Perinatal Use of Citrulline Rescues Hypertension in Adult Male Offspring Born to Pregnant Uremic Rats.

The growing recognition of the association between maternal chronic kidney disease (CKD) and fetal programming highlights the increased vulnerability of hypertension in offspring. Potential mechanisms involve oxidative stress, dysbiosis in gut microbiota, and activation of the renin-angiotensin system (RAS). Our prior investigation showed that the administration of adenine to pregnant rats resulted in the development of CKD, ultimately causing hypertension in their adult offspring. Citrulline, known for enhancing nitric oxide (NO) production and possessing antioxidant and antihypertensive properties, was explored for its potential to reverse high blood pressure (BP) in offspring born to CKD dams. Male rat offspring, both from normal and adenine-induced CKD models, were randomly assigned to four groups (8 animals each): (1) control, (2) CKD, (3) citrulline-treated control rats, and (4) citrulline-treated CKD rats. Citrulline supplementation successfully reversed elevated BP in male progeny born to uremic mothers. The protective effects of perinatal citrulline supplementation were linked to an enhanced NO pathway, decreased expression of renal (pro)renin receptor, and changes in gut microbiota composition. Citrulline supplementation led to a reduction in the abundance of Monoglobus and Streptococcus genera and an increase in Agothobacterium Butyriciproducens. Citrulline's ability to influence taxa associated with hypertension may be linked to its protective effects against maternal CKD-induced offspring hypertension. In conclusion, perinatal citrulline treatment increased NO availability and mitigated elevated BP in rat offspring from uremic mother rats.

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.

Multi-dose enteral L-citrulline administration in premature infants at risk of developing pulmonary hypertension associated with bronchopulmonary dysplasia.

OBJECTIVE: Information is needed to guide the design of randomized controlled trials (RCTs) evaluating L-citrulline therapy for premature infants with pulmonary hypertension associated with bronchopulmonary dysplasia (BPD-PH). Based on our single-dose pharmacokinetic study, we evaluated the ability of a multi-dose enteral L-citrulline strategy to achieve a target trough steady-state L-citrulline plasma concentration and its tolerability in premature infants. STUDY DESIGN: Plasma L-citrulline concentrations were measured in six premature infants receiving 60 mg/kg L-citrulline every 6 h for 72 h before the first and last L-citrulline doses. L-citrulline concentrations were compared to concentration-time profiles from our previous study. RESULTS: Target trough plasma L-citrulline concentrations were achieved in 2/6 subjects. No serious adverse events occurred. CONCLUSIONS: Multi-dose L-citrulline was well tolerated. These results will assist in the design of phase II RCTs evaluating L-citrulline dosage strategies to achieve target plasma L-citrulline concentrations in infants at risk for BPD-PH. CLINICAL TRIALS: gov ID: NCT03542812.

Enteral citrulline supplementation versus placebo on SOFA score on day 7 in mechanically ventilated critically ill patients: the IMMUNOCITRE randomized clinical trial.

BACKGROUND: Restoring plasma arginine levels through enteral administration of L-citrulline in critically ill patients may improve outcomes. We aimed to evaluate whether enteral L-citrulline administration reduced organ dysfunction based on the Sequential Organ Failure Assessment (SOFA) score and affected selected immune parameters in mechanically ventilated medical intensive care unit (ICU) patients. METHODS: A randomized, double-blind, multicenter clinical trial of enteral administration of L-citrulline versus placebo for critically ill adult patients under invasive mechanical ventilation without sepsis or septic shock was conducted in four ICUs in France between September 2016 and February 2019. Patients were randomly assigned to receive enteral L-citrulline (5 g) every 12 h for 5 days or isonitrogenous, isocaloric placebo. The primary outcome was the SOFA score on day 7. Secondary outcomes included SOFA score improvement (defined as a decrease in total SOFA score by 2 points or more between day 1 and day 7), secondary infection acquisition, ICU length of stay, plasma amino acid levels, and immune biomarkers on day 3 and day 7 (HLA-DR expression on monocytes and interleukin-6). RESULTS: Of 120 randomized patients (mean age, 60 ± 17 years; 44 [36.7%] women; ICU stay 10 days [IQR, 7-16]; incidence of secondary infections 25 patients (20.8%)), 60 were allocated to L-citrulline and 60 were allocated to placebo. Overall, there was no significant difference in organ dysfunction as assessed by the SOFA score on day 7 after enrollment (4 [IQR, 2-6] in the L-citrulline group vs. 4 [IQR, 2-7] in the placebo group; Mann‒Whitney U test, p = 0.9). Plasma arginine was significantly increased on day 3 in the treatment group, while immune parameters remained unaffected. CONCLUSION: Among mechanically ventilated ICU patients without sepsis or septic shock, enteral L-citrulline administration did not result in a significant difference in SOFA score on day 7 compared to placebo. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT02864017 (date of registration: 11 August 2016).

Anti-citrullinated protein antibodies as biomarkers in rheumatoid arthritis.

INTRODUCTION: The serological biomarker anti-citrullinated protein antibodies (ACPAs) may have several functions but is especially important for the diagnosis of rheumatoid arthritis (RA) along with clinical symptoms. AREAS COVERED: This review provides an overview of ACPAs, which are useful in RA diagnostics and may improve our understanding of disease etiology. PubMed was searched with combinations of words related to antibodies recognizing epitopes containing the post-translationally modified amino acid citrulline in combination with rheumatoid arthritis; cyclic citrullinated peptide, CCP, anti-CCP, anti-citrullinated protein antibodies, ACPA, citrullination, peptide/protein arginine deiminase, PAD, filaggrin, vimentin, keratin, collagen, perinuclear factor, EBNA1, EBNA2, and others. From this search, we made a qualitative extract of publications relevant to the discovery, characterization, and clinical use of these antibodies in relation to RA. We highlight significant findings and identify areas for improvement. EXPERT OPINION: ACPAs have high diagnostic sensitivity and specificity for RA and recognize citrullinated epitopes from several proteins. The best-performing single epitope originates from Epstein-Barr Virus nuclear antigen 2 and contains a central Cit-Gly motif, which is recognized by ACPAS when located in a flexible peptide structure. In addition, ACPAs may also have prognostic value, especially in relation to early treatment, although ACPAs' main function is to aid in the diagnosis of RA.

Citrulline in the management of patients with urea cycle disorders.

BACKGROUND: Treatment recommendations for urea cycle disorders (UCDs) include supplementation with amino acids involved in the urea cycle (arginine and/or citrulline, depending on the enzyme deficiency), to maximize ammonia excretion through the urea cycle, but limited data are available regarding the use of citrulline. This study retrospectively reviewed clinical and biological data from patients with UCDs treated with citrulline and/or arginine at a reference center since 1990. The aim was to describe the prescription, impact, and safety of these therapies. Data collection included patient background, treatment details, changes in biochemical parameters (plasma ammonia and amino acids concentrations), decompensations, and patient outcomes. RESULTS: Overall, 79 patients (median age at diagnosis, 0.9 months) received citrulline and/or arginine in combination with a restricted protein diet, most with ornithine transcarbamylase (n = 57, 73%) or carbamoyl phosphate synthetase 1 (n = 15, 19%) deficiencies. Most patients also received ammonium scavengers. Median follow-up was 9.5 years and median exposure to first treatment with arginine + citrulline, citrulline monotherapy, or arginine monotherapy was 5.5, 2.5, or 0.3 years, respectively. During follow-up, arginine or citrulline was administered at least once (as monotherapy or in combination) in the same proportion of patients (86.1%); the overall median duration of exposure was 5.9 years for arginine + citrulline, 3.1 years for citrulline monotherapy, and 0.6 years for arginine monotherapy. The most common switch was from monotherapy to combination therapy (41 of 75 switches, 54.7%). During treatment, mean ammonia concentrations were 35.9 µmol/L with citrulline, 49.8 µmol/L with arginine, and 53.0 µmol/L with arginine + citrulline. Mean plasma arginine concentrations increased significantly from the beginning to the end of citrulline treatment periods (from 67.6 µmol/L to 84.9 µmol/L, P < 0.05). At last evaluation, mean height and weight for age were normal and most patients showed normal or adapted behavior (98.7%) and normal social life (79.0%). Two patients (2.5%) experienced three treatment-related gastrointestinal adverse reactions. CONCLUSIONS: This study underlines the importance of citrulline supplementation, either alone or together with arginine, in the management of patients with UCDs. When a monotherapy is considered, citrulline would be the preferred option in terms of increasing plasma arginine concentrations.

Variant in the allosteric domain of CPS1 protein associated with effectiveness of N-carbamoyl glutamate therapy in neonatal onset CPS1 deficiency.

OBJECTIVES: Carbamoyl phosphate synthetase 1 (CPS1) deficiency is a severe urea cycle disorder. Patients can present with hyperammonemic coma in the first days of life. Treatment includes nitrogen scavengers, reduced protein intake and supplementation with L-arginine and/or L-citrulline. N-carbamoyl glutamate (NCG) has been hypothesized to stimulate the residual CPS1 function, although only few patients are reported. CASE PRESENTATION: We report a patient with neonatal-onset CPS1 deficiency who received NCG in association with nitrogen scavenger and L-citrulline. The patient carried the novel variants CPS1-c.2447A>G p.(Gln816Arg) and CPS1-c.4489T>C p.(Tyr1497His). The latter is localized in the C-terminal allosteric domain of the protein, and is implicated in the binding of the natural activator N-acetyl-L-glutamate. NCG therapy was effective in controlling ammonia levels, allowing to increase the protein intake. CONCLUSIONS: Our data show that the response to NCG can be indicated based on the protein structure. We hypothesize that variants in the C-terminal domain may be responsive to NCG therapy.

Pathophysiology of Preeclampsia and L-Arginine/L-Citrulline Supplementation as a Potential Strategy to Improve Birth Outcomes.

In preeclampsia, the shallow invasion of cytotrophoblast cells to uterine spiral arteries, leading to a reduction in placental blood flow, is associated with an imbalance of proangiogenic/antiangiogenic factors to impaired nitric oxide (NO) production. Proangiogenic factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), require NO to induce angiogenesis through antioxidant regulation mechanisms. At the same time, there are increases in antiangiogenic factors in preeclampsia, such as soluble fms-like tyrosine kinase type 1 receptor (sFIt1) and toll-like receptor 9 (TLR9), which are mechanism derivates in the reduction of NO bioavailability and oxidative stress in placenta.Different strategies have been proposed to prevent or alleviate the detrimental effects of preeclampsia. However, the only intervention to avoid the severe consequences of the disease is the interruption of pregnancy. In this scenario, different approaches have been analysed to treat preeclamptic pregnant women safely. The supplementation with amino acids is one of them, especially those associated with NO synthesis. In this review, we discuss emerging concepts in the pathogenesis of preeclampsia to highlight L-arginine and L-citrulline supplementation as potential strategies to improve birth outcomes. Clinical and experimental data concerning L-arginine and L-citrulline supplementation have shown benefits in improving NO availability in the placenta and uterine-placental circulation, prolonging pregnancy in patients with gestational hypertension and decreasing maternal blood pressure.

A Randomized Feasibility Trial of Stereotactic Prostate Radiation Therapy With or Without Elective Nodal Irradiation in High-Risk Localized Prostate Cancer (SPORT Trial).

PURPOSE: The aim of this study was to establish the feasibility of a randomized clinical trial comparing SABR with prostate-only (P-SABR) or with prostate plus pelvic lymph nodes (PPN-SABR) in patients with unfavorable intermediate- or high-risk localized prostate cancer and to explore potential toxicity biomarkers. METHODS AND MATERIALS: Thirty adult men with at least 1 of the following features were randomized 1:1 to P-SABR or PPN-SABR: clinical magnetic resonance imaging stage T3a N0 M0, Gleason score ≥7 (4+3), and prostate-specific antigen >20 ng/mL. P-SABR patients received 36.25 Gy/5 fractions/29 days, and PPN-SABR patients received 25 Gy/5 fractions to pelvic nodes, with the final cohort receiving a boost to the dominant intraprostatic lesion of 45 to 50 Gy. Phosphorylated gamma-H2AX (γH2AX) foci numbers, citrulline levels, and circulating lymphocyte counts were quantified. Acute toxicity information (Common Terminology Criteria for Adverse Events, version 4.03) was collected weekly at each treatment and at 6 weeks and 3 months. Physician-reported late Radiation Therapy Oncology Group (RTOG) toxicity was recorded from 90 days to 36 months postcompletion of SABR. Patient-reported quality of life (Expanded Prostate Cancer Index Composite and International Prostate Symptom Score) scores were recorded with each toxicity time point. RESULTS: The target recruitment was achieved, and treatment was successfully delivered in all patients. A total of 0% and 6.7% (P-SABR) and 6.7% and 20.0% (PPN-SABR) experienced acute grade ≥2 gastrointestinal (GI) and genitourinary (GU) toxicity, respectively. At 3 years, 6.7% and 6.7% (P-SABR) and 13.3% and 33.3% (PPN-SABR) had experienced late grade ≥2 GI and GU toxicity, respectively. One patient (PPN-SABR) had late grade 3 GU toxicity (cystitis and hematuria). No other grade ≥3 toxicity was observed. In addition, 33.3% and 60% (P-SABR) and 64.3% and 92.9% (PPN-SABR) experienced a minimally clinically important change in late Expanded Prostate Cancer Index Composite bowel and urinary summary scores, respectively. γH2AX foci numbers at 1 hour after the first fraction were significantly higher in the PPN-SABR arm compared with the P-SABR arm (P = .04). Patients with late grade ≥1 GI toxicity had significantly greater falls in circulating lymphocytes (12 weeks post-radiation therapy, P = .01) and a trend toward higher γH2AX foci numbers (P = .09) than patients with no late toxicity. Patients with late grade ≥1 bowel toxicity and late diarrhea experienced greater falls in citrulline levels (P = .05). CONCLUSIONS: A randomized trial comparing P-SABR with PPN-SABR is feasible with acceptable toxicity. Correlations of γH2AX foci, lymphocyte counts, and citrulline levels with irradiated volume and toxicity suggest potential as predictive biomarkers. This study has informed a multicenter, randomized, phase 3 clinical trial in the United Kingdom.

Metabolic effects of L-citrulline in type 2 diabetes.

The prevalence of type 2 diabetes (T2D) is increasing worldwide. Decreased nitric oxide (NO) bioavailability is involved in the pathophysiology of T2D and its complications. L-citrulline (Cit), a precursor of NO production, has been suggested as a novel therapeutic agent for T2D. Available data from human and animal studies indicate that Cit supplementation in T2D increases circulating levels of Cit and L-arginine while decreasing circulating glucose and free fatty acids and improving dyslipidemia. The underlying mechanisms for these beneficial effects of Cit include increased insulin secretion from the pancreatic ß cells, increased glucose uptake by the skeletal muscle, as well as increased lipolysis and ß-oxidation, and decreased glyceroneogenesis in the adipose tissue. Thus, Cit has antihyperglycemic, antidyslipidemic, and antioxidant effects and has the potential to be used as a new therapeutic agent in the management of T2D. This review summarizes available literature from human and animal studies to explore the effects of Cit on metabolic parameters in T2D. It also discusses the possible mechanisms underlying Cit-induced improved metabolic parameters in T2D.

The effects of L-citrulline adjunctive treatment of Toxoplasma gondii RH strain infection in a mouse model.

Toxoplasma gondii is a zoonotic intracellular protozoan parasite and its therapeutic limitations are one of its major problems. L-citrulline is an organic compound that has beneficial effects on many diseases. The purpose of this study was to assess the impact of L-citrulline, alone or in combination with sulfamethoxazole-trimethoprim (SMZ-TMP) on acute toxoplasmosis caused by Toxoplasma gondii RH virulent strain. In our study, 60 Swiss albino mice were divided into two main groups; the control group and the infected treated group, which was subdivided into group IIa: infected treated with L-citrulline, group IIb: infected treated with SMZ-TMP, and group IIc: infected treated with L-citrulline combined with SMZ-TMP. The effects of treatment were assessed by parasitological study, electron microscopic study of tachyzoites, and histopathological study of the liver. Moreover, ELISA measurement of the serum level of Interferon-gamma, Interleukin 10, Nitric oxide, and apoptotic markers was used. It was noticed that L-citrulline combined with SMZ-TMP significantly increased the survival time of infected mice with a significant decrease in the number of tachyzoites compared to the other groups. Moreover, it increased the levels of measured cytokines and serum anti-apoptotic proteins Bcl-2 and improved the extent of liver cell damage associated with a decrease in inflammatory infiltration. In conclusion, L-citrulline supplementation was found to be effective against acute toxoplasmosis, especially when combined with SMZ-TMP as it has multifactorial mechanisms; nitric oxide production, anti-inflammatory, anti-apoptotic, and immune stimulator.

Pharmacokinetics of L-Citrulline in Neonates at Risk of Developing Bronchopulmonary Dysplasia-Associated Pulmonary Hypertension.

BACKGROUND: Options to treat pulmonary hypertension (PH) in neonates with bronchopulmonary dysplasia (BPD) are few and largely ineffective. Improving the bioavailability of nitric oxide (NO) might be an efficacious treatment for BPD-PH. When administered orally, the NO-L-arginine precursor, L-citrulline, increases NO production in children and adults, however, pharmacokinetic (PK) studies of oral L-citrulline have not been performed in infants and children. OBJECTIVES: This study characterized the PK of enterally administered L-citrulline in neonates at risk of developing BPD-PH to devise a model-informed dosing strategy. METHODS AND RESULTS: Ten premature neonates (≤ 28 weeks gestation) were administered a single dose of 150 mg/kg (powder form solubilized in sterile water) oral L-citrulline at 32 ± 1 weeks postmenstrual age. Due to the need to limit blood draws, time windows were used to maximize the sampling over the dosing interval by assigning neonates to one of two groups (ii) samples collected pre-dose and at 1- and 2.5-h post-dose, and (ii) pre-dose and 0.25- and 3-h post-dose. The L-arginine concentrations (µmol/L) and the L-citrulline (µmol/L) plasma concentration-time data were evaluated using non-compartmental analysis (Phoenix WinNonlin version 8.1). Optimal dosage strategies were derived using a simulation-based methodology. Simulated doses of 51.5 mg or 37.5 mg/kg given four times a day produced steady-state concentrations close to a target of 50 µmol/L. The volume of distribution (V/F) and clearance (CL/F) were 302.89 ml and 774.96 ml/h, respectively, with the drug exhibiting a half-life of 16 minutes. The AUC from the time of dosing to the time of last concentration was 1473.3 h*µmol/L, with Cmax and Tmax of 799 µmol/L and 1.55 h, respectively. CONCLUSION: This is the first PK study in neonates presenting data that can be used to inform dosing strategies in future randomized controlled trials evaluating enteral L-citrulline as a potential treatment to reduce PH associated with BPD in premature neonates. REGISTRATION: Clinical trials.gov Identifier: NCT03542812.

The impact of L-citrulline on murine intestinal cell integrity, immune response, and arginine metabolism in the face of Giardia lamblia infection.

Giardiasis is an intestinal protozoal disease caused by Giardia lamblia (G. lamblia) which is a major worldwide health problem due to development of resistance to commonly used drugs. Therefore, it is necessary to identify an effective drug for giardiasis. This study aimed to assess the therapeutic role of L-citrulline against giardiasis in experimental animals. 40 male Swiss Albino weaned rats were used in this study, divided into four groups. Group I: normal control; group II: infected un-treated; group III: infected and treated with L-citrulline and Group IV: infected and treated with metronidazole. The efficacy was evaluated by counting Giardia trophozoites in the intestinal mucosa and cysts in the stool of infected rats. Histopathological analyses, immunohistochemistry expression of inducible nitric oxide synthase (iNOS) in the small intestine tissues were performed. Along with, serum IL6, the intestinal arginase enzyme level and giardial flavohemoglobin (flavoHb) expression were measured. L-citrulline administration reduced the mean number of G. lamblia cysts and trophozoites, serum IL-6, and intestinal arginase enzyme levels. Furthermore, the intestinal brush border was restored, with a reduction in the inflammatory infiltrate and an increase in iNOS activity. Moreover, there was a significant decrease in flavoHb gene expression in both the L-citrulline and metronidazole treated groups. Thus L-citrulline is effective in NO production therefore it has a therapeutic potential in controlling giardiasis.

Antitumor Effects of L-citrulline on Hela Cervical Cancer Cell Lines.

AIM: Cervical cancer is the deadliest gynecological malignancy. This study aims to examine the anticancer effects of L-citrulline on HeLa cell culture. MATERIALS AND METHODS: HeLa cells were cultured in complete Eagle's minimum essential medium. HeLa cells were seeded in 96-well plates and incubated with L-citrulline. After incubation, MTT dye was added and incubated. Annexin- V technique was used to test the apoptosis. The activated caspases of HeLa cells by L-citrulline exposure were measured with the Caspase 3/7 technique. One-way variance analysis was conducted for statistical analysis by using GraphPad Prism 6.0 for Windows. RESULTS: L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner in application times of 24 and 48 hours. The IC50 dose of L-citrulline was 0.19 and 0.16 mg/mL at 24 and 48 hours, respectively. When HeLa cells were exposed to an IC50 dose of L-citrulline for 24 hours, the percentages of the dead, early apoptotic, and late apoptotic cells were detected to be 0.75%, 23.05%, and 12.75%, respectively. The differences in the wideness of the scratch area were observed at the initial stage and after 24 hours of applying L-citrulline. CONCLUSION: L-citrulline showed its toxicity on HeLa cells in a dose-dependent manner. Based on Annexin and Caspase findings, it can be concluded that L-citrulline exerted a pro-apoptotic effect on HeLa cells in only a short exposure time. L-citrulline also showed a migration inhibitory effect. The findings of this study indicate L-citrulline to be worthy of investigation for its anticancer activities in vitro and in vivo, and as a candidate for cancer therapy.

l-Citrulline ameliorates pathophysiology in a rat model of superimposed preeclampsia.

BACKGROUND AND PURPOSE: Preeclampsia, characterized by hypertension, proteinuria and restriction of fetal growth, is one of the leading causes of maternal and perinatal mortality. So far, there is no effective pharmacological therapy for preeclampsia. The present study was conducted to investigate the effects of supplementation with l-citrulline in Dahl salt-sensitive rats, a model of superimposed preeclampsia. EXPERIMENTAL APPROACH: Parental Dahl salt-sensitive rats were treated with l-citrulline (2.5 g·L-1 in drinking water) from the day of mating to the end of lactation period. Blood pressure was monitored throughout pregnancy and markers of preeclampsia were assessed. Endothelial function of the pregnant Dahl salt-sensitive rats was assessed by wire myograph. KEY RESULTS: In Dahl salt-sensitive rats, l-citrulline supplementation significantly reduced maternal blood pressure, proteinuria and levels of circulating soluble fms-like tyrosine kinase 1. l-Citrulline improved maternal endothelial function by augmenting the production of nitric oxide in the aorta and improving endothelium-derived hyperpolarizing factor-mediated vasorelaxation in resistance arteries. l-Citrulline supplementation improved placental insufficiency and fetal growth, which were associated with an enhancement of angiogenesis and reduction of fibrosis and senescence in the placentas. In addition, l-citrulline down-regulated genes involved in the TLR4 and NF-κB signalling pathways. CONCLUSION AND IMPLICATIONS: This study shows that l-citrulline supplementation reduced gestational hypertension and improved placentation and fetal growth in a rat model of superimposed preeclampsia. l-Citrulline supplementation may provide an effective and safe therapeutic strategy for preeclampsia that benefits both the mother and the fetus.

Dysregulated Arginine Metabolism in Young Patients with Chronic Persistent Asthma and in Human Bronchial Epithelial Cells.

BACKGROUND: Recent metabolomics studies have found circulatory metabolism alterations in patients with asthma, indicating that altered metabolites played a significant role in asthma. However, the regulatory mechanisms in asthma, especially in young chronic persistent asthma remain underexplored. METHODS: In this study, a prospective cohort of 162 patients diagnosed of asthma admitted to the First Affiliated Hospital of Xi'an Jiaotong University from January 2018 to December 2019 was used to perform a nested case-control study. Among them, we included 30 patients with chronic persistent asthma between 20 to 35 years old; 30 health control with evenly distributed age and sex were then recruited. Nontargeted metabolomics was applied to identify serum metabolic profiles and altered metabolic pathways. RESULTS: In vitro, human bronchial epithelial cells (HBECs) line BEAS-2B with the addition of L-citrulline and/or asymmetric dimethylarginine (ADMA) model was utilized and the concentrations of nitric oxide (NO) metabolites were tested to evaluate the therapeutic potential of L-citrulline. The young patients with chronic persistent asthma displayed dysregulated serum metabolic profiles, especially enriched in arginine metabolism. The ratio of L-citrulline to ornithine is associated with blood eosinophil count. In vitro, adding L-citrulline could reverse ADMA-mediated reduction of NOx at lower L-arginine concentration (25 µM), but was ineffective in the higher L-arginine concentration (100 µM) media. CONCLUSIONS: The arginine metabolism balance is of vital importance during the pathogenesis and progression of chronic asthma. L-citrulline could be a powerful approach to restore airway NO production, potentially exhibiting therapeutic benefits among young patients with chronic asthma.

Folic acid, either solely or combined with L-citrulline, improves NO signaling and ameliorates chronic hypoxia-induced pulmonary hypertension in newborn pigs.

Concomitant with developing pulmonary hypertension (PH), newborn piglets exposed to chronic hypoxia develop pulmonary vascular NO signaling impairments. PH is reduced and NO signaling is improved in chronically hypoxic piglets treated with the NO-arginine precursor, L-citrulline. Folic acid positively impacts NO signaling. We evaluated whether the effect on NO signaling and PH is greater using co-treatment with folic acid and L-citrulline than either alone. From day 3 to day 10 of hypoxia, piglets were treated solely with folic acid, solely with L-citrulline, or co-treated with both. Catheters were placed to measure in vivo hemodynamics. NO production was measured in vitro in dissected pulmonary arteries. Compared to normoxic piglets, pulmonary vascular resistance (PVR) was elevated and NO production was reduced in untreated hypoxic piglets. Regardless of treatment strategy, PVR was less in all three treated groups of hypoxic piglets when compared to the untreated hypoxic group. In addition, for all three groups of treated hypoxic piglets, NO production was higher than the untreated group. Improvements in PVR and NO production did not differ between piglets co-treated with folic acid and L-citrulline and those treated solely with either. Thus, the impact on NO production and PVR was not augmented by combining folic acid and L-citrulline treatments. Nonetheless, treatment with folic acid, either singly or when combined with L-citrulline, increases NO production and inhibits PH in chronically hypoxic newborn piglets. Folic acid merits consideration as a therapy for PH in human infants with chronic heart and lung conditions that are associated with chronic hypoxia.

Treatment with L-Citrulline in patients with post-polio syndrome: A single center, randomized, double blind, placebo-controlled trial.

This single-centered, randomized, double-blind, placebo-controlled study reports the results of L-Citrulline treatment for 24 weeks in patients with post-polio syndrome (PPS). Twenty-nine patients were randomized and assigned into receiving a treatment of 15 g L-Citrulline or placebo. The primary endpoint was the change of the 6 min walking distance test. Secondary endpoints included motor function measure, quantitative muscle strength, quantitative MRI and self-reported impairment questionnaires. Patients receiving L-Citrulline walked 17.5 longer in the 6 min walking distance test when compared to placebo group, however not statistically significant (95% CI = -14.69; 49.68, p = 0.298). None of the secondary endpoints showed a statistically significant change in the L-Citrulline group when compared to placebo group. The motor function measure showed a change of -0.78 (95% CI= [-3.39; 1.83] p = 0.563). Muscle degeneration of leg muscles assessed with quantitative MRI indicated no significant change (estimate= -0.01, 95% CI =-0.13; 0.11, p = 0.869). L-Citrulline was safe and well tolerated. In conclusion, administration of 15 g L-Citrulline daily for 24 weeks to patients with PPS showed no beneficial treatment effect in timed muscle function.

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.

PEG-G-CSF and L-Citrulline Combination Therapy for Mitigating Skin Wound Combined Radiation Injury in a Mouse Model.

Radiation combined injury (RCI, radiation exposure coupled with other forms of injury, such as burn, wound, hemorrhage, blast, trauma and/or sepsis) comprises approximately 65% of injuries from a nuclear explosion, and greatly increases the risk of morbidity and mortality when compared to that of radiation injury alone. To date, no U.S. Food and Drug Administration (FDA)-approved countermeasures are available for RCI. Currently, three leukocyte growth factors (Neupogen®, Neulasta® and Leukine®) have been approved by the FDA for mitigating the hematopoietic acute radiation syndrome. However these granulocyte-colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) products have failed to increase 30-day survival of mice after RCI, suggesting a more complicated biological mechanism is in play for RCI than for radiation injury. In the current study, the mitigative efficacy of combination therapy using pegylated (PEG)-G-CSF (Neulasta) and -citrulline was evaluated in an RCI mouse model. L-citrulline is a neutral alpha-amino acid shown to improve vascular endothelial function in cardiovascular diseases. Three doses of PEG-G-CSF at 1 mg/kg, subcutaneously administered on days 1, 8 and 15 postirradiation, were supplemented with oral -citrulline (1 g/kg), once daily from day 1 to day 21 postirradiation. The combination treatment significantly improved the 30-day survival of mice after RCI from 15% (vehicle-treated) to 42%, and extended the median survival time by 4 days, as compared to vehicle controls. In addition, the combination therapy significantly increased body weight and bone marrow stem and progenitor cell clonogenicity in RCI mice, and accelerated recovery from RCI-induced intestinal injury, compared to animals treated with vehicle. Treatment with -citrulline alone also accelerated skin wound healing after RCI. In conclusion, these data indicate that the PEG-G-CSF and -citrulline combination therapy is a potentially effective countermeasure for mitigating RCI, likely by enhancing survival of the hematopoietic stem/progenitor cells and accelerating recovery from the RCI-induced intestinal injury and skin wounds.