Citrulline protects human retinal pigment epithelium from hydrogen peroxide and iron/ascorbate induced damages.

Oxidative stress plays an important role in the ageing of the retina and in the pathogenesis of retinal diseases such as age-related macular degeneration (ARMD). Hydrogen peroxide is a reactive oxygen species generated by the photo-excited lipofuscin that accumulates during ageing in the retinal pigment epithelium (RPE), and the age-related accumulation of lipofuscin is associated with ARMD. Iron also accumulates with age in the RPE that may contribute to ARMD as an important source of oxidative stress. The aim of this work was to investigate the effects of L-Citrulline (CIT), a naturally occurring amino acid with known antioxidant properties, on oxidative stressed cultured RPE cells. Human RPE (ARPE-19) cells were exposed to hydrogen peroxide (H2 O2 ) or iron/ascorbate (I/A) for 4 h, either in the presence of CIT or after 24 h of pretreatment. Here, we show that supplementation with CIT protects ARPE-19 cells against H2 O2 and I/A. CIT improves cell metabolic activity, decreases ROS production, limits lipid peroxidation, reduces cell death and attenuates IL-8 secretion. Our study evidences that CIT is able to protect human RPE cells from oxidative damage and suggests potential protective effect for the treatment of retinal diseases associated with oxidative stress.

Glutamate: A Safe Nutrient, Not Just a Simple Additive.

BACKGROUND: In 2017, a European Food Safety Authority (EFSA) opinion on the use of glutamate and its salts as food additives led to an Acceptable Daily Intake (ADI) of 30 mg/kg body weight/day. Then, in 2021, an EFSA statement presented a proposal for harmonizing the establishment of Health-Based Guidance Values for nutrients that are also regulated substances (including food additives). The present review argues that the 2017 glutamate ADI is unsuitable because safety of glutamate should firstly consider its status as a nutrient and not only as an additive. SUMMARY: Glutamate is a non-essential amino acid playing a key role in nitrogen homeostasis. The dietary exposure to glutamate in adults is extensive, due to its ubiquitous presence in foods, under three forms: bound to proteins, naturally free and free form added as an additive. Glutamate naturally included in proteins is the major source of dietary glutamate. Thus, since it plays a role in nitrogen homeostasis, it is a nutrient before being an additive. Its pharmacokinetics are largely impacted by concomitant food intake, but the extent to which plasma glutamate concentration must rise to have deleterious effects is never encountered in humans consuming glutamate in their daily diets. This is due to the fact that glutamate is highly metabolized in the splanchnic area. KEY MESSAGE: Glutamate should be considered as a safe nutrient before being considered as an additive by risk assessor.

Is N-Carbamoyl Putrescine, the Decarboxylation Derivative of Citrulline, a Regulator of Muscle Protein Metabolism in Rats?

N-carbamoyl putrescine (NCP), the decarboxylation derivative of citrulline, metabolically related to polyamines, may exert biological effects in mammals. The aim of this study was (i) to evaluate the nutritional properties of NCP in healthy rats and (ii) to determine the effect of NCP administration on muscle metabolism in malnourished old rats. The nutritional properties of NCP were first evaluated in 20 8-week-old male rats randomized to receive for two weeks a standard diet either alone (C group) or supplemented with NCP, 5 or 50 mg/kg/d. In a second study, 29 malnourished 18-month-old male rats were studied either before or after a 4-day refeeding with a standard diet either alone (REN group) or supplemented with NCP, 1 or 10 mg/kg/d. NCP had no effect on weight gain and body composition in either of the two studies. In healthy rats, muscle protein content was significantly increased in the soleus with NCP 5 mg/kg/d. A decrease in plasma glutamine and kidney spermine was observed at the 50 mg/kg/d dose; otherwise, no significant changes in plasma chemistry and tissue polyamines were observed. In malnutrition-induced sarcopenic old rats, refeeding with NCP 10 mg/kg/d was associated with higher tibialis weight and a trend for increased protein content in extensor digitorum longus (EDL). While the muscle protein synthesis rate was similar between groups, ribosomal protein S6 kinase was increased in tibialis and higher in the EDL in NCP-treated rats. The muscle RING-finger protein-1 expression was decreased in tibialis and urinary 3-methyl-histidine to creatinine ratio slightly lower with the supply of NCP. However, this initial period of refeeding was also associated with elevated fasted plasma triglycerides and glucose, significant in NCP groups, suggesting glucose intolerance and possibly insulin resistance. NCP was well-tolerated in healthy young-adults and in malnourished old rats. In healthy adults, NCP at 5 mg/kg/d induced a significant increase in protein content in the soleus, a type I fiber-rich muscle. In malnourished old rats, NCP supply during refeeding, may help to preserve lean mass by limiting protein breakdown; however, these effects may be limited in our model by a possible immediate refeeding-associated glucose intolerance.

An oligomeric diet limits the response to injury in traumatic brain-injured rats.

Adequate nutritional support is a major challenge in brain injury patients, because malnutrition cannot be reversed by standard enteral nutrition. We hypothesized that an oligomeric formula could improve nutritional status by restoring intestinal trophicity. Eighteen male Sprague-Dawley rats (300-330 g) underwent gastrostomy on day-7 (D-7) and traumatic brain injury (TBI) by hydraulic percussion (D0) and were then fed for 4 days with either a polymeric formula (Sondalis® HP, TBIP, n = 9), or an oligomeric formula (Peptamen® HN, TBIO, n = 9). In addition, a control group of healthy gastrostomized rats was fed the polymeric diet (control, n = 8). All rats were weighed daily. On D+4, the rats were euthanized. Blood was collected for plasma amino acid determination. Organs were removed and weighed. Intestinal morphometry was studied. Protein content was assessed on intestine and muscles. Enterobacterial translocation and dissemination were evaluated. Results were expressed as means ± SEM and compared using analysis of variance+Newman-Keuls test. TBI induced a significant decrease in whole body weight (TBIP vs. control, p < 0.05) that was totally blunted by the oligomeric diet (TBIP vs. TBIO, p < 0.01). Thymus weight significantly decreased after TBI (TBIP vs. control, p < 0.05) and was restored by the oligomeric formula (TBIO vs. TBIP, p < 0.05). Glutamine (GLN) concentration was improved by the oligomeric diet in both plasma (TBIO: 688 ± 19 vs. control: 591 ± 45 and TBIP: 615 ± 42 µmol/L, p < 0.05) and soleus muscle. These results show that the use of an oligomeric diet may limit response to injury after brain injury and could be a simple nutritional strategy in this setting.

Increasing plasma glutamine in postoperative patients fed an arginine-rich immune-enhancing diet--a pharmacokinetic randomized controlled study.

OBJECTIVE: Immune-enhancing diets (IEDs) rich in arginine (ARG) reduce morbi-mortality in trauma and surgical patients. Among the pharmaconutrients inducing these effects, ARG may be involved by generating active metabolites such as glutamine (GLN). However, the ability of an ARG-enriched diet to normalize GLN plasma levels in intensive care unit (ICU) patients has never been documented. To analyze plasma GLN and related amino acid (AA) kinetics in response to an ARG-enriched IED in ICU surgical patients. DESIGN: This prospective, randomized, single-blind, comparative study was performed on 22 patients randomized to receive total enteral nutrition for 7 days with either an ARG-enriched IED or a standard formula (rendered isonitrogenous to the IED, S group, n = 11), providing 30 kcal/kg/day and 0.3 g N/kg/day. MEASUREMENTS: Plasma AA concentrations were measured on day 5 after a 3-hour washout period (basal values = T0) and after 30, 60, 90, 120, 180, and 360 minutes of enteral nutrition. The primary end point was the variation in plasma GLN from T0 to T90. RESULTS: Only the IED-fed patients showed an increase in plasma levels of GLN (differences [T90 - T0]: +40 +/- 6 vs. -35 +/- 18 micromol/L, mean +/- sem, p < 0.05, two-way analysis of variance), ARG (+35 +/- 5 vs.+1 +/- 4 micromol/L, p < 0.05), ornithine (+23 +/- 6 vs. -2 +/- 2 micromol/L, p < 0.05), and proline (+36 +/- 10 vs. -6 +/- 11 micromol/L, p < 0.05). CONCLUSION: To our knowledge, this is the first reported pharmacokinetic study on an IED even though these products have been on the market for 20 years. Our main result is that administering an ARG-enriched IED causes a significant increase in plasma GLN probably from de novo GLN synthesis from ARG. This suggests that the ARG present in IED can serve to supply GLN to ICU patients, who are usually depleted in this conditionally essential AA during injury.

Tolerance and efficacy of a new enteral formula specifically designed for elderly persons: an experimental study in the aged rat.

For the first time, a formula was specifically designed for the nutritional support of tube-fed elderly patients (Elderly-Specific Formula [ESF], Nestlé, Switzerland). It was tested against a standard formula (Sondalis Iso [SI], Nestlé Clinical Nutrition, Marne la Vallée, France) in sixteen 22-month-old Sprague Dawley rats fed by total continuous enteral infusion for 7 days. Body weight, stool weight, and nitrogen balance were measured daily. After death, muscle weight, plasma levels of amino acids, tissue protein, and amino acid content were measured. The ESF curbed weight loss, improved cumulative nitrogen balance, and increased jejunum protein content. Plasma levels of threonine, leucine, and isoleucine and the sum of total amino acids were higher in ESF-fed than in SF-fed rats. Threonine and isoleucine content in the soleus and gastrocnemius were higher in ESF-fed rats than SI-fed ones. ESF improved intestinal transit. Thus, in old rats, the ESF favored nutritional status more than a standard formula.

Does the ornithine-alpha-ketoglutarate ratio influence ornithine alpha-ketoglutarate metabolism in healthy rats?

Ornithine alpha-ketoglutarate (OKG) is a salt composed of 2 molecules of ornithine (ORN) and one molecule of alpha-ketoglutarate (alphaKG). OKG has been used successfully via oral, enteral, and parenteral routes to improve protein status in patients with chronic and acute protein depletion, but its mechanism of action, which is probably multifactorial, is still unclear. A specific metabolic interaction between alphaKG and ORN has been shown to be a key factor in the effects of OKG, but the impact of the ORN/alphaKG ratio (2 molecules of ORN for 1 molecule of alphaKG) has never been discussed. To clarify this point, young (3 weeks old) male Wistar rats in the postabsorptive state received 5 g/kg of either OKG or a mono-ornithine alphaKG (MOKG) salt (ORN/alphaKG ratio = 1:1) in amounts that were either isonitrogenous or isomolar to OKG, or a saline solution (controls) and were killed 1 hour later. In a second experiment, a kinetic study was performed in which rats were killed 1, 2, 3, or 6 hours after OKG, MOKG, or saline administration. Amino acid contents were analyzed in the plasma, liver, jejunal and ileal mucosae, and the extensor digitorum longus (EDL) muscle. The major metabolites detected after intake of OKG or MOKG (ie, ORN, proline [PRO], and glutamate; OKG and MOKG vs control, P < .05) together with the absence of increased arginine and citrulline levels suggested that ORN was mainly metabolized by the ORN aminotransferase pathway, leading to glutamate and PRO production with accumulation persisting at 6 hours postadministration. This study provides new and important data on the influence of the ORN/alphaKG ratio on OKG metabolism: MOKG-treated rats presented less intestinal ORN than OKG-treated rats (MOKG vs OKG, P < .05), suggesting that ORN/alphaKG ratio influences the rate of ORN availability and metabolism. In addition, the metabolic interaction between ORN and alphaKG (ie, in the presence of alphaKG, ORN metabolism is partially diverted toward PRO production), which is characteristic of OKG metabolism, still takes place even if the salt contains only 1 molecule of ORN instead of two.

Evaluation of a glycine-rich amino acid solution for parenteral nutrition in endotoxemic rats.

OBJECTIVE: It has been shown recently that high amounts of glycine might have some pharmacologic effects (reduction of injury and mortality in endotoxemic rats), but its effects on the nutritional status and protein metabolism during injury are still unknown. The aim of this study was to compare the nutritional effects of a glycine-rich amino acid solution for parenteral nutrition (AFD) with a standard one (Vintene) (glycine, 15 vs. 9 g/L) in endotoxemic rats. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: Male Wistar rats (198 +/- 11 g). INTERVENTIONS: Rats were operated to receive total parenteral nutrition (250 kcal/kg/day, 2 g N/kg/day) with amino acids supplied by either AFD (n = 9) or Vintene (V, n = 6). One day after surgery, corresponding to day 0 of the experiment and to the first day of full-strength total parenteral nutrition, the AFD and V group rats received an endotoxemic shock by intraperitoneal injection of lipopolysaccharide (Escherichia coli, 8 mg/kg). The rats were then studied over 3 days and compared with a healthy ad libitum-fed group (AL, n = 10). MEASUREMENTS AND MAIN RESULTS: The rats were weighed and urine was collected daily to determine nitrogen balance and 3-methylhistidine excretion. On day 3, the thymus, spleen, liver, intestinal mucosa, and muscles were weighed, and amino acids from plasma and tissues were analyzed. Lipopolysaccharide caused the classic endotoxemic shock, of similar intensity in the V and AFD groups (V and AFD not equal AL, p < .05): no weight gain, decreased nitrogen balance (day 3, AL 558 +/- 21, V 83 +/- 28, AFD 123 +/- 25 mg N/day), increased urinary 3-methylhistidine/creatinine excretion (day 3, AL 51 +/- 2, V 91 +/- 13, AFD 87 +/- 14 mumol/mmol), soleus (V -15% and AFD -26 % vs. AL) and thymus atrophy (V -36% and AFD -33%), and spleen hypertrophy (V 51% and AFD 83%). Compared with V solution, AFD has a reduced content of some essential amino acids and proline and an elevated content of glycine, aspartate, and glutamate. These differences were not reflected in tissue or plasma amino acids, except for plasma glycine, which in the AFD group was restored to the level of the AL group (AL 426 +/- 12 and AFD 379 +/- 50 vs. V 251 +/- 31 mumol/L, p < .05). CONCLUSIONS: In endotoxemic rats, the nutritional effects of a glycine-rich AFD solution are similar to those of a standard amino acid solution for parenteral nutrition.

Ornithine alpha-ketoglutarate metabolism in the healthy rat in the postabsorptive state.

To gain further insight into the ability of ornithine alpha-ketoglutarate (OKG) to generate key metabolites, the aim of this work was to study the short-term metabolism, that is, 1 hour after administration, of OKG in plasma and tissues. Particular attention was paid to keto acids (alpha-ketoglutarate and branched-chain keto acids). Young (3 weeks old) male Wistar rats in the postabsorptive state received either 1.5 g/kg of monohydrated OKG (OKG group, n = 8) diluted in distilled water or an equivalent volume of saline solution at 0.9% (control group, n = 8) by gavage and were killed 1 hour later. Plasma, liver, jejunal and ileal mucosa, and the extensor digitorum longus muscle were removed to analyze amino and keto acid contents. Major metabolites detected after OKG ingestion (ornithine [ORN], alpha-ketoglutarate, proline and glutamate; OKG vs control, P < .05) and the absence of increased arginine (and even a decrease in jejunum and muscle) and citrulline levels suggested that ORN was mainly metabolized by the ORN aminotransferase pathway. In addition, significantly decreased plasma branched-chain keto acids and increased hepatic branched-chain amino acids (OKG vs control, P < .05) were observed upon OKG ingestion. Finally, glutamine accumulation restricted to the intestine, as evidenced in this short-term study, suggests that the effects of OKG on glutamine pools in other tissues in various pathological states after several days of treatment, as observed in previous studies, may be related to a long-term induction of glutamine synthetase.