Microcirculatory Function during Endotoxemia-A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation.

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 µg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3-/- compared to Nos2-/- mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2-/- mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3-/- or Nos2-/-/Nos3-/- mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2-/- mice), as this beneficial effect was absent in Nos3-/- or Nos2-/-/Nos3-/- mice.

Dietary citrulline does not modify rat colon tumor response to chemotherapy, but failed to improve nutritional status.

During cancer therapy many patients experience significant malnutrition, leading to decreased tolerance to chemotherapy and decreased survival. Dietary citrulline supplementation improves nutritional status in situations such as short bowel syndrome and aging, and is of potential interest in oncology. However, a mandatory prerequisite is to test this amino acid for interaction with tumor growth and chemotherapy response. Dietary citrulline (Cit; 2%), or an isonitrogenous mix of non-essential amino acids (control), was given to Ward colon tumor-bearing rats the day before chemotherapy initiation. Chemotherapy included 2 cycles, one week apart, each consisting of one injection of CPT-11 (50 mg/kg) and of 5-fluorouracil (50 mg/kg) the day after. Body weight, food intake and tumor volume were measured daily. The day after the last injection, rats were killed, muscles (EDL, gastrocnemius), intestinal mucosa, tumor, spleen and liver were weighed. Muscle and intestinal mucosa protein content were measured. Phosphorylated 4E-BP1 was measured in muscle and tumor as a surrogate for biosynthetic activation. FRAPS (Ferric Reducing Ability of Plasma) and thiols in plasma, muscle and tumor were evaluated and plasma amino acids and haptoglobin were measured. Numerous parameters did not differ by diet overall: a) response of tumor mass to treatment, b) tumor antioxidants and phosphorylated 4E-BP1 levels, c) relative body weight and relative food intake, d) weight of EDL, gastrocnemius, intestinal mucosa, spleen and liver and e) plasma haptoglobin concentrations. Moreover, plasma citrulline concentration was not correlated to relative body weight, only cumulated food intake and plasma haptoglobin concentrations were correlated to relative body weight. Citrulline does not alter the tumor response to CPT-11/5FU based therapy but, has no effect on nutritional status, which could be due to the anorexia and the low amount of citrulline and protein ingested.

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.

Contrast-enhanced ultrasound for determining muscular perfusion after oral intake of L-citrulline, L-arginine, and galloylated epicatechines: A study protocol.

INTRODUCTION: The market for dietary supplements in the sports sector has been growing rapidly for several years, though there is still lacking evidence regarding their claimed benefits. One group is that of nitric oxide increasing supplements, so-called "NO-boosters," which are claimed to improve the supply of oxygen and nutrients to the muscle by enhancing vasodilation.The aim of this study was to investigate 3 of these supplements in healthy male athletes for their muscle perfusion-enhancing potential using contrast-enhanced ultrasound (CEUS). METHODS: This placebo-controlled, double-blind, randomized cross-over trial will be carried out at the Center for Orthopedics, Trauma Surgery and Spinal Cord Injury of the University Hospital Heidelberg. Three commercial NO enhancing products including 300 mg of the specific green tea extract VASO6 and a combination of 8 g L-citrulline malate and 3 g L-arginine hydrochloride will be examined for their potential to increase muscular perfusion in 30-male athletes between 18 and 40 years and will be compared with a placebo. On each of the 3 appointments CEUS of the dominant biceps muscle will be performed at rest and after a standardized resistance training. Every athlete receives each of the 3 supplements once after a wash-out period of at least 1 week. Perfusion will be quantified via VueBox quantification software. The results of CEUS perfusion measurements will be compared intra- and interindividually and correlated with clinical parameters. DISCUSSION: The results of this study may help to establish CEUS as a suitable imaging modality for the evaluation of potentially vasodilatory drugs in the field of sports. Other supplements could also be evaluated in this way to verify the content of their advertising claims. TRIAL REGISTRATION: German Clinical Trials Register (DRKS), ID: DRKS00016972, registered on 25.03.2019.

Inflammatory markers response to citrulline supplementation in patients with non-alcoholic fatty liver disease: a randomized, double blind, placebo-controlled, clinical trial.

OBJECTIVES: The aim of this study was to investigate the effects of citrulline (Cit) supplementation on inflammatory markers and liver histopathology in patients with non-alcoholic fatty liver disease (NAFLD). In this clinical trial, fifty NAFLD patients were assigned to receive 2 g/day Cit or placebo for 3 months. RESULTS: At the end of study, serum high sensitive C-reactive protein (hs-CRP) and activity of nuclear factor kappa B (NF-κB) were reduced in Cit group significantly more than placebo group (P-value = 0.02 and < 0.01 respectively). Serum concentrations of tumor necrosis factor-α (TNF-α) was reduced in Cit group significantly more than placebo after adjusting for levels of baseline (P-value < 0.001). Moreover, Cit supplementation decreased serum alanine aminotransferase (ALT) and hepatic steatosis significantly (P = 0.04). Anthropometric measurements and hepatic enzymes did not change significantly in any group (P ≥ 0.05). In conclusion, our results showed that 12 weeks supplementation with 2 g/day Cit improved inflammatory markers in patients with NAFLD. Further studies with longer period of supplementation and different dosages of Cit are needed to be able to conclude. Trial registration IRCT201703194010N18 on 2017-10-13.

Effect of oral citrulline supplementation on whole body protein metabolism in adult patients with short bowel syndrome: A pilot, randomized, double-blind, cross-over study.

BACKGROUND & AIMS: As citrulline is produced by small intestine, plasma citrulline concentration is decreased and may become essential in patients with short bowel syndrome (SBS). In a rat model of SBS, citrulline supplementation enhanced muscle protein synthesis. The aim of the study was to determine whether citrulline impacts whole body protein metabolism in patients with SBS. METHODS: Nine adults with non-malignant SBS (residual small bowel 90 ± 48 cm; mean ± SD) who were in near-normal nutritional status without any artificial nutrition, were recruited long after surgery. They received 7-day oral supplementation with citrulline (0.18 g/kg/day), or an iso-nitrogenous placebo in a randomized, double-blind, cross-over design with a 13-day wash-out between regimens, and an intravenous 5-h infusion of L-[1-13C]-leucine in the postabsorptive state to assess protein metabolism after each regimen. RESULTS: Plasma citrulline concentration rose 17-fold (25 ± 9 vs. 384 ± 95 µmol/L) and plasma arginine 3-fold after oral citrulline supplementation (both p < 4 × 10-6). Supplementation did not alter leucine appearance rate (97 ± 5 vs. 97 ± 5 µmol kg-1.h-1; p = 0.88), leucine oxidation (14 ± 1 vs. 12 ± 1 µmol kg-1.h-1; p = 0.22), or non-oxidative leucine disposal (NOLD), an index of whole-body protein synthesis (83 ± 4 vs. 85 ± 5 µmol kg-1.h-1; p = 0.36), nor insulin or IGF-1 plasma concentrations. In each of the 3 patients with baseline citrulline<20 µmol/L, citrulline supplementation increased NOLD. Among the 7 patients with plasma citrulline <30 µmol/L, the effect of supplementation on NOLD correlated inversely (r2 = 0.81) with baseline plasma citrulline concentration. CONCLUSION: 1) Oral citrulline supplementation enhances citrulline and arginine bioavailability in SBS patients. 2) Oral citrulline supplementation does not have any anabolic effect on whole body protein metabolism in patients with SBS in good nutritional status, in the late phase of intestinal adaptation, and with near-normal baseline citrulline homeostasis. 3) Whether oral citrulline would impact whole body protein anabolism in severely malnourished SBS patients in the early adaptive period, and with baseline plasma citrulline below 20 µmol/L, warrants further study. Registered under ClinicalTrials.gov Identifier no. NCT01386034.

Kidney Mass Reduction Leads to l-Arginine Metabolism-Dependent Blood Pressure Increase in Mice.

BACKGROUND: Uninephrectomy (UNX) is performed for various reasons, including kidney cancer or donation. Kidneys being the main site of l-arginine production in the body, we tested whether UNX mediated kidney mass reduction impacts l-arginine metabolism and thereby nitric oxide production and blood pressure regulation in mice. METHODS AND RESULTS: In a first series of experiments, we observed a significant increase in arterial blood pressure 8 days post-UNX in female and not in male mice. Further experimental series were performed in female mice, and the blood pressure increase was confirmed by telemetry. l-citrulline, that is used in the kidney to produce l-arginine, was elevated post-UNX as was also asymmetric dimethylarginine, an inhibitor of nitric oxide synthase that competes with l-arginine and is a marker for renal failure. Interestingly, the UNX-induced blood pressure increase was prevented by supplementation of the diet with 5% of the l-arginine precursor, l-citrulline. Because l-arginine is metabolized in the kidney and other peripheral tissues by arginase-2, we tested whether the lack of this metabolic pathway also compensates for decreased l-arginine production in the kidney and/or for local nitric oxide synthase inhibition and consecutive blood pressure increase. Indeed, upon uninephrectomy, arginase-2 knockout mice (Arg-2-/-) neither displayed an increase in asymmetric dimethylarginine and l-citrulline plasma levels nor a significant increase in blood pressure. CONCLUSIONS: UNX leads to a small increase in blood pressure that is prevented by l-citrulline supplementation or arginase deficiency, 2 measures that appear to compensate for the impact of kidney mass reduction on l-arginine metabolism.

Nitric oxide production by peritoneal macrophages from aged rats: A short term and direct modulation by citrulline.

Citrulline has anti-inflammatory properties and exerts beneficial effects on various impaired functions in aging. However, there are few data on citrulline action on immune function in aged populations. The objective of the study was to evaluate citrulline ability, after in vivo and in vitro administration, to modulate macrophage functions in aged rats and the possible pathways involved. Twenty-one-month-old Sprague-Dawley rats (n = 27) received a citrulline supplementation at 5 g/kg/d for 5 days, or an isonitrogenous diet, and peritoneal macrophages were cultured with or without LPS. In the in vitro study, macrophages from 22-month-old rats (n = 16) were cultured with or without LPS, citrulline and inhibitors of different inflammatory pathways (n = 8/conditions). Nitric oxide (NO) and tumor necrosis factor α (TNFα) production were measured in both in vivo and in vitro studies. Citrulline decreased NO production variability by peritoneal macrophages after in vivo administration (p = 0.0034) and downregulated NO production by 22% after in vitro administration (95% CI: [6%; 35%]; p = 0.0394), without any direct effect on TNFα production. None of the transductional pathways explored seem to be involved. Citrulline slightly modulates NO production in vivo and in vitro, suggesting a possible action through modulation of arginine metabolism in macrophages rather than a direct transductional effect. The pleiotropic effects of citrulline in aging could be due, at least in part, to the anti-inflammatory effect of citrulline.

Does Citrulline Have Protective Effects on Liver Injury in Septic Rats?

Citrulline (Cit) supplementation was proposed to serve as a therapeutic intervention to restore arginine (Arg) concentrations and improve related functions in sepsis. This study explored whether citrulline had positive effects on liver injury and cytokine release in the early stages of sepsis. The cecal ligation and puncture (CLP) model was utilized in our study. Rats were divided into four groups: normal, Cit, CLP, and CLP+Cit. The CLP group and CLP+Cit group were separated into 6-, 12-, and 24-hour groups, according to the time points of sacrifice after surgery. Intragastric administration of L-citrulline was applied to rats in Cit and CLP+Cit groups before surgery. Serum AST and ALT levels and levels of MDA, SOD, NO, and iNOS in the liver tissues were evaluated. Plasma concentrations of Cit and Arg were assessed using HPLC-MS/MS. Serum concentrations of cytokines and chemokines were calculated by Luminex. Results showed SOD activities of CLP+Cit groups were significantly higher than that of CLP groups, contrasting with the MDA and NO levels which were significantly lower in CLP+Cit groups than in CLP groups. In addition, plasma concentrations of TNF-α, IL-6, and IL-1ß were significantly lower in the CLP+Cit 6-hour group than in the CLP 6-hour group.

Combined L-citrulline and glutathione supplementation increases the concentration of markers indicative of nitric oxide synthesis.

BACKGROUND: Nitric oxide (NO) is endogenously synthesized from L-arginine and L-citrulline. Due to its effects on nitric oxide synthase (NOS), reduced glutathione (GSH) may protect against the oxidative reduction of NO. The present study determined the effectiveness of L-citrulline and/or GSH on markers indicative of NO synthesis in in vivo conditions with rodents and humans and also in an in vitro condition. METHODS: In phase one, human umbilical vein endothelial cells (HUVECs) were treated with either 0.3 mM L-citrulline, 1 mM GSH (Setria®) or a combination of each at 0.3 mM. In phase two, Sprague-Dawley rats (8 weeks old) were randomly assigned to 3 groups and received either purified water, L-citrulline (500 mg/kg/day), or a combination of L-citrulline (500 mg/kg/day) and GSH (50 mg/kg/day) by oral gavage for 3 days. Blood samples were collected and plasma NOx (nitrite + nitrate) assessed. In phase three, resistance-trained males were randomly assigned to orally ingest either cellulose placebo (2.52 g/day), L-citrulline (2 g/day), GSH (1 g/day), or L-citrulline (2 g/day) + GSH (200 mg/day) for 7 days, and then perform a resistance exercise session involving 3 sets of 10-RM involving the elbow flexors. Venous blood was obtained and used to assess plasma cGMP, nitrite, and NOx. RESULTS: In phase one, nitrite levels in cells treated with L-citrulline and GSH were significantly greater than control (p < 0.05). In phase two, plasma NOx with L-citrulline + GSH was significantly greater than control and L-citrulline (p < 0.05). In phase three, plasma cGMP was increased, but not significantly (p > 0.05). However, nitrite and NOx for L-citrulline + GSH were significantly greater at 30 min post-exercise when compared to placebo (p < 0.05). CONCLUSIONS: Combining L-citrulline with GSH augments increases in nitrite and NOx levels during in vitro and in vivo conditions.

The effect of l-citrulline and watermelon juice supplementation on anaerobic and aerobic exercise performance.

Citrulline has been proposed as an ergogenic aid, leading to an interest in watermelon given its high citrulline concentration. The aim of this study was to determine the effects of a single, pre-exercise dose of l-citrulline, watermelon juice, or a placebo on the total maximum number of repetitions completed over 5 sets, time to exhaustion, maximal oxygen consumption (VO2max), anaerobic threshold, and flow-mediated vasodilation. A randomised double-blind within-participants study design was used to examine these effects among 22 participants (n = 11 males). Supplementation included either a 7.5% sucrose drink containing 6 g of l-citrulline, 710 mL of watermelon juice (~1.0 g citrulline), or a 7.5% sucrose placebo drink. Supplementation was administered 1 or 2 h before exercise testing to investigate a timing effect. There was no significant effect between the three supplements for the total number of repetitions, time to exhaustion, VO2max, anaerobic threshold, or flow-mediated vasodilation. There was also no interaction observed relative to gender or supplement timing (P > 0.05). A single dose of l-citrulline or watermelon juice as a pre-exercise supplement appears to be ineffective in improving exercise performance; however, greater doses of l-citrulline have been shown to be safe and are currently left unexamined.

Oral L-citrulline supplementation improves erectile function and penile structure in castrated rats.

OBJECTIVES: To investigate the efficacy of oral L-citrulline for erectile dysfunction and penile structure disruption in a rat model. METHODS: Male Wistar-ST rats aged 15 weeks were randomly divided into three groups as follows: sham-operated rats (control group), surgically castrated rats (castrated group) and surgically castrated rats subsequently treated with 2% L-citrulline water (castrated + L-citrulline). At 4 weeks postoperative, erectile function was assessed based on intracavernous pressure changes, followed by electrostimulation of cavernous nerves and calculation of maximum intracavernous pressure/mean arterial pressure. Penile structure was evaluated by Masson's trichrome staining and the smooth muscle-to-collagen ratio was calculated. The serum bioavailable testosterone, L-arginine, L-citrulline, N(G),N(G) -dimethylarginine and nitrogen oxide levels were evaluated. RESULTS: The bioavailable testosterone concentrations were decreased in the castrated and castrated + L-citrulline groups compared with the control group at 4 weeks after surgery. The intracavernous pressure-to-mean arterial pressure and smooth muscle-to-collagen ratios were significantly decreased in the castrated group compared with the control group, but significantly increased in the castrated + L-citrulline group compared with the castrated group. The serum L-citrulline, L-arginine and N(G),N(G)-dimethylarginine levels, and the L-arginine-to-N(G),N(G)-dimethylarginine ratios were significantly increased in the castrated +L-citrulline group compared with the castrated group. The serum nitrogen oxide levels were increased in the castrated + L-citrulline group compared with the castrated group. CONCLUSIONS: Oral L-citrulline can improve the erectile response to electric stimulation of cavernous nerve and penile structure in castrated rats.

Oral L-citrulline supplementation improves erectile function in rats with acute arteriogenic erectile dysfunction.

INTRODUCTION: Oral L-citrulline supplementation increases serum L-arginine levels more efficiently than L-arginine itself and increases nitric oxide (NO) production. AIM: To investigate whether oral L-citrulline supplementation improves erectile function in rats with acute arteriogenic erectile dysfunction (ED). METHODS: We divided 8-week-old male Wistar-ST rats into 3 groups: sham-operated rats (control group), arteriogenic ED rats who underwent ligation of both internal iliac arteries (ligation group), and arteriogenic ED rats receiving oral 2% L-citrulline water supplementation (citrulline group). Citrulline water was given to arteriogenic ED rats for 3 weeks from 1 week after surgery. Erectile function was evaluated by maximum intracavernous pressure/mean arterial pressure (ICP/MAP) ratios via cavernous nerve stimulation at 4 weeks after surgery. Then, the penises were resected, stained with Masson's trichrome, and observed microscopically. Serum nitrogen oxides (NOx) levels were measured by high-performance liquid chromatography. Bonferroni's multiple t-test was used for statistical analysis. MAIN OUTCOME MEASURES: The main outcome measures were changes in ICP/MAP, smooth muscle (SM)/collagen ratios, and NOx levels following L-citrulline supplementation. RESULTS: The ICP/MAP ratio in the ligation group was significantly lower than that in the control group (P<0.05), denoting ED. The ICP/MAP ratio of the citrulline group was significantly higher than that of the ligation group (P<0.05), indicating ED amelioration. Levels of NOx in the ligation group were significantly lower than in the control group (P<0.05), while those in the citrulline group were significantly higher than in the ligation group (P<0.05). SM/collagen ratios in the ligation group were significantly lower than in the control group (P<0.05), while ratios in the citrulline group were significantly higher than those in the ligation group (P<0.05). CONCLUSIONS: Oral L-citrulline supplementation improved ICP/MAP and SM/collagen ratios and increased NOx. Therefore, oral L-citrulline supplementation might be a useful novel therapy for acute arteriogenic ED.

Novel cathepsin B-sensitive paclitaxel conjugate: Higher water solubility, better efficacy and lower toxicity.

In order to realize the targeted delivery of paclitaxel (PTX) to tumor through an environment-sensitive mechanism, increase its solubility in water and reformulate without toxic excipients, a novel PTX conjugate, PEG-VC-PABC-PTX was designed and synthesized in this study, using p-aminobenzylcarbonyl (PABC), a spacer, and valine-citrulline (VC), a substrate of cathepsin B (C(B)), to link polyethylene glycol (PEG) and PTX. Pegylated PTX (PEG-PTX) which was synthesized and Taxol formulation were prepared as controls. The conjugates were purified and characterized by melting points, (1)H-NMR, ESI-MS or MALDI-TOF-MS. The two conjugates were similar in particle size, water solubility and their effects on MCF-7 cell line in vitro, and both of them induced no obvious toxicity in vivo. The release of PTX from PEG-PTX was faster due to its ester bond, while PEG-VC-PABC-PTX was proved to be C(B)-sensitive in terms of PTX release and its effect on cell cycle. Additionally, PEG-VC-PABC-PTX exhibited significant effects of antitumor, anti-angiogenesis and anti-proliferation in vivo, while the control conjugate was almost inefficacious at the same in vivo test. On the other hand, PTX conjugates demonstrated a thousand-time or more improvement in water solubility compared to PTX, suggesting a very easy way in the preparation and use of its injection. Without involvement of Cremophore EL and ethanol, the PTX conjugate will guarantee less adverse effects as frequently reported for Taxol formulation. Taxol formulation had a higher cytoxicity in vitro than PEG-VC-PABC-PTX likely because of toxic additives. Importantly, the C(B)-sensitive conjugate indicated a similar in vivo efficacy with the Taxol control, but much lower in vivo toxicity at the same doses evidenced by body weight, animal status, liver toxicity and blood count. Moreover, at the tolerant dose, this novel conjugate exhibited significantly better antitumor effect than that of Taxol formulation. In general, the PEG-VC-PABC-PTX conjugate designed in this study demonstrated significant advantages in terms of high water solubility, no toxic surfactant or organic solvent, tumor environment-sensitivity and high therapeutic index.

Decreased glutamate, glutamine and citrulline concentrations in plasma and muscle in endotoxemia cannot be reversed by glutamate or glutamine supplementation: a primary intestinal defect?

Endotoxemia affects intestinal physiology. A decrease of circulating citrulline concentration is considered as a reflection of the intestinal function. Citrulline can be produced in enterocytes notably from glutamate and glutamine. The aim of this work was to determine if glutamate, glutamine and citrulline concentrations in blood, intestine and muscle are decreased by endotoxemia, and if supplementation with glutamate or glutamine can restore normal concentrations. We induced endotoxemia in rats by an intraperitoneal injection of 0.3 mg kg(-1) lipopolysaccharide (LPS). This led to a rapid anorexia, negative nitrogen balance and a transient increase of the circulating level of IL-6 and TNF-α. When compared with the values measured in pair fed (PF) animals, almost all circulating amino acids (AA) including citrulline decreased, suggesting a decrease of intestinal function. However, at D2 after LPS injection, most circulating AA concentrations were closed to the values recorded in the PF group. At that time, among AA, only glutamate, glutamine and citrulline were decreased in gastrocnemius muscle without change in intestinal mucosa. A supplementation with 4% monosodium glutamate (MSG) or an isomolar amount of glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscle. However, MSG supplementation led to an accumulation of glutamate in the intestinal mucosa. In conclusion, endotoxemia rapidly but transiently decreased the circulating concentrations of almost all AA and more durably of glutamate, glutamine and citrulline in muscle. Supplementation with glutamate or glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscles. The implication of a loss of the intestinal capacity for AA absorption and/or metabolism in endotoxemia (as judged from decreased citrulline plasma concentration) for explaining such results are discussed.

Effects of leucine and citrulline versus non-essential amino acids on muscle protein synthesis in fasted rat: a common activation pathway?

Leucine (LEU) is recognized as a major regulator of muscle protein synthesis (MPS). Citrulline (CIT) is emerging as a potent new regulator. The aim of our study was to compare MPS modulation by CIT and LEU in food-deprived rats and to determine whether their action was driven by similar mechanisms. Rats were either freely fed (F, n = 10) or food deprived for 18 h. Food-deprived rats were randomly assigned to one of four groups and received per os, i.e., gavage, saline (S, n = 10), L: -leucine (1.35 g/kg, LEU, n = 10), L: -citrulline (1.80 g/kg CIT, n = 10) or isonitrogenous non-essential amino acids (NEAA, n = 10). After gavage, the rats were injected with a flooding dose of [(13)C] valine to determine MPS. The rats were killed 50 min after the injection of the flooding dose. Blood was collected for amino acid, glucose and insulin determinations. Tibialis anterior muscles were excised for determination of MPS and for Western blot analyses of the PI3K/Akt, mTORC1, ERK1/2/MAPK pathways and AMP kinase component. MPS was depressed by 61% in starved rats (Saline vs. Fed, P < 0.05). Administration of amino acids (NEAA, LEU or CIT) completely abolished this decrease (NEAA, CIT, LEU vs. Fed, NS). Food deprivation affected the phosphorylation status of the mTORC1 pathway and AMP kinase (Saline vs. Fed, P < 0.05). LEU and CIT administration differently stimulated the mTORC1 pathway (LEU > CIT). LEU but not CIT increased the phosphorylation of rpS6 at serine 235/236. Our findings clearly demonstrated that both CIT and LEU were able to stimulate MPS, but this effect was likely related to the nitrogen load. LEU, CIT and NEAA may have different actions on MPS in this model as they share different mTORC1 regulation capacities.

Inhaled nitric oxide and related therapies.

Children with congenital heart defects are at risk for perioperative pulmonary hypertension if they require corrective or palliative surgery in the first week of life or if they have defects associated with significant pulmonary overcirculation. In addition, children undergoing cavopulmonary connections for single ventricle lesions require low pulmonary vascular resistance for surgical success. Treatment of perioperative pulmonary hypertension with inhaled nitric oxide has become standard therapy in many centers. Related drugs that increase nitric oxide synthesis, including arginine and citrulline, have also been studied in the perioperative period. In this article, previous clinical trials of inhaled nitric oxide, intravenous arginine, and intravenous and oral citrulline in children with perioperative pulmonary hypertension or elevated pulmonary vascular resistance after a cavopulmonary connection are reviewed. In addition, recommendations are presented for each agent on the clinical use in the perioperative setting including clinical indications, assessment of clinical effect, and length of therapy.

Pharmacokinetics and safety of intravenously administered citrulline in children undergoing congenital heart surgery: potential therapy for postoperative pulmonary hypertension.

OBJECTIVE: Pulmonary hypertension may complicate surgical correction of congenital heart defects, resulting in increased morbidity and mortality. We have previously shown that plasma levels of the nitric oxide precursors citrulline and arginine drop precipitously after congenital cardiac surgery and that oral citrulline supplementation may be protective against the development of pulmonary hypertension. In this study, we assessed the safety and pharmacokinetic profile of intravenous citrulline as a potential therapy for postoperative pulmonary hypertension. METHODS: The initial phase of this investigation was a dose-escalation study of intravenously administered citrulline in infants and children undergoing one of five congenital cardiac surgical procedures (phase 1). The primary safety outcome was a 20% drop in mean arterial blood pressure from the baseline pressure recorded after admission to the intensive care unit. Based on our previous work, the target circulating plasma citrulline trough was 80 to 100 micromol/L. Each patient was given two separate doses of citrulline: the first in the operating room immediately after initiation of cardiopulmonary bypass and the second 4 hours later in the pediatric intensive care unit. Stepwise dose escalations included 50 mg/kg, 100 mg/kg, and 150 mg/kg. After model-dependent pharmacokinetic analysis, we enrolled an additional 9 patients (phase 2) in an optimized dosing protocol that replaced the postoperative dose with a continuous infusion of citrulline at 9 mg/(kg.h) for 48 hours postoperatively. RESULTS: The initial stepwise escalation protocol (phase 1) revealed that an intravenous citrulline dose of 150 mg/kg given after initiation of cardiopulmonary bypass yielded a trough level of in the target range of approximately 80 to 100 micromol/L 4 hours later. The postoperative dose revealed that the clearance of intravenously administered citrulline was 0.6 L/(h.kg), with a volume of distribution of 0.9 L/kg and estimated half-life of 60 minutes. Because of the short half-life, we altered the protocol to replace the postoperative dose with a continuous infusion of 9 mg/(kg.h). An additional 9 patients were studied with this continuous infusion protocol (phase 2). Mean plasma citrulline levels were maintained at approximately 125 mumol/L, with a calculated clearance of 0.52 L/(h.kg). None of the 17 patients studied had a 20% drop in mean arterial blood pressure from baseline. CONCLUSIONS: In this first report of the use of intravenous citrulline in humans, we found citrulline to be both safe and well tolerated in infants and young children undergoing congenital cardiac surgery. Because of the rapid clearance, the optimal dosing regimen was identified as an initial bolus of 150 mg/kg given at the initiation of cardiopulmonary bypass, followed 4 hours later by a postoperative infusion of 9 mg/(kg.h) continued up to 48 hours. Using this regimen, plasma arginine, citrulline, and nitric oxide metabolite levels were well maintained. Intravenous citrulline needs to be studied further as a potential therapy for postoperative pulmonary hypertension.

Intestinal and hepatic metabolism of glutamine and citrulline in humans.

Glutamine plays an important role in nitrogen homeostasis and intestinal substrate supply. It has been suggested that glutamine is a precursor for arginine through an intestinal-renal pathway involving inter-organ transport of citrulline. The importance of intestinal glutamine metabolism for endogenous arginine synthesis in humans, however, has remained unaddressed. The aim of this study was to investigate the intestinal conversion of glutamine to citrulline and the effect of the liver on splanchnic citrulline metabolism in humans. Eight patients undergoing upper gastrointestinal surgery received a primed continuous intravenous infusion of [2-(15)N]glutamine and [ureido-(13)C-(2)H(2)]citrulline. Arterial, portal venous and hepatic venous blood were sampled and portal and hepatic blood flows were measured. Organ specific amino acid uptake (disposal), production and net balance, as well as whole body rates of plasma appearance were calculated according to established methods. The intestines consumed glutamine at a rate that was dependent on glutamine supply. Approximately 13% of glutamine taken up by the intestines was converted to citrulline. Quantitatively glutamine was the only important precursor for intestinal citrulline release. Both glutamine and citrulline were consumed and produced by the liver, but net hepatic flux of both amino acids was not significantly different from zero. Plasma glutamine was the precursor of 80% of plasma citrulline and plasma citrulline in turn was the precursor of 10% of plasma arginine. In conclusion, glutamine is an important precursor for the synthesis of arginine after intestinal conversion to citrulline in humans.