The importance and dosage of amino acids in nutritional support of various pathological conditions in ICU patients

Background. Normal adults require twenty L-amino acids (AA) for protein synthesis. Functional AA regulate key metabolic pathways that are necessary for maintenance, growth, reproduction and immunity. Dietary supplementation with one or a mixture of these AA may be beneficial for ameliorating health problems at various stages of the life cycle and for optimizing of the efficiency of metabolic transformations. During disease, other amino acids also become essential. The principal goal of protein/amino acid administration in various pathological conditions in intensive care unit (ICU) patients is to provide the precursors of protein synthesis in tissues with high turnover and to protect skeletal muscle mass and function. Amino acid requirements in parenteral nutrition (PN) are higher when the patient is stressed/ traumatized/infected than in the unstressed state. In severely ill ICU patients a higher provision of protein and amino acids has been associated with a lower mortality. Methods and Results. An overview of the effects and dosage of amino acids in nutritional support of various pathological conditions in ICU patients is presented. Conclusion. It was demonstrated that 2.0–2.5 g protein substrate/kg normal body weight/day is safe and could be optimal for the most critically ill adults to decrease the risk of morbidity and mortality in some pathological conditions.


INTRODUCTION
Amino acids (AA) are not only cell signaling molecules but are also regulators of gene expression and the protein phosphorylation cascade.AA are key precursors for the synthesis of hormones and low-molecular weight nitrogenous substances.Some AA regulate key metabolic pathways are necessary for maintenance, growth, reproduction, and immunity.They are called functional AA, which include arginine, cysteine, glutamine, leucine, proline, and tryptophan.Dietary supplementation with one or a mixture of these AA may be beneficial for (1) ameliorating health problems at various stages of the life cycle (e.g., fetal growth impairment, neonatal morbidity and mortality, weaning-associated intestinal dysfunction and wasting syndrome, obesity, diabetes, cardiovascular disease, the metabolic syndrome, and infertility); (2) optimizing efficiency of metabolic transformations to enhance muscle growth and athletic performance, while preventing excess fat deposition and reducing adiposity1.
Normal adults require twenty L-amino acids for protein synthesis, although only leucine, isoleucine, valine, lysine, threonine, phenylalanine, methionine and tryptophan cannot be synthesised and so are essential (Table 1).The minimal daily requirement in a normal healthy adult is about 0.25 g/day of tryptophan (the requirement for tryptophan decreases with age, the minimum daily requirement for an adult is 3 mg/kg/day or about 160 mg/ day in females and 250 mg/day in males 2,3 ).Assuming a mean total protein requirement of 0.66 g/kg/day, intakes of about 0.18 g/kg/day and 0.48 g/kg/day of indispensable and dispensable amino acids, respectively, or preformed α-amino nitrogen (28 mg nitrogen/kg/day and 78 mg nitrogen/kg/day, respectively), should be sufficient to maintain body nitrogen homeostasis in healthy adults 4 .
The branched-chain amino acids (BCAA) of leucine, isoleucine and valine are unique in that they bypass the liver and are metabolised almost exclusively by the skeletal muscle.Leucine also stimulates skeletal muscle protein synthesis and inhibits muscle proteolysis (even during sepsis).During disease, other amino acids are also essential (e.g.histidine, cystine/cysteine, glutamine, arginine and tyrosine).Histidine is essential in infants and in patients who have renal failure.Cysteine is essential in premature infants and in critically ill patients.Glutamine is a precursor for renal ammonia production and a crucial substrate for the rapidly dividing cells of both the gastrointestinal mucosa and the immune system.It is also required for the production of the major cellular antioxidant glutathione, a requirement which is increased in the critically ill patient.Arginine is required in sufficient amounts to convert ammonia to urea and is the precursor for endo-thelium derived relaxing factor (i.e., nitric oxide); it may also enhance cell mediated immunity5.
The hypoaminoacidemia of critical illness appears to represent a state of increased amino acid uptake by the rapid turn over of central proteins, which is constrained by the maximum rate of amino acids released from the muscle.This is a portrayal of acute central protein deficiency, and it suggests that sufficient exogenous amino acid provisions could improve clinical outcomes, both early by increasing central protein synthesis, optimizing the inflammatory response, and mitigating the extensive loss of muscle protein characteristic of the first week of catabolic critical illness and in the long term by minimizing the muscle atrophy that commonly occurs in protracted critical illness.The recommendation for metabolically normal hospitalized adults is the same as for healthy people: 0.8 g of protein/kg normal body weight/day.The most common recommendation in critical illness lies between 1.2 and 1.5 g protein/kg normal body weight/day.What is the real need of sufficient protein/amino acids which will reduce the risk of morbidity and mortality of critical illness patients?We carried out a review of the clinical literature of amino acids with the optimum and safe upper limit of protein provision in nutritional support in various pathological conditions in ICU patients.

The conditionally essentials amino acids
Nutrients such as arginine (Arg), refined menhaden oil, and RNA have been found to have immune-stimulating properties 7 .There is compelling evidence that Arg regulates interorgan metabolism of energy substrates and the function of multiple organs.The results of both experimental and clinical studies indicate that Arg is a nutritionally essential AA for spermatogenesis, embryonic survival, fetal and neonatal growth, as well as maintenance of vascular tone and hemodynamics 8 .Arginine plays an important role in many physiologic and biologic processes beyond its role as a protein-incorporated amino acid 9 .It has multiple metabolic fates and thus is one of the most versatile amino acids.Not only is it metabolically interconvertible with the amino acids proline and glutamate, but it also serves as a precursor for the synthesis of protein, nitric oxide (NO), creatine, polyamines, agmatine, and urea.These processes do not all occur within each cell but are differentially expressed according to cell type, age and developmental stage, diet, and state of health or disease 10 .
The speculation that arginine may pose a threat to critically ill patients is mainly based on the concept that critically ill patients are often hemodynamically unstable and that this population is in a state in which Table 1.Protein L-amino acid requirements in adults (modified by ref. 4,5,6  Consequently, delivering supplemental arginine as the substrate for upregulated iNOS will result in increased NO.This increased NO could result in vasodilation and hypotension, leading to greater hemodynamic instability.An alternate, equally valid argument would be that controlled vasodilation would be beneficial in critical illness and sepsis.Unfortunately, few human studies have evaluated arginine as a single agent in critically ill populations.It can be extrapolated from several studies that the 15-30 g of enteral supplemental arginine, which is the amount commonly given when a critically ill patient is being fed enterally at goal rates with immune-modulating formula, is safe and appears to meet the needs of the patient.The appropriate and safe level in the critically ill or hypermetabolic patient in which a proinflammatory state exists is much more difficult to determine.From review of the available animal and human data, arginine appears to be safe and potentially beneficial at doses delivered in immune modulation formulas for most of the hemodynamically stable ICU populations able to tolerate enteral feeding.This would include medical and surgical ICU patients, trauma patients, major surgical patients, postmyocardial infarction, and those with pulmonary hypertension.In major elective surgical patients expected to be admitted to the ICU postoperatively, arginine given before the surgical procedure has been shown to be beneficial.Hemodynamically unstable ICU patients with poor gut perfusion are not candidates for supplemental arginine.If enteral feeding is pursued in patients during hemodynamically unstable periods, it should be done with extreme caution, so as not to result in mesenteric ischemic injury 11 .Dietary supplementation of Arg can enhance wound healing, regulate endocrine activity and potentiate immune activity (Table 2).These finding have led to the use of Arg supplementation as part of an immune-enhancing dietary regimen to help combat the immune suppression seen in such patients 9 .Moreover, a growing body of evidence clearly indicates that dietary supplementation or intravenous administration of Arg is beneficial in improving reproductive, cardiovascular, pulmonary, renal, gastrointestinal, liver and immune functions, as well as facilitating wound healing, enhancing insulin sensitivity, and maintaining tissue integrity (Table 3) (ref. 8).Arginine supplementation may enhance or preserve immune function in high-risk surgical patients and theoretically improve the host's capacity to resist infection 12 .Critically ill patients fed a high-protein diet enriched with Arg, fiber, and antioxidants had a significantly lower catheter-related sepsis rate than patients fed a standard high-protein diet.There were no differences in mortality or ICU and hospital length of stay 13 .Another study suggests that postoperative enteral nutrition with supplemental Arg, RNA, and omega-3 fatty acids instead of a standard enteral diet significantly improved immunologic, metabolic, and clinical outcomes in patients with upper gastrointestinal malignancies who were undergoing major elective surgery 14 .

Author
Effect of supplementation Efron et al. 9 Enhancing wound healing, regulating endocrine activity and potentiating immune activity Wu et al. 8 Beneficial in improving reproductive, cardiovascular, pulmonary, renal, gastrointestinal, liver and immune functions, as well as facilitating wound healing, enhancing insulin sensitivity, and maintaining tissue integrity Daly et al. 12 Enhancing or preserving immune function in high-risk surgical patients and theoretically improving the host's capacity to resist infection Caparrós et al. 13 Significantly lower catheter-related sepsis rate Daly et al. 14 Improving immunologic, metabolic, and clinical outcomes Table 3.Studies in which arginine was given i.v. for various disease conditions (modified by ref. 15 ).

Author Disease I.v. dose Outcome
Barbul et al. 16 Surgical wound 28 g/day ↑ Collagen deposition Facchinetti et al. 17 Preterm labor 1 g/min for 30 min ↓ Uterine contractions Campisi et al. 18 Cardiac 0.66 g/min for 45 min Normalized vasomotor tone in smokers Mehta et al. 19 Pulmonary hypertension 0. Taurine is a sulfonated β -amino acid derived from methionine and cysteine metabolism.It is present in high concentrations in most tissues and in particular in proinflammatory cells such as polymorphonuclear phagocytes.Role for this amino acid has been found in membrane stabilization, bile salt formation, antioxidation, calcium homeostasis, growth modulation, and osmoregulation 23 .Intracellular and plasma taurine levels are high and although cellular taurine is tightly regulated, plasma levels are known to decrease in response to surgical injury and numerous pathological conditions including cancer, trauma and sepsis.Decreased plasma concentrations can be restored with supplementary taurine 24 .In human sepsis, levels of taurine are directly and significantly related to levels of glutamate, aspartate, β-alanine and phosphoethanolamine (and unrelated to other amino acids).Levels of these amino acids increased simultaneously with increasing doses of leucine, isoleucine and valine in total parenteral nutrition (TPN).Decreasing taurine was associated with increasing lactate, arteriovenous O 2 concentration difference and respiratory index, and with decreasing cho-lesterol and cardiac index 25 .Taurine plays a role in the modulation of intracellular free calcium concentration, and although it is one of the few amino acids not incorporated into proteins, taurine is one of the most abundant amino acids in the brain, retina, muscle tissue, and organs throughout the body.All ocular tissues contain taurine.In the retina, taurine is critical for photoreceptor development and acts as a cytoprotectant against stressrelated neuronal damage and other pathological conditions 26 .Taurine supplementation might help forestall the age-related decline in cognitive functions through interaction with the GABAergic system 27 .Gonzales-Contreras's study shows hepatoprotective effect of i.v.dose 22.41 +/-3.57mg/kg/day of taurine 28 .
L-cysteine is now widely recognized as a conditionally essential (or indispensible) sulphur amino acid.Cysteinerich proteins, such as keratin, may have advantages over the simple amino acid or its derivatives, such as nutraceuticals, to safely and beneficially improve antioxidant status in health and disease.Cysteine and glutathione (GSH) metabolism is impaired in neonates and the critically ill.

Author
Effect of supplementation Osowska et al. 36 Improving protein status Smith et al. 31 Increasing protein synthesis and decreasing protein degradation in skeletal muscle and stimulating glycogen synthesis in the liver Houdijk et al. 35 Decreasing the number of infections, a low frequency of pneumonia, sepsis, and bacteraemia Bollhalder et al. 37 Reducing infections, length of stay and mortality Enteral nutrition enriched with cysteine can decrease cysteine catabolism and improve GSH status 29 .Cysteine is essential in premature infants and in critically ill patients 5 .Glutamine (Gln) is normally an abundant amino acid in the body.It has many important metabolic roles, which may protect or promote tissue integrity and enhance the immune system 30 .It is the most abundant free amino acid in circulation and in intracellular pools and a precursor for the synthesis of amino acids, proteins, nucleotides, and many other biologically important molecules.It is the most important precursor for ammoniagenesis in the kidney, the major end product of ammonia-trapping pathways in the liver, a substrate for gluconeogenesis, and an oxidative fuel in rapidly proliferating cells and tissues 31 .
Gln has many important metabolic roles that may protect or promote tissue integrity and enhance the immune system 32 .Gln also has a number of important regulatory roles, such as increasing protein synthesis and decreasing protein degradation in skeletal muscle and stimulating glycogen synthesis in the liver.When glutamine concentrations decrease, the tissue glutamine metabolism increases markedly in many catabolic, stressful disease states and has led to a reconsideration of the classification of glutamine from a nonessential amino acid to the alternative hypothesis that glutamine may be a conditionally essential nutrient 31 .
During stress the body's requirements for Gln appear to exceed the individual's ability to produce sufficient amounts of this AA.Standard formulation of enteral nutrition contains 2-4 g/l of glutamine.However, this dose is insufficient to normalize glutamine plasma concentration.Plasma concentration of glutamine is low in many patients with critical illness and a low level is an independent risk factor for mortality 33 .
Provision of supplemental glutamine in specialized enteral or parenteral feeding may improve protein status and immunocompetence, enhance nutritional management, reduce the number of infections and augment recovery of the seriously ill while minimizing hospital stay (Table 4) (ref. 30,32,34,35,37).The effect of Gln is dose dependent (Table 5).High doses of glycyl-glutamine or alanylglutamine dipeptide are feasible and safe in patients with polytrauma and are not associated with any relevant renal substrate loss 38,39 .
Of all studies involving glutamine supplementation by enteral or parenteral route, there are virtually no reports of adverse or harmful effects.The only condition known to be accompanied by very high plasma glutamine concentrations is acute liver failure, whereas chronic liver failure is not accompanied by high plasma glutamine levels.The pathophysiology behind these high concentrations is not well characterized.There is no literature reporting, that high glutamine concentrations are associated with any of the symptoms of acute liver failure or the prognosis 40 .

The conditionally essential amino acids with omega-3 fatty acids
Immunonutrition in composition of the "conditionally essential" amino acids arginine, glutamine, cysteine, and taurine with omega-3 fatty acids can enhance the immune response in critically ill patients.This is due to the immunomodulating properties of these nutrients 52 .
Omega-3 fatty acids increase bleeding time; decrease platelet aggregation, blood viscosity, and fibrinogen; and increase erythrocyte deformability, thus decreasing the tendency of thrombus formation 53 .It has been increasingly reported that administration of omega-3 fatty acids is beneficial in patiens with inflammatory processes.This effect is most likely caused by different biological characteristics, including an immunomodulating effect of the products derived from omega-3 fatty acids through eicosanoid metabolism.Weiss et al. observed shorter postoperative periods in the intensive care unit and on the regular medical wards, as well as lower rates of severe infections.The results suggest that perioperative administration of omega-3 fatty acids may have a favourable effect on the outcome of patients with severe surgical interventions by lowering the magnitude of inflammatory response and by modulating the immune response 54 .Fish oil -supplemented PN initiated at the onset of sepsis to improve survival, beneficially altering the lipid profile in plasma and erythrocyte membrane, modulate immune function, and regulate inflammatory response in a rat model 55 .

Branched-chain amino acids
The metabolic response to severe tissue injury and sepsis releases a flood of amino acids from their muscle reservoir, especially ketogenic amino acids valine, leucine, isoleucine.The supplementation of BCAA appears to be beneficial.
BCAA of isoleucine, leucine and valine are unique in that they bypass the liver and are principally metabolized extrahepatically in the skeletal muscle.Leucine also stimulates skeletal muscle protein synthesis and inhibits muscle proteolysis -even during sepsis 5,56,57 .Plasma concentrations of the BCAA are more prominently affected than the concentrations of other amino acids by changes in dietary-caloric, protein, fat, and carbohydrate-intake in man 58 .It is concluded that amino acids, particularly the branched-chain ones, increase the sensitivity of muscle protein synthesis to insulin 59,60 .But in critically ill septic patients, une modulation consistently improve either survival or morbidity.BCAA are recommended in treatment of liver failure.The rationale for recommendation is based on their unique pharmacologic properties, stimulatory effect on ammonia detoxification to glutamine, and decreased concentrations in liver cirrhosis 61 .Potential areas of further research may include the combination of BCAA supplements with other anabolic factors (e.g.growth hormone (GH)) in managing patients with catabolic disease states 56 .Specific amino acids, such as arginine, lysine and ornithine, can stimulate GH release when infused intravenously or administered orally 62 .The chronic BCAA effects on plasma levels of GH and plasma levels of GH binding protein might suggest an improvement of muscle activity through protein synthesis 63 .For a list of clinical trials evaluating branched-chain amino acids therapy, see Table 6.
Studies both in vivo and in vitro have shown that leucine at a very high dose can stimulate muscle protein synthesis, an effect that is enhanced in vivo by insulin secreted in response to the leucine dose.High levels of leucine can also inhibit protein degradation in skeletal muscle, as well as in the liver.In contrast, at normal physiological levels, increasing leucine concentration by infusion stimulates muscle protein synthesis by enhancing its sensitivity to insulin 73 .Leucine was demonstrated to positively affect protein synthesis in an experimental model of sepsis or burn.BCAA supplementation in septic patients also demonstrated an improvement in patients' nutritional status and outcome 57 .
Doi et al. reports of isoleucine stimulating glucose uptake in rat skeletal muscle in vivo, and these results indicate that there might be a relation between the reduction in blood glucose and the increase in skeletal muscle glucose uptake that occur with isoleucine administration in rats.The alterations in glucose metabolism caused by isoleucine may result in an improvement of the availability of ATP in the absence of increases in AMP-activated protein kinase activity in skeletal muscle 74 .

The dosing of amino acids in critically ill patiens
6] ).
Other studies suggest that a protein intake of 1.2 g/ kg/day is currently recommended for inactive healthy individuals, whereas guidelines recommend up to 1.5 g/kg/ day in patients with severe systemic inflammation, such as those affected by critical illness or cancer (Table 8).High protein intake accelerates progression of chronic renal insufficiency but does not affect renal function in healthy individuals 79 .During chronic renal failure (CRF), the aims of nutritional interventions are to minimize uremic toxicity, avoid malnutrition and delay progression of kidney disease.BCAA and Branched chain keto acids (BCKA) supplements have been proposed to decrease further protein intake while maintaining satisfactory nutritional status.Protein restriction together with keto acids and/or essential AAs has been reported to improve insulin sensitivity and hyperparathyroidism and to be compatible with a preservation of nutritional status 80 .Acute renal failure (ARF) is associated with fundamental alterations of metabolism and immunocompetence with the induction of a pro-oxidative and proinflammatory state.A nutritional program for a patient with ARF must consider not only the specific metabolic consequences associated with renal failure and with the underlying disease process but also the profound alterations in nutrient balances induced by replacement therapy.For most patients with ARF requiring nutrition support, evidence suggests that The length of stay in the ICU did not change.Reduced mortality rate, plasma levels of leucine and isoleucine levels increased both essential and nonessential amino acids should be employed.However, there appears to be a therapeutic role for small quantities of essential amino acids, without nonessential amino acids, in selected patients.Data supports the importance of proactive measures to prevent fluid and electrolyte imbalances in patients with ARF (ref. 81,82

ESPEN Guidelines for adult parenteral nutrition
Intensive care -a balanced amino acid mixture should be infused at approximately 1.3-1.5 g/kg/day (ideal body weight) -in ICU patients the amino acid solution should contain 0.2-0.4g/kg/day of L-glutamine (ref. 75)

Surgery
In illness/stressed conditions a daily nitrogen delivery equivalent to a protein intake of 1.5 g/kg ideal body weight (or approximately 20% of total energy requirements) is generally effective to limit nitrogen losses (ref. 76).

ESPEN Guidelines on adult enteral nutrition
Intensive care -during the acute and initial phase of critical illness: in excess of 20-25 kcal/kg/day may be associated with a less favourable outcome -during the anabolic recovery phase, the aim should be to provide 25-30 kcal/kg/day (ref. 77) Surgery -use enteral nutrition preferably with immuno-modulating substrates (arginine, omega-3 fatty acids and nucleotides) perioperatively independent of the nutritional risk for patiens after severe trauma (ref. 78) ARF patients and acutely ill CRF patients on renal replacement therapy, substrate requirements depend on disease severity, type and extent/frequency of extracorporeal renal replacement therapy, nutritional status, underlying disease and complications occurring during the course of the disease.Patients under HD have a higher risk of developing malnutrition.Intradialytic PN should be used if causes of malnutrition cannot be eliminated and other interventions fail 84 .Strack van Schijndel and colleagues have observed an improvement in survival when patients reach the calorie target according to indirect calorimetry and a protein intake of greater than 1.2 g/kg/day (ref. 85).And the study of Atkinson and Worthley suggest that in the acutely ill patient, the minimal quantity of protein required is unknown, although it is unlikely to exceed 50-60 g/day, in patients who have no external protein loss 5 .The metaanalysis of Hoffer and Bistrian showed that 1.2-1.5 g protein/kg normal body weight/day is low and it was demonstrated that 2.0-2.5 g protein substrate/kg normal body weight/day is safe and could be optimum for most critically ill adults to decrease the risk of morbidity and mortality in some pathological conditions.The main conclusion of this systematic review is that nitrogen balance improves with increasing protein provision up to the highest studied dose of 2.5 g/kg/day.Some studies suggest that higher levels of protein provision increase the rate of wholebody protein synthesis.During the early phase of critical illness, the body's priority is central protein synthesis at the expense of protein loss from the skeletal muscle compartment.Exogenous protein could increase protein synthesis in the small, but crucial, central compartment, with benefit to the patient, without necessarily requiring a commensurate reduction in muscle protein catabolism.Sufficient protein provision mitigates nitrogen loss by promoting central and peripheral protein synthesis.This is clearly preferable when the body's priority is to mobilize peripheral amino acids in support of central protein synthesis 86 .

CONCLUSION
The principal goal of protein/amino acid administration in critical illness is to provide precursors for protein synthesis in tissues with a high turnover and to protect skeletal muscle mass and function.In physiological conditions, intravenous amino acid administration leads to stimulation of the whole body and muscle protein synthesis, while insulin and glucose infusions preferentially inhibit proteolysis.In critical illness, stress hormones and inflammatory mediators inhibit insulin and amino acid anabolic efficiency, and lean tissue loss is unavoidable in patients with severe trauma or sepsis despite aggressive nutritional support.Acceleration of muscle proteolysis plays a pivotal role in the catabolic response to critical illness 75 .In severely ill ICU patients, a higher provision of protein and amino acids has been associated with a lower mortality 87 .
Dietary supplementation of arginine can enhance wound healing, regulate endocrine activity and potentiate immune activity 9 .Cysteine is essential in critically ill patients 5 .Supplementation of glutamine may improve protein status and immunocompetence, enhance nutritional management, reduce the number of infections and augment recovery of the seriously ill while minimizing hospital stay 30,32,[34][35][36][37] .Leucin stimulates skeletal muscle protein synthesis and inhibits muscle proteolysis, even during sepsis 5,56,57 .
The prevalent opinion in modern critical care nutrition is that 1.2-1.5 g protein/kg normal body weight/day is sufficient and hence not usually exceeded.At the present time, most critically ill adults receive less than half the current recommendation, 1.5 g protein/day, for the first week or longer of their ICU stay.The limited amount and poor quality of the available evidence preclude conclusions or clinical recommendations but it has been demonstrated, that 2.0-2.5 g of protein/kg normal body weight/ day is safe and could be optimum for most critically ill adults to decrease the risk of morbidity and mortality in some pathological conditions.It is important to note, that it is commonly assumed, that the weight of the amino ac-ids in a parenteral amino acid mixture equals the amount of protein they provide, although the molecular weight of free amino acids is 18mass units greater than when they are protein bound.Hoffer concluded that amino acid mixtures provided 17% less protein and energy than is now widely assumed.Clinicians who aim to provide 0.8-1.5 g protein/kg must administer 1.0-1.8g mixed amino acids/ kg (ref. 88).

Table 4 .
Effect of supplementation of glutamine on organ fuction.

Table 5 .
Different effects of various doses of glutamine (modified by ref.41).

Table 7 .
The ESPEN's guidelines for enteral and parenteral nutrition in critically ill patients.