l-Citrulline supplementation during pregnancy improves perinatal and postpartum maternal vascular function in a mouse model of preeclampsia.

Preeclampsia is a spontaneously occurring pregnancy complication diagnosed by new-onset hypertension and end-organ dysfunction with or without proteinuria. This pregnancy-specific syndrome contributes to maternal morbidity and mortality and can have detrimental effects on fetal outcomes. Preeclampsia is also linked to increased risk of maternal cardiovascular disease throughout life. Despite intense investigation of this disorder, few treatment options are available. The aim of this study was to investigate the potential therapeutic effects of maternal l-citrulline supplementation on pregnancy-specific vascular dysfunction in the male C57BL/6J × female C57BL/6J C1q-/- preeclampsia-like mouse model. l-Citrulline is a nonessential amino acid that is converted to l-arginine to promote smooth muscle and blood vessel relaxation and improve nitric oxide (NO)-mediated vascular function. To model a preeclampsia-like pregnancy, female C57BL/6J mice were mated to C1q-/- male mice, and a subset of dams was supplemented with l-citrulline throughout pregnancy. Blood pressure, systemic vascular glycocalyx, and ex vivo vascular function were investigated in late pregnancy, and postpartum at 6 and 10 mo of age. Main findings show that l-citrulline reduced blood pressure, increased vascular glycocalyx volume, and rescued ex-vivo vascular function at gestation day 17.5 in this preeclampsia-like model. The vascular benefit of l-citrulline also extended postpartum, with improved vascular function and glycocalyx measures at 6 and 10 mo of age. l-Citrulline-mediated vascular improvements appear, in part, attributable to NO pathway signaling. Taken together, l-citrulline supplementation during pregnancy appears to have beneficial effects on maternal vascular health, which may have translational implications for improved maternal cardiovascular health.

Prospective diagnosis of MT-ATP6-related mitochondrial disease by newborn screening.

Elevated citrulline and C5-OH levels are reported as part of the newborn screening of core and secondary disorders on the Recommended Uniform Screening Panel (RUSP). Additionally, some state laboratory newborn screening programs report low citrulline levels, which may be observed in proximal urea cycle disorders. We report six patients who were found on newborn screening to have low citrulline and/or elevated C5-OH levels in whom confirmatory testing showed the combination of these two abnormal analytes. Mitochondrial sequencing revealed known pathogenic variants in MT-ATP6 at high heteroplasmy levels in all cases. MT-ATP6 at these heteroplasmy levels is associated with Leigh syndrome, a progressive neurodegenerative disease. Patients were treated with supplemental citrulline and, in some cases, mitochondrial cofactor therapy. These six patients have not experienced metabolic crises or developmental regression, and early diagnosis and management may help prevent the neurological sequelae of Leigh syndrome. The affected mothers and siblings are asymptomatic or paucisymptomatic (e.g. intellectual disability, depression, migraines, obsessive-compulsive disorder, and poor balance) despite high heteroplasmy or apparent homoplasmy of the familial variant, thus expanding the clinical spectrum seen in pathogenic variants of MT-ATP6. Confirmatory plasma amino acid analysis and acylcarnitine profiling should be ordered in a patient with either low citrulline and/or elevated C5-OH, as this combination appears specific for pathogenic variants in MT-ATP6.

L-Citrulline increases nitric oxide and improves control in obese asthmatics.

BACKGROUNDThe airways of obese asthmatics have been shown to be NO deficient, and this contributes to airway dysfunction and reduced response to inhaled corticosteroids. In cultured airway epithelial cells, L-citrulline, a precursor of L-arginine recycling and NO formation, has been shown to prevent asymmetric dimethyl arginine-mediated (ADMA-mediated) NO synthase (NOS2) uncoupling, restoring NO and reducing oxidative stress.METHODSIn a proof-of-concept, open-label pilot study in which participants were analyzed before and after treatment, we hypothesized that 15 g/d L-citrulline for 2 weeks would (a) increase the fractional excretion of NO (FeNO), (b) improve asthma control, and (c) improve lung function. To this end, we recruited obese (BMI >30) asthmatics on controller therapy, with a baseline FeNO of ≤30 ppb from the University of Colorado Medical Center and Duke University Health System.RESULTSA total of 41 subjects with an average FeNO of 17 ppb (95% CI, 15-19) and poorly controlled asthma (average asthma control questionnaire [ACQ] 1.5 [95% CI, 1.2-1.8]) completed the study. Compared with baseline, L-citrulline increased whereas ADMA and arginase concentration did not (values represent the mean Δ and 95% CI): plasma L-citrulline (190 µM, 84-297), plasma L-arginine (67 µM, 38-95), and plasma L-arginine/ADMA (ratio 117, 67-167). FeNO increased by 4.2 ppb (1.7-6.7 ppb); ACQ decreased by -0.46 (-0.67 to 0.27 points); the forced vital capacity and forced exhalation volume in 1 second, respectively, changed by 86 ml (10-161 ml) and 52 ml (-11 to 132 ml). In a secondary analysis, the greatest FEV1 increments occurred in those subjects with late-onset asthma (>12 years) (63 ml [95% CI, 1-137]), in females (80 ml [95% CI, 5-154]), with a greater change seen in late-onset females (100 ml, [95% CI, 2-177]). The changes in lung function or asthma control were not significantly associated with the changes before and after treatment in L-arginine/ADMA or FeNO.CONCLUSIONShort-term L-citrulline treatment improved asthma control and FeNO levels in obese asthmatics with low or normal FeNO. Larger FEV1 increments were observed in those with late-onset asthma and in females.TRIAL REGISTRATIONClinicalTrials.gov NCT01715844.FUNDINGNIH NHLBI R01 HL146542-01.

The iridoid loganic acid and anthocyanins from the cornelian cherry (Cornus mas L.) fruit increase the plasma l-arginine/ADMA ratio and decrease levels of ADMA in rabbits fed a high-cholesterol diet.

BACKGROUND: Although fruit and vegetable-rich diets have beneficial effects on cardiovascular diseases, we have little knowledge of the impact of fruits and their constituents, iridoids and anthocyanins, on the l-arginine-ADMA-DDAH pathway. Our previous study demonstrated the modulation of those factors by the oral administration of the cornelian cherry fruit. HYPOTHESIS/PURPOSE: We have assessed the effects of the oral administration of two main constituents isolated from the cornelian cherry fruit, iridoid loganic acid and anthocyanins, on l-arginine, its derivatives (ADMA, SDMA), metabolites (DMA, l-citrulline), and the hepatic DDAH activity and its isoform expression in rabbits fed a high-cholesterol diet. We have also analyzed eNOS expression in the thoracic aorta as well as the redox status in blood. STUDY DESIGN: In the present study, we used an animal model of diet induced atherosclerosis. For 60 days, white New Zealand rabbits were fed a standard diet, a 1% cholesterol enriched diet, or concomitantly with the investigated substances. l-arginine, ADMA, SDMA, DMA, and l-citrulline were assessed using the LC-MS/MS method. DDAH activity and redox parameters were analyzed spectrophotometrically. DDAH1 and DDAH2 isoform expressions were assessed by western blotting, mRNA expression of eNOS was quantified by real-time PCR. RESULTS: We demonstrated that the administration of loganic acid (20 mg/kg b.w.), and to a lesser extent of anthocyanins (10 mg/kg b.w.), caused an increase in the l-arginine level and the l-arginine/ADMA ratio. Also, both substances decreased ADMA, DMA, and l-citrulline, but not SDMA levels. Anthocyanins, but not loganic acid, enhanced the activity of DDAH in the liver. Anthocyanins also significantly enhanced both DDAH1 and DDAH2 expression, while loganic acid to a lesser extent enhanced DDAH1 but not DDAH2 expression. Both loganic acid and anthocyanins pronouncedly increased mRNA expression of eNOS in thoracic aortas. Both loganic acid and anthocyanins reversed the blood glutathione level depleted by dietary cholesterol. Cholesterol feeding decreased the blood GPx level, and the change was not reversed by anthocyanins or loganic acid. We did not observe any significant differences in the blood levels of MDA or SOD among the groups. CONCLUSION: Iridoids and anthocyanins may modulate the l-arginine-ADMA pathway in subjects fed a high-cholesterol diet.

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.

Bioelectrical impedance analysis (BIA)-derived phase angle (PA) is a practical aid to nutritional assessment in hospital in-patients.

BACKGROUND: Nutritional status can be difficult to assess. Bioelectrical impedance analysis (BIA)-derived phase angle (PA), and the plasma markers citrulline and transthyretin (pre-albumin) have the potential to assist, but the protocol of fasting and resting for BIA renders the investigation impractical for routine use, especially so in populations at high risk of malnutrition. AIMS: 1 To clarify whether starving and resting are necessary for reliable measurement of PA. 2 To identify whether PA, citrulline and transthyretin correlate with nutritional status. METHODS: Eighty consenting adult in-patients were recruited. Nutritional status was determined by subjective global assessment (SGA) used as gold standard. The Malnutrition Universal Screening Tool (MUST) was used and anthropometric measurements were performed. Serum was analysed for citrulline and transthyretin. PA was measured using Bodystat 4000. The PA was considered to define malnutrition when lower than reference ranges for sex and age, and severe malnutrition if more than 2 integers below the lower limit. Anthropometric measurements were categorised according to WHO reference centiles. Ordinal logistic regression estimated the strength of association of PA, citrulline and transthyretin with SGA. PA values in the different metabolic states were compared using paired t tests. RESULTS: All 80 subjects completed the BIA and the nutritional assessments in the 3 different states; 14 declined to provide blood samples for the biochemical assays. Malnutrition was identified in 32 cases, severe malnutrition in 14 cases, the remaining 34 cases were deemed not to be malnourished. PA was strongly inversely associated with SGA (Odds Ratio [OR] per unit increase = 0.21, CI 0.12-0.37, p < 0.001). PA was not influenced by exercise (p = 0.134) or food intake (p = 0.184). Transthyretin was inversely associated with malnourished/severely malnourished states (OR = 0.98, 95% CI 0.97-0.99, p = 0.001), but had poorer predictive values than PA. There was no significant association between citrulline concentration and SGA (OR = 1.01, 95% CI 0.99-1.04, p = 0.348). CONCLUSIONS: The BIA-derived PA reliably identifies malnutrition. It is strongly associated with SGA but requires less skill and experience, and out-performs circulating transthyretin, rendering it a promising and less operator-dependent tool for assessing nutritional status in hospital patients. Our novel demonstration that fasting and bed-rest are unnecessary consolidates that position.

Plasma citrulline concentration, a marker for intestinal functionality, reflects exercise intensity in healthy young men.

BACKGROUND & AIMS: Plasma citrulline concentration is considered to be a marker for enterocyte metabolic mass and to reflect its reduction as may occur during intestinal dysfunction. Strenuous exercise can act as a stressor to induce small intestinal injury. Our previous studies suggest that this comprises the intestinal ability to produce citrulline from a glutamine-rich protein bolus. In this study we investigated the effects of different exercise intensities and hydration state on citrulline and iFABP levels following a post-exercise glutamine bolus in healthy young men. METHODS: Fifteen healthy young men (20-35 yrs, VO2 max 56.9 ± 3.9 ml kg-1 min-1) performed in a randomly assigned cross-over design, a rest (protocol 1) and four cycle ergometer protocols. The volunteers cycled submaximal at different percentages of their individual pre-assessed maximum workload (Wmax): 70% Wmax in hydrated (protocol 2) and dehydrated state (protocol 3), 50% Wmax (protocol 4) and intermittent 85/55% Wmax in blocks of 2 min (protocol 5). Immediately after 1 h exercise or rest, subjects were given a glutamine bolus with added alanine as an iso-caloric internal standard (7.5 g of each amino acid). Blood samples were collected before, during and after rest or exercise, up to 24 h post onset of the experiment. Amino acids and urea were analysed as metabolic markers, creatine phosphokinase and iFABP as markers of muscle and intestinal damage, respectively. Data were analysed using a multilevel mixed linear statistical model. p values were corrected for multiple testing. RESULTS: Citrulline levels already increased before glutamine supplementation during normal hydrated exercise, while this was not observed in the dehydrated and rest protocols. The low intensity exercise protocol (50% Wmax) showed the highest increase in citrulline levels both during exercise (43.83 µmol/L ± 2.63 (p < 0.001)) and after glutamine consumption (50.54 µmol/L ± 2.62) compared to the rest protocol (28.97 µmol/L ± 1.503 and 41.65 µmol/L ± 1.96, respectively, p < 0.05). However, following strenuous exercise at 70% Wmax in the dehydrated state, citrulline levels did not increase during exercise and less after the glutamine consumption when compared to the resting condition and hydrated protocols. In line with this, serum iFABP levels were the highest with the strenuous dehydrated protocol (1443.72 µmol/L ± 249.9, p < 0.001), followed by the high intensity exercise at 70% Wmax in the hydrated condition. CONCLUSIONS: Exercise induces an increase in plasma citrulline, irrespective of a glutamine bolus. The extent to which this occurs is dependent on exercise intensity and the hydration state of the subjects. The same holds true for both the post-exercise increase in citrulline levels following glutamine supplementation and serum iFABP levels. These data indicate that citrulline release during exercise and after an oral glutamine bolus might be dependent on the intestinal health state and therefore on intestinal functionality. Glutamine is known to play a major role in intestinal physiology and the maintenance of gut health and barrier function. Together, this suggests that in clinical practice, a glutamine bolus to increase citrulline levels after exercise might be preferable compared to supplementing citrulline itself. To our knowledge this is the first time that exercise workload-related effects on plasma citrulline are reported in relation to intestinal damage.

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.

Nutritional therapy complications in children with ultra-short bowel syndrome include growth deficiency but not cholestasis.

AIM: Children with ultra-short bowel syndrome (USBS) have not been extensively studied to date because the condition is rare. The aim of the study was to assess the nutritional status of children with USBS receiving home parenteral nutrition, using citrulline serum concentration and cholestasis. METHODS: We studied 17 patients with USBS, with a median age of 6.6 years and median duration of parenteral nutrition of 6.6 years. The study was carried out at The Children's Memorial Health Institute, Warsaw, from January 2014 to January 2015. RESULTS: The median standard deviation score (SDS) was -1.2 for body mass according to chronological age, -1.72 according to height and -0.59 according to height for age. Patients requiring seven days per week parenteral nutrition had a citrulline concentration below 10 µmol/L. Decreased bone-mineral density was observed in 87% of the patients. Low values of 25-hydroxyvitamin D were found in 53% of the children. None of the patients had elevated conjugated bilirubin levels above 34.2 µmol/L. CONCLUSION: Children with USBS were growth deficient according to their chronological age, with frequent abnormal bone mineralisation and vitamin D deficiency. Children requiring parenteral nutrition seven days a week had citrulline concentrations below 10 µmol/L. Cholestasis was not seen.

Plasma l-citrulline concentrations in l-arginine-supplemented healthy dogs.

INTRODUCTION: To determine whether oral l-arginine increases plasma [l-citrulline] in dogs. ANIMALS: Eleven healthy staff-owned dogs were used in this study. MATERIALS AND METHODS: Dogs (n = 3) were given l-arginine (50mg/kg PO q8h) for 7 days, and plasma [l-arginine] and [l-citrulline] were analyzed by high performance liquid chromatography at baseline (BL), steady state trough, and 0.5, 1, 1.5, 2, 4, 6, and 8 h after final dosing on day 7. Eleven dogs were then treated with 100mg/kg l-arginine PO q8h for 7 days, and [l-arginine] and [l-citrulline] were measured at BL, steady state trough, and at peak 4 hrs after dosing (T4 hrs). RESULTS: - Plasma [l-arginine] and [l-citrulline] peaked at T4 hrs on the 50mg/kg dosage. Target outcome, modeled after human study results, of a doubling of [l-arginine] and a 25-30% increase in [l-citrulline] from BL were not reached. After the 100mg/kg dosage, plasma [l-arginine] increased from a BL median of 160.1 µM (range, 100.2-231.4 µM) to a peak of 417.4 µM (206.5-807.3 µM) at T4 hrs, and plasma [l-citrulline] increased from a BL median of 87.8 µM (59.1-117.1 µM) to peak of 102.2 µM (47.4-192.6 µM) at T4 hrs. Ten of eleven dogs showed a doubling of plasma [l-arginine] and 4/11 dogs achieved 25-30% or greater increases in plasma [l-citrulline]. No adverse effects on heart rate or blood pressure were noted. CONCLUSIONS: - Oral l-arginine dosage of 100mg/kg q8h doubles plasma [l-arginine] in healthy dogs, but conversion to l-citrulline is quite variable. Further evaluation of this dosage regimen in dogs with pulmonary hypertension is warranted.

p.Val452Ile mutation of the SLC25A13 gene in a Turkish patient with citrin deficiency.

Seker-Yilmaz B, Kör D, Tümgör G, Ceylaner S, Önenli-Mungan N. p.Val452Ile mutation of the SLC25A13 gene in a Turkish patient with citrin deficiency. Turk J Pediatr 2017; 59: 311-314. Citrin deficiency is an autosomal recessive metabolic disorder, which is caused by pathogenic mutations in the SLC25A13 gene on chromosome 7q21.3, as the causative gene that encodes the liver type aspartate/glutamate carrier isoform 2 (AGC2). One of the main clinical presentations is neonatal intrahepatic cholestatic hepatitis caused by citrin deficiency. We report a Turkish child presented with prolonged neonatal jaundice associated with elevated plasma citrulline and galactosuria. NICCD was suspected at this point and mutation study of SLC25A13 showed that she was homozygous for the missense NM_014251.2:c.1354G > A (NP_055066.1:p.Val452Ile) (dbSNP: rs143877538) mutation. Dramatic response was observed to the dietary treatment with medium-chain triglycerides containing formula, ursodeoxycholic acid and fat-soluble vitamin supplementation. The minor allele frequency of this variant was given as nearly as 0.01 in the South Asian population; it seems like a disease causing variant. This is the first report of this variant in the Turkish and European population.

Two weeks of watermelon juice supplementation improves nitric oxide bioavailability but not endurance exercise performance in humans.

This study tested the hypothesis that watermelon juice supplementation would improve nitric oxide bioavailability and exercise performance. Eight healthy recreationally-active adult males reported to the laboratory on two occasions for initial testing without dietary supplementation (control condition). Thereafter, participants were randomly assigned, in a cross-over experimental design, to receive 16 days of supplementation with 300 mL·day(-1) of a watermelon juice concentrate, which provided ∼3.4 g l-citrulline·day(-1) and an apple juice concentrate as a placebo. Participants reported to the laboratory on days 14 and 16 of supplementation to assess the effects of the interventions on blood pressure, plasma [l-citrulline], plasma [l-arginine], plasma [nitrite], muscle oxygenation and time-to-exhaustion during severe-intensity exercise. Compared to control and placebo, plasma [l-citrulline] (29 ± 4, 22 ± 6 and 101 ± 23 µM), [l-arginine] (74 ± 9, 67 ± 13 and 116 ± 9 µM) and [nitrite] (102 ± 29, 106 ± 21 and 201 ± 106 nM) were higher after watermelon juice supplementation (P < 0.01). However, systolic blood pressure was higher in the watermelon juice (130 ± 11) and placebo (131 ± 9) conditions compared to the control condition (124 ± 8 mmHg; P < 0.05). The skeletal muscle oxygenation index during moderate-intensity exercise was greater in the watermelon juice condition than the placebo and control conditions (P < 0.05), but time-to-exhaustion during the severe-intensity exercise test (control: 478 ± 80, placebo: 539 ± 108, watermelon juice: 550 ± 143 s) was not significantly different between conditions (P < 0.05). In conclusion, while watermelon juice supplementation increased baseline plasma [nitrite] and improved muscle oxygenation during moderate-intensity exercise, it increased resting blood pressure and did not improve time-to-exhaustion during severe-intensity exercise. These findings do not support the use of watermelon juice supplementation as a nutritional intervention to lower blood pressure or improve endurance exercise performance in healthy adults.

Oral L-citrulline supplementation enhances cycling time trial performance in healthy trained men: Double-blind randomized placebo-controlled 2-way crossover study.

BACKGROUND: Many human studies report that nitric oxide (NO) improves sport performance. This is because NO is a potential modulator of blood flow, muscle energy metabolism, and mitochondrial respiration during exercise. L-Citrulline is an amino acid present in the body and is a potent endogenous precursor of L-arginine, which is a substrate for NO synthase. Here, we investigated the effect of oral L-citrulline supplementation on cycling time trial performance in humans. METHODS: A double-blind randomized placebo-controlled 2-way crossover study was employed. Twenty-two trained males consumed 2.4 g/day of L-citrulline or placebo orally for 7 days. On Day 8 they took 2.4 g of L-citrulline or placebo 1 h before a 4-km cycling time trial. Time taken to complete the 4 km cycle, along with power output/VO2 ratio (PO/VO2), plasma nitrite and nitrate (NOx) and amino acid levels, and visual analog scale (VAS) scores, was evaluated. RESULTS: L-Citrulline supplementation significantly increased plasma L-arginine levels and reduced completion time by 1.5 % (p < 0.05) compared with placebo. Moreover, L-citrulline significantly improved subjective feelings of muscle fatigue and concentration immediately after exercise. CONCLUSIONS: Oral L-citrulline supplementation reduced the time take to complete a cycle ergometer exercise trial. TRIAL REGISTRATION: Current Controlled Trials UMIN000014278.

Acute Citrulline-Malate Supplementation and High-Intensity Cycling Performance.

Cunniffe, B, Papageorgiou, M, O'Brien, B, Davies, NA, Grimble, GK, and Cardinale, M. Acute citrulline-malate supplementation and high-intensity cycling performance. J Strength Cond Res 30(9): 2638-2647, 2016-Dietary L-citrulline-malate (CM) consumption has been suggested to improve skeletal muscle metabolism and contractile efficiency, which would be expected to predispose exercising individuals to greater fatigue resistance. The purpose of this study was to examine the effects of CM supplementation on acid-base balance and high-intensity exercise performance. In a double-blind, placebo-controlled, crossover study, 10 well-trained males consumed either 12 g of CM (in 400 ml) or lemon sugar-free cordial (placebo [PL]) 60 minutes before completion of 2 exercise trials. Each trial consisted of subjects performing 10 (×15 seconds) maximal cycle sprints (with 30-second rest intervals) followed by 5 minutes recovery before completing a cycle time-to-exhaustion test (TTE) at 100% of individual peak power (PP). Significant increases in plasma concentrations of citrulline (8.8-fold), ornithine (3.9-fold), and glutamine (1.3-fold) were observed 60 minutes after supplementation in the CM trial only (p ≤ 0.05) and none of the subjects experienced gastrointestinal side-effects during testing. Significantly higher exercise heart rates were observed in CM condition (vs. PL) although no between trial differences in performance related variables (TTE: [120 ± 61 seconds CM vs. 113 ± 50 seconds PL]), PP or mean power, ([power fatigue index: 36 ± 16% CM vs. 28 ± 18% PL]), subjective rating of perceived exertion or measures of acid-base balance (pH, lactate, bicarbonate, base-excess) were observed (p > 0.05). This study demonstrated that acute supplementation of 12 g CM does not provide acute ergogenic benefits using the protocol implemented in this study in well-trained males.

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

High salt exacerbates programmed hypertension in maternal fructose-fed male offspring.

BACKGROUND AND AIMS: Consumption of food and drinks containing high fructose (HF), which is associated with hypertension, is increasing steeply. Moreover, increased salt intake significantly increases hypertension risk. We examined whether maternal HF and postnatal high salt (HS) intake had synergistic effects on blood pressure (BP) elevation in adult offspring and determined the underlying mechanisms. METHODS AND RESULTS: Pregnant Sprague-Dawley rats received regular chow or chow supplemented with 60% fructose during the entire pregnancy and lactation periods. Half of the male offspring received 1% NaCl in drinking water from weaning to 3 months of age. Male offspring were assigned to 4 groups (control, HF, HS, and HF + HS) and were sacrificed at 12 weeks of age. Offspring in HF and HS groups developed hypertension, indicating that HF and HS synergistically increased BP. Postnatal HS intake increased Ace expression and decreased Agtr1b and Mas1 expression in the kidneys. Renal mRNA levels of Ace and Agtr1a were significantly higher in HF + HS group than in control group. Renal levels of Na-K-2Cl cotransporter, type 3 sodium hydrogen exchanger, and Na(+)/Cl(-) cotransporter were higher in HS and HF + HS groups than in control group. CONCLUSION: Postnatal HS intake exacerbated prenatal HF-induced programmed hypertension. HF and HS induced programmed hypertension by differentially inducing renin-angiotensin system and sodium transporters in the kidneys. Better understanding of the effect of the relationship between HF and HS on hypertension development will help prevent hypertension in mothers and children exposed to HF and HS.

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.

l-Citrulline supplementation improves O2 uptake kinetics and high-intensity exercise performance in humans.

The purpose of this study was to compare the effects of l-citrulline (Cit) and l-arginine (Arg) supplementation on nitric oxide (NO) biomarkers, pulmonary O2 uptake (V̇o2) kinetics, and exercise performance. In a randomized, placebo (Pla)-controlled, crossover study, 10 healthy adult men completed moderate- and severe-intensity cycling exercise on days 6 and 7 of a 7-day supplementation period with Pla, Arg (6 g/day), and Cit (6 g/day). Compared with Pla, plasma Arg concentration was increased by a similar magnitude with Arg and Cit supplementation, but plasma Cit concentration was only increased (P < 0.001) with Cit supplementation. Plasma nitrite (NO2 (-)) concentration was increased with Arg supplementation (P < 0.05) and tended to increase with Cit supplementation (P = 0.08) compared with Pla (83 ± 25, 106 ± 41, and 100 ± 38 nM with Pla, Arg, and Cit, respectively); however, mean arterial blood pressure was only lower (P < 0.05) after Cit supplementation. The steady-state V̇o2 amplitude during moderate-intensity cycle exercise was not significantly different between supplements, but Cit lowered the V̇o2 mean response time (59 ± 8 and 53 ± 5 s with Pla and Cit, respectively, P < 0.05) during severe-intensity exercise, improved tolerance to severe-intensity exercise (589 ± 101 and 661 ± 107 s with Pla and Cit, respectively), and increased the total amount of work completed in the exercise performance test (123 ± 18 and 125 ± 19 kJ with Pla and Cit, respectively, P < 0.05). These variables were not altered by Arg supplementation (P > 0.05). In conclusion, these results suggest that short-term Cit, but not Arg, supplementation can improve blood pressure, V̇o2 kinetics, and exercise performance in healthy adults.

Bovine Colostrum Modulates Myeloablative Chemotherapy-Induced Gut Toxicity in Piglets.

BACKGROUND: Intensive chemotherapy frequently results in gut toxicity, indicated by oral and intestinal mucositis, resulting in poor treatment outcomes and increased mortality. There are no effective preventive strategies against gut toxicity and the role of diet is unknown. OBJECTIVE: We hypothesized that the severity of chemotherapy-induced gut toxicity in early life is diet-dependent, and that intake of bovine colostrum (BC) provides better gut protection than an artificial milk replacer (MR). METHODS: A total of 37 3-d-old pigs received for 6 d either intravenous saline control or myeloablative treatment with busulfan and cyclophosphamide, and were fed either BC or MR, resulting in the following 4 treatments (n = 8-10/group): bovine colostrum plus saline control (Ctr-BC), milk replacer plus saline control (Ctr-MR), bovine colostrum plus busulfan and cyclophosphamide chemotherapy (BUCY-BC), and milk replacer plus busulfan and cyclophosphamide chemotherapy (BUCY-MR). The gut was collected for analysis 11 d after the start of chemotherapy. RESULTS: Relative to the control groups, both busulfan and cyclophosphamide chemotherapy (BUCY) groups showed signs of gut toxicity, with oral ulcers, reduced intestinal dimensions, and hematologic toxicity. Diet type did not affect mucosal structure on day 11, but BUCY-BC pigs had less vomiting than BUCY-MR pigs (1 of 10 vs. 10 of 10, P < 0.05). Markers of intestinal function were higher (up to 20-fold greater galactose absorption and 2-3-fold greater brush border enzyme activity, all P < 0.05), and tissue inflammatory cytokine concentrations and serum liver enzyme values were lower in BUCY-BC than in BUCY-MR pigs (30-50% reductions in interleukin 6 and 8, aminotransferase, and bilirubin concentrations, P < 0.05). Gut colonization was not significantly affected except that BUCY pigs had lower microbial diversity with a higher abundance of Lactobacilli. CONCLUSION: BC may reduce gut toxicity during myeloablative chemotherapy in piglets by preserving intestinal function and reducing inflammation. Whether similar effects occur in children remains to be tested.