Determination of N-Carbamylglutamate in Feeds and Animal Products by High Performance Liquid Chromatography Tandem Mass Spectrometry.
ABSTRACT: N-carbamylglutamate (NCG), a synthetic analogue of N-acetylglutamate, is an activator of blood ammonia conversion and endogenous arginine synthesis. Here, we established an accurate quantitative determination of NCG in feeds, animal tissues, and body fluids using the high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The sample pretreatment procedures included extraction with 0.5% of formic acid in water/methanol (80/20, v/v), and purification using an anionic solid phase extraction cartridge. Satisfactory separation of NCG was achieved in 20 min with the application of an Atlantis T3 column, and a confirmative detection of NCG was ensured by multiple reaction monitoring of positive ions. NCG spiked in feeds, tissues, and body fluids were evaluated in regard to linearity, sensitivity, recovery, and repeatability. Recoveries for different sample matrices were in the range of 88.12% to 110.21% with relative standard deviations (RSDs) less than 8.8%. Limits of quantification were within the range of 0.012 to 0.073 mg kg-1 and 0.047 to 0.077 ?g mL-1 for solid and liquid samples, respectively. This study will provide a solid foundation for the evaluation of availability and metabolic mechanism of NCG in animals.
Project description:N-carbamylglutamate (NCG), an analogue of N-acetyl-L-glutamate (NAG), can increase arginine synthesis in mammals and improve the reproductive performance. However, the effect of NCG on poultry laying performance is still unclear. This study investigated the effect of dietary NCG on development of chicken ovarian follicles. The dosage and timing for NCG administration were evaluated for its effect on follicular development. Results showed that supplementation with 1% NCG in the diet for 14 D led to accelerated development of growing follicles (over 60 ?m in oocyte diameter) and significantly increased feed intake and feed efficiency. Plasma amino acids (AA) analysis showed that feeding with 1% NCG significantly increased of plasma AA levels. RNA-seq analysis revealed that NCG supplementation upregulated expression of genes related to angiogenesis and cell proliferation, but downregulated expression of apoptosis-related genes. Meanwhile, RT-qPCR and Western blot analysis validated the RNA-seq results. Moreover, NCG enhanced plasma NO level; upregulated expression of PKG-I, Raf1, and p-p38; and increased angiogenesis of the ovaries. In conclusion, dietary NCG (1% for 14 D) can promote development of ovarian follicles by increasing angiogenesis in ovaries of the chicken.
Project description:The detoxification of ammonia to urea requires a functional hepatic urea cycle, which consists of six enzymes and two mitochondrial membrane transporters. The initial step of the urea cycle is catalyzed by carbamyl phosphate synthetase 1 (CPS1). CPS1 deficiency (CPS1D) is a rare autosomal recessive disorder. N-Carbamylglutamate (NCG), a deacylase-resistant analogue of N-acetylglutamate, can activate CPS1. We describe the therapeutic course of a patient suffering from neonatal onset CPS1D with compound heterozygosity for the c.2359C > T (p.Arg787*) and c.3559G > T (p.Val1187Phe) variants in CPS1, treated with NCG. She presented with hyperammonemia, which reached 944 ?mol/L at the age of 2 days. The ammonia concentration decreased after treatment with continuous hemodiafiltration, NCG, sodium benzoate, sodium phenylbutyrate, L-arginine, vitamin cocktail (vitamin B1, vitamin B12, vitamin C, vitamin E, biotin), l-carnitine, coenzyme Q10, and parenteral nutrition. Her ammonia and glutamine levels remained low; thus, protein intake was increased to 1.2 g/kg/day. Furthermore, the amount of sodium benzoate and sodium phenylbutyrate were reduced. She remained metabolically stable and experienced no metabolic crisis following treatment with oral NCG, sodium benzoate, sodium phenylbutyrate, citrulline, vitamin cocktail, l-carnitine, and coenzyme Q10 until she underwent liver transplantation at 207 days of age. She had no neurological complications at the age of 15 months. Ammonia and glutamine levels of the patient were successfully maintained at a low level via NCG treatment with increased protein intake, which led to normal neurological development. Thus, undiagnosed urea cycle disorders should be treated rapidly with acute therapy including NCG, which should be maintained until a genetic diagnosis is reached. It is essential to prevent metabolic crises in patients with CPS1D until liver transplantation to improve their prognoses.
Project description:Organic acidemias and urea cycle disorders are ultra-rare inborn errors of metabolism characterized by episodic acute decompensation, often associated with hyperammonemia, resulting in brain edema and encephalopathy. Retrospective reports and translational studies suggest that N-carbamylglutamate (NCG) may be effective in reducing ammonia levels during acute decompensation in two organic acidemias, propionic and methylmalonic acidemia (PA and MMA), and in two urea cycle disorders, carbamylphosphate synthetase 1 and ornithine transcarbamylase deficiency (CPSD and OTCD). We established the 9-site N-carbamylglutamate Consortium (NCGC) in order to conduct two randomized double-blind, placebo-controlled trials of NCG in acute hyperammonemia of PA, MMA, CPSD and OTCD. Conducting clinical trials is challenging in any disease, but poses unique barriers and risks in the ultra-rare disorders. As the number of clinical trials in orphan diseases increases, evaluating the successes and opportunities for improvement in such trials is essential. We summarize herein the design, methods, experiences, challenges and lessons from the NCGC-conducted trials.
Project description:Administration of N-carbamylglutamate (NCG), an analogue of endogenous N-acetyl-glutamate (an activator of arginine synthesis) has been shown to enhance neonatal growth by increasing circulating arginine levels. However, the effect of NCG on pregnancy remains unknown. This study examined the effects of NCG on pregnancy outcome and evaluated potential mechanisms involved. Reproductive performance, embryo implantation, and concentration of amino acids in serum and uterine flushing, were determined in rats fed control or NCG supplemented diets. Ishikawa cells and JAR cells were used to examine the mechanism by which NCG affects embryo implantation. Dietary NCG supplementation increased serum levels of arginine, onithine, and proline, as well as uterine levels of arginine, glutamine, glutamate, and proline. Additionally, it stimulated LIF expression, and enhanced the activation of signal transduction and activator of transcription 3 (Stat3), protein kinase B (PKB), and 70-kDa ribosomal protein S6 kinase (S6K1) during the periimplantation period, resulting in an increase in litter size but not birth weight. In uterine Ishikawa cells, LIF expression was also enhanced by treatment with arginine and its metabolites. In trophoblast JAR cells, treatment with arginine and its metabolites enhanced Stat3, PKB, and S6K1 activation and facilitated cellular adhesion activity. These effects were abolished by pretreatment with inhibitors of phosphatidylinositol 3-kinase (wortmannin) and mammalian target of rapamycin (rapamycin). The results demonstrate that NCG supplementation enhances pregnancy outcome and have important implications for the pregnancy outcome of mammalian species.
Project description:This study evaluated the effects of arginine (Arg) or N-carbamylglutamate (NCG) on inflammation, antioxidant property, and antioxidant-related gene expression in rat spleen under oxidative stress. A total of 52 rats were randomly distributed into 4 treatment groups with 13 replicates per group. Rats were fed a basal diet (BD) or BD supplemented with Arg or NCG for 30 days. On day 28, half of the BD-fed rats were intraperitoneally injected with sterile saline (control group), and the other half with 12 mg/kg body weight of diquat (DT; DT group). The other 2 diet groups were intraperitoneally injected with 12 mg/kg body weight of DT with either Arg (1%) (DT + Arg) or NCG (0.1%) (DT + NCG). Rat spleen samples were collected for analysis at 48 h after DT injection. Results showed that DT damaged the antioxidant defense in rats compared with the control group (P < 0.05). Compared with the DT group, the DT + Arg and DT + NCG groups manifested improved anti-hydroxyl radical, catalase, and total superoxide dismutase (T-SOD) activities, increased glutathione content (P < 0.05), and decreased malondialdehyde content (P < 0.05). Moreover, compared with the DT group, the DT + Arg and DT + NCG groups enhanced mRNA expression of superoxide dismutase (SOD), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1(Keap-1), and mammalian target of rapamycin (mTOR) (P < 0.05). Both NCG and Arg significantly increased anti-inflammatory cytokine mRNA level but suppressed the pro-inflammatory cytokine mRNA expression under oxidative stress (P < 0.05). In summary, NCG and Arg effectively alleviated oxidative stress, improved the antioxidant capacity and regulated the antioxidant-related signaling molecular expression in rat spleen. N-carbamylglutamate and Arg reduced the inflammation in the spleen by mediating the gene expression of anti-inflammatory and pro-inflammatory cytokines and transforming growth factor-? (TGF-?).
Project description:Egg quality defects seriously reduce the quality grade and increase egg breakage in egg marketing activities. In this study, the effect of N-carbamylglutamate (NCG) on eggshell quality was investigated by evaluating calcium absorption and calcification in laying hens. A total of 30 newly hatched female Hy-Line chicks were randomly assigned to the control group (basal diet) and treatment group (basal diet supplemented with 1% NCG). At 25 wk, eggs from each group were obtained to assess egg quality parameters. Blood samples were collected for analysis of mineral, hormone, and amino acids levels at 16 h after laying egg. Uterine tissues were removed and fixed in 4% neutral paraformaldehyde solution or kept in liquid nitrogen for mineral determination, quantitative PCR, and Western blot. Results showed that the egg quality (eggshell thickness, strength and percentage, egg specific gravity, and eggshell effective thickness) was significantly increased while effective thickness of mastoid layer, width of mastoid gap, and mammillary knobs were significantly decreased by dietary NCG supplementation (P < 0.05). The levels of minerals (Ca, P, Fe, Mg, Na, K) in eggshell, plasma, and uterus were remarkably elevated (P < 0.05). Meanwhile, the concentrations of calcium metabolism-related hormones (17?-estradiol, parathyroid hormone, and calcitonin) were increased in the NCG group (P < 0.05). Moreover, expression of calbindin 1, carbonic anhydrase 2, ovalbumin, ovotransferrin, ovocleidin-17, ovocleidin-116, and clusterin mRNAs, as well as calbindin 1 and ATP2A1 proteins in uterus, duodenum, and kidney, was all upregulated in hens fed with NCG (P < 0.05). In addition, the number of blood vessels in the uterus, height of uterine mucosal folds, villus length in endometrium, and areas of uterine mucosal folds were significantly increased in the NCG group (P < 0.05). In conclusion, dietary 1% NCG supplementation during 0 to 25 wk can improve eggshell quality through changes in endometrial morphology, expression of calcium metabolism-related genes, and secretion of related hormones to promote eggshell formation in the laying hens.
Project description:L-arginine (Arg) is a semiessential amino acid with several physiological functions. N-Carbamylglutamate (NCG) can promote the synthesis of endogenous Arg in mammals. However, the roles of Arg or NCG on hepatic inflammation and apoptosis in suckling lambs suffering from intrauterine growth restriction (IUGR) are still unclear. The current work is aimed at examining the effects of dietary Arg and NCG on inflammatory and hepatocyte apoptosis in IUGR suckling lambs. On day 7 after birth, 48 newborn Hu lambs were selected from a cohort of 432 twin lambs. Normal-birthweight and IUGR Hu lambs were allocated randomly (n = 12/group) to control (CON), IUGR, IUGR+1% Arg, or IUGR+0.1% NCG groups. Lambs were fed for 21 days from 7 to 28 days old. Compared with CON lambs, relative protein 53 (P53), apoptosis antigen 1 (Fas), Bcl-2-associated X protein (Bax), caspase-3, cytochrome C, tumor necrosis factor alpha (TNF-?), nuclear factor kappa-B (NF-?B) p65, and NF-?B pp65 protein levels were higher (P < 0.05) in liver from IUGR lambs, whereas those in liver from IUGR lambs under Arg or NCG treatment were lower than those in IUGR lambs. These findings indicated that supplementing Arg or NCG reduced the contents of proinflammatory cytokines at the same time when the apoptosis-related pathway was being suppressed, thus suppressing the IUGR-induced apoptosis of hepatic cells.
Project description:High-altitude pulmonary hypertension (HAPH) is a life-threating condition for animals in high altitude, and disturbance of endothelial nitric oxide (NO) synthesis contributes to its pathogenesis. N-carbamylglutamate (NCG), which enhances arginine synthesis, promotes endogenous synthesis of NO. In this study, we determined the effects of NCG on alleviating HAPH in Holstein heifers that ascended to Tibet (Lhasa, 3,658 m).Exp. 1, 2,000 Holstein heifers were transported from low elevation (1,027 m) to Lhasa. After being exposed to hypoxia for 1 yr, Holstein heifers were assigned to a healthy group (Control, n?=?6) with mean pulmonary hypertension (mPAP)?<?41 mmHg, and an HAPH affected group (HAPH, n?=?6) with mPAP >?49 mmHg. Lung tissues were collected to evaluate histopathological changes and the expression of endothelial nitric oxide synthase (eNOS). Exp. 2, ten healthy heifers and 10 HAPH affected heifers were supplemented with NCG (20 g/d per heifer) for 4 wk. Physiological parameters were determined and blood samples were collected on d?-?1 and d 28 of the feeding trial.Expression of eNOS in small pulmonary arteriole intima was higher in the healthy than HAPH group (P?=?0.006), whereas HAPH group had significantly thicker media and adventitia than healthy group (all P?<?0.05). The mRNA of eNOS and protein level of eNOS were higher in the lungs of heifers in the healthy group than in the HAPH group (both P?<?0.001), whereas endothelin-1 protein levels were higher in HAPH group than in the healthy group (P?=?0.025). NCG supplementation decreased mPAP and ammonia (both P?=?0.001), whereas it increased the expression of eNOS, arginine, and plasma NO (all P?<?0.05).The expression of eNOS was decreased in Holstein heifers with HAPH. NCG supplementation decreased mPAP through the restoration of eNOS and endogenous NO synthesis.
Project description:N-acetylglutamate synthase (NAGS) catalyzes the conversion of glutamate and acetyl-CoA to NAG, the essential allosteric activator of carbamyl phosphate synthetase I, the first urea cycle enzyme in mammals. A 17-year-old female with recurrent hyperammonemia attacks, the cause of which remained undiagnosed for 8 years in spite of multiple molecular and biochemical investigations, showed markedly enhanced ureagenesis (measured by isotope incorporation) in response to N-carbamylglutamate (NCG). This led to sequencing of the regulatory regions of the NAGS gene and identification of a deleterious single-base substitution in the upstream enhancer. The homozygous mutation (c.-3064C>A), affecting a highly conserved nucleotide within the hepatic nuclear factor 1 (HNF-1) binding site, was not found in single nucleotide polymorphism databases and in a screen of 1,086 alleles from a diverse population. Functional assays demonstrated that this mutation decreases transcription and binding of HNF-1 to the NAGS gene, while a consensus HNF-1 binding sequence enhances binding to HNF-1 and increases transcription. Oral daily NCG therapy restored ureagenesis in this patient, normalizing her biochemical markers, and allowing discontinuation of alternate pathway therapy and normalization of her diet with no recurrence of hyperammonemia. Inc.
Project description:Glutamine and N-carbamylglutamate can enhance growth performance and health in animals, but the underlying mechanisms are not yet elucidated. This study aimed to investigate the effect of glutamine and N-carbamylglutamate supplementation in rat metabolism. Thirty rats were fed a control, glutamine, or N-carbamylglutamate diet for four weeks. Urine samples were analyzed by nuclear magnetic resonance (NMR)-based metabolomics, specifically high-resolution ¹H NMR metabolic profiling combined with multivariate data analysis. Glutamine significantly increased the urine levels of acetamide, acetate, citrulline, creatinine, and methymalonate, and decreased the urine levels of ethanol and formate (p < 0.05). Moreover, N-carbamylglutamate significantly increased the urine levels of creatinine, ethanol, indoxyl sulfate, lactate, methymalonate, acetoacetate, m-hydroxyphenylacetate, and sarcosine, and decreased the urine levels of acetamide, acetate, citrulline, creatine, glycine, hippurate, homogentisate, N-acetylglutamate, phenylacetyglycine, acetone, and p-hydroxyphenylacetate (p < 0.05). Results suggested that glutamine and N-carbamylglutamate could modify urinary metabolome related to nitrogen metabolism and gut microbiota metabolism. Moreover, N-carbamylglutamate could alter energy and lipid metabolism. These findings indicate that different arginine precursors may lead to differences in the biofluid profile in rats.