J.-P. De Bandt

Aliphatic polyamines in physiology and diseases

Aliphatic polyamines are a family of polycationic molecules derived from decarboxylation of the amino acid ornithine that classically comprise three molecules: putrescine, spermidine and spermine. In-cell polyamine homeostasis is tightly controlled at key steps of cell metabolism. Polyamines are involved in an array of cellular functions from DNA stabilization, and regulation of gene expression to ion channel function and, particularly, cell proliferation. As such, aliphatic polyamines play an essential role in rapidly dividing cells such as in the immune system and digestive tract. Because of their role in cell proliferation, polyamines are also involved in carcinogenesis, prompting intensive research into polyamine metabolism as a target in cancer therapy. More recently, another aliphatic polyamine, agmatine, the decarboxylated derivative of arginine, has been identified as a neurotransmitter in mammals, and investigations have focused on its effects in the CNS, notably as a neuroprotector in brain injury.

Simultaneous determination of retinol and α-tocopherol in polymeric diets for enteral nutrition

Existing methods for simultaneous measurements of retinol and alpha-tocopherol in enteral formulas require large sample and solvent volumes and are time-consuming and costly. We have developed a simple, sensitive, cost-effective method for the determination of these vitamins in polymeric diets that can easily be applied to standard quality control of large numbers of samples. Our analytical procedure comprises deproteinization with pure ethanol, saponification with a 3.6M KOH solution in a sonicator for 30 min at 65 degrees C under a nitrogen atmosphere, solubilization of samples in phosphate buffer and extraction with hexane. Vitamins are separated by reversed-phase HPLC and quantified by dual-wavelength spectrophotometry. The method gives satisfactory results, with recovery rates of 106.3+/-1.5% for retinol and 102.3+/-1.5% for alpha-tocopherol and RSDs ranging between 1.2 and 4.8% for precision. This method is suitable for the quality control of enteral formulas.

Adaptative response of nitrogen metabolism in early endotoxemia: role of ornithine aminotransferase.

Arginine (Arg) and glutamine (Gln) utilization is greatly increased during catabolic stress. While the supply of both amino acids has been advocated in this situation, arginine administration is possibly associated with deleterious effects. From a metabolic point of view, these two amino acids are reciprocal precursors via ornithine aminotransferase (OAT). We hypothesized that OAT plays a key role in the interconversion between Arg and Gln. To test this hypothesis, we evaluated the influence of OAT activity in a model of septic shock induced by intraperitoneal injection of lipopolysaccharide (LPS) in wild-type (WT) and transgenic mice overexpressing OAT (OAT) in the liver, kidney and intestine, i.e. the three main organs of OAT expression. Plasma and tissue amino acid concentrations and tissue OAT expression and activity were measured. Five hours after LPS injection, WT and OAT mice showed a similar response to LPS in terms of inflammatory cytokine production and protein catabolism, suggesting that the interconversion between Arg and Gln through this pathway remains limited. Endotoxemia led to a significant decrease in plasma Orn levels and an increase in liver Orn levels. Of note, Orn levels were always lower in OAT mice. While only plasma Arg and Gln remained unaffected by LPS treatment, hepatic Gln was significantly increased without any difference between the two genotypes. In this model of early endotoxemia, arginine and glutamine maintained their metabolic homeostasis. Our results show an inhibition of OAT activity and expression in the liver following LPS treatment. These data highlight the importance of OAT in ornithine metabolism, especially in the liver, and suggest a post-transcriptional regulation of OAT by LPS in the liver.

An in silico model of enterocytic glutamine to citrulline conversion pathway.

Enterocyte is one of the main sites of amino acids metabolism and particularly of the citrulline biosynthesis. Working at the cellular scale and applying ordinary differential equations (ODEs) formalism, we have built a mathematical model of the enterocytic glutamine to citrulline conversion in the fasting state. This model enables us to test different physiopathological scenarios of enzyme activity loss. Results from two different approaches were compared: a standard approach (KA) based on the Michaelis–Menten assumptions and an association–dissociation approach (VH) based on the kinetic mass action law. For both approaches, ODEs system was numerically solved using Mathematica™. In both cases, the model correctly predicts the physiological plasma citrulline steady-state, but the two approaches present clear differences for metabolites of enzymes having a complex mechanism, challenging the validity of the KA approach in such cases. When physiopathological scenarios of enzyme activity loss are simulated, both approaches predict a very sharp transition from the physiological citrulline plasma level to the lack of its production: the concentration profiles of these simulations show a clear threshold of which characteristics vary with the involved enzyme. Moreover, amongst all enzymes included in the model, the ornithine aminotransferase (OAT) shows the highest sensitivity in the system whatever the approach used. This model points out the limits of the Michaelis–Menten approach to model complex enzyme mechanisms. It highlights the key role of OAT in the studied citrulline synthesis pathway and also suggests an order of magnitude about the optimal ratio of enzyme concentrations in this pathway.

Evidence for a role of the ileum in the control of nitrogen homeostasis via the regulation of arginine metabolism.

As arginine plays a key role in the regulation of liver ureagenesis, we hypothesised that a modulation of enzymes involved in arginine metabolism within the intestine contributes to the regulation of N homeostasis according to protein supply. Our aim was to study the influence of variations in protein or amino acid (AA) supply on intestinal arginase, glutaminase, ornithine aminotransferase (OAT), argininosuccinate lyase and argininosuccinate synthetase. We evaluated in vivo in rats the responses of these enzymes to short-term (ST, 16 h) and long-term (LT, 15 d) variations in dietary protein (10, 17 or 25 % protein diet). In addition, in order to test whether these responses could involve a direct action of AA on the gene expression and activity of these enzymes, Caco-2/TC7 cells were cultured for 3 d with increasing AA concentrations. In vivo, in the ST, both high- and low-protein diets increased arginase activity in the intestinal mucosa (ST25 %: 46 (sem 2) μmol/g per min and ST10 %: 46 (sem 2) μmol/g per min v. ST17 %: 36 (sem 3) μmol/g per min, P < 0.05). In the LT, OAT expression was increased in the LT10 % group (+277 %, P < 0.05) compared with the LT17 % group. Caco-2/TC7 cells showed inverse relationships between AA supply and arginase (P = 0.058) and OAT (P = 0.035) expressions. The present study demonstrates the regulation of intestinal arginase and OAT expressions in response to protein supply. Our in vitro experiments further indicate a direct AA-induced regulation of the mRNA abundance of these enzymes. In situations of limited protein supply, this regulation would increase intestinal arginine catabolism and, possibly via a decrease in arginine portal release, decrease hepatic AA oxidation, thus promoting N sparing.

N-Carbamoylputrescine, a citrulline-derived polyamine, is not a significant citrulline metabolite in rats.

Citrulline, a key amino acid of the urea cycle, has been shown to play a regulatory role in protein and energy metabolism in mammals. We questioned whether N-carbamoyl-putrescine (NCP), the decarboxylated derivative of citrulline, could play a role in the biological properties of this amino acid. To evidence the presence of NCP in mammalian tissues, we developed a sensitive reverse-phase high-performance liquid chromatography (HPLC) with fluorimetric detection method with precolumn dansyl derivatization and solid-phase extraction for the determination of NCP together with polyamines in biological samples. Dansyl NCP was identified with a 5.85-min retention time. Linearity was obtained in a concentration range of 0.125 to 12.5 μM. Intraday and day-to-day relative coefficients of variation ranged from 8.9% to 12.3% and from 14% to 14.3%, respectively. Recovery rates in serum ranged from 75% to 83%. Thereafter, we used this method to search for the presence of NCP in serum, muscle, liver, jejunum, and ileum in rats after both short-term intraperitoneal injection and long-term oral citrulline supplementation. We failed to detect NCP in these animals. These data suggest that NCP is not a significant citrulline metabolite in rats.

Effect of recombinant human interleukin 1β (rhIL-1β) on amino acid flux in the isolated perfused rat liver

SummaryWe studied the effect of recombinant human IL-1β (rhIL-1β) on hepatic amino acid (AA) flux in the isolated perfused rat liver model. Two experimental groups were used — a control group (n = 5) and a rhIL-1β-treated group (n = 5). IL-1 was added to the perfusate in two successive boluses of 0.1µg and 0.9µg, respectively 35 min (final concentration 0.67 ng/ml) and 60 min (6 ng/ml) after beginning the perfusion. In the IL-1 treated group, a reduction in flux was observed for only three AA, alanine, phenylalanine and serine. Glucose and urea production in the IL-1-treated group was slightly but not-significantly lower than in the controls.rhIL-1β thus has only minor direct effects on AA flux and gluconeogenesis in the liver and cannot therefore be held responsible for the increase in hepatic amino acid uptake during stress.

PP054-SUN: Immunonutrition: Effect of age on Arginine and Related Amino Acids Systemic Bioavailability After Surgical Stress

gastrointestinal disorders. However, inappropriate chronic exposure and/or prescription have been recently associated with a number of adverse events, especially in the elderly. Among known drug-class effects of PPI, hypomagnesaemia has been recently shown by a growing number of case reports and series. However, epidemiological studies addressing this topic, especially in older subjects, are still needed. Methods: We cross-sectionally investigated the relationship between PPI use and magnesium status in a large cohort of community-dwelling older volunteers from the Baltimore Longitudinal Study of Aging (BLSA). 4017 older subjects 65 years or older (1983 women and 2034 men) with complete data on serum magnesium levels and PPI use were evaluated. Subjects were categorized according to PPI use. Linear regression models adjusted for age and sex (Model 1) and for additional confounders including BMI, mineral and magnesium supplements, creatinine, calcium serum levels, TSH, use of diuretics, digitalis, antibiotics, calcineurin inhibitors, presence of chronic disease (type 2 diabetes, cardiovascular diseases, cancer) (Model 2) were used to address the relationship between PPI use and serum magnesium levels. Results: 505 subjects (12.6%) were PPI users. After adjustment for age and sex, PPI users exhibited significantly lower magnesium levels than non-users counterpart (1.99±0.22 vs 2.03±0.20mg/ml, p < 0.001). PPI use was negatively associated with serum magnesium levels independent of multiple confounders ( 0.041±0.009, p < 0.0001). Conclusion: In community-dwelling older subjects the use of PPIs is negatively and independently associated with serum magnesium levels.

PP143-SUN: Effect of Dietary Amino Acids on Liver Function of Fructose Fed Rats

PP142-SUN HOME ENTERAL NUTRITION (HEN) HELPS TO REDUCE COMPLICATIONS, LENGTH OF STAY AND HEALTH-CARE COSTS IN ADULTS S. Klek1, A. Hermanowicz2, G. Dziwiszek3, K. Matysiak4, K. Szczepanek1, P. Szybinski1, T. Kowalczyk1, K. Figula1, R. Choruz1, A. Galas5. 1General and Oncology Surgery Unit, Stanley Dudrick’s Memorial Hospital, Skawina, 2Department of Pediatric Surgery, Medical University of Bialystok, Bialystok, 3Home Enteral Nutrition Unit, Stomed, Ostroleka, 4Chair of General, Gastroenterology and Oncology and Plastic Surgery, Medical University of Poznan, Poznan, 5Chair of Epidemiology and Preventive Medicine, Jagiellonian University Medical College, Krakow, Poland

P015 Le niveau d’apport azoté module l’expression des enzymes du carrefour arginine-ornithine-citrulline dans les cellules Caco-2/TC7

Introduction et but de l’etude Le flux portal d’Arginine (Arg) est un element cle de la regulation de l’ureogenese hepatique. L’intestin est l’organe qui controle la disponibilite de l’Arg apportee par voie digestive. Cela dependrait d’une modulation de l’expression de differentes enzymes (arginase, glutaminase, ornithine ami-notransferase (OAT) et argininosuccinate lyase (ASL) et synthetase (ASS)) par les apports en proteines de la ration alimentaire. Le but de ce travail a ete d’etudier l’influence d’apports croissants en acides amines (AA) sur l’expression de ces enzymes dans un modele de cellules intestinales, les cellules Caco-2/TC7. Materiel et methodes Des cellules Caco-2/TC7 ont ete cultivees sur filtre semi-permeable pendant 15 jours en milieu standard puis ont ete reparties en 4 groupes (n=3). Les cellules ont alors ete exposees pendant 3 jours a differents milieux depourvu d’AA (0), ou contenant des concentrations croissantes en AA, correspondant a une (1X), deux (2X) et quatre (4X) fois les concentrations physiologiques plasmatiques post-prandiales. Les ARNm codant pour les enzymes d’interet ont ete quantifies par PCR en temps reel. Les resultats ont ete analyses par regression lineaire (test de Student sur la pente). Resultats Il existe une relation inverse entre les apports en AA et l’expression de la glutaminase (p= 0,028), de l’OAT (p= 0,035), de l’ASS (p= 0,028) et de l’arginase (p= 0,058). Conclusions Cette etude montre pour la premiere fois un effet direct des AA sur la regulation au niveau transcriptionnel des enzymes du carrefour intestinal de l’arginine en fonction de l’apport Tableau 1 [AA] Glutaminase Arginase OAT ASS O 1,03 ± 0,34 2,85 ± 1,81 ,33 ± 1,44 1,49±0,48 1X 1,05 ± 0,19 0,96 ± 0,01 1,02 ± 0,01 0,91 ± 0,10 2X 0,34 ± 0,05 1,11 ± 0,15 0,42 ± 0,07 0,55 ± 0,02 4X 0,37 ± 0,10 0,27 ± 0,07 0,29 ± 0,04 0,52 ± 0,11 Les niveaux d’expression des differentes enzymes, rapportees a la quantite d’ARNr 18S azote. Cette regulation permettrait ainsi, dans des situations de faibles apports azotes, de favoriser la synthese intestinale de citrulline et par consequent la neosynthese renale d’Arg. Lors d’apports azotes plus importants, cela favoriserait le passage de l’Arg absorbee dans le systeme porte et donc une stimulation adaptee de l’ureoge-nese hepatique.