L. Cynober

Action of ornithine alpha-ketoglutarate, ornithine hydrochloride, and calcium alpha-ketoglutarate on plasma amino acid and hormonal patterns in healthy subjects.

Ornithine alpha-ketoglutarate (OKG) has been useful as an adjuvant of enteral and parenteral nutrition. However, its metabolism and mechanism of action remain unclear although it is known that alpha-ketoglutarate (alpha KG) and ornithine (ORN) follow, in part, common metabolic pathways. Six fasting healthy male subjects underwent three separate oral load tests: (i) they received 10 g of OKG (i.e., 3.6 g of alpha KG and 6.4 g of ORN); (ii) 6.4 g of ORN as ornithine hydrochloride, and (iii) 3.6 g of alpha KG as calcium alpha-ketoglutarate. Blood was drawn 15 times over a five-hour period for measurements of plasma amino acids, alpha KG, insulin, and glucagon. After OKG and ORN administration, plasma ORN peaked at 60-75 min (494 +/- 91 and 541 +/- 85 mumol/L). The increase in plasma alpha KG was very small. OKG, alpha KG, and ORN all increased glutamate concentrations at 60 min (mean: +43%, +68%, +68%, respectively, p less than 0.05 compared to basal values). However, only OKG increased proline and arginine levels at 60 min (mean: +35%, p less than 0.01 and mean: +41%, p less than 0.05). Furthermore, glutamate, proline, and arginine concentrations correlated linearly with ornithine levels at 60 min. Finally, OKG increased insulinemia and glucagonemia (mean: +24% at 15 min, p less than 0.05 and +30% at 60 min, p less than 0.01, respectively). These data provide evidence that the combination of ORN and alpha KG modifies amino acid metabolism in a way which is not observed when they are administered separately. In addition, the OKG-mediated increase in insulin levels probably does not appear to result from a direct action of ORN on pancreatic secretion.

Hepatic preservation, liposomally entrapped adenosine triphosphate and nitric oxide production: a study of energy state and protein metabolism in the cold-stored rat liver.

Background: Liposomally entrapped adenosine triphosphate (ATP) has been demonstrated to improve energy state and function of the cold-stored liver. The increased nitrite release associated with liposome administration led us to investigate the interactions between liposome supply and nitric oxide (NO) production through the use of L-NAME, a non-selective inhibitor of NO synthesis. Methods: Twenty-four livers from fasted rats were stored for 18 h at +4°C in University of Wisconsin solution directly (control group) or after infusion with ATP-containing liposomes (Lip-ATP), L-NAME (L-NAME) or both (Lip-ATP-L-NAME). Metabolic fluxes, cell volume and energy state were studied during reperfusion. Results: After storage, nitrite release was increased by 61% in the Lip-ATP group, markedly decreased in the Lip-ATP-L-NAME group and almost abolished in the L-NAME group. The ATP content was increased by 20% in the Lip-ATP group ( P < 0.05 versus control) and on reperfusion this was associated with an increase in cell volume (17%; P < 0.05) and a decrease in branched-chain amino acid release (21%; P < 0.01). The simultaneous addition of L-NAME did not affect these results, but induced a large (6-fold) increase in glucose production, possibly related to the metabolism of glycerol supplied by the liposomes. In the L-NAME group, global amino acid release was 50% lower and was associated with a dramatic decrease in urea production while the energy state deteriorated rapidly. Conclusions: The improvement in energy state and anabolic cell swelling induced by ATP-containing liposomes seems to be independent of NO synthesis. On the other hand, inhibition of NO synthesis appears to exert a detrimental effect on the liver, presumably through the decrease in hepatic energy content.

Amino acid metabolism in isolated perfused rat liver

Conflicting evidence concerning hepatic amino acid (AA) metabolism in the isolated perfused rat liver (IPRL) led us to investigate the response of IPRL using perfusates with various AA contents. Perfusion (n = 4) with whole rat blood diluted in Krebs buffer (1:3, v/v) led to acute proteolysis on account of AA deprivation, as shown by the large release of AA (approximately 1400 mumoles in 120 min), especially branched-chain AA (BCAA) (e.g., Leu, 35.4 +/- 10.4 nmole.min-1.g-1 the first hour, 34.3 +/- 5.5 nmole.min-1.g-1 the second hour). In a first attempt to prevent proteolysis, livers (n = 4) were perfused with the previous medium supplemented with AA known for their antiproteolytic activity, at twice their physiological concentrations. Results during the first hour showed uptake of several AA (mainly alanine, glutamine, and proline), reduced release of BCAA (leucine, 12.5 +/- 6.3 nmole.min-1.g-1), and an increase in glucose and urea production. However, during the second hour, because of the use of a recirculating system, progressive AA depletion induced a reappearance of proteolysis. A two-step AA loading technique, i.e., the addition of antiproteolytic AA at the beginning of the perfusion and the addition of a balanced AA mixture at 60 min caused a further decrease in proteolysis during the 2 hr of perfusion (n = 6). Under these conditions, most AA were taken up by the liver with uptake values comparable to those observed in vivo.

Effect of apoE/ATP-containing liposomes on hepatic energy state

Background/Aims: ATP‐containing liposomes partially prevent ATP depletion in the cold‐stored liver. As hepatocytes can specifically bind apoE, we investigated whether the addition of apoE to large (200 nm) ATP‐containing liposomes increases their uptake by the liver and further improves hepatic energy stores.

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.

O021 Méthode simple et rapide pour le dosage simultané des vitamines A et E dans les mélanges polymériques pour nutrition entérale

Introduction et but de l’etude Dans les melanges polymeriques destines a la nutrition enterale (MPE), les vitamines liposolubles sont presentes sous forme de differents esters. Le dosage du contenu total en ces vitamines necessite de les liberer de ces liaisons esters par une premiere etape de saponification, etape delicate du fait de la sensibilite de ces molecules a l’oxydation. Les methodes existantes sont peu sensibles, fastidieuses, couteuses et peu adaptees a une utilisation en routine. Le but de cette etude a ete de developper une methode simple et rapide pour doser simultanement les vitamines A et E dans les MPE. Materiel et methodes Apres optimisation des etapes de saponification et d’extraction, la procedure analytique suivante a ete retenue. A 100 μl de MPE (Sondalis ® Iso, Nestle Clinical Nutrition) sont ajoutes 50 |il d’un etalon interne (Tocol 64 |JM). L’echantillon est ensuite deproteinise par addition de 900 μl d’ethanol absolu contenant 1 % de pyrogallol (antioxydant) et saponifie par 200 μl d’une solution de KOH (3,6 M) dans un sonicateur (Branson 2210), 30 min a 65°C et sous atmosphere d’azote. Les echantillons sont alors tamponnes par 500 μl de NaH2PO4 (0,2 M, pH 7,8) et une double extraction a l’hexane est realisee. La separation et la quantification des vitamines sont realisees par chromatographie liquide haute performance en phase inverse (colonne DiscoveryTM C8, 25 cm × 4,6 mm, 5 μm) (avec une phase mobile methanol-acetonitrile-eau 75/15/10, v/v) et une detection spectrophotometrique a deux longueurs d’onde (292 et 325 nm). La linearite, la repetabilite (n = 10) et la reproductibilite (n = 20 en 4 series) ont ete determinees et leurs coefficients de variation (CV) ont ete calcules. Resultats Les temps de retention des vitamines A et E sont respectivement de 5,7 et 14,1 min. Les parametres de validation sont les suivants : Tableau 1 Vit A Vit A Vit E Vit E Linearite min : 0,2 μ M max : 10,0 μ M min : 3,0 μ M max : 84,4 μ M Repetabilite 4,7 ± 0,2 μ M CV : 4,0 % 30,6 ±1,5 μ M CV: 4,9 % Reproductibilite 5,2 ± 0,3 μ M CV : 4,7 % 27,2 ± 1,4 μ M CV: 5,0 % Conclusions Cette procedure validee est bien adaptee en termes de sensibilite. Elle est simple a realiser, peu couteuse et applicable en routine.

Pharmaconutriments azotés: Glutamine, arginine, α-cétoglutarate ďornithine

Ľapport de substrats azotes en nutrition artificielle a longtemps ete defini en termes de synthese proteique, afin de promouvoir ľaccretion proteique ou tout au moins de preserver la masse maigre en situation catabolique. Il se fondait essentiellement sur deux criteres: ľapport quantitatif, permettant ďameliorer ou de positiver le bilan ďazote, et ľapport qualitatif tenant compte de la necessite de fournir les acides amines dits essentiels. Si la notion ďessentialite de certains acides amines, reposant sur ľincapacite de ľorganisme humain a les synthetiser, est bien etablie, certains aspects metaboliques ont ete sous-estimes. Tout ďabord, le fait qu’un acide amine soit dit non essentiel ou, pour ľarginine, semi-essentiel (car indispensable chez ľenfant en croissance) chez un individu sain, ne signifie pas que la capacite de ľorganisme a le synthetiser soit suffisante quelles que soient les circonstances pathologiques. Ensuite, en dehors de leur role de substrat pour les syntheses proteiques, certains acides amines sont impliques dans la modulation de ľetat nutritionnel du fait de leurs proprietes particulieres.