Diagnostic accuracy of des-gamma-carboxy prothrombin for hepatocellular carcinoma in a French cohort using the Lumipulse® G600 analyzer.

The increasing incidence of hepatocellular carcinoma (HCC) in Western countries requests reliable tumour markers for preclinical diagnosis. We evaluated the diagnostic accuracy of des-gamma-carboxy prothrombin (DCP), in comparison with alpha-fetoprotein (AFP) in a French cohort using a new analyser. One hundred and sixty-two patients with virus-related cirrhosis (46 HCC patients and 116 controls) were recruited in this retrospective proof-of-concept study. DCP was measured on new Lumipulse® G600 analyzer and AFP on usual Cobas e602 analyzer in serum samples that were collected at the time of HCC diagnosis for HCC patients or during follow-up for controls. DCP and AFP levels were higher in HCC patients. The area under receiver operating characteristic curve was larger for DCP than for AFP (0.89 vs 0.77, P=.03). At the cut-off value of 128 mAU/mL, sensitivity and specificity for DCP were 74% and 92%. At the cut-off value of 20 µg/L, sensitivity and specificity for AFP were 63% and 82%. NRI>0 for the association of "AFP+DCP" were 101%, P<.0001, and 23%, P=.03, compared to "AFP" or "DCP" alone, respectively. We conclude that DCP outperformed AFP for the detection of HCC.

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 Blood was withdrawn over an 8-h period to measure plasma AA concentrations.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

Granulocytic myeloid-derived suppressor cells inversely correlate with plasma arginine and overall survival in critically ill patients.

Critically ill patients display a state of immunosuppression that has been attributed in part to decreased plasma arginine concentrations. However, we and other authors have failed to demonstrate a clinical benefit of L-arginine supplementation. We hypothesize that, in these critically ill patients, these low plasma arginine levels may be secondary to the presence of granulocytic myeloid-derived suppressor cells (gMDSC), which express arginase known to convert arginine into nitric oxide (NO) and citrulline. Indeed, in a series of 28 non-surgical critically ill patients, we showed a dramatic increase in gMDSC compared to healthy subjects (P = 0·0002). A significant inverse correlation was observed between arginine levels and gMDSC (P = 0·01). As expected, gMDSC expressed arginase preferentially in these patients. Patients with high gMDSC levels on admission to the medical intensive care unit (MICU) presented an increased risk of death at day 7 after admission (P = 0·02). In contrast, neither plasma arginine levels, monocytic MDSC levels nor neutrophil levels were associated with overall survival at day 7. No relationship was found between body mass index (BMI) or simplified acute physiology score (SAPS) score, sequential organ failure assessment (SOFA) score or gMDSC levels, eliminating a possible bias concerning the direct prognostic role of these cells. As gMDSC exert their immunosuppressive activity via multiple mechanisms [production of prostaglandin E2 (PGE2 ), interleukin (IL)-10, arginase, etc.], it may be more relevant to target these cells, rather than simply supplementing with L-arginine to improve immunosuppression and its clinical consequences observed in critically ill patients.

Citrulline and nitrogen homeostasis: an overview.

Citrulline (Cit) is a non-essential amino acid whose metabolic properties were largely ignored until the last decade when it began to emerge as a highly promising nutrient with many regulatory properties, with a key role in nitrogen homeostasis. Because Cit is not taken up by the liver, its synthesis from arginine, glutamine, ornithine and proline in the intestine prevents the hepatic uptake of the two first amino acids which activate the urea cycle and so prevents amino acid catabolism. This sparing effect may have positive spin-off for muscle via increased protein synthesis, protein content and functionality. However, the mechanisms of action of Cit are not fully known, even if preliminary data suggest an implication of mTOR pathway. Further exploration is needed to gain a complete overview of the role of Cit in the control of nitrogen homeostasis.

Effect of citrulline on muscle functions during moderate dietary restriction in healthy adult rats.

Low calorie diets are designed to reduce body weight and fat mass, but they also lead to a detrimental loss of lean body mass, which is an important problem for overweight people trying to lose weight. In this context, a specific dietary intervention that preserves muscle mass in people following a slimming regime would be of great benefit. Leucine (LEU) and Citrulline (CIT) are known to stimulate muscle protein synthesis (MPS) in post-prandial and post-absorptive state, respectively. This makes them interesting bioactive components to test in the context of dietary restriction. We tested the concept of combining LEU and CIT in adult female rats. We postulated that the sequential administration of LEU (mixed in chow) and CIT (given in drinking water before a rest period) could be beneficial for preservation of muscle function during food restriction. Sixty female rats (22 weeks old) were randomized into six groups: one group fed ad libitum with a standard diet (C) and five food-restricted groups (60 % of spontaneous intake for 2 weeks) receiving a standard diet (R group), a CIT-supplemented diet (0.2 or 1 g/kg/day, CIT0.2 group and CIT1 group, respectively), a LEU-supplemented diet (1.0 g/kg/day) or a CIT + LEU-supplemented diet (CIT + LEU 1.0 g/kg/day each). At the end of the experiment, body composition, muscle contractile properties and muscle protein synthesis (MPS) rate were studied in the tibialis anterior muscle. Dietary restriction tended to decrease MPS (R: 2.5 ± 0.2 vs. C: 3.4 ± 0.4 %/day, p = 0.06) and decrease muscle strength (R: 3,045 ± 663 vs. C: 5,650 ± 661 A.U., p = 0.03). Only CIT administration (1 g/kg) was able to restore MPS (CIT1: 3.4 ± 0.3 vs. R: 2.5 ± 0.2 %/day, p = 0.05) and increase muscle maximum tetanic force (CIT1: 441 ± 15 vs. R: 392 ± 22 g, p = 0.05) and muscle strength (CIT1: 4,259 ± 478 vs. R: 3,045 ± 663 A.U., p = 0.05). LEU had no effect and CIT + LEU supplementation had few effects, limited to adipose mass and fatigue force. The results of this study highlight the ability of CIT alone to preserve muscle function during dietary restriction. Surprisingly, LEU antagonized some effects of CIT. The mechanisms involved in this antagonistic effect warrant further study.

Arginine plus proline supplementation elicits metabolic adaptation that favors wound healing in diabetic rats.

Diabetic patients with wounds are at risk of protein malnutrition, have low arginine plasma levels, and suffer from delayed wound healing. We sought to determine the efficacy of arginine plus proline supplementation on protein and amino acid metabolism and on wound repair in a model of diabetic rats. Eighteen 11-wk-old Zucker diabetic fatty fa/fa male rats underwent a 7-cm abdominal skin incision with implantation of sponges and daily excision of full-thickness round sections of dorsal skin for 5 days. They were randomized to be fed with either a standard formula (S group, Clinutren Iso), a high-protein and arginine (ARG) plus proline (PRO)-enriched formula (ARG+PRO group, Clinutren Repair), or an isonitrogenous isoenergetic control formula (IC group). Nitrogen balance was calculated daily. The rats were euthanized on day 5, and plasma glucose, insulin, amino acids, skin epithelialization, and angiogenesis were measured. In macrophages, we assessed inducible nitric oxide synthase (iNOS) and arginase expression, production of nitric oxide (NO) and amino acid metabolism. Both the ARG+PRO and IC groups showed improved nitrogen balance. ARG plus PRO supplementation increased proline and branched-chain amino acid plasma concentrations and improved angiogenesis. Arginase and iNOS expressions in macrophages were reduced, together with NO and citrulline production. In diabetic rats, ARG plus PRO supplementation improves wound angiogenesis and favors whole body protein metabolism. Low macrophage iNOS expression at day 5 may reflect a low inflammatory state in the wounds, favoring wound closure.

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.

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.

Effect of age, stress and protein supply on plasma amino acids during continuous enteral nutrition; a pragmatic study in rats.

BACKGROUND & AIMS: As life expectancy increases, an increasing older population may require surgery with perioperative nutritional management. While little is known about the combined effect of age and stress on amino acid metabolism during enteral nutrition, we hypothesized that blood amino acid bioavailability may be influenced not only by the characteristics of the ingested protein but also by intestinal ageing and splanchnic sequestration of amino acids. Plasma amino acid kinetics were thus evaluated in aged and adult rats receiving continuous enteral nutrition before and after standardized surgical stress. METHODS: Sixteen 5-month-old and sixteen 21-month-old male rats were used. After a gastrostomy, the insertion of a jugular vein catheter and a one-week recovery, the animals were enterally fed with commercially available formulas containing whole milk proteins or a whey hydrolysate for 24 h before (healthy state) and 18 h after a standardized laparotomy (surgical stress). Data were analyzed by 3-factor ANOVA. RESULTS: In all rats, enteral nutrition was associated with a marked increase in plasma alanine, threonine, lysine and proline (+50 to +150 µmol/L; p < 0.001), and a decrease in glycine (≈-80 µmol/L; p < 0.01). For most amino acids, their availability depended first on the amino acid composition of each protein and second on surgical stress. Aging was only associated with higher tyrosine and threonine availability (p < 0.001). There was only limited statistical interaction between age and surgical stress. CONCLUSION: In rats, plasma amino acid availability during continuous enteral nutrition is determined by the nature of the protein source and the occurrence of stress. The effects of aging on plasma amino acid availability seem very limited. Commonly used formulas therefore appear to be as suitable for elderly patients as for adult patients.

Dietary citrulline does not modify rat colon tumor response to chemotherapy, but failed to improve nutritional status.

During cancer therapy many patients experience significant malnutrition, leading to decreased tolerance to chemotherapy and decreased survival. Dietary citrulline supplementation improves nutritional status in situations such as short bowel syndrome and aging, and is of potential interest in oncology. However, a mandatory prerequisite is to test this amino acid for interaction with tumor growth and chemotherapy response. Dietary citrulline (Cit; 2%), or an isonitrogenous mix of non-essential amino acids (control), was given to Ward colon tumor-bearing rats the day before chemotherapy initiation. Chemotherapy included 2 cycles, one week apart, each consisting of one injection of CPT-11 (50 mg/kg) and of 5-fluorouracil (50 mg/kg) the day after. Body weight, food intake and tumor volume were measured daily. The day after the last injection, rats were killed, muscles (EDL, gastrocnemius), intestinal mucosa, tumor, spleen and liver were weighed. Muscle and intestinal mucosa protein content were measured. Phosphorylated 4E-BP1 was measured in muscle and tumor as a surrogate for biosynthetic activation. FRAPS (Ferric Reducing Ability of Plasma) and thiols in plasma, muscle and tumor were evaluated and plasma amino acids and haptoglobin were measured. Numerous parameters did not differ by diet overall: a) response of tumor mass to treatment, b) tumor antioxidants and phosphorylated 4E-BP1 levels, c) relative body weight and relative food intake, d) weight of EDL, gastrocnemius, intestinal mucosa, spleen and liver and e) plasma haptoglobin concentrations. Moreover, plasma citrulline concentration was not correlated to relative body weight, only cumulated food intake and plasma haptoglobin concentrations were correlated to relative body weight. Citrulline does not alter the tumor response to CPT-11/5FU based therapy but, has no effect on nutritional status, which could be due to the anorexia and the low amount of citrulline and protein ingested.

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.

Citrulline and muscle protein homeostasis in three different models of hypercatabolism.

Supplementation of enteral nutrition (EN) by specific amino acids (AAs) has been proposed to prevent muscle protein loss in intensive care unit (ICU) patients. Citrulline (Cit), which has been shown to stimulate muscle protein synthesis in other situations, may be of interest in this setting. Our aim was to assess the effect of Cit in three catabolic models relevant to critical illness: endotoxemia (LPS), traumatic brain injury (TBI), and TBI with infectious complications (TBI-Ec), which are characterized by different alterations in protein homeostasis. Fifty-eight male Sprague-Dawley rats (200-220 g) were randomized to receive a standard diet ad libitum (CON, n = 9) or to undergo catabolic injuries on day 0 (D0, n = 49), and EN (Sondalis HP energy® 290 kcal/kg/d) from day 1 (D1) combined with Cit (2 g/kg/d) or isonitrogenous non-essential AAs (NEAAs) until day 3 (D3). Endotoxemia was induced by IP injection of LPS from E. coli (3 mg/kg), TBI by hydraulic percussion, and infectious complications (TBI-Ec) by administration of luminescent E. coli on D1. Nitrogen balance (ΔN) and 3-methylhistidine (3-MHis) were measured daily. Muscle protein synthesis (MPS, measured by the SUnSET method) and mTORC1 activation (S6K-1 and 4E-BP1 phosphorylation) were measured on D3 2 h after the arrest of enteral nutrition in soleus, extensor digitorum longus (EDL), gastrocnemius and tibialis muscles. ΔN was lower (p < 0.001) in all three models of injury compared with basal and CON from D1 to D3, and more negative in the LPS-CIT (p < 0.05) than in the LPS group. The 3-MHis/creatinine ratio was significantly increased on D1 in all groups compared with CON, and on D2 only in the LPS and TBI groups (p < 0.0001, LPS and TBI vs. CON). MPS was similar in all groups in soleus and tibialis but significantly higher in EDL in LPS-CIT [LPS-CIT: 4.5 ± 1.7 (mean ± SD) vs. CON: 2.3 ± 1.2; and vs. LPS-NEAA: 3.1 ± 2.3] and in gastrocnemius (LPS-CIT vs. CON; p = 0.05). S6K-1 phosphorylation in the EDL was also higher in LPS-CIT vs. CON (LPS-CIT: 0.94 ± 0.51 CON: 0.42 ± 0.28), but not in gastrocnemius. IL-6 plasma level was significantly higher in all the catabolic groups vs. CON (p < 0.005) with no difference between treatments (Cit or NEAAs). In conclusion, the TBI model showed only a rise in muscle proteolysis, whereas the LPS model displayed a rise in both protein synthesis and proteolysis. Secondly, our results show that the Cit effect varies according to the type of injury and to the muscle under study. The stimulation of MPS rate and the mTOR pathway in LPS-treated rats contrasts with degraded ΔN, suggesting that the Cit effect on protein metabolism in critically ill rats is limited at the whole-body level.

Effect of aging on the availability of amino acids from an immune-enhancing diet (IED) after a surgical stress in rats.

BACKGROUND & AIMS: Dietary amino acid (AA) requirements increase after a surgical stress while the systemic AA availability from the diet decreases with age, due to splanchnic sequestration. While immune-enhancing diets (IEDs) have been recommended for the nutritional management of surgical patients, the systemic bioavailability of their AA supply has not been evaluated in elderly surgical patients. This was determined in surgically-stressed IED-fed aged rats. METHODS: Thirty-four 5-month- or 21-month-old male Sprague-Dawley rats were used. After a gastrostomy and placement of a jugular vein catheter and a one-week recovery period, the animals underwent two 24 h-enteral feedings with an arginine-enriched IED (Impact®, Nestlé Health Science) before (healthy state) and 18 h after a standardized laparotomy, used as a model of surgical stress. During enteral nutrition, blood samples were repeatedly collected to measure plasma AA bioavailability (incremental areas under the curve) at 2, 5 and 24 h. Surgical stress was evaluated from urinary catecholamines and plasma protein profile. RESULTS: Whatever the age or stress situation, IED feeding was associated with decreased plasma glycine and increased alanine, proline and arginine. Aging was mainly associated with a delayed plasma AA accumulation in the first hours after the initiation of enteral nutrition. Stress was associated with higher plasma arginine increase and lower histidine, methionine, phenylalanine and tyrosine accumulation. Age and stress interactions seem limited. CONCLUSIONS: AA bioavailability from an arginine-enriched IED seems to be maintained whatever age and stress situation. Aging appears to be mainly associated with a delay in plasma AA accumulation probably related to age-associated splanchnic sequestration of AAs. Additional effects of surgical stress per se seem limited.

Malnutrition with hypoaminoacidemia in a 22-year-old pregnant patient masking a likely ornithine transcarbamylase deficiency.

BACKGROUND: Symptoms and clinical presentations of OTC deficiency vary widely according to the remaining activity of the enzyme. Three factors determine the residual enzyme activity. First, as the OTC gene is carried on the X chromosome, a complete inactivation of this enzyme in a newborn boy results an acute ammonia intoxication. Second, the female mosaicism due to lyonization (differential randomized X-inactivation) leads to differential OTC expression in hepatocytes. Third, the degree of severity depends on the mutation and the level of remaining activity it leaves to the protein. Published cases of OTC deficiency during pregnancy are scant. Most often, diagnosis of the metabolic disease is made before pregnancy or during the post-partum period. METHODS: We report the case of a 22-year-old woman's successful pregnancy with a moderate form of ornithine transcarbamylase (OTC) deficiency, unsuspected before pregnancy, biochemically consistent with plasma aminoacidogram and orotic acid analysis, and initially masked by malnutrition. RESULTS - CONCLUSION: Although maternal ammonia was subnormal and the neonate was safe, an OTC deficiency was revealed by stress factors such as the pregnancy itself and infection, and associated with uncontrollable maternal vomiting and psychiatric syndrome. However, this metabolic disease, revealed by aminoacidogram and urine orotic acid analysis, fortunately did not prevent a successful pregnancy. Even if infrequent, this situation deserves to be highlighted.

Current data on ATP-containing liposomes and potential prospects to enhance cellular energy status for hepatic applications.

The pharmacological use of adenosine triphosphate (ATP), although promising, is restricted due to poor cellular penetration and drastic hydrolysis that is markedly accelerated in vivo by ectoenzymes. In the literature, liposomes have proven efficient in offering a physical barrier to extracellular enzymes and favor penetration into cells. First, this review addresses the issues raised by ATP development in pharmaceutics. Second, studies conducted with ATP liposomally entrapped (lipo-ATP) are described, including pharmaco-technical formulation engineering and related models of assessment. Finally, potential directions for research to better target ATP penetration into the liver are considered. Lipo-ATP were formulated for a number of applications, including sepsis-related disorders; spermatozoid alteration; brain ischemia episodes; and ophthalmic, cardiac, and hepatic use. Key formulation parameters need to be carefully considered to optimize stability and entrapment yield value, and to define the manufacturing process. Positive lipids, such as stearylamine, increase entrapment yield value by electrostatic interaction with negatively charged ATP. A freezing-thawing step in the manufacturing process considerably increases entrapment yield value. Lipo-ATP were assessed using cell culture, isolated organs, and animal experimental models. Very promising results were obtained with antimyosin PEGylated immunoliposomes using isolated rat hearts and experimental myocardial infarction in rabbits. In hepatic applications, lipo-ATP are effective in preventing liver injury during shock and to improve the energy status of cold-stored rat liver, in particular, if liposomes are loaded with apolipoprotein E (ApoE). For liver delivery, liposome size needs to be lower than 100 nm to allow diffusion through the Disse space, but liposome flexibility and lipid content may also influence liver uptake. The role of the liposome charge remains unclear. ApoE and the ligand for the asialoglycoprotein receptor [ASGPr) were both used in the literature, but the ASGPr seems more promising. Ligand-ASGPr interaction is based on the sugar preference (N-acetylgalactosamine>>galactose), the antennary structure (tetra>tri>di>monoantennary), and sugar spacing. Numerous high-affinity ligands have been extracted or designed to target hepatocytes, which can be classified according to their origin (i.e., natural, hemisynthetic, or synthetic). Synthetic ASGPr, such as Gal-C4-Chol (cholesten-5-yloxy-N-(4-((1-imino-2-D-thiogalactosylethyl)formamide), are composed of a lipid anchor (e.g., cholesteryl), a spacer (C2 to C6 chain), and a sugar head (galactose or lactose). The formulation includes ligand incorporation, by either simple preincubation or covalent graft, onto preformulated liposomes or direct mixing with other lipids. The ligand-loaded liposomes encapsulated pharmacological agents, markers, or plasmid DNA. Interesting results were obtained with antitumor or antioxidant agents to promote drug penetration in cell culture (e.g., primary rat hepatocyte or HepG2) and specific targeting to hepatocyte in isolated perfused liver (pharmacokinetic studies). Effectiveness was demonstrated in experimental models (e.g., tumor-bearing animals and hepatotoxic models). These targeted formulations were less toxic than standard formulations and controls. A development scheme that can be applied to other drugs, which may benefit from improved hepatic targeting, is proposed to optimize liposome characteristics and ligand structure, including verifications such as the displacement-binding test, ligand incorporation, cell internalization, tissue diffusion, organ and receptor specificity, and efficiency in experimental models.

Simultaneous determination of retinol and alpha-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.

Effect of ornithine alpha-ketoglutarate on glutamine pools in burn injury: evidence of component interaction.

BACKGROUND: Ornithine alpha-ketoglutarate (OKG) has proved to be efficient in restoring glutamine (Gln) pools which are strongly depleted in hypercatabolic patients. Since its two components, alpha-ketoglutarate (alphaKG) and ornithine (Orn), give rise to glutamate (Glu), they are both considered as Gln precursors. The aim of this study was to assess the relative contributions of Orn and alphaKG to Gln generation in a rat model of burn injury. METHODS: Forty-eight young Wistar rats were scalded to give a 20% burn surface area. They were fasted for 24 h and then refed by enteral nutrition for 48 h by gavages with Osmolite (Abbott-Ross, 210 kcal/kg day(-1), 1.18 N/kg day(-1)) supplemented with either 5 g OKG/kg day(-1) (B-OKG), Orn (isomolar to OKG; B-Orn), alphaKG (isomolar to OKG; B-KG) or glycine (as an isonitrogenous control; B-Gly). Rats in the B-KG group also received glycine to make all the groups isonitrogenous. Amino acid concentrations were determined in plasma, muscles, jejunal mucosa and liver. RESULTS: The alpha-KG-enriched diet had no effect on plasma Glu content or plasma and muscle Gln content compared with the burn-injured controls. The Orn-enriched diet significantly increased (p<0.01) muscle Glu and Gln contents but not plasma Gln content. In OKG-treated animals, plasma Gln as well as muscle Glu and Gln were significantly higher than in the control (p<0.01), alpha-KG-treated (p< 0.01) and Orn-treated (p<0.05 for muscle Gln and p<0.01 for plasma Gln) animals. CONCLUSION: OKG was more efficient than Orn or alphaKG alone in restoring Gln pools in plasma and muscle, which is evidence of metabolic interaction between the two components of this molecule.

Is transthyretin a good marker of nutritional status?

The assay of plasma transthyretin (TTR), also known as prealbumin, is a key step in the assessment of nutritional status. However, it remains unclear whether it really is a useful nutrition marker, and when and how to use it and interpret TTR levels and variations. Risk of malnutrition, malnutrition severity, prognosis associated with malnutrition and effectiveness of refeeding are four parameters in nutritional assessment, and need clear separation to understand the associated utility of TTR. TTR does not have the same impact and potential on each of these parameters: it can be helpful but not essential for evaluating the risk of malnutrition, and it can diagnose malnutrition and its severity in patients with no inflammation syndrome. TTR is a good marker for prognosis associated with malnutrition, and is even better for monitoring refeeding efficacy despite inflammation. Thresholds depend on the purpose for which it is used. We propose a simple algorithm to guide the interpretation of TTR levels as a helpful tool for day-to-day practice.

Defining malnutrition: A plea to rethink.

In a recent consensus report in Clinical Nutrition the undernourished category of malnutrition was proposed to be defined and diagnosed on the basis of a low BMI or unintentional weight loss combined with low BMI or FFMI with certain cut off points. The definition was endorsed by ESPEN despite recent endorsement of a very different definition. The approach aims to assess whether nutritional intake is sufficient but is imprecise because a low BMI does not always indicate malnutrition and individuals with increasing BMI's may have decreasing FFM's. The pathophysiology of individuals, considered to be malnourished in rich countries and in areas with endemic malnutrition, results predominantly from deficient nutrition combined with infection/inflammation. Both elements jointly determine body composition and function and consequently outcome of disease, trauma or treatment. When following the consensus statement only an imprecise estimate is acquired of nutritional intake without knowing the impact of inflammation. Most importantly, functional abilities are not assessed. Consequently it will remain uncertain how well the individual can overcome stressful events, what the causes are of dysfunction, how to set priorities for treatment and how to predict the effect of nutritional support. We therefore advise to consider the pathophysiology of malnourished individuals leading to inclusion of the following elements in the definition of malnutrition: a disordered nutritional state resulting from a combination of inflammation and a negative nutrient balance, leading to changes in body composition, function and outcome. A precise diagnosis of malnutrition should be based on assessment of these elements.

Modulation of insulin action on 2-deoxyglucose uptake by chloroquine in chick embryo fibroblasts.

Blazar et al. recently found that chloroquine therapy decreased intravenous insulin requirements in a case of extreme insulin resistance. However, no relationship has been shown to exist between insulin degradation and the stimulation of glucose uptake. In this study we investigate the action of insulin on glucose uptake by the ability of this hormone to stimulate 2-deoxyglucose. The effect on alpha-aminoisobutyrate uptake, which is known to be insulin sensitive, is also investigated. Cell-associated 125I-labeled insulin and trichloroacetic acid-soluble and -precipitable substances were measured in parallel. Chloroquine increased insulin-stimulated uptake of 2-deoxyglucose and alpha-aminoisobutyrate. Three hours were required for this effect to appear, and it did not depend on DNA synthesis. Chloroquine also increased cell-associated insulin and slightly decreased the percentage of trichloroacetic acid-soluble products. Methylamine affected neither nutrient uptake processes nor insulin binding and degradation; however, it did abolish the effect of chloroquine on these parameters. These data suggest that in chick embryo fibroblasts a relationship may exist between the increase in undegraded cell-associated insulin and the ability of the hormone to stimulate sugar and amino acid uptake.