Effects of improved amino acid balance diet on lysine mammary utilization, whole body protein turnover and muscle protein breakdown on lactating sows.

BACKGROUND: The study objective was to test the hypothesis that low crude protein (CP) diet with crystalline amino acids (CAA) supplementation improves Lys utilization efficiency for milk production and reduces protein turnover and muscle protein breakdown. Eighteen lactating multiparous Yorkshire sows were allotted to 1 of 2 isocaloric diets (10.80 MJ/kg net energy): control (CON; 19.24% CP) and reduced CP with "optimal" AA profile (OPT; 14.00% CP). Sow body weight and backfat were recorded on d 1 and 21 of lactation and piglets were weighed on d 1, 14, 18, and 21 of lactation. Between d 14 and 18, a subset of 9 sows (CON = 4, OPT = 5) was infused with a mixed solution of 3-[methyl-2H3]histidine (bolus injection) and [13C]bicarbonate (priming dose) first, then a constant 2-h [13C]bicarbonate infusion followed by a 6-h primed constant [1-13C]lysine infusion. Serial blood and milk sampling were performed to determine plasma and milk Lys enrichment, Lys oxidation rate, whole body protein turnover, and muscle protein breakdown. RESULTS: Over the 21-d lactation period, compared to CON, sows fed OPT had greater litter growth rate (P < 0.05). Compared to CON, sows fed OPT had greater efficiency of Lys (P < 0.05), Lys mammary flux (P < 0.01) and whole-body protein turnover efficiency (P < 0.05). Compared to CON, sows fed OPT tended to have lower whole body protein breakdown rate (P = 0.069). Muscle protein breakdown rate did not differ between OPT and CON (P = 0.197). CONCLUSION: Feeding an improved AA balance diet increased efficiency of Lys and reduced whole-body protein turnover and protein breakdown. These results imply that the lower maternal N retention observed in lactating sows fed improved AA balance diets in previous studies may be a result of greater partitioning of AA towards milk rather than greater body protein breakdown.

Pediatric Swine Model of Methicillin-Resistant Staphylococcus aureus Sepsis-Induced Coagulopathy, Disseminated Microvascular Thrombosis, and Organ Injuries.

Sepsis-induced coagulopathy leading to disseminated microvascular thrombosis is associated with high mortality and has no existing therapy. Despite the high prevalence of Gram-positive bacterial sepsis, especially methicillin-resistant Staphylococcus aureus (MRSA), there is a paucity of published Gram-positive pediatric sepsis models. Large animal models replicating sepsis-induced coagulopathy are needed to test new therapeutics before human clinical trials. HYPOTHESIS: Our objective is to develop a pediatric sepsis-induced coagulopathy swine model that last 70 hours. METHODS AND MODELS: Ten 3 weeks old piglets, implanted with telemetry devices for continuous hemodynamic monitoring, were IV injected with MRSA (n = 6) (USA300, Texas Children's Hospital 1516 strain) at 1 × 109 colony forming units/kg or saline (n = 4). Fluid resuscitation was given for heart rate greater than 50% or mean arterial blood pressure less than 30% from baseline. Acetaminophen and dextrose were provided as indicated. Point-of-care complete blood count, prothrombin time (PT), activated thromboplastin time, d-dimer, fibrinogen, and specialized coagulation assays were performed at pre- and post-injection, at 0, 24, 48, 60, and 70 hours. Piglets were euthanized and necropsies performed. RESULTS: Compared with the saline treated piglets (control), the septic piglets within 24 hours had significantly lower neurologic and respiratory scores. Over time, PT, d-dimer, and fibrinogen increased, while platelet counts and activities of factors V, VII, protein C, antithrombin, and a disintegrin and metalloproteinase with thrombospondin-1 motifs (13th member of the family) (ADAMTS-13) decreased significantly in septic piglets compared with control. Histopathologic examination showed minor focal organ injuries including microvascular thrombi and necrosis in the kidney and liver of septic piglets. INTERPRETATIONS AND CONCLUSIONS: We established a 70-hour swine model of MRSA sepsis-induced coagulopathy with signs of consumptive coagulopathy, disseminated microvascular thrombosis, and early organ injuries with histological minor focal organ injuries. This model is clinically relevant to pediatric sepsis and can be used to study dysregulated host immune response and coagulopathy to infection, identify potential early biomarkers, and to test new therapeutics.

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model.

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-2H2]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-2H2]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

Blood Flow Within Bioengineered 3D Printed Vascular Constructs Using the Porcine Model.

Recently developed biofabrication technologies are enabling the production of three-dimensional engineered tissues containing vascular networks which can deliver oxygen and nutrients across large tissue volumes. Tissues at this scale show promise for eventual regenerative medicine applications; however, the implantation and integration of these constructs in vivo remains poorly studied. Here, we introduce a surgical model for implantation and direct in-line vascular connection of 3D printed hydrogels in a porcine arteriovenous shunt configuration. Utilizing perfusable poly(ethylene glycol) diacrylate (PEGDA) hydrogels fabricated through projection stereolithography, we first optimized the implantation procedure in deceased piglets. Subsequently, we utilized the arteriovenous shunt model to evaluate blood flow through implanted PEGDA hydrogels in non-survivable studies. Connections between the host femoral artery and vein were robust and the patterned vascular channels withstood arterial pressure, permitting blood flow for 6 h. Our study demonstrates rapid prototyping of a biocompatible and perfusable hydrogel that can be implanted in vivo as a porcine arteriovenous shunt, suggesting a viable surgical approach for in-line implantation of bioprinted tissues, along with design considerations for future in vivo studies. We further envision that this surgical model may be broadly applicable for assessing whether biomaterials optimized for 3D printing and cell function can also withstand vascular cannulation and arterial blood pressure. This provides a crucial step toward generated transplantable engineered organs, demonstrating successful implantation of engineered tissues within host vasculature.

Pegylated arginine deiminase depletes plasma arginine but maintains tissue arginine availability in young pigs.

Pegylated arginine deiminase (ADI-PEG20) results in the depletion of arginine with the production of isomolar amounts of citrulline. This citrulline has the potential to be utilized by the citrulline recycling pathway regenerating arginine and sustaining tissue arginine availability. The goal of this research was to test the hypothesis that ADI-PEG20 depletes circulating arginine in pigs but maintains tissue arginine concentration and function, and to characterize the kinetics of citrulline and arginine. Two multitracer approaches (bolus dose and primed-continuous infusion) were used to investigate the metabolism of arginine and citrulline in Control (n = 7) and ADI-PEG20 treated (n = 8) pigs during the postprandial period. In addition, blood pressure was monitored by telemetry, and multiple tissues were collected to determine arginine concentration. Plasma arginine was depleted immediately after ADI-PEG20 administration, with an increase in plasma citrulline concentration (P < 0.01). The depletion of arginine did not affect (P > 0.10) blood pressure, whole body protein synthesis, or urea production. Despite the lack of circulating arginine in ADI-PEG20-treated pigs, most tissues were able to maintain concentrations similar (P > 0.10) to those in Control animals. The kinetics of citrulline and arginine indicated the high citrulline turnover and regeneration of arginine through the citrulline recycling pathway. ADI-PEG20 administration resulted in an absolute and almost instantaneous depletion of circulating arginine, thus reducing global availability without affecting cardiovascular parameters and protein metabolism. The citrulline produced from the deimination of arginine was in turn utilized by the citrulline recycling pathway restoring local tissue arginine availability.NEW & NOTEWORTHY Pegylated arginine deiminase depletes circulating arginine, but the citrulline generated is utilized by multiple tissues to regenerate arginine and sustain local arginine availability. Preempting the arginine depletion that occurs as result of sepsis and trauma with arginine deiminase offers the possibility of maintaining tissue arginine availability despite negligible plasma arginine concentrations.

Modeling age-dependent developmental changes in the expression of genes involved in citrulline synthesis using pig enteroids.

BACKGROUND: Age-dependent changes in the intestinal gene expression of enzymes involved in the metabolism of citrulline and arginine are well characterized. Enteroids, a novel ex-vivo model that recreates the three-dimensional structure of the intestinal crypt-villus unit, have shown to replicate molecular and physiological profiles of the intestinal segment from where they originated ("location memory"). OBJECTIVE: The present study tested the hypothesis that enteroids recapitulate the developmental changes observed in vivo regarding citrulline production in pigs ("developmental memory"). METHODS: Preterm (10- and 5-d preterm) and term pigs at birth, together with 7- and 35-d-old pigs were studied. Gene expression was measured in jejunal samples and in enteroids derived from this segment. Whole body citrulline production was measured by isotope dilution and enteroid citrulline production by accumulation in the media. RESULTS: With the exception of arginase I and inducible nitric oxide synthase, all the genes investigated expressed in jejunum were expressed by enteroids. In the jejunum, established markers of development (lactase and sucrase-isomaltase), as well as genes that code for enzymes involved in the production and utilization of citrulline and arginine, underwent the ontogenic changes described in the literature. However, enteroid expression of these genes, as well as citrulline production, failed to recapitulate the changes observed in vivo. CONCLUSIONS: Under culture conditions used in our study, enteroids derived from jejunal crypts of pigs at different ages failed to replicate the gene expression observed in whole tissue and whole body citrulline production. Additional extracellular cues may be needed to reproduce the age-dependent phenotype.

Arginase II Plays a Central Role in the Sexual Dimorphism of Arginine Metabolism in C57BL/6 Mice.

BACKGROUND: Sex differences in plasma concentration of arginine and arginase activity of different tissues have been reported in mice. In addition, male but not female C57BL/6 mice have a dietary arginine requirement for growth. OBJECTIVE: The goal of this research was to test the hypothesis that arginase II is a key factor in the sexual dimorphism of arginine metabolism. METHODS: Young adult male and female wild type (WT), and heterozygous and arginase II knockout mice on a C57BL/6 background mice were infused with labeled citrulline, arginine, ornithine, phenylalanine, and tyrosine to determine the rates of appearance and interconversion of these amino acids. Tissue arginase activity was measured in the liver, heart, jejunum, kidney, pancreas, and spleen with an arginine radioisotope. The effect of genotype, sex, and their interaction was tested. RESULTS: Female mice produced ∼36% more citrulline than their male littermates, which translated into a greater arginine endogenous synthesis, flux, and plasma concentration (42, 6, and 27%, respectively; P < 0.001). Female mice also had a greater phenylalanine flux (10%) indicating a greater rate of whole protein breakdown; however, they had a lower protein synthesis rate than males (18%; P < 0.001). The ablation of arginase II reduced the production of citrulline and the de novo synthesis of arginine in females and increased the rate of appearance of arginine and plasma arginine concentration in male mice (16 and 22%, respectively; P < 0.001). No effect of arginase II deletion, however, was observed for whole-body protein kinetics. Arginase II activity was present in the pancreas, kidney, jejunum, and spleen; WT females had a ∼2-fold greater renal arginase activity than their WT counterparts. CONCLUSIONS: A clear sexual dimorphism exists in the endogenous synthesis of arginine and its disposal. Female mice have a greater arginine availability than their male littermates. The ablation of arginase II increases arginine availability in male mice.

Derangement of Arginine and Related Amino Acids in Children Undergoing Surgery for Congenital Heart Disease With Cardiopulmonary Bypass.

Arginine is a conditionally essential amino acid, the precursor for nitric oxide and a key factor in cell proliferation, protein synthesis, and energy metabolism. When there is increased demand in the setting of inflammation, ischemia-reperfusion injury, and organ dysfunction, endogenous arginine production falls short, and external supplementation may be necessary. The goal of this study was to assess changes in concentrations of plasma arginine, citrulline, ornithine, glutamine, and plasma arginase in infants and children undergoing surgery for congenital heart disease with cardiopulmonary bypass. DESIGN: Prospective observational study. SETTING: The study was conducted in the Heart Center at Texas Children's Hospital. SUBJECTS: Children undergoing surgery for congenital heart disease with cardiopulmonary bypass. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Serial perioperative blood samples were collected for quantification of amino acids, arginase, nitric oxide metabolites, and markers of organ function (lactate, Pao2/Fio2 ratio, and creatinine clearance). Thirty children (18 males) were included in the study; median (interquartile range) age 0.5 years (0.3-0.9 yr). The mean ± sd for plasma amino acid concentrations before cardiopulmonary bypass: arginine 62 ± 20 µmol/L, citrulline 24 ± 6 µmol/L, ornithine 53 ± 32 µmol/L, and glutamine 591 ± 126 µmol/L. Arginine concentration was decreased within the first 24 hours (43 ± 15 µmol/L; p = 0.004), citrulline and glutamine concentrations decreased over the first 48 hours (11 ± 4 µmol/L; p < 0.001 and 493 ± 131 µmol/L; p = 0.019, respectively) and were associated with an increase in arginase (3.8 ± 3 µg/mL; p < 0.05). There was an increase in Vasoactive-Inotropic Score (5.9 ± 19 vs 0.5 ± 2; p < 0.001), decrease in creatinine clearance (76 ± 24 vs 93 ± 31; p = 0.002), and Pao2/Fio2 ratio (243 ± 138 vs 374 ± 200; p = 0.007) comparing to baseline. CONCLUSIONS: A widely variable degree of arginine, citrulline, and glutamine depletion occurs in children after surgery for congenital heart disease. These findings were associated with increased arginase and coincide with some of the markers of organ perfusion.

Developmental changes in the utilization of citrulline by neonatal pigs.

Developmental changes in the renal expression and activity of argininosuccinate synthase (ASS1) and argininosuccinate lyase (ASL), enzymes that use citrulline for the production of arginine, have been reported. Thus, the ability of neonates, and especially premature neonates, to produce arginine may be compromised. To determine the utilization of citrulline in vivo, we measured renal expression of ASS1 and ASL and conducted citrulline compartmental and noncompartmental kinetics using [15N]citrulline in pigs of five different ages (from 10 days preterm to 5 wk of age). The tracer was given in substrate amounts to also test the ability of neonatal pigs to use exogenous citrulline. Preterm and term pigs at birth had lower ASS1 and ASL expression than older animals, which was reflected in the longer half-life of citrulline in the neonatal groups. The production and utilization of citrulline by 1-wk-old pigs was greater than in pigs of other ages, including 5-wk-old animals. Plasma citrulline concentration was not able to capture these differences in citrulline production and utilization. In conclusion, the developmental changes in renal ASS1 and ASL gene expression are reflected in the ability of the pigs to use citrulline. However, it seems that there is an excess capacity to use citrulline at all ages, including during prematurity, since the bolus dose of tracer did not result in an increase in endogenous citrulline. Our results support the idea that citrulline supplementation in neonatal, including premature, pigs is a viable option to increase arginine availability.

Sex Dependent Dysregulation of Hepatic Glucose Production in Lean Type 2 Diabetic Rats.

We have characterized a lean type 2 diabetic rat model by gestational low protein programming. We aimed to identify if the regulation of hepatic glucose production (HGP) via gluconeogenesis and glycogenolysis is affected and if there are any sex differences. Fasting (6-7 months old) type 2 diabetic rats received 2H2O followed by a primed constant rate infusion of [6,6-2H2] glucose. Blood samples were drawn during steady states after 4 h of fasting and following a euglycemic hyperinsulinemic clamp. HGP and the fraction of glucose derived from gluconeogenesis under fasting and euglycemic states were measured from steady state glucose enrichments after the infusion of [6,6-2H2]glucose and 2H2O tracers. Glycogenolysis was determined by calculating the difference between total HGP and gluconeogenesis rates. Hepatic gene expression of enzymes involved in HGP were quantified using qPCR. HGP rates was similar during fasting in both groups and sexes. However, under simulated fed condition, HGP rate was suppressed in controls but not in type 2 diabetic rats. They also showed inefficient HGP suppression in a simulated fed state. Differential analysis showed that suppression of both gluconeogenesis and glycogenolysis under simulated fed state was affected in these low protein programmed type 2 diabetic rats. These effects were greater in females when compared to males. Further, key genes involved in these processes like G6Pase, Pepck, pyruvate carboxylase, and glycogen phosphorylase in liver were dysregulated. Our data shows impaired suppression of HGP via gluconeogenesis and glycogenolysis in type 2 diabetic rats with greater effects on females.

Cardiac-specific ablation of glutaredoxin 3 leads to cardiac hypertrophy and heart failure.

Growing evidence suggests that redox-sensitive proteins including glutaredoxins (Grxs) can protect cardiac muscle cells from oxidative stress-induced damage. Mammalian Grx3 has been shown to be critical in regulating cellular redox states. However, how Grx3 affects cardiac function by modulating reactive oxygen species (ROS) signaling remains unknown. In this study, we found that the expression of Grx3 in the heart is decreased during aging. To assess the physiological role of Grx3 in the heart, we generated mice in which Grx3 was conditionally deleted in cardiomyocytes (Grx3 conditional knockout (CKO) mice). Grx3 CKO mice were viable and grew indistinguishably from their littermates at young age. No difference in cardiac function was found comparing Grx3 CKO mice and littermate controls at this age. However, by the age of 12 months, Grx3 CKO mice exhibited left ventricular hypertrophy with a significant decrease in ejection fraction and fractional shortening along with a significant increase of ROS production in cardiomyocytes compared to controls. Deletion of Grx3 also impaired Ca2+ handling, caused enhanced sarcoplasmic reticulum (SR) calcium (Ca2+ ) leak, and decreased SR Ca2+ uptake. Furthermore, enhanced ROS production and alteration of Ca2+ handling in cardiomyocytes occurred, prior to cardiac dysfunction in young mice. Therefore, our findings demonstrate that Grx3 is an important factor in regulating cardiac hypertrophy and heart failure by modulating both cellular redox homeostasis and Ca2+ handling in the heart.

Differential action of TGR5 agonists on GLP-2 secretion and promotion of intestinal adaptation in a piglet short bowel model.

Enteroendocrine L cells and glucagon-like peptide 2 (GLP-2) secretion are activated in the intestinal adaptation process following bowel resection in patients with short bowel syndrome. We hypothesized that enteral activation of Takeda G protein-coupled receptor 5 (TGR5), expressed in enteroendocrine L cells, could augment endogenous GLP-2 secretion and the intestinal adaptation response. Our aim was to assess the efficacy of different TGR5 agonists to stimulate GLP-2 secretion and intestinal adaptation in a piglet short-bowel model. In study 1, parenterally fed neonatal pigs (n = 6/group) were gavaged with vehicle, olive extract (OE; 10 or 50 mg/kg), or ursolic acid (UA; 10 mg/kg), and plasma GLP-2 was measured for 6 h. In study 2, neonatal pigs (n = 6-8/group) were subjected to transection or 80% mid-small intestine resection and, after 2 days, assigned to treatments for 10 days as follows: 1) transection + vehicle (sham), 2) resection + vehicle (SBS), 3) resection + 30 mg UA (SBS + UA), and 4) resection + 180 mg/kg OE (SBS + OE). We measured plasma GLP-2, intestinal histology, cell proliferation, and gene expression, as well as whole body citrulline-arginine kinetics and bile acid profiles. In study 1, GLP-2 secretion was increased by UA and tended to be increased by OE. In study 2, SBS alone, but not additional treatment with either TGR5 agonist, resulted in increased mucosal thickness and crypt cell proliferation in remnant jejunum and ileum sections. SBS increased biliary and ileal concentration of bile acids and expression of inflammatory and farnesoid X receptor target genes, but these measures were suppressed by UA treatment. In conclusion, UA is an effective oral GLP-2 secretagogue in parenterally fed pigs but is not capable of augmenting GLP-2 secretion or the intestinal adaptation response after massive small bowel resection. NEW & NOTEWORTHY Therapeutic activation of endogenous glucagon-like peptide 2 (GLP-2) secretion is a promising strategy to improve intestinal adaptation in patients with short bowel syndrome. This study in neonatal pigs showed that oral supplementation with a selective Takeda G protein-coupled receptor 5 (TGR5) agonist is an effective approach to increase GLP-2 secretion. The results warrant further study to establish a more potent oral TGR5 agonist that can effectively improve intestinal adaptation in pediatric patients with SBS.

Citrulline Generation Test: What Does It Measure?

BACKGROUND: The citrulline generation test (CGT) has been proposed as a tool to determine gut function. However, the increase in plasma citrulline concentration that follows a bolus dose of alanyl-glutamine may also result from a reduction in citrulline clearance due to competition with glutamine for transport. MATERIALS AND METHODS: A swine model was developed, and stable isotope tracers were used to determine the mechanism behind the increase in plasma citrulline that follows a bolus dose of alanyl-glutamine. Plasma concentrations and enrichments were determined, and a non-steady-state model was used to calculate rates of appearance, disappearance, and conversion. RESULTS: The pig model recapitulated the increase in plasma citrulline observed in humans after a dose of alanyl-glutamine. The dipeptide was rapidly hydrolyzed to its constitutive amino acids. Both citrulline plasma concentration and citrulline rate of appearance increased by ≈45% after the bolus dose of alanyl-glutamine. The conversion of citrulline to arginine and the rate of appearance of arginine also increased. Glutamine contributed up to 25% ± 2% of the rate of appearance of citrulline. No changes in the rate of disappearance of citrulline were observed. CONCLUSION: Our results indicate that a single bolus dose of alanyl-glutamine increases plasma citrulline concentration by increasing citrulline production without any effect on citrulline disposal. Our findings strongly indicate that the CGT assesses the metabolic response of the gut and that CGT can become a useful tool to evaluate gut mass and function.

Rapid Disruption of Genes Specifically in Livers of Mice Using Multiplex CRISPR/Cas9 Editing.

BACKGROUND & AIMS: Despite advances in gene editing technologies, generation of tissue-specific knockout mice is time-consuming. We used CRISPR/Cas9-mediated genome editing to disrupt genes in livers of adult mice in just a few months, which we refer to as somatic liver knockouts. METHODS: In this system, Fah-/- mice are given hydrodynamic tail vein injections of plasmids carrying CRISPR/Cas9 designed to excise exons in Hpd; the Hpd-edited hepatocytes have a survival advantage in these mice. Plasmids that target Hpd and a separate gene of interest can therefore be used to rapidly generate mice with liver-specific deletion of nearly any gene product. RESULTS: We used this system to create mice with liver-specific knockout of argininosuccinate lyase, which develop hyperammonemia, observed in humans with mutations in this gene. We also created mice with liver-specific knockout of ATP binding cassette subfamily B member 11, which encodes the bile salt export pump. We found that these mice have a biochemical phenotype similar to that of Abcb11-/- mice. We then used this system to knock out expression of 5 different enzymes involved in drug metabolism within the same mouse. CONCLUSIONS: This approach might be used to develop new models of liver diseases and study liver functions of genes that are required during development.

Ex Vivo Enteroids Recapitulate In Vivo Citrulline Production in Mice.

Background: The endogenous production of arginine relies on the synthesis of citrulline by enteral ornithine transcarbamylase (OTC). Mutations in the gene coding for this enzyme are the most frequent cause of urea cycle disorders. There is a lack of correlation between in vivo metabolic function and DNA sequence, transcript abundance, or in vitro enzyme activity. Objective: The goal of the present work was to test the hypothesis that enteroids, a novel ex vivo model, are able to recapitulate the in vivo citrulline production of wild-type (WT) and mutant mice. Methods: Six-week-old male WT and OTC-deficient mice [sparse fur and abnormal skin (spf-ash) mutation] were studied. Urea and citrulline fluxes were determined in vivo, and OTC abundance was measured in liver and gut tissue. Intestinal crypts were isolated and cultured to develop enteroids. Ex vivo citrulline production and OTC abundance were determined in these enteroids. Results: Liver OTC abundance was lower (mean ± SE: 0.16 ± 0.01 compared with 1.85 ± 0.18 arbitrary units; P < 0.001) in spf-ash mice than in WT mice, but there was no difference in urea production. In gut tissue, OTC was barely detectable in mutant mice; despite this, a lower but substantial citrulline production (67 ± 3 compared with 167 ± 8 µmol · kg-1 · h-1; P < 0.001) was shown in the mutant mice. Enteroids recapitulated the in vivo findings of a very low OTC content accompanied by a reduced citrulline production (1.07 ± 0.20 compared with 4.64 ± 0.44 nmol · µg DNA-1 · d-1; P < 0.001). Conclusions: Enteroids recapitulate in vivo citrulline production and offer the opportunity to study the regulation of citrulline production in a highly manipulable system.

Prematurity reduces citrulline-arginine-nitric oxide production and precedes the onset of necrotizing enterocolitis in piglets.

Necrotizing enterocolitis (NEC) is associated with low plasma arginine and vascular dysfunction. It is not clear whether low intestinal citrulline production, the precursor for arginine synthesis, occurs before and thus predisposes to NEC or if it results from tissue damage. This study was designed to test the hypothesis that whole body rates of citrulline, arginine, and nitric oxide synthesis are low in premature pigs and that they precede NEC. Piglets delivered by cesarean section at 103 days [preterm (PT)], 110 days [near-term (NT)], or 114 days [full-term (FT)] of gestation were given total parenteral nutrition and after 2 days orogastrically fed infant formula for 42 h to induce NEC. Citrulline and arginine fluxes were determined before and during the feeding protocol. Gross macroscopic and histological NEC scores and plasma fatty acid binding protein (iFABP) concentration were determined as indicators of NEC. Intestinal gene expression for enzymes of the arginine pathway were quantitated. A lower ( P < 0.05) survival rate was observed for PT (8/27) than for NT (9/9) and FT pigs (11/11). PT pigs had higher macroscopic gross ( P < 0.05) and histological NEC ( P < 0.05) scores and iFABP concentration ( P < 0.05) than pigs of more advanced gestational age. PT pigs had lower citrulline production and arginine fluxes ( P < 0.05) throughout and a reduced gene expression in genes of the citrulline-arginine pathway. In summary, intestinal enzyme expression and whole body citrulline and arginine fluxes were reduced in PT pigs compared with animals of more advance gestational age and preceded the development of NEC. NEW & NOTEWORTHY Arginine supplementation prevents necrotizing enterocolitis (NEC), the most common gastrointestinal emergency of prematurity. Citrulline (precursor for arginine) production is reduced during NEC, and this is believed to be a consequence of intestinal damage. In a swine model of NEC, we show that intestinal gene expression of the enzymes for citrulline production and whole body citrulline and arginine fluxes are reduced and precede the onset of NEC in premature pigs. Reduced citrulline production during prematurity may be a predisposition to NEC.

The Citrulline Recycling Pathway Sustains Cardiovascular Function in Arginine-Depleted Healthy Mice, but Cannot Sustain Nitric Oxide Production during Endotoxin Challenge.

Background: The recycling of citrulline by argininosuccinate synthase 1 (ASS1) and argininosuccinate lyase (ASL) is crucial to maintain arginine availability and nitric oxide (NO) production. Pegylated arginine deiminase (ADI-PEG20) is a bacterial enzyme used to deplete circulating arginine. Objective: The goal of this research was to test the hypothesis that citrulline is able to sustain intracellular arginine availability for NO production in ADI-PEG20 arginine-depleted mice. Methods: Six- to 8-wk-old male C57BL/6J mice injected with ADI-PEG20 (5 IU) or saline (control) were used in 4 different studies. Arginine, citrulline, and NO kinetics were determined by using stable isotopes in unchallenged (study 1) and endotoxin-challenged (study 2) mice. Blood pressure was determined by telemetry for 6 d after ADI-PEG20 administration (study 3), and vasomotor activity and ASS1 and ASL gene expression were determined in mesenteric arteries collected from additional mice (study 4). Results: ADI-PEG20 administration resulted in arginine depletion (<1 compared with 111 ± 37 µmol/L) but in greater plasma citrulline concentrations (900 ± 123 compared with 76 ± 8 µmol/L; P < 0.001) and fluxes (402 ± 17 compared with 126 ± 4 µmol ⋅ kg-1 ⋅ h-1; P < 0.001) compared with controls. Endotoxin-challenged ADI-PEG20-treated mice produced less NO than controls (13 ± 1 compared with 27 ± 2 µmol ⋅ kg-1 ⋅ h-1; P < 0.001). No differences (P > 0.50) were observed for cardiovascular variables (heart rate, blood pressure) between ADI-PEG20-treated and control mice. Furthermore, no ex vivo vasomotor differences were observed between the 2 treatments. ADI-PEG20 administration resulted in greater gene expression of ASS1 (∼3-fold) but lower expression of ASL (-30%). Conclusion: ADI-PEG20 successfully depleted circulating arginine without any effect on cardiovascular endpoints in healthy mice but limited NO production after endotoxin challenge. Therefore, the citrulline recycling pathway can sustain local arginine availability independently from circulating arginine, satisfying the demand of arginine for endothelial NO production; however, it is unable to do so when a high demand for arginine is elicited by endotoxin.

A randomized trial to study the comparative efficacy of phenylbutyrate and benzoate on nitrogen excretion and ureagenesis in healthy volunteers.

PURPOSE: Benzoate and phenylbutyrate are widely used in the treatment of urea cycle disorders, but detailed studies on pharmacokinetics and comparative efficacy on nitrogen excretion are lacking. METHODS: We conducted a randomized, three-arm, crossover trial in healthy volunteers to study pharmacokinetics and comparative efficacy of phenylbutyrate (NaPB; 7.15 g•m-2BSA•day-1), benzoate (NaBz; 5.5 g•m-2BSA•day-1), and a combination of two medications (MIX arm; 3.575 g NaPB and 2.75 g NaBz•m-2BSA•day-1) on nitrogen excretion. Stable isotopes were used to study effects on urea production and dietary nitrogen disposal. RESULTS: The conjugation efficacy for both phenylbutyrate and benzoate was 65%; conjugation was superior at the lower dose used in the MIX arm. Whereas NaPB and MIX treatments were more effective at excreting nitrogen than NaBz, nitrogen excretion as a drug conjugate was similar between phenylbutyrate and MIX arms. Nitrogen excreted per USD was higher with combination therapy compared with NaPB. CONCLUSION: Phenylbutyrate was more effective than benzoate at disposing nitrogen. Increasing phenylbutyrate dose may not result in higher nitrogen excretion due to decreased conjugation efficiency at higher doses. Combinatorial therapy with phenylbutyrate and benzoate has the potential to significantly decrease treatment cost without compromising the nitrogen disposal efficacy.