Nutritional Intervention High in Vitamins, Protein, Amino Acids, and ω3 Fatty Acids Improves Protein Metabolism During the Hypermetabolic State After Thermal Injury FREE

THE RESPONSE to injury, known as the hypermetabolic response, occurs after a severe thermal injury.^{1 - 2} Increases in cardiac output, oxygen consumption, nitrogen loss, lipolysis, body weight loss, and metabolic rate are directly proportional to the size of the burn.¹ The pathophysiology of this state is associated with the inflammatory cascade of cytokines, glucocorticoids, and prostaglandins.³ The metabolic rate postinjury is extremely high and energy requirements are immense. Increased protein turnover, degradation, and negative nitrogen balance are characteristics of this critical state.^{4 - 5} These energy requirements are met by mobilization of carbohydrates, fat, and protein stores. However, energy stores, particularly glycogen stores, are quickly depleted and the energy needs are met by increased glycogenesis from amino acid mobilization originating from the skeletal muscle, which leads to loss of muscle tissue and malnutrition.⁶ Consequently, the structure and function of essential organs are compromised, such as skeletal muscle, skin, and the immune system, and cellular membrane transport functions are altered.^{7 - 8} Another important step in the hypermetabolic cascade is growth hormone resistance and decreased production and action of insulin-like growth factor I (IGF-I).^{9 - 10}

Malnutrition in thermally injured patients has been suggested to be subverted to some extent by exogenous nutritional support¹¹ and several modifications of the nutrient composition have undergone basic and clinical investigations. The results are somewhat contrary. An arginine-supplemented diet has been suggested to decrease proinflammatory cytokines and to improve survival in burned rats,¹² while the administration of an ω3 fatty acid–enriched diet has been found to enhance the immune system.¹³ However, there have been studies that have found no beneficial effects of arginine or ω3 fatty acids.¹⁴ The effect of high-protein supplementation has also been described with controversy. Nelson et al¹⁵ found that a high-protein diet is associated with increased bacterial translocation in septic guinea pigs, while Saffle et al¹⁴ reported improved survival of burned rats receiving a high-protein diet. Demling and colleagues¹⁶ studied the effect of a high-calorie, high-protein diet on weight gain and muscle function in the recovery phase after severe burns and found that increased protein intake by adding a protein hydrolysate increases the rate of restoration of body weight and muscle function.

The purpose of our study was to determine whether early enteral administration of a diet high in protein, vitamins, ω3 fatty acids, and amino acids improves protein metabolism and the hepatic acute-phase response during the posttrauma hypermetabolic state. Protein metabolism was evaluated by measuring hepatic protein synthesis, hepatic, serum, and muscle protein levels; IGF-I concentration; and dermal wound healing in the form of reepithelization.

Twenty-two adult male Sprague-Dawley rats (350-375 g) were placed in wire-bottomed cages housed in a temperature-controlled room with a 12-hour light-dark cycle. Rats were acclimatized to their environment for 7 days before the study. All rats received water ad libitum throughout the study. Four days before the injury, rats were randomized into 2 groups: rats fed standard rat chow (control, n = 11) or rats fed a diet rich in vitamins, ω3 fatty acids, protein, and carbohydrates (enriched diet, n = 11).

Each rat received a 60% total body surface area full-thickness scald burn under general anesthesia (pentobarbital 50 mg/kg of body weight) and analgesia (buprenorphine, 1 mg/kg body weight) following a modified procedure as previously described.¹⁷ Rats were anesthetized, shaved, and scalded with 99° water over 60% of their total body surface area (10 seconds of contact to the back and 1.5 seconds to the belly). This model ensures that both groups are similar in metabolic rates. After the thermal injury, rats were immediately resuscitated by intraperitoneal injection of Ringer's lactate (50 mL/kg of body weight).

The study period lasted 5 weeks after the injury. Animals were euthanized by decapitation. Blood was collected into serum and plasma separators, spun at 1000g for 15 minutes, and the supernatant and pellet were separated and stored at −73°C. Samples of liver, muscle, and skin from the back were harvested, snap-frozen in liquid nitrogen, and stored at −73°C for analysis.

Rat chow was composed of 64% carbohydrates, 5% fat, and 14% protein (Harlan Teklad 2014; Harlan, Madison, Wis). The mean ± SD food intake of rats receiving chow was 26 ± 3 g/d. The liquid diet (Sustacal; Mead Johnson Nutritionals, Evansville, Ind), rich in vitamins, protein, and carbohydrates, had a caloric distribution of 24% protein, 21% fat, and 55% carbohydrates, resulting in an energy intake of 1.01 cal/mL. The diet had a renal solute load of 510 mOsm/L, an osmolality of 650 mOsm/kg of water, and was lactose free. The nutrient values for Sustacal and chow are presented in Table 1. Nutrient compositions were provided by the manufacturers.

Both groups of rats were pair-fed according to the caloric intake. The feeding protocol was as follows: 25 calories on the day of the burn, 51 calories on the first day postburn, 76 calories on the second, and 101 calories from the third day on. Nutritional intake was the same in all groups.

Serum total protein, albumin, and transferrin as constitutive hepatic proteins were determined to be nutritional markers. Protein levels were determined by a Behring nephelometer (Behring, Deerfield, Ill). Liver and muscle protein concentrations were determined by protein assay. Wet-dry ratios were determined in liver and muscle using the formula:

Grahic Jump Location

in which w indicates wet weight; and d, dry weight.

Insulin-like growth factor I protein concentrations were measured by rat radioimmunoassay in serum, liver, and muscle, taken 35 days postburn. Proteins in tissues were extracted by pulverizing approximately 40 mg of tissue in liquid nitrogen, adding an extraction buffer (phosphate-buffered isotonic sodium chloride solution, 0.25 mL of PMSF, 50 mg of leupeptin, 100 mg of aprotinin, and 50 mg of antipain) in a 1:7 mixture (7 mL buffer per gram of tissue), and homogenizing the mixture. To allow proteins to recover, samples were frozen overnight at −80°C. After thawing, 50 µL of the homogenate was added to 150 µL of the extraction solution and centrifuged at 13 500 rpm for 5 minutes. One hundred microliters of supernatant was added to 400 µL of neutralization solution and the rat IGF-I radioimmunoassay was performed as described in the kit guidelines (Diagnostic System Laboratories, Webster, Tex).

Wound healing, defined here as reepithelization, was determined as follows: The wound eschar was left intact for the first 28 days and then removed by gentle traction, caution being taken not to disturb or destroy the healing edge along the periphery. After removing the eschar, the animals were placed on a standard surface and the wound area was traced onto acetate sheets along the well-demarcated reepithelized and nonburned interface and the leading edge of the neoepithelium. The areas of these tracings were calculated by computerized planimetry. The area of reepithelization was calculated by the following formula:

Grahic Jump Location

in which o indicates outer area 4 or 5 weeks postburn; i, inner area 4 or 5 weeks postburn; and b, original area at the time of the burn.

Values in Figure 1 are expressed as percent reepithelization from the original burn wound.

These studies were reviewed and approved by the Animal Care and Use Committee of the University of Texas Medical Branch, Galveston, assuring that all animals received humane care according to the criteria of the National Institutes of Health. Statistical comparisons were made by analysis of variance and the t test with a Bonferroni correction. Data are expressed as mean ± SEM. Significance was accepted at P<.05.

Both groups of rats lost body weight after the burn. Rats that received the enriched diet demonstrated progressive improvement in body weight 1 through 5 weeks postinjury compared with rats fed standard chow (P<.05). Rats receiving the enriched diet had a 0.5% increase in body weight (compared with the body weight preburn) 5 weeks after the burn, whereas rats receiving normal chow lost approximately 10% of their preburn weight (P<.05) (Figure 2).

Rats that were fed the standard chow had significantly lower total serum protein concentrations (P<.001), lower serum albumin concentrations (P<.001), and lower serum transferrin concentrations (P<.003) 5 weeks after the thermal injury than rats that received the diet (Table 2).

Rats receiving Sustacal had an increased muscle wet-dry ratio (P<.01) and total protein content in the muscle compared with rats receiving chow (P<.02). Similarly, the Sustacal group had increased hepatic protein content as shown by increased hepatic wet-dry ratio (P<.001) and total hepatic protein content (P<.02) compared with the chow group (Table 2).

Animals fed Sustacal had higher serum, hepatic, and muscle IGF-I concentrations (Table 3) compared with animals fed standard chow (P<.05).

Reepithelization was determined 28 and 33 days after the burn. Rats fed chow had a reepithelization of 17.3% ± 0.7% 28 days postburn and 18.4% ± 0.5% 33 days postburn, respectively. Rats fed the diet had a reepithelization of 23.3% ± 0.8% 28 days postburn and 24.0% ± 0.9% 33 days postburn (Figure 2). Therefore, rats fed Sustacal had a significantly more accelerated wound reepithelization compared with rats fed regular chow (P<.001).

The hypermetabolism and negative nitrogen balance associated with thermal injury were recognized by Cuthbertson and Zagreb.¹⁸ Since that time there has been a lot of progress in therapeutic strategies to improve the hypermetabolic response and therefore, mortality, of severely burned patients, such as maintaining patients in a warm environment (approximately 30°C), providing pain control, early wound excision, grafting using biological and synthetic wound dressings, and the prevention of sepsis.

The characteristics of critically injured patients are increased protein turnover and negative nitrogen balance.^{4 - 5} The specific pathophysiology has been recently described.⁷ After trauma in skeletal muscle, the rates of proteolysis and synthesis are accelerated. However, despite the increased protein synthesis rate, proteolysis dominates, leading to a negative protein net balance. Intramuscular glutamine concentration is decreased because of increased efflux and decreased de novo synthesis. As a consequence, tissues and organs characterized by rapidly replicating cells, such as the gut with its enterocytes, the skin with its granulation tissues consisting of fibroblasts and keratinocytes, the skeletal muscle, and the immune system, exhibit early decreased protein synthesis. Protein depletion results in the need for prolonged respiratory support, delayed mobilization of the patient, delayed wound healing, and compromised immune function.⁸

Several therapeutic approaches have been undertaken to lessen the massive protein waste, one of which is adequate nutritional support. The aims of nutritional support are to maintain and improve organ function, prevent calorie malnutrition, and improve morbidity and mortality. The change from total parenteral nutrition to early enteral feeding has been shown to decrease the incidence of sepsis and improve the hypermetabolic state.¹¹ An arginine-supplemented diet has been found to decrease proinflammatory cytokines and improve survival rates in burned rats¹² and administration of ω3 fatty acids has been reported to improve the immune system functions.¹⁴

The hepatic acute-phase response is a cascade of events initiated to prevent tissue damage and to activate repair processes. The acute-phase response is initiated by activated phagocytic cells, fibroblasts, and endothelial cells, which release proinflammatory cytokines, leading to the systemic phase of the acute-phase response. A crucial step in this cascade of reactions involves the interaction between the site of injury and the liver, which is the principal organ responsible for producing acute-phase proteins and modulating the systemic inflammatory response. After major trauma such as a severe burn, hepatic protein synthesis shifts from hepatic constitutive proteins, such as albumin, prealbumin, transferrin, and retinol-binding proteins to acute-phase proteins.^{19 - 21} After a thermal injury, albumin and transferrin decrease by 50% to 70% below normal levels.^{20 - 22} Albumin and transferrin, however, have important physiologic functions since they serve as transporter proteins and contribute to osmotic pressure and plasma pH.²³ In the postinjury state their synthesis has been used as a predictor of mortality, nutritional status, severity of stress, and as an indicator of recovery.^{22 - 24} Several other studies hypothesized that an exaggerated increase in the hepatic acute-phase response can be detrimental.^{25 - 27} In our study we showed that nutritional intervention high in protein, vitamins, ω3 fatty acids, and amino acids improves the hypermetabolic and hepatic acute-phase response by increasing constitutive hepatic protein synthesis of albumin, transferrin, and total protein 33 days after a severe thermal injury. In addition, hepatic protein concentration and wet-dry weight were significantly increased in rats receiving the intervention, indicating a reduction in organ protein catabolism.

Muscle protein concentration and wet-dry weight ratios in muscles were increased in rats receiving the diet compared with rats receiving standard rat chow. The reduction of protein loss was further reflected in the pattern of loss of body weight. Rats fed Sustacal showed an improvement in body weight compared with rats receiving chow. A possible mechanism of improved protein metabolism in our burn model could be the dermal regeneration growth hormone (GH)–IGF-I axis. Thermally injured skin is a major source of proinflammatory cytokines early after the injury occurs, such as tumor necrosis factor α or interleukin 1, which are systemically released.²⁸ Tumor necrosis factor α and interleukin 1 enhance and support the protein catabolism and decrease the anabolic hormones GH and IGF-I.²⁹ Growth hormone and IGF-I are anabolic agents that accelerate protein synthesis rates.³⁰ During the hypermetabolic state, catabolism is enhanced because of GH resistance and decreased production and action of IGF-I.^{9 - 10} This leads to the hypothesis that 2 augmenting factors decrease GH and IGF-I–GH resistance and the release of proinflammatory cytokines from the burn wound. Accelerated wound healing would therefore lead to decreased release of proinflammatory cytokines, which is followed by a decreased hypermetabolic response, a negative nitrogen balance, and increased GH and IGF-I concentrations. We found that animals receiving Sustacal had an accelerated dermal regeneration compared with animals receiving regular chow and increased IGF-I concentrations in serum, liver, and muscle. It is, however, unclear whether IGF-I was increased because of accelerated wound healing or whether the diet increased IGF-I, leading to improved protein synthesis and accelerated wound healing. Furthermore, it has been shown that dietary supplements of antioxidant vitamins A, C, and E, which serve as radical scavengers, are associated with improved resistance to oxidative stress and enhanced and accelerated wound regeneration.³¹

We showed that nutritional intervention that is high in protein, vitamins, amino acids, and ω3 fatty acids improves protein metabolism during the hypermetabolic response to a thermal injury. Thermally injured rats receiving the diet had an improvement in body weight, increased protein concentrations in serum, liver, and muscle, and increased IGF-I levels compared with animals fed standard rat chow. Rats receiving the diet had accelerated wound healing in terms of reepithelization. Because the diet used in our study was varied in overall composition, it is difficult to determine the specific factor causing the positive effects. However, diets that are used clinically are often composed of a variety of elements. Another concern is the bioavailability of the different diets we used. We have no data on the bioavailability but speculate that resorption and utilization of the 2 diets are similar.

This study was supported by grants 8010 from the Shriners Hospital for Children, and DFG Je 233/1-2 from the Deutsche Forschungsgemeinschaft, Bonn, Germany.

Corresponding author: Marc G. Jeschke, MD, Klinik und Poliklinik für Chirurgie, Klinikum der Universität Regensburg, Franz-Joseph-Strauss Allee 11, 93053 Regensburg, Germany (e-mail: mcjeschke@hotmail.com).