Effect of food sources of nitrate, polyphenols, L-arginine and L-citrulline on endurance exercise performance: a systematic review and meta-analysis of randomised controlled trials.

BACKGROUND: Increasing nitric oxide bioavailability may induce physiological effects that enhance endurance exercise performance. This review sought to evaluate the performance effects of consuming foods containing compounds that may promote nitric oxide bioavailability. METHODS: Scopus, Web of Science, Ovid Medline, EMBASE and SportDiscus were searched, with included studies assessing endurance performance following consumption of foods containing nitrate, L-arginine, L-citrulline or polyphenols. Random effects meta-analysis was conducted, with subgroup analyses performed based on food sources, sex, fitness, performance test type and supplementation protocol (e.g. duration). RESULTS: One hundred and eighteen studies were included in the meta-analysis, which encompassed 59 polyphenol studies, 56 nitrate studies and three L-citrulline studies. No effect on exercise performance following consumption of foods rich in L-citrulline was identified (SMD=-0.03, p=0.24). Trivial but significant benefits were demonstrated for consumption of nitrate and polyphenol-rich foods (SMD=0.15 and 0.17, respectively, p<0.001), including performance in time-trial, time-to-exhaustion and intermittent-type tests, and following both acute and multiple-day supplementation, but no effect of nitrate or polyphenol consumption was found in females. Among nitrate-rich foods, beneficial effects were seen for beetroot, but not red spinach or Swiss chard and rhubarb. For polyphenol-rich foods, benefits were found for grape, (nitrate-depleted) beetroot, French maritime pine, Montmorency cherry and pomegranate, while no significant effects were evident for New Zealand blackcurrant, cocoa, ginseng, green tea or raisins. Considerable heterogeneity between polyphenol studies may reflect food-specific effects or differences in study designs and subject characteristics. Well-trained males (V̇O2max ≥65 ml.kg.min-1) exhibited small, significant benefits following polyphenol, but not nitrate consumption. CONCLUSION: Foods rich in polyphenols and nitrate provide trivial benefits for endurance exercise performance, although these effects may be food dependent. Highly trained endurance athletes do not appear to benefit from consuming nitrate-rich foods but may benefit from polyphenol consumption. Further research into food sources, dosage and supplementation duration to optimise the ergogenic response to polyphenol consumption is warranted. Further studies should evaluate whether differential sex-based responses to nitrate and polyphenol consumption are attributable to physiological differences or sample size limitations. OTHER: The review protocol was registered on the Open Science Framework ( https://osf.io/u7nsj ) and no funding was provided.

Analysis of repeated measures data in nutrition research.

Amino acid nutrition studies often involve repeated measures data. An example is that the concentrations of plasma citrulline in steers are repeatedly measured from the same animals. The standard repeated measures ANOVA method does not detect significant time changes in the concentrations of plasma citrulline within 6 hours after steers consumed rumen-protected citrulline, while a graphical analysis indicates that there exists a time effect. Here we describe three mixed model analyses that capture the time effect in a statistically significant way, while accounting for the correlations of measurements over time from the same steers. First, we allow flexible variance-covariance structures on our model. Second, we use baseline measurements as a covariate in our model. Third, we use percent-change from baseline as a data normalization method. In our data analysis, all these three approaches can lead to meaningful statistical results that oral administration of rumen-protected citrulline enhances the concentrations of plasma citrulline over time in ruminants. This supports the notion that rumen-protected citrulline can bypass the rumen to effectively enter the blood circulation.

Platform model describing pharmacokinetic properties of vc-MMAE antibody-drug conjugates.

Antibody-drug conjugates (ADCs) developed using the valine-citrulline-MMAE (vc-MMAE) platform, consist of a monoclonal antibody (mAb) covalently bound with a potent anti-mitotic toxin (MMAE) through a protease-labile vc linker. Recently, clinical data for a variety of vc-MMAE ADCs has become available. The goal of this analysis was to develop a platform model that simultaneously described antibody-conjugated MMAE (acMMAE) pharmacokinetic (PK) data from eight vc-MMAE ADCs, against different targets and tumor indications; and to assess differences and similarities of model parameters and model predictions, between different compounds. Clinical PK data of eight vc-MMAE ADCs from eight Phase I studies were pooled. A population PK platform model for the eight ADCs was developed, where the inter-compound variability (ICV) was described explicitly, using the third random effect level (ICV), and implemented using LEVEL option of NONMEM 7.3. The PK was described by a two-compartment model with time dependent clearance. Clearance and volume of distribution increased with body weight; volume was higher for males, and clearance mildly decreased with the nominal dose. Michaelis-Menten elimination had only minor effect on PK and was not included in the model. Time-dependence of clearance had no effect beyond the first dosing cycle. Clearance and central volume were similar among ADCs, with ICV of 15 and 5%, respectively. Thus, PK of acMMAE was largely comparable across different vc-MMAE ADCs. The model may be applied to predict PK-profiles of vc-MMAE ADCs under development, estimate individual exposure for the subsequent PK-pharmacodynamics (PD) analysis, and project optimal dose regimens and PK sampling times.

Ablation of Arginase II Spares Arginine and Abolishes the Arginine Requirement for Growth in Male Mice.

Background: Arginine is considered a semiessential amino acid in many species, including humans, because under certain conditions its demand exceeds endogenous production. Arginine availability, however, is determined not only by its production but also by its disposal. Manipulation of disposal pathways has the potential to increase availability and thus abolish the requirement for arginine.Objective: The objective of the study was to test the hypothesis that arginase II ablation increases arginine availability for growth.Methods: In a completely randomized design with a factorial arrangement of treatments, postweaning growth was determined for 3 wk in male and female wild-type (WT) mice and arginase II knockout mice (ARGII) on a C57BL/6J background fed arginine-sufficient [Arg(+); 8 g arginine/kg] or arginine-free [Arg(-)] diets. Tracers were used to determine citrulline and arginine kinetics.Results: A sex dimorphism in arginine metabolism was detected; female mice had a greater citrulline flux (∼30%, P < 0.001), which translated to greater de novo synthesis of arginine (∼31%, P < 0.001). Female mice also had greater arginine fluxes (P < 0.015) and plasma arginine concentrations (P < 0.01), but a reduced arginine clearance rate (P < 0.001). Ablation of arginase II increased plasma arginine concentrations in both sexes (∼27%, P < 0.01) but increased arginine flux only in males (P < 0.01). The absence of arginine in the diet limited the growth of male WT mice (P < 0.01), but had no effect on male ARGII mice (P = 0.12). In contrast, WT females on the Arg(-) diet grew at the same rate and achieved final weight similar to that of female WT mice fed the Arg(+) diet (P = 0.47).Conclusion: The ablation of arginase II in male mice spares arginine that can then be used for growth and to meet other metabolic functions, thus abolishing arginine requirements.

Supplemental Citrulline Is More Efficient Than Arginine in Increasing Systemic Arginine Availability in Mice.

Background: Arginine is considered to be an essential amino acid in various (patho)physiologic conditions of high demand. However, dietary arginine supplementation suffers from various drawbacks, including extensive first-pass extraction. Citrulline supplementation may be a better alternative than arginine, because its only fate in vivo is conversion into arginine.Objective: The goal of the present research was to determine the relative efficiency of arginine and citrulline supplementation to improve arginine availability.Methods: Six-week-old C57BL/6J male mice fitted with gastric catheters were adapted to 1 of 7 experimental diets for 2 wk. The basal diet contained 2.5 g l-arginine/kg, whereas the supplemented diets contained an additional 2.5, 7.5, and 12.5 g/kg diet of either l-arginine or l-citrulline. On the final day, after a 3-h food deprivation, mice were continuously infused intragastrically with an elemental diet similar to the dietary treatment, along with l-[13C6]arginine, to determine the splanchnic first-pass metabolism (FPM) of arginine. In addition, tracers were continuously infused intravenously to determine the fluxes and interconversions between citrulline and arginine. Linear regression slopes were compared to determine the relative efficiency of each supplement.Results: Whereas all the supplemented citrulline (105% ± 7% SEM) appeared in plasma and resulted in a marginal increase of 86% in arginine flux, supplemental arginine underwent an ∼70% FPM, indicating that only 30% of the supplemental arginine entered the peripheral circulation. However, supplemental arginine did not increase arginine flux. Both supplements linearly increased (P < 0.01) plasma arginine concentration from 109 µmol/L for the basal diet to 159 and 214 µmol/L for the highest arginine and citrulline supplementation levels, respectively. However, supplemental citrulline increased arginine concentrations to a greater extent (35%, P < 0.01).Conclusions: Citrulline supplementation is more efficient at increasing arginine availability than is arginine supplementation itself in mice.

La cinética de la citrulina y la arginina y su valor como factor pronóstico en pacientes pediátricos críticamente enfermos / The value of the kinetics of citrulline and arginine as a prognostic factor in critically ill paediatric patients

No disponible

Impaired nitric oxide production in children with MELAS syndrome and the effect of arginine and citrulline supplementation.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is one of the most frequent maternally inherited mitochondrial disorders. The pathogenesis of this syndrome is not fully understood and believed to result from several interacting mechanisms including impaired mitochondrial energy production, microvasculature angiopathy, and nitric oxide (NO) deficiency. NO deficiency in MELAS syndrome is likely to be multifactorial in origin with the decreased availability of the NO precursors, arginine and citrulline, playing a major role. In this study we used stable isotope infusion techniques to assess NO production in children with MELAS syndrome and healthy pediatric controls. We also assessed the effect of oral arginine and citrulline supplementations on NO production in children with MELAS syndrome. When compared to control subjects, children with MELAS syndrome were found to have lower NO production, arginine flux, plasma arginine, and citrulline flux. In children with MELAS syndrome, arginine supplementation resulted in increased NO production, arginine flux, and arginine concentration. Citrulline supplementation resulted in a greater increase of these parameters. Additionally, citrulline supplementation was associated with a robust increase in citrulline concentration and flux and de novo arginine synthesis rate. The greater effect of citrulline in increasing NO production is due to its greater ability to increase arginine availability particularly in the intracellular compartment in which NO synthesis takes place. This study, which is the first one to assess NO metabolism in children with mitochondrial diseases, adds more evidence to the notion that NO deficiency occurs in MELAS syndrome, suggests a better effect for citrulline because of its greater role as NO precursor, and indicates that impaired NO production occurs in children as well as adults with MELAS syndrome. Thus, the initiation of treatment with NO precursors may be beneficial earlier in life. Controlled clinical trials to assess the therapeutic effects of arginine and citrulline on clinical complications of MELAS syndrome are needed.

Circadian and postprandial variation in plasma citrulline concentration in healthy dogs.

OBJECTIVE: To evaluate circadian and postprandial variations in plasma citrulline concentration in healthy dogs. ANIMALS: 8 healthy Beagles. PROCEDURES: Blood samples were collected from dogs after 12 hours of food withholding (0 hours; 8:00 am) and then every 2 hours for 12 hours (until 8:00 pm) and again at 24 hours (8:00 am the next day). The same protocol was repeated, with the only difference being that a meal was given immediately after the 0-hour sample collection point. Plasma citrulline concentration was measured by ion exchange chromatography. RESULTS: No significant difference in plasma citrulline concentration was identified among measurement points when food was withheld. Mean ± SD plasma citrulline concentration at 4 hours (72.2 ± 12.7 µmol/L) and 24 hours (56.1 ± 12.5 µmol/L) after dogs were fed was significantly different from that at 0 hours (64.4 ± 12.7 µmol/L). CONCLUSIONS AND CLINICAL RELEVANCE: Plasma citrulline concentration had no circadian variation in unfed dogs but increased significantly in fed dogs 4 hours after a meal. Therefore, food should be withheld from dogs for 8 to 12 hours before blood sample collection for measurement of citrulline concentration.

Arginine behaviour after arginine or citrulline administration in older subjects.

Arginine (ARG) and its precursor citrulline (CIT) are popular dietary supplements, especially for the elderly. However, age-related reductions in lean body mass and alterations in organ functions could change their bioavailability. Pharmacokinetics and tolerance to amino acid (AA) loads are poorly documented in elderly subjects. The objective here was to characterise the plasma kinetics of CIT and ARG in a single-dosing study design. Eight fasting elderly men underwent two separate isomolar oral loading tests (10 g of CIT or 9·94 g of ARG). Blood was withdrawn over an 8-h period to measure plasma AA concentrations. Only CIT, ornithine and ARG plasma concentrations were changed. Volume of distribution was not dependent on AA administered. Conversely, parameters related to ARG kinetics were strongly dependent on AA administered: after ARG load, elimination was higher (ARG>CIT; P=0·041) and admission period+time at peak concentration was lower (ARG

La cinética de la citrulina y la arginina y su valor como factor pronóstico en pacientes pediátricos críticamente enfermos / Citrulline and arginine kinetics and its value as a prognostic factor in pediatric critically ill patients

INTRODUCCIÓN: Se han documentado bajas concentraciones de citrulina y arginina plasmáticas en niños en diversas condiciones patológicas. Hipótesis: La cinética de citrulina y arginina plasmáticas durante la enfermedad crítica pediátrica se correlaciona con parámetros evolutivos clínicos y bioquímicos. PACIENTES Y MÉTODOS: Estudio observacional unicéntrico prospectivo en pacientes de 7 días a 14 años ingresados en Unidad de Cuidados Intensivos Pediátricos (PICU). Los datos bioquímicos y clínicos fueron recogidos al ingreso, a las 12 h, a las 24 h, al 3.er y al 7.° día. RESULTADOS: Un total de 44 pacientes críticamente enfermos fueron incluidos y un grupo control de 51 niños sanos. La citrulina desciende de forma significativa (p < 0,05) a las 12 h de ingreso con niveles bajos mantenidos hasta el día 7, comenzando un aumento progresivo después. La arginina ya está descendida a las 6h, aunque tiene una subida más precoz (día 3). La disminución de citrulina al tercer día se correlaciona directamente con la arginina. Hay correlación entre la elevación de la citrulina al 7.° día con menor duración de ventilación mecánica, menor estancia en PICU y menos complicaciones. Los niveles de citrulina bajos al 7° día aún descendidos el día 7 se asocian con un mayor aumento de PCR y procalcitonina en primeras 24 h. La disminución de arginina en las primeras 12 h se correlaciona inversamente con estancia más larga, mayor número de complicaciones y aumento de reactantes de fase aguda en día 3. CONCLUSIONES: Hay disminución de arginina y citrulina en los primeros días de la enfermedad crítica, con recuperación al 3.er y 7.° día, respectivamente, y existe una relación entre mayor disminución y peor evolución

INTRODUCTION: Low concentrations of plasma citrulline and arginine have been reported in children under various pathological conditions. Hypothesis: Plasma citrulline and arginine levels undergo different kinetics during the early days of critical illness in children according to the severity of symptoms and can be correlated with other clinical and laboratory parameters associated with the SIR. PATIENTS AND METHODS: A single-center prospective observational study in patients 7 days to 14 years admitted to pediatric intensive care unit (PICU). Citrulline and arginine blood levels (blood in dry paper, analysis by mass spectrometry in tandem), acute phase reactants and clinical data were collected on admission, at 12 h, 24 h, 3 and 7 days. RESULTS: A total of 44 critically ill patients were included and control group was formed by 42 healthy children. The citrulline and arginine kinetic analysis showed: 1) Citrulline falls significantly (P<.05) at 12 h of admission; levels remain low until day 7 and begin progressive increase again. 2) Arginine is already lowered at 6h, although an earlier rise occurs (3rd day). 3. The decrease of citrulline in the first 3 days of admission positively correlates with arginine kinetics. Bivariate analysis showed: 1) Correlation of elevated citrulline on the 7th day with shorter duration of mechanical ventilation, lower PICU stay and lower occurrence of complications. The levels of citrulline still descended at day 7 are associated with increased CRP/procalcitonin elevation at first 24 h. 2) The greatest decrease of arginine in the first 12 h is associated with a longer PICU stay and greater number of complications and increase of acute phase reactants at 3 days. CONCLUSIONS: There are decreased levels of arginine and citrulline in the first days at PICU, with recovery at the 3rd and 7th day respectively, and a relationship between a greater decrease and a worse outcome and between a longer income and a higher serum CRP/procalcitonin

Circulating blood endothelial nitric oxide synthase contributes to the regulation of systemic blood pressure and nitrite homeostasis.

OBJECTIVE: Mice genetically deficient in endothelial nitric oxide synthase (eNOS(-/-)) are hypertensive with lower circulating nitrite levels, indicating the importance of constitutively produced nitric oxide (NO•) to blood pressure regulation and vascular homeostasis. Although the current paradigm holds that this bioactivity derives specifically from the expression of eNOS in endothelium, circulating blood cells also express eNOS protein. A functional red cell eNOS that modulates vascular NO• signaling has been proposed. APPROACH AND RESULTS: To test the hypothesis that blood cells contribute to mammalian blood pressure regulation via eNOS-dependent NO• generation, we cross-transplanted wild-type and eNOS(-/-) mice, producing chimeras competent or deficient for eNOS expression in circulating blood cells. Surprisingly, we observed a significant contribution of both endothelial and circulating blood cell eNOS to blood pressure and systemic nitrite levels, the latter being a major component of the circulating NO• reservoir. These effects were abolished by the NOS inhibitor L-NG-nitroarginine methyl ester and repristinated by the NOS substrate L-arginine and were independent of platelet or leukocyte depletion. Mouse erythrocytes were also found to carry an eNOS protein and convert (14)C-arginine into (14)C-citrulline in NOS-dependent fashion. CONCLUSIONS: These are the first studies to definitively establish a role for a blood-borne eNOS, using cross-transplant chimera models, that contributes to the regulation of blood pressure and nitrite homeostasis. This work provides evidence suggesting that erythrocyte eNOS may mediate this effect.

Citrulline: from metabolism to therapeutic use.

Citrulline possesses a highly specific metabolism that bypasses splanchnic extraction because it is not used by the intestine or taken up by the liver. The administration of citrulline may be used to deliver available nitrogen for protein homeostasis in peripheral tissues and as an arginine precursor synthesized de novo in the kidneys and endothelial and immune cells. Fresh research has shown that citrulline is efficiently transported across the intestinal luminal membrane by a set of transporters belonging to the B°,⁺, L, and b°,⁺ systems. Several pharmacokinetic studies have confirmed that citrulline is efficiently absorbed when administered orally. Oral citrulline could be used to deliver arginine to the systemic circulation or as a protein anabolic agent in specific clinical situations, because recent data have suggested that citrulline, although not a component of proteins, stimulates protein synthesis in skeletal muscle through the mammalian target of rapamycin signaling pathway. Hence, citrulline could play a pivotal role in maintaining protein homeostasis and is a promising pharmaconutrient in nutritional support strategies for malnourished patients, especially in aging and sarcopenia.

Citrulline a more suitable substrate than arginine to restore NO production and the microcirculation during endotoxemia.

BACKGROUND: Impaired microcirculation during endotoxemia correlates with a disturbed arginine-nitric oxide (NO) metabolism and is associated with deteriorating organ function. Improving the organ perfusion in endotoxemia, as often seen in patients with severe infection or systemic inflammatory response syndrome (SIRS) is, therefore, an important therapeutic target. We hypothesized that supplementation of the arginine precursor citrulline rather than arginine would specifically increase eNOS-induced intracellular NO production and thereby improve the microcirculation during endotoxemia. METHODOLOGY/PRINCIPAL FINDINGS: To study the effects of L-Citrulline and L-Arginine supplementation on jejunal microcirculation, intracellular arginine availability and NO production in a non-lethal prolonged endotoxemia model in mice. C57/Bl6 mice received an 18 hrs intravenous infusion of endotoxin (LPS, 0.4 µg • g bodyweight(-1) • h(-1)), combined with either L-Citrulline (6.25 mg • h-1), L-Arginine (6.25 mg • h(-1)), or L-Alanine (isonitrogenous control; 12.5 mg • h(-1)) during the last 6 hrs. The control group received an 18 hrs sterile saline infusion combined with L-Alanine or L-Citrulline during the last 6 hrs. The microcirculation was evaluated at the end of the infusion period using sidestream dark-field imaging of jejunal villi. Plasma and jejunal tissue amino-acid concentrations were measured by HPLC, NO tissue concentrations by electron-spin resonance spectroscopy and NOS protein concentrations using Western blot. CONCLUSION/SIGNIFICANCE: L-Citrulline supplementation during endotoxemia positively influenced the intestinal microvascular perfusion compared to L-Arginine-supplemented and control endotoxemic mice. L-Citrulline supplementation increased plasma and tissue concentrations of arginine and citrulline, and restored intracellular NO production in the intestine. L-Arginine supplementation did not increase the intracellular arginine availability. Jejunal tissues in the L-Citrulline-supplemented group showed, compared to the endotoxemic and L-Arginine-supplemented endotoxemic group, an increase in degree of phosphorylation of eNOS (Ser 1177) and a decrease in iNOS protein level. In conclusion, L-Citrulline supplementation during endotoxemia and not L-Arginine reduced intestinal microcirculatory dysfunction and increased intracellular NO production, likely via increased intracellular citrulline and arginine availability.

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.

Endotoxemia affects citrulline, arginine and glutamine bioavailability.

BACKGROUND: Sepsis considerably alters the intestinal barrier functions, which in turn modify the absorption and bioavailability of nutrients. However, the effects of septic shock on aminoacid (AAs) bioavailability are poorly documented. The aim of this study was to compare the bioavailability of citrulline, arginine and glutamine during endotoxemia. MATERIALS AND METHODS: Thirty-six rats were randomised into two groups: control and lipopolysaccharides (LPS). The LPS group received an intraperitoneal injection of endotoxins (7·5 mg/kg). After 12 h, each group was again randomised into three subgroups, each of which received an oral bolus of citrulline, arginine or glutamine (5·7 mmol/kg). Blood samples were collected at various times from 0 to 600 min after AA administration. The concentrations of citrulline, arginine, glutamine and their metabolites arginine and ornithine were measured to determine pharmacokinetic parameters Area Under Curve (AUC), C(max) and T(max). RESULTS: The AUC values of citrulline decreased in LPS rats [citrulline, control: 761 ± 67 and LPS: 508 ± 72 µmol min/mL (P = 0·02)]. Maximum concentrations of citrulline were also significantly decreased by endotoxemia (P = 0·01). The pharmacokinetic parameters of arginine and glutamine were not significantly modified by endotoxemia. The AUC value of arginine from citrulline conversion was diminished in endotoxemic rats. The other pharmacokinetic parameters of arginine were not significantly modified after arginine or citrulline supply in either group (control or LPS). CONCLUSION: Endotoxemia affects the bioavailability of AAs differently according to the amino acid considered. This feature may be important for nutritional strategy in ICU patients.

Simultaneous bioanalysis of L-arginine, L-citrulline, and dimethylarginines by LC-MS/MS.

PURPOSE: L-Arginine (ARG) is converted to nitric oxide (NO) and L-citrulline (CIT) by endothelial nitric oxide synthase which is competitively inhibited by asymmetric dimethylarginine (ADMA). We have developed a liquid chromatography-mass spectrometric method for the simultaneous determination of endogenous ARG, labeled ARG (¹5N4-ARG), CIT, ADMA, and its inactive isomer, symmetric dimethylarginine (SDMA) in biological samples. METHODS: Concentrations of unlabeled ARG, ¹5N4-ARG, CIT, ADMA, and SDMA in EA.hy926 human endothelial cell lysate, cell incubation media, rat plasma or rat urine were measured by hydrophilic-interaction liquid chromatography electrospray tandem mass spectrometry. ¹³C6-ARG, D4-CIT and D7-ADMA were used as internal standards for ARG and ¹5N4-ARG, CIT, and dimethylarginines, respectively. RESULTS: The calibration curves of ARG, ¹5N4-ARG, CIT, ADMA, and SDMA were linear and independent of several sample matrices. Intra- and inter-day variabilities for the quantification of all the compounds were below 15% in quality control samples. Application of this method to determine the uptake as well as efflux of these compounds was illustrated through in vitro cell study by exposing human endothelial cells to ¹5N4-ARG, which allowed the observation of generation of ¹5N3-CIT and ¹5N3-ARG in the cell lyate. Use of these isotopes adds insights into the cellular handling of endogenous vs. exogenous ARG. Application of this method for rat plasma and rat urine assays was demonstrated after ARG oral supplementation in rats. CONCLUSION: An LC-MS/MS method was developed to quantify 6 ARG-related compounds simultaneously, utilizing 3 separate internal standards. This assay allows concurrent monitoring of uptake, efflux and metabolic processes when isotope-labeled ARG and CIT are measured, and can be applied for determination of these compounds in rat plasma and rat urine.

Plasma citrulline kinetics and prognostic value in critically ill patients.

PURPOSE: Multiple organ failure is a leading cause of death in critically ill patients and could be secondary to early gut ischemia. Plasma citrulline is a biomarker of enterocyte mass, and critically ill patients may have enterocyte mass reduction. The objectives of this study were to assess plasma citrulline kinetics and its prognostic value in critically ill patients. METHODS: This prospective observational study included adults without small bowel disease and without chronic renal failure consecutively admitted to a single intensive care unit. Prognostic variables as well as plasma citrulline concentrations were studied at admission, 12, 24, 48 h, and the 7th day after admission. Univariate and multivariate analyses including plasma citrulline (0-10, 11-20, and >20 micromol l(-1)) and other variables were performed. RESULTS: Sixty-seven patients were included, and the 28-day mortality was 34%. During the 1st day mean plasma citrulline decreased from 18.8 to 13.5 micromol l(-1). Low plasma citrulline at 24 h was associated with low plasma glutamine and arginine (p = 0.01 and 0.04), and high plasma CRP concentration, nosocomial infection rate, and 28-day mortality (p = 0.008, 0.03, and 0.02, respectively). In multivariate analysis plasma citrulline < or = 10 micromol l(-1) at 24 h and SOFA score > or =8 at 24 h were associated with 28-day mortality(odds ratios 8.70 and 15.08). CONCLUSIONS: In critically ill patients, low plasma citrulline at 24 h is an independent factor of mortality and could be a marker of acute intestinal failure.

Intravenous administration of L-citrulline to pregnant ewes is more effective than L-arginine for increasing arginine availability in the fetus.

L-arginine administration may be useful for the treatment of intrauterine growth restriction, but concerns remain about effective precursors for administration into pregnant dams. Therefore, we used an ovine model to test the hypothesis that infusion of L-citrulline into the maternal circulation increases L-arginine availability to the fetus. On d 135 +/- 1 of gestation, ewes received an i.v. bolus dose of L-citrulline (155 micromol/kg body weight) or the same dose of L-arginine-HCl. Maternal and fetal arterial blood samples were obtained simultaneously at -120, -60, 0, 5, 15, 30, 60, 120, 180, and 240 min relative to the time of amino acid administration. Concentrations of arginine in maternal plasma increased to peak values within 5 min after its injection in ewes and declined rapidly thereafter, whereas concentrations of arginine in fetal plasma increased between 15 and 30 min and returned to baseline values by 60 min. In contrast, administration of citrulline increased concentrations of citrulline and arginine in maternal and fetal plasma between 5 and 60 min and values remained elevated thereafter. The differential pharmacokinetics for arginine compared with citrulline infusion was consistent with the observation that the half-life of citrulline was twice that of arginine in ewes. We conclude that i.v. administration of citrulline is more effective than arginine in sustaining high concentrations of arginine in the maternal and fetal circulations of pregnant ewes. These novel findings provide support for studies of the clinical use of arginine and citrulline as therapeutic means to prevent or ameliorate fetal growth retardation in mammals.

Mechanisms and kinetics of citrulline uptake in a model of human intestinal epithelial cells.

BACKGROUND & AIMS: Citrulline is a major precursor of arginine by de novo synthesis in the kidneys. Oral citrulline supplementation may be beneficial in some clinical conditions. However, citrulline bioavailability depends on its intestinal absorption. Since the mechanism of citrulline transport across the intestine has not been established yet, this study was designed to characterize L-[(14)C]-citrulline uptake by Caco-2 cells. METHODS: Caco-2 cells were cultured in a bicameral insert system. Inhibition studies were conducted in the presence of neutral, cationic, acidic and non-metabolized amino acids. We performed control inhibition studies for arginine uptake. RESULTS: Citrulline uptake was pH-independent whereas the uptake rate was reduced in the absence of Na(+). Kinetic analysis indicated the involvement of Na(+)-dependent and Na(+)-independent saturable transport components. For competition studies, both the transport components were markedly inhibited by large, small neutral and cationic amino acids. It was also noticed that specific inhibitor of system lBCH inhibited uptake. The inhibition profile of arginine transport was different from that of citrulline transport as arginine uptake was insensitive to BCH. CONCLUSIONS: These characteristics suggest that system B(0,+) might be responsible for the Na(+)-dependent uptake of citrulline, whereas Na(+)-independent uptake may include systems L and b(0,+). Our results show that systems involved in citrulline transport are partly different from those involved in arginine transport.