Citrulline In Urine Research Articles

Pharmacometabolomics Profiling of Preterm Infants Validates Patterns of Metabolism Associated With Response to Dexamethasone Treatment for Bronchopulmonary Dysplasia.

Bronchopulmonary dysplasia (BPD) is one of the most common health complications of premature birth. Corticosteroids are commonly used for treatment of BPD, but their use is challenging due to variability in treatment response. Previous pharmacometabolomics study has established patterns of metabolite levels with response to dexamethasone. We obtained additional patient samples for metabolomics analysis to find associations between the metabolome and dexamethasone response in a validation cohort. A total of 14 infants provided 15 plasma and 12 urine samples. The measure of treatment response was the calculated change in respiratory severity score (deltaRSS) from pre-to-post treatment. Each metabolite was assessed with paired analysis of pre and post-treatment samples using Wilcoxon signed rank test. Correlation analysis was conducted between deltaRSS and pre-to-post change in metabolite level. Paired association analysis identified 20 plasma and 26 urine metabolites with significant level difference comparing pre to post treatment samples (p < 0.05). 4 plasma and 4 urine metabolites were also significant in the original study. Pre-to-post treatment change in metabolite analysis identified 4 plasma and 8 urine metabolites significantly associated with deltaRSS (p < 0.05). Change in urine citrulline levels showed a similar correlation pattern with deltaRSS in the first study, with increasing level associated with improved drug response. These results help validate the first major findings from pharmacometabolomics of BPD including key metabolites within the urea cycle and trans-4-hydroxyproline as a potential marker for lung injury. Ultimately, this study furthers our understanding of the mechanisms of steroid response in BPD patients and helps to design future targeted metabolomics studies in this patient population.

Plasma citrulline correlates with basolateral amino acid transporter LAT4 expression in human small intestine

Background & aimsPlasma citrulline, a non-protein amino acid, is a biochemical marker of small intestine enterocyte mass in humans. Indeed, citrulline is highly correlated with residual bowel length in patients with short bowel syndrome. It is known to be synthesised in epithelial cells of the small intestine from other amino acids (precursors). Citrulline is then released into systemic circulation and interconverted into arginine in kidneys. If plasma citrulline concentration depends on abundance of intestinal amino acid transporters is not known. The aim of the present study was to explore whether plasma citrulline concentration correlates with the expression of intestinal amino acid transporters. Furthermore, we assessed if arginine in urine correlates with plasma citrulline.MethodsDuodenal samples, blood plasma and urine were collected from 43 subjects undergoing routine gastroduodenoscopy. mRNA expression of seven basolateral membrane amino acid transporters/transporter subunits were assessed by real-time PCR. Plasma and urine amino acid concentrations of citrulline, its precursors and other amino acids were analysed using High Performance Liquid Chromatography measurements. Amino acid transporter mRNA expression was correlated with blood plasma and urine levels of citrulline and its precursors using Spearman's rank correlation. Likewise, urine arginine was correlated with plasma citrulline.ResultsPlasma citrulline correlated with the mRNA expression of basolateral amino acid transporter LAT4 (Spearman's r = 0.467, p = 0.028) in small intestine. None of the other basolateral membrane transporters/transporter subunits assessed correlated with plasma citrulline. Plasma citrulline correlated with urinary arginine, (Spearman's r = 0.419, p = 0.017), but not with urinary citrulline or other proteinogenic amino acids in the urine.ConclusionsIn this study, we showed for the first time that small intestinal basolateral LAT4 expression correlates with plasma citrulline concentration. This finding indicates that LAT4 has an important function in mediating citrulline efflux from enterocytes. Furthermore, urine arginine correlated with plasma citrulline, indicating arginine in the urine as possible additional marker for small intestine enterocyte mass. Finally, basolateral LAT4 expression along the human small intestine was shown for the first time.

Quantitative Metabolomic Analysis of Urinary Citrulline and Calcitroic Acid in Mice after Exposure to Various Types of Ionizing Radiation.

With the safety of existing nuclear power plants being brought into question after the Fukushima disaster and the increased level of concern over terrorism-sponsored use of improvised nuclear devices, it is more crucial to develop well-defined radiation injury markers in easily accessible biofluids to help emergency-responders with injury assessment during patient triage. Here, we focused on utilizing ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify and quantitate the unique changes in the urinary excretion of two metabolite markers, calcitroic acid and citrulline, in mice induced by different forms of irradiation; X-ray irradiation at a low dose rate (LDR) of 3.0 mGy/min and a high dose rate (HDR) of 1.1 Gy/min, and internal exposure to Cesium-137 (137Cs) and Strontium-90 (90Sr). The multiple reaction monitoring analysis showed that, while exposure to 137Cs and 90Sr induced a statistically significant and persistent decrease, similar doses of X-ray beam at the HDR had the opposite effect, and the LDR had no effect on the urinary levels of these two metabolites. This suggests that the source of exposure and the dose rate strongly modulate the in vivo metabolomic injury responses, which may have utility in clinical biodosimetry assays for the assessment of exposure in an affected population. This study complements our previous investigations into the metabolomic profile of urine from mice internally exposed to 90Sr and 137Cs and to X-ray beam radiation.

Low urinary citrulline/arginine ratio associated with blood pressure abnormalities and arterial stiffness in childhood chronic kidney disease

Arginine (ARG) and citrulline (CIT) are essential for nitric oxide (NO) synthesis. Their metabolites are interrelated, and involved in blood pressure (BP) control, chronic kidney disease (CKD), and cardiovascular disease (CVD). Although CVD is the leading cause of mortality in CKD, little is known about subclinical CVD in early-stage childhood CKD. Twenty-four–hour ambulatory BP monitoring and arterial stiffness assessment allows the earlier possible detection of subclinical CVD. We investigated whether urinary CIT and ARG metabolites and their ratios are correlated with BP load and vascular abnormalities in children and adolescents with early-stage CKD. We enrolled 55 pediatric patients with mild-to-moderate CKD. Seventy percent (30/43) had at least one out of BP load abnormality on ambulatory BP monitoring, mainly increased asleep systolic BP (SBP) load (40%), asleep SBP or diastolic BP load > 95th percentile (40%), and nocturnal SBP nondipping (35%). Low urinary CIT level and CIT/ARG ratio were associated with BP load abnormalities in children with early CKD. Urinary CIT/ARG ratio was correlated with arterial stiffness, represented as pulse-wave velocity and augmentation index. SBP and diastolic BP loads were negatively correlated with urinary CIT, ARG, asymmetric dimethylarginine (an endogenous NO synthase inhibitor), and CIT/ARG ratio, while positively associated with dimethylamine/asymmetric dimethylarginine ratio and pulse-wave velocity. Early assessments of BP load abnormalities, urinary biomarkers in the CIT-ARG-NO pathway, and arterial stiffness parameters should increase early preventive care toward decreasing hypertension and CV remodeling in pediatric CKD.

Citrin deficiency in a Romanian child living in Spain highlights the worldwide distribution of this defect and illustrates the value of nutritional therapy

We report citrin deficiency in a neonatal non-East-Asian patient, the ninth Caucasian reported with this disease. The association of intrahepatic cholestasis, galactosuria, very high alpha-fetoprotein and increased plasma and urine citrulline, tyrosine, methionine and threonine levels suggested citrin deficiency. Identification of a protein-truncating mutation (c.1078C>T; p.Arg360*) in the SLC25A13 gene confirmed the diagnosis. An immediate response to a high-protein, lactose-free, low-carbohydrate formula was observed. Our report illustrates the need for awareness on citrin deficiency in Western countries.

Urinary citrulline in very low birth weight preterm infants receiving intravenous nutrition

As gut immaturity precludes full enteral feeding, very low birth weight (VLBW) preterm infants receive parenteral nutrition (PN) during the first few weeks of life. Weaning VLBW infants off PN, however, is a top priority since PN is associated with a high risk of complications. The decision making is purely empirical, as there is currently no suitable index of gastrointestinal (GI) maturity. Plasma citrulline concentration is considered an index of GI function in conditions such as short-bowel syndrome and coeliac disease in adults. To identify the factors determining urinary citrulline excretion, and determine whether urinary citrulline excretion could be used as a non-invasive index of GI tolerance to enteral feeding, nutritional intake and urinary citrulline were monitored bi-weekly in forty-seven preterm infants<1500g (interquartiles 880-1320g), during their stay in the Neonatology unit. Median urinary citrulline was 24·7μmol/mmol creatinine (14·5-38·6μmol/mmol creatinine). No relationship was observed with the percentage of energy tolerated enterally. In multivariate regression analysis, weak correlations were found with post-conceptional age (P=0·001), parenteral amino acid supply (P=0·001) and the daily volume of enteral mixture administered (P=0·043). A significant correlation was found with urinary nitrite+nitrate excretion (r 0·47; P<0·001). We conclude that in preterm infants: (1) one of the major determinants of urinary citrulline may be the biosynthesis of citrulline from arginine by NO-synthase; (2) urinary citrulline cannot be used to predict GI tolerance. This is consistent with the observations that, in neonatal gut, citrulline is converted to arginine in situ rather than exported towards the kidneys as observed in adults.

Serum and urine nitric oxide levels in children with Henoch–Schonlein purpura during activity and remission: a study from North India

The objective of this study is to compare serum and urine reactive nitrogen intermediates (RNI) and citrulline levels in children with Henoch-Schonlein purpura (HSP) during activity and remission. The study group consisted of 14 children with HSP. We measured serum and urine RNI and citrulline levels by spectrophotometry in the active phase of the disease and then during remission. Serum RNI levels were 303.95 +/- 221.44 nmol/ml in children with active HSP and 72.57 +/- 26.56 nmol/ml during remission, the differences being statistically significant (P = 0.002). Mean urine RNI levels in children with active HSP were significantly higher than that seen during remission (3.25 +/- 1.80 vs. 1.68 +/- 0.65 nmol/ml; P = 0.003). Similarly, serum citrulline levels during disease activity were 790.65 +/- 707.87 nmol/ml as compared to 281.49 +/- 307.29 nmol/ml at the time of remission, the differences being statistically significant (P = 0.002). Mean urine citrulline levels in children with active disease was 1,969.94 +/- 1655.42 nmol/ml as compared to 1,099.34 +/- 955.82 nmol/ml in children with remission, (P = 0.007). Serum and urine RNI and citrulline levels were significantly higher during the acute phase of HSP as compared to the levels obtained during the phase of disease remission. These findings suggest that nitric oxide may perhaps have a role in the pathogenesis of HSP. Further, these laboratory parameters could be of value in monitoring disease activity. To the best of our knowledge, this study is the most comprehensive work published on the subject so far. Our findings, however, need to be confirmed on a larger study sample before firm conclusions can be drawn.

Serum and urine nitric oxide levels in children with Henoch-Schonlein Purpura during activity and remission – a study from North India

The objective of this study is to compare serum and urine reactive nitrogen intermediates (RNI) and citrulline levels in children with Henoch-Schonlein purpura (HSP) during activity and remission. The study group consisted of 14 children with HSP. We measured serum and urine RNI and citrulline levels by spectrophotometry in the active phase of the disease and then during remission. Serum RNI levels were 303.95 ± 221.44 nmol/ml in children with active HSP and 72.57 ± 26.56 nmol/ml during remission, the differences being statistically significant (P = 0.002). Mean urine RNI levels in children with active HSP were significantly higher than that seen during remission (3.25 ± 1.80 vs. 1.68 ± 0.65 nmol/ml; P = 0.003). Similarly, serum citrulline levels during disease activity were 790.65 ± 707.87 nmol/ml as compared to 281.49 ± 307.29 nmol/ml at the time of remission, the differences being statistically significant (P = 0.002). Mean urine citrulline levels in children with active disease was 1,969.94 ± 1655.42 nmol/ml as compared to 1,099.34 ± 955.82 nmol/ml in children with remission, (P = 0.007). Serum and urine RNI and citrulline levels were significantly higher during the acute phase of HSP as compared to the levels obtained during the phase of disease remission. These findings suggest that nitric oxide may perhaps have a role in the pathogenesis of HSP. Further, these laboratory parameters could be of value in monitoring disease activity. To the best of our knowledge, this study is the most comprehensive work published on the subject so far. Our findings, however, need to be confirmed on a larger study sample before firm conclusions can be drawn.

Manipulation of citrulline availability in humans

To determine whether circulating citrulline can be manipulated in vivo in humans, and, if so, whether citrulline availability affects the levels of related amino acids, nitric oxide, urinary citrulline, and urea nitrogen, 10 healthy volunteers were studied on 3 separate days: 1) under baseline conditions; 2) after a 24-h treatment with phenylbutyrate (0.36 g.kg(-1).day(-1)), a glutamine "trapping" agent; and 3) during oral L-citrulline supplementation (0.18 g.kg(-1).day(-1)), in randomized order. Plasma, erythrocyte (RBC), and urinary citrulline concentrations were determined by gas chromatography-mass spectrometry at 3-h intervals between 1100 and 2000 on each study day. Regardless of treatment, RBC citrulline was lower than plasma citrulline, with an RBC-to-plasma ratio of 0.60 +/- 0.04, and urinary citrulline excretion accounted for <1% of the citrulline load filtered by kidney. Phenylbutyrate induced an approximately 7% drop in plasma glutamine (P = 0.013), and 18 +/- 14% (P < 0.0001) and 19 +/- 17% (P < 0.01) declines in plasma and urine citrulline, respectively, with no alteration in RBC citrulline. Oral L-citrulline administration was associated with 1) a rise in plasma, urine, and RBC citrulline (39 +/- 4 vs. 225 +/- 44 micromol/l, 0.9 +/- 0.3 vs. 6.2 +/- 3.8 micromol/mmol creatinine, and 23 +/- 1 vs. 52 +/- 9 micromol/l, respectively); and 2) a doubling in plasma arginine level, without altering blood urea or urinary urea nitrogen excretion, and thus enhanced nitrogen balance. We conclude that 1) depletion of glutamine, the main precursor of citrulline, depletes plasma citrulline; 2) oral citrulline can be used to enhance systemic citrulline and arginine availability, because citrulline is bioavailable and very little citrulline is lost in urine; and 3) further studies are warranted to determine the mechanisms by which citrulline may enhance nitrogen balance in vivo in humans.

Insight into a “new disorder”—Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD)

Citrullinemia is an autosomal recessive disease caused by a deficiency of argininosuccinate synthetase and is characterized by elevated serum and urine citrulline levels. Classic citrullinemia (CTLN1) is caused by mutations in the gene encoding argininosuccinate synthetase (ASS). Citrullinemia type II (CTLN2) is now recognized to occur in two different phenotypes: adult-onset type II citrullinemia and neonatal-onset type II citrullinemia (also known as Neonatal Intrahepatic Cholestasis Caused By Citrin Deficiency (NICCD). Both are caused by mutation in the SLC25A13 gene (gene map locus 7q21.3) (Nature Genet 1999;22:159). There have been multiple recent reports of Japanese neonates with NICCD. Affected infants presented with intrahepatic cholestasis associated with hypercitrullinemia, hypermethioninemia, or hypergalactosemia detected by a neonatal mass screening. Liver biopsy findings consisted of marked fatty changes and variable degrees of fibrosis. Without specific treatment other than feeding formula containing medium-chain triglycerides or a lactose-free formula, most patients had favorable clinical courses, with amino acid profiles and clinical signs of cholestasis normalizing within the first year. It was found that citrin deficiency causes NICCD and adult-onset CTLN2 with an epigenetic effect of genetic or environmental modifiers. Since citrin is a mitochondrial aspartate glutamate carrier, NICCD and CTLN2 are presumably related to defective aspartate export from the mitochondria to the cytosol along with defects in the malate aspartate shuttle (J Hum Genet 2002;47:333). It is commonly reported that CTLN2, both the neonatal and the adult onset forms, are found exclusively in Japanese subjects. In this issue of The Journal, Yeh et al report NICCD in Taiwanese infants. This disorder accounted for ∼15% of the infants in their clinic being evaluated for idiopathic intrahepatic cholestasis. This observation is an important milestone in the emerging history of this “new disease” and should lead to greater recognition. This disorder should also be a consideration–in any infant–when elevated amino acid profiles are detected by neonatal screening programs.

Anesthesia for Living Related Liver Transplantation in Classic Citrullinemia: A case report

Citrullinemia is an autosomal recessive disorder resulting in a deficiency of the urea cycle enzyme, argininosuccinate synthetase, which is mainly found in the liver. Despite the improvement in a dietary therapy during the past 20 years for the treatment of urea cycle disorders with the systematic adjunction of sodium benzoate, sodium phenylbutyrate and arginine, the overall outcome of severe forms of hyperammonemia often remains disappointing. As the liver is the only organ in which ammonia is transformed into urea, liver transplantation has been considered as an elegant and radical alternative therapy to classical dietetic and medical therapy. A child with classical citrullinemia was treated at age 34 months by a living related liver transplantation. The levels of plasma and urinary citrulline decreased slightly after transplantation, but serum ammonia levels and amino acid concentrations returned to the normal range without protein restriction. We describe this case and include a brief review of the literature.

Serum and Urine Nitrite and Citrulline Levels among Patients with Systemic Lupus Erythematosus: A Possible Addition to Activity Parameters?

Nitric oxide (NO) plays a significant role in the inflammatory process and has been implicated in several autoimmune disorders. This study was carried out prospectively to estimate the levels of nitrite and citrulline in the serum and urine, as surrogate markers of NO production, among patients with systemic lupus erythematosus (SLE). Forty-seven patients and 44 age- and sex-matched, healthy volunteers were studied. Nitrite and citrulline were measured in serum and urine by spectrophotometry.Median serum nitrite and citrulline levels and urine citrulline levels were higher among patients as compared with controls (p < 0.05). Patients with skin involvement stood out and had higher median serum and urine citrulline levels (p < 0.05). Disease activity correlated with steroid dosage, serum nitrite levels, and serum and urine citrulline levels (p < 0.05). Steroid dosage correlated with serum citrulline level (p < 0.05). Serum and urine citrulline levels correlated with each other (p < 0.01). In the subset of 13 individuals with renal involvement, serum and urine citrulline levels correlated with each other (p < 0.01) as did urine nitrite and citrulline levels (p < 0.05).NO production is increased among patients with SLE, and this increase correlates with disease activity and dosage of steroids used. The addition of a urine test to measure NO production as a marker of disease activity using simple spectrophotometry can be a valuable adjunct to other tests, can obviate the need for drawing a blood sample for this purpose, and can be repeated as often as necessary.

Elevated urinary nitrite and citrulline levels in patients with rheumatoid arthritis

The objective of this research was to determine, if NO production, as measured in the serum and urine, is increased in patients with rheumatoid arthritis. Forty-seven patients with RA were recruited in the study and subdivided into inactive and active disease (24 and 23 patients, respectively). Twenty-eight healthy individuals served as controls and nine patients with gastroenteritis were studied to validate the technique of measurement of NO production. Nitrite and citrulline were measured by spectrophotometry, as surrogate markers of NO production. It was found that serum nitrite and citrulline levels of patients with gastroenteritis were not significantly different from controls and the two subgroups of RA. Urine nitrite and citrulline levels were significantly higher in patients with gastroenteritis as compared to the two subgroups of RA and controls (p > 0.001). Serum and urine nitrite levels of patients with active RA were higher than controls and patients with inactive disease (p > 0.05). Serum citrulline levels were not significantly different among the two subgroups of patients with RA. However, they were significantly higher in patients with active disease as compared with controls (p > 0.05). Urinary citrulline levels were significantly higher among patients with active disease as compared to controls and patients with inactive RA (p > 0.05). It is therefore suggested that urinary nitrite and citrulline levels can be useful for the measurement of NO production and are associated with active disease in patients with RA.

Lysinuric protein intolerance. Basolateral transport defect in renal tubuli.

In patients with an autosomal recessive diamino acid transport disorder, lysinuric protein intolerance (LPI), we measured plasma and urinary amino acids basally, and during intravenous infusion of citrulline at two rates. Compared with controls, the patients' plasma citrulline concentrations rose similarly, but urinary citrulline excretion increased excessively. Their plasma arginine and ornithine levels rose subnormally, but massive argininuria and moderate ornithinuria appeared. The excretion rates of the third diamino acid lysine and other amino acids remained practically unaltered, thus excluding mutual competition as the cause for the increases. The results suggest that (a) in the normal kidney reabsorption involves partial conversion of citrulline to arginine and ornithine (metabolic run-out), (b) in LPI, the diamino acid transport defect is located at the basolateral cell membrane of the renal tubules; this inhibits the efflux of arginine and ornithine, increasing their cellular concentration, which in turn inhibits the metabolic disposal of citrulline, and causes leakage of arginine, ornithine, and citrulline into the tubular lumen.

Citrullinemia: Phenotypic Variations

An 18-month-old female infant was found to have citrullinemia on routine plasma screening by the Scriver Method at 5 days of age. At 10 days of age, plasma citrulline concentration was 0.704mumol/ml (normal, 0.010 to 0.030mumol/ml) and has remained 60 to 80 times higher than normal. Urine citrulline concentration was markedly elevated. Hyperammonemia occurred at 1 month of age. The serum ammonia concentration was 473mug/100 ml (normal, 50 to 250 mug/100 ml) and rose to 770mug/100 ml at 4 months of age. Dietary protein was restricted to 1.6 gm/kg/day. Without further change in protein intake, the serum ammonia concentration decreased to 280mug/100 ml and, since then, it has returned to normal. The addition of three synthetic L-amino acids was required for a short time during dietary therapy. At 10 months of age, the infant was given a normal diet. At 18 months of age, her physical and mental development is normal. Activity of argininosuccinic acid synthetase measured in skin fibroblasts was 0.0037mumol of radioactive carbon dioxide per milligram of protein per hour. To demonstrate heterozygosity, fasting plasma citrulline concentrations were measured in five members of the family. Comparison of findings in this patient with those reported in the literature suggests phenotypical variation of the disease, probably due to genetic heterogeneity.

Differential assay for urea and citrulline Automated and manual procedures

1. A simple, quick and sensitive method, manual and automated, for the assay of urea and citrulline in urine or serum is presented. 2. It may be used by clinical wards as a screening test for citrullinemia.

Enzyme studies in a patient with saccharopinuria: a defect of lysine metabolism.

Extract: Elevated levels of saecharopine, lysine, and citrulline in urine and plasma were observed in a patient suffering from saccharopinuria. Using radioisotope methods the lysine-degradative enzymes, lysine-oxoglutarate reductase and saecharopine dehyrogenase, were studied in skin fibroblasts grown from this patient and from healthy subjects. The results show that, in contrast to healthy individuals (range 177–320 pmol formed/min/mg protein), the paitent's fibroblasts are completely lacking in saecharopine dehydrogenase activity, which accounts for the presence of the high levels of saccharopine. The patient also has a reduced level of lysine-oxoglutarate reductase activity (333 pmol saccharopine formed/min/mg protein; range 550–1,570 nmol), which may in part explain the hyperlysinemia. A further enzyme saecharopine oxidoreductase which metabolizes saecharopine to lysine was found to be present in the patient's fibroblasts (63 pmol lysine formed/min/mg protein) but absent from those of healthy control subjects. This indicated induction of this enzyme by the patient in an attempt to reduce the high levels of saecharopine in her tissues and body fluid. Speculation: There are two interesting features of this patient with saccharopinuria which the present studies have not clearly defined, namely the presence of high levels of lysine and citrulline found in plasma and urine. There are three possible explanations for the hyperlysinemia which may be operating singly or in combination. Depression in activity of lysine-oxoglutarate reductase (the patient possesses 37% of the normal activity). Inhibitory effect of saecharopine on lysine-oxoglutarate reductase (in vitro studies). Induction by the patient of saccharopine oxidoreductase which metabolizes saccharopine to lysine. No explanation has yet been found for the high levels of citrulline in plasma and urine.

CITRULLINURIA

A heretofore undescribed disorder manifested by mental retardation, citrullinuria, hypercitrullinemia, and hyperammonemia is reported. Dietary studies show a direct relationship between protein intake and urinary citrulline excretion. Citrulline excretion occurs as an overflow mechanism with plasma citrulline being maintained at about 25 mg/100 ml. Loading studies suggest that the ornithine urea cycle is functioning; high post-absorptive blood ammonia levels indicate that little reserve capacity for urea formation is present.