.036), while VO2peak and maximum heart rate remained similar. CMJ jumping test results showed no between-condition differences, and blood samples were consistent (P > 0.200). CONCLUSION: Supplementation with 3.5 g of BR extract plus 6 g of CIT for 7 days improved lactate clearance after Wingate test and peak power in a performance test. No further improvements were found, suggesting longer period of supplementation might be needed to show greater benefits.

Antenatal Growth, Gestational Age, Birth, Enteral Feeding, and Blood Citrulline Levels in Very Low Birth Weight Infants.

Early enteral nutrition using reliable biomarkers of intestinal function must be established to improve neurodevelopmental outcomes in very low birth weight infants (VLBWIs). Serum citrulline levels reflect the intestinal function in adults. To elucidate the relationship among antenatal growth, postnatal enteral nutrition, and blood citrulline levels, a retrospective single-center observational study was conducted on 248 VLBWIs born between April 2014 and March 2021. A mixed effect model and post hoc simple slope analysis were used to estimate the correlations between clinical variables and citrulline levels at Early (day 5.1) and Late (day 24.3) postnatal ages. Greater gestational age, birth weight, and amount of enteral nutrition at the time of blood sampling were associated with lower citrulline levels at the Early postnatal age and higher citrulline levels at the Late postnatal age. Provided that Early citrulline levels predominantly reflect the consequence of antenatal citrulline metabolism, it is suggested that fetal growth and maturation are likely to promote citrulline catabolism in utero and its synthesis after birth. With additional insights into the temporal transition point wherein the maturation-dependent balance of citrulline metabolism shifts from catabolism-dominant to synthesis-dominant, citrulline emerges as a potential biomarker for assessing intestinal function and gastrointestinal disorders.

The roles of gut microbiome and metabolites associated with skin photoaging in mice by intestinal flora sequencing and metabolomics.

Photoaging of skin, a chronic disease, can produce the appearance changes and cancer lesions of skin. Therefore, it is of great significance to investigate the mechanisms and explore effective methods to treat the disorder. Gut microbiota and intestinal metabolisms have critical roles in a variety of diseases. However, their roles on photoaging of skin were not well tested. In the present work, the results showed that compared with control group, the levels of MDA, SOD and CAT associated with oxidative stress, the levels of COL I, CER, and HA associated with skin function, and the mRNA levels of IL-1ß, IL-6, TNF-α associated with inflammation after long-term exposure to ultraviolet radiation in mice were significantly changed. Skin pathological tissue was also seriously damaged. The protein levels of AQP3 and FLG were significantly decreased. Ultraviolet exposure also promoted skin photoaging by activating TNFR1/TRAF2-mediated MAPK pathway, in which the protein levels of P38/P-P38, c-FOS/P-c-FOS, MMP1, TNFR1 and TRAF2 were significantly increased in model mice compared with control group. In fecal microbiota transplantation (FMT) experiment, we found that the intestinal microbiome of control mice alleviated skin photoaging via adjusting the protein levels of P38/P-P38, c-FOS/P-c-FOS, MMP1, TNFR1 and TRAF2. 16S rRNA sequencing found that 1639 intestinal bacteria were found, in which 15 bacteria including norank_f_Ruminococcaceae, Lachnospirac -eae_NK4A136_group, Lachnoclostridium, etc., were significantly different at the genus level. Untargeted GC-TOF/MS and UHPLC-MS/MS metabolomics showed 72 and 188 metabolites including taurine, ornithine, L-arginine, L-histidine, sucrose with significant differences compared with control group. Then, amino acid targeting assay showed 10 amino acids including L-ornithine, L-arginine and L-citrulline with higher levels in control group compared with model group. In addition, we also found that the variation of Lachnoclostridium abundance may regulate L-arginine metabolism to affect skin photoaging. Some intestinal bacteria and metabolites including amino acids may be closely related to skin photoaging, which should provide new methods to treat skin photoaging in the future.