Christian Aussel

Citrulline: from metabolism to therapeutic use.

Citrulline possesses a highly specific metabolism that bypasses splanchnic extraction because it is not used by the intestine or taken up by the liver. The administration of citrulline may be used to deliver available nitrogen for protein homeostasis in peripheral tissues and as an arginine precursor synthesized de novo in the kidneys and endothelial and immune cells. Fresh research has shown that citrulline is efficiently transported across the intestinal luminal membrane by a set of transporters belonging to the B⁰,⁺, L, and b⁰,⁺ systems. Several pharmacokinetic studies have confirmed that citrulline is efficiently absorbed when administered orally. Oral citrulline could be used to deliver arginine to the systemic circulation or as a protein anabolic agent in specific clinical situations, because recent data have suggested that citrulline, although not a component of proteins, stimulates protein synthesis in skeletal muscle through the mammalian target of rapamycin signaling pathway. Hence, citrulline could play a pivotal role in maintaining protein homeostasis and is a promising pharmaconutrient in nutritional support strategies for malnourished patients, especially in aging and sarcopenia.

Fat mass protects hospitalized elderly persons against morbidity and mortality

BACKGROUND It is well established that the combination of protein-energy malnutrition and low body mass index (BMI) increases the risk of death in elderly patients, but recent studies indicate that the risk of death may decrease with higher body weight. However, these studies did not perform direct, separate, and reliable measurements of fat and lean mass by using a reference technique. OBJECTIVE Our objective was to evaluate the relation between body composition, based on the 4-compartment model, and morbidity and mortality in hospitalized elderly patients. DESIGN This prospective study enrolled 125 elderly patients evaluated at admission for body composition on the basis of BMI plus fat mass, lean mass, appendicular skeletal muscle mass, and body cell mass indexes (calculated as the ratio of the mass to the square of the height) measured by X-ray absorptiometry and bioelectrical impedance analysis. Outcomes were assessed 6 mo later by using a score system that takes into account complications (pressure ulcers and/or infections) and 6-mo mortality. RESULTS The fat mass index correlated positively (r = 0.19 and P = 0.023 adjusted for sex; r = 0.18 and P = 0.043 adjusted for sex, albuminemia, and C-reactive protein) with outcome score (1: death, 2: complications, 3: no complications). There was no significant correlation between outcome score and BMI, lean mass, appendicular skeletal muscle mass, and body cell mass indexes. CONCLUSIONS This study clearly indicates that the generally accepted rule that overweight is associated with morbidity and mortality does not apply to hospitalized elderly patients, for whom fat mass is associated with a decreased risk of adverse events.

Evidence that albumin is not a suitable marker of body composition-related nutritional status in elderly patients

OBJECTIVE Serum albumin has long been used in clinical practice as a marker of protein-energy undernutrition, but very few studies have focused on its relation with dual-energy X-ray absorptiometry-assessed lean mass measurements, which is the current reference method in routine for body composition-related nutritional status. Serum albumin concentration is also affected by non-nutrition-related factors, and there is published evidence on the relation between serum albumin concentration and morbidity/mortality in the elderly. This study was designed to examine the relationship between serum albumin and lean mass and nutrition-related risk of morbidity/mortality in geriatric patients. Our objective was to clarify whether serum albumin in geriatric patients is a marker of body composition-related nutritional status, risk of morbidity/mortality, neither, or both. METHODS This prospective study enrolled 125 elderly patients hospitalized in a rehabilitation unit [83.8 (SD 7.7) y]. Subjects were evaluated for serum albumin concentration and nutritional status at admission [body mass index, lean mass, appendicular skeletal muscle mass index, and body cell mass index (calculated as the ratio of the mass to the square of the height), evaluated by dual-energy X-ray absorptiometry combined with bioelectrical impedance analysis]. Outcome scores were assessed 6 mo later, taking into account complications (pressure ulcers and/or infections) and 6-mo mortality. RESULTS Serum albumin concentration was not correlated with the lean mass, appendicular skeletal muscle mass, or body cell mass indexes. Serum albumin concentration was, however, correlated with outcome score (r = 0.22, P = 0.02). CONCLUSION This study clearly demonstrates that albumin is not suitable as a marker of body composition in elderly patients.

Influence of enterally administered ornithine α-ketogluta rate on hormonal patterns in burn patients

Abstract Plasma concentrations of glucose, insulin, C-peptide, glucagon, cortisol and hGH were measured in burn patients (mean burn surface area 21 percent) treated or not with ornithine α-ketoglutarate (OKG). An increase in basal values of glucose, insulin, C-peptide and cortisol was demonstrated in both groups, whereas hGH values diminished. OKG modified neither insulin nor hGH values 24 h after its enteral administration nor insulin levels within the first 4h after intake. On the other hand, 60 min after enteral nutrition was restarted the hyperglycaemia observed in untreated subjects was reduced by OKG whereas a hyperinsulinism was observed in both groups. These results suggest that: (i) the anticataboliclanabolic action of OKG in burn patients is not mediated by insulin or hGH, (ii) OKG probably induces an increase in glucose tolerance in burn patients, in whom there is a state of insulin resistance. The mechanism of this action requires further study.

Citrulline enhances myofibrillar constituents expression of skeletal muscle and induces a switch in muscle energy metabolism in malnourished aged rats

Citrulline (Cit) actions on muscle metabolism remain unclear. Those latter were investigated using a proteomic approach on Tibialis muscles from male Sprague‐Dawley rats. At 23 months of age, rats were either fed ad libitum (AL group) or subjected to dietary restriction for 12 weeks. At the end of the restriction period, one group of rats was euthanized (R group) and two groups were refed for one week with a standard diet supplemented with nonessential amino acids group or Cit (CIT group). Results of the proteomic approach were validated using targeted Western blot analysis and assessment of gene expression of the related genes. Maximal activities of the key enzymes involved in mitochondrial functioning were also determined. Cit supplementation results in a significant increase in the protein expression of the main myofibrillar constituents and of a few enzymes involved in glycogenolysis and glycolysis (CIT vs. AL and R, p < 0.05). Conversely, the expression of oxidative enzymes from Krebs cycle and mitochondrial respiratory chain was significantly decreased (CIT vs. AL, p < 0.05). However, maximal activities of key enzymes of mitochondrial metabolism were not significantly affected, except for complex 1 which presented an increased activity (CIT vs. AL and R, p < 0.05). In conclusion, Cit supplementation increases expression of the main myofibrillar proteins and seems to induce a switch in muscle energy metabolism, from aerobia toward anaerobia.

Citrulline stimulates muscle protein synthesis in the post-absorptive state in healthy people fed a low-protein diet – A pilot study

BACKGROUND & AIMS Amino acid (AA) availability is critical to maintain protein homeostasis and reduced protein intake causes a decline in protein synthesis. Citrulline, an amino acid metabolite, has been reported to stimulate muscle protein synthesis in malnourished rats. METHODS To determine whether citrulline stimulates muscle protein synthesis in healthy adults while on a low-protein diet, we studied 8 healthy participants twice in a cross-over study design. Following a 3-days of low-protein intake, either citrulline or a non-essential AA mixture (NEAA) was given orally as small boluses over the course of 8 h. [ring-(13)C6] phenylalanine and [(15)N] tyrosine were administered as tracers to assess protein metabolism. Fractional synthesis rates (FSR) of muscle proteins were measured using phenylalanine enrichment in muscle tissue fluid as the precursor pool. RESULTS FSR of mixed muscle protein was higher during the administration of citrulline than during NEAA (NEAA: 0.049 ± 0.005; citrulline: 0.060 ± 0.006; P = 0.03), while muscle mitochondrial protein FSR and whole-body protein turnover were not different between the studies. Citrulline administration increased arginine and ornithine plasma concentrations without any effect on glucose, insulin, C-peptide, and IGF-1 levels. Citrulline administration did not promote mitochondria protein synthesis, transcripts, or citrate synthesis. CONCLUSIONS Citrulline ingestion enhances mixed muscle protein synthesis in healthy participants on 3-day low-protein intake. This anabolic action of citrulline appears to be independent of insulin action and may offer potential clinical application in conditions involving low amino acid intake.

Features, Causes and Consequences of Splanchnic Sequestration of Amino Acid in Old Rats

Rationale In elderly subjects, splanchnic extraction of amino acids (AA) increases during meals in a process known as splanchnic sequestration of amino acids (SSAA). This process potentially contributes to the age-related progressive decline in muscle mass via reduced peripheral availability of dietary AA. SSAA mechanisms are unknown but may involve an increased net utilization of ingested AA in the splanchnic area. Objectives Using stable isotope methodology in fed adult and old rats to provide insight into age-related SSAA using three hypotheses: 1) an increase in protein synthesis in the gut and/or the liver, 2) an increase in AA oxidation related to an increased ureagenesis, and 3) Kupffer cell (KC) activation consequently to age-related low-grade inflammation. Findings Splanchnic extraction of Leu (SPELeu) was doubled in old rats compared to adult rats and was not changed after KC inactivation. No age-related effects on gut and liver protein synthesis were observed, but urea synthesis was lower in old rats and negatively correlated to liver Arg utilization. Net whole-body protein synthesis and arterial AA levels were lower in old rats and correlated negatively with SPELeu. Conclusion SSAA is not the consequence of age-related alterations in ureagenesis, gut or liver protein synthesis or of KC activity. However, SSAA may be related to reduced net whole-body protein synthesis and consequently to the reduced lean body mass that occurs during aging.

Mechanisms and kinetics of citrulline uptake in a model of human intestinal epithelial cells

BACKGROUND & AIMS Citrulline is a major precursor of arginine by de novo synthesis in the kidneys. Oral citrulline supplementation may be beneficial in some clinical conditions. However, citrulline bioavailability depends on its intestinal absorption. Since the mechanism of citrulline transport across the intestine has not been established yet, this study was designed to characterize L-[(14)C]-citrulline uptake by Caco-2 cells. METHODS Caco-2 cells were cultured in a bicameral insert system. Inhibition studies were conducted in the presence of neutral, cationic, acidic and non-metabolized amino acids. We performed control inhibition studies for arginine uptake. RESULTS Citrulline uptake was pH-independent whereas the uptake rate was reduced in the absence of Na(+). Kinetic analysis indicated the involvement of Na(+)-dependent and Na(+)-independent saturable transport components. For competition studies, both the transport components were markedly inhibited by large, small neutral and cationic amino acids. It was also noticed that specific inhibitor of system lBCH inhibited uptake. The inhibition profile of arginine transport was different from that of citrulline transport as arginine uptake was insensitive to BCH. CONCLUSIONS These characteristics suggest that system B(0,+) might be responsible for the Na(+)-dependent uptake of citrulline, whereas Na(+)-independent uptake may include systems L and b(0,+). Our results show that systems involved in citrulline transport are partly different from those involved in arginine transport.

Effect of aging on liver functions—an experimental study in a perfused rat liver model

BACKGROUND Aging is associated with marked changes in the physiology of many organs. Aging of the liver has been little studied and findings are inconclusive. The purpose of this work was to determine the effect of aging on transport and metabolic functions of the liver as assessed by extraction ratio of indocyanine green (ICG) and urea flux respectively. Bile flow was also recorded. As ICG is removed exclusively by the liver without bioconversion, its clearance reflects hepatic functional mass. METHODS Livers from adult (3-month old) or old (24-month old) rats were perfused in a recirculating system for 90 min. At time 30 min, a bolus of 0.125 mg of indocyanine green was introduced in the perfusion buffer. At least every 10 minutes, the perfusion buffer was sampled for the measurements of ICG and urea. Bile flow was closely monitored throughout the experiment. RESULTS Extraction ratio of ICG was increased in livers from old rats (9.49 +/- 2.84 vs 3.70 +/- 1.56% of ICG extracted), whereas urea flux was diminished (0.33 +/- 0.06 vs 1.33 +/- 0.65 micromol/min/% of ICG extracted) and bile flow was unchanged (4.03 +/- 1.02 vs 3.57 +/- 1.34 microl/min). CONCLUSIONS Aging does not affect the different functions of the liver in the same way. It increases hepatocellular uptake function, but decreases the metabolic function of hepatocytes and does not change excretion function. These discrepancies are likely to be of some importance in the study of drug metabolism and action, and so we suggest that results should be corrected for ICG extraction.

Long-lasting improved amino acid bioavailability associated with protein pulse feeding in hospitalized elderly patients: a randomized controlled trial.

OBJECTIVE Aging is associated with a blunted anabolic response to dietary intake, possibly related to a decrease in systemically available amino acids (AAs), which in turn may stem from increased splanchnic AA metabolism. Splanchnic sequestration can be saturated by pulse feeding (80% of daily protein intake in a single meal), enabling increased protein synthesis. The aim of this study was to explore whether protein pulse feeding increased postprandial AA concentrations, and if so whether this increase persisted after 6 wk of dietary treatment. METHODS This prospective randomized study enrolled 66 elderly malnourished or at-risk patients in an inpatient rehabilitation unit. All were given a controlled diet for 6 wk. In a spread diet (SD) group (n = 36), dietary protein was spread over the four daily meals. In a pulse diet (PD) group (n = 30), 72% of dietary protein (averaging 1.31 g/kg body weight daily) was consumed in one meal at noon. The patients were evaluated on day 1 and at 6 wk for plasma postprandial (five times from 0 to +180 min) AA concentrations (expressed as area under the curve above baseline). RESULTS Protein pulse feeding was more efficient than protein spread feeding at increasing plasma postprandial AA concentrations, notably of essential AAs. This increased postprandial AA bioavailability was maintained after 6 wk. CONCLUSIONS This study demonstrates that increased postprandial AA bioavailability induced by protein pulse feeding persists after 6 wk (i.e., that there is no metabolic adaptation blunting AA bioavailability).