Introduction
The stress response to burn injury is similar to severe trauma or critical care but differs in its severity and duration. The inflammatory response starts immediately after trauma and persists for almost 5 weeks postburn [
1]. The hypermetabolic response after a major burn is characterized by a hyperdynamic response with increased body temperature, increased oxygen and glucose consumption, increased CO
2 production, increased glycogenolysis, increased proteolysis, increased lipolysis, and increased futile substrate cycling [
2]. This response begins on the fifth day postinjury and continues up to 24 months postburn, causing the loss of lean body mass, the loss of bone density, muscle weakness, and poor wound healing [
2‐
4]. The increased metabolic requirements cause tissue catabolism, leading to nitrogen loss and a potentially lethal depletion of essential protein stores [
5]. The energy requirements are met by the mobilization of proteins and amino acids. Increased protein turnover, degradation and negative nitrogen balance are all characteristic of this severe critical illness [
2,
5]. As a consequence, the structure and function of essential organs, such as the heart, the liver, skeletal muscle, the skin, the immune system and cellular membrane transport functions, are compromised [
6‐
8].
Numerous studies show that an increased burn size leads to increased mortality of burned patients [
9,
10]. Furthermore, it was speculated that the burn size determines the extent of the hypermetabolic response. Whether mortality is due to inflammation, hypermetabolism or other pathophysiologic contributing factors is not entirely determined. The purpose of the present study was to determine in a large prospective clinical trial whether different burn sizes are associated with differences in inflammation, in body composition, in protein synthesis, and in organ function.
Patients and methods
Participants
All thermally injured children over a time period of 9 years who were admitted to our burns unit and required at least one surgical intervention were included in the study. Patients were resuscitated according to the Galveston formula using Ringer's lactate. Within 48 hours of admission all patients underwent total burn wound excision, in which the wounds were covered with the available autograft skin and an allograft was used to cover any remaining open areas. After the first operative procedure it was 5–10 days until the donor site was healed, and patients were then taken back to the operating theater. The skin graft procedure was repeated until all open wound areas were covered with autologous skin material.
All patients underwent the same nutritional treatment to a standardized protocol. We used the Galveston formulas – Galveston Infant, Galveston Revised, and Galveston Adolescent. The formula changes with age based on the body surface alterations that occur with growth. The intake is approximately calculated as 1,500 kcal/m2 body surface area + 1,500 kcal/m2 area burn. The composition of the nutritional supplement is also important. The optimal dietary composition contains 1–2 g/kg/day protein, which provides a calorie-to-nitrogen ratio of approximately 100:1 with the suggested caloric intakes. Nonprotein calories can be given either as carbohydrate or as fat, with clinical advantages for the carbohydrates. The diet was delivered by enteral nutrition, if possible, in all our patients. Total parenteral nutrition was only used as a supplemental form of nutrition when the calculated intake could not be achieved.
Patient demographics (age, date of burn and admission, sex, burn size and depth of burn) and concomitant injuries, such as inhalation injury, sepsis, morbidity and mortality, were recorded. Sepsis was defined as a blood culture identifying the pathogen during hospitalization or at autopsy, in combination with leucocytosis or leucopenia, hyperthermia or hypothermia, and tachycardia. Wound healing was evaluated from the time of donor site healing, and therefore from the time between operative interventions.
Indirect calorimetry
As part of our routine clinical practice, all patients underwent resting energy expenditure (REE) measurements within 1 week following hospital admission, at 2–4 weeks after hospital admission, at discharge, and at 6 months postburn. Measurements of REE were performed between midnight and 5:00 am while the patients were asleep and receiving continuous feeding. The REE was measured using a Sensor-Medics Vmax 29 metabolic cart (Sensor-Medics, Yorba Linda, CA, USA). Subjects were tested in a supine position while under a large, clear, ventilated hood. The REE was calculated from the oxygen consumption and carbon dioxide production using equations described by Weir [
11] All REE measurements were made at ambient temperatures of 30°C, which is the standard environmental setting for all patient rooms in our acute burn intensive care unit.
The REE measurements were used to guide nutritional management and to assess the level of metabolism. The discharge REE measurement was used to determine the level of hypermetabolism when the burn wounds were 95% healed and was included as part of the study. Measured values were compared with predicted norms based upon the Harris–Benedict equation [
12]. The REE studies were repeated at 6, 9 and 12 months postburn when the patients returned for outpatient surgery. Assessments of the REE at these time points were completed utilizing the methodology and environmental settings as described above. For statistical comparison, energy expenditure was expressed both as the absolute REE and as the percentage of the basal metabolic rate predicted by the Harris–Benedict equation.
Muscle protein net balance
The muscle protein net balance was calculated from the product of the amino acid concentration difference and the blood flow, as previously published [
13‐
16].
Body composition
The height and the body weight were determined clinically 5 days after admission and at discharge. The total body lean mass, fat, bone mineral density, and bone mineral content were measured by dual-energy X-ray absorptiometry. The Hologic model QDR-4500W DEXA (Hologic Inc., Waltham, MA, USA) was used to measure the body composition. To minimize systematic deviations, the Hologic system was calibrated daily against a spinal phantom in the anteroposterior, lateral, and single-beam modes. Individual pixels were calibrated against a tissue bar phantom to determine whether the pixel was reading bone, fat, lean tissue, or air. Plain anterior–posterior and lateral tibia–fibula X-rays were taken of each subject at each follow-up period to evaluate for possible premature closure of epiphyseal plates induced by anabolic agents.
Serum hormones, proteins, and cytokines
Blood or urine was collected from the burn patients at the time of admission, preoperatively, and 5 days postoperatively for 4 weeks for serum hormone, protein, cytokine and urine hormone analysis. Blood was drawn in a serum-separator collection tube (BD, Franklin Lakes, NJ, USA) and was centrifuged for 10 minutes at 1,300 × g. The serum was then removed and stored at -70°C until assayed.
Serum hormones and acute phase proteins were determined using high-performance liquid chromatography and ELISA techniques. The Bio-Plex Human Cytokine 17-Plex panel was used with the Bio-Plex Suspension Array System (Bio-Rad, Hercules, CA, USA) to profile expression of 17 inflammatory mediators: IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17, granulocyte colony-stimulating factor, granulocyte–macrophage colony-stimulating factor, IFNγ, monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and TNF. The assay was performed according to the manufacturer's instructions. Briefly, serum samples were thawed and then centrifuged at 1,300 × g for 3 minutes at 4°C. Serum samples were then incubated with microbeads labeled with specific antibodies to one of the aforementioned cytokines for 30 minutes. Following a washing step, the beads were incubated with the detection antibody cocktail (each antibody specific to a single cytokine). After another washing step, the beads were incubated with streptavidin–phycoerythrin for 10 minutes, were washed again, and the concentrations of each cytokine were then determined using the array reader.
Urine cortisol was determined by standard laboratory techniques, measuring for the urine amount, creatinine and creatinine clearance.
Liver and cardiac changes
Ultrasound measurements in this study were made with the HP Sonos 100 CF echocardiogram (Hewlett Packard Imaging Systems, Andover, MA, USA). To obtain the ultrasound liver weight, a 3.5 MHz transducer was placed directly below the midline of the rib cage on the right upper quadrant on a vertical line running through the right nipple with the patient in the supine position. Once the liver was visualized, measurements were made by scanning in a plane perpendicular to the base of the liver. The base of the liver, as well as the free edge hepatic dome, was marked on the display screen and the distance between these two points was automatically measured.
The formula used for estimating the liver weight from the single longitudinal scan along the right nipple line was weight = (1.15 l)3d, where l3 represents the volume of a cube cut in half diagonally to visualize the approximate shape of the normal liver in situ. A factor of 1.15 was used to correct for the portion of the liver (15%) lateral to the left nipple line and representing the most inferior point of the liver. This correction was estimated from the liver at autopsy. The density (d) of the liver was measured on several sections by water displacement. Determining the right nipple line was not problematic unless the nipple was obliterated by a severe burn to the thorax. In these cases an approximation was made and recorded as such. The actual size was then compared with the predicted size.
M-mode echocardiograms were completed as follows. At the time of the study, none of the patients presented with or previously suffered from other concomitant diseases affecting cardiac function, such as diabetes mellitus, coronary artery disease, longstanding hypertension, or hyperthyroidism. The study variables included the resting cardiac output, the cardiac index, the stroke volume, the resting heart rate and the left ventricular ejection fraction. The stroke volume and cardiac output were adjusted for body surface area and were expressed as indexes. All ultrasound measurements were made with the Sonosite Titan echocardiogram, with a 3.5 MHz transducer. Recordings were performed with the subjects in a supine position and breathing freely. M-mode tracings were obtained at the level of the tips of the mitral leaflets in the parasternal long axis position and measurements were performed according to the American Society of Echocardiography recommendations. Left ventricular volumes determined at end diastole and end systole were used to calculate the ejection fraction, the stroke volume, the resting cardiac output and the cardiac index. Three measurements were performed and averaged for data analysis.
Ethics and statistics
The study was reviewed and approved by the Institutional Review Board of the University Texas Medical Branch, Galveston, TX, USA. Prior to the study, each subject, parent or child's legal guardian signed a written informed consent form. Analysis of variance with post-hoc Bonferroni correction, paired and unpaired Student's t tests, chi-square analysis, and Mann–Whitney tests were used. Data are expressed as the mean ± standard deviation in the tables or as the mean ± standard error of the mean in the figures. Significance was accepted at P < 0.05.
Discussion
The metabolic rate in burns is extremely high, and energy requirements are immense and are met by the mobilization of proteins and amino acids [
5]. As a consequence, the structure and function of essential organs such as skeletal muscle, skin, immune system, and cellular membrane transport functions are compromised [
17‐
19]. Catecholamines involved in the hypermetabolic response to burn injury are released from sympathetic nerve ends and the adrenal medulla, and are raised two-fold to 10-fold in proportion to the burn size [
20‐
23]. In the present study, by comparing four different burn sizes, we showed that an increase in burn size is associated with increased hypermetabolism, with persistent inflammation, with catabolism, with changes in body composition, with increased stress hormone production, and with organ dysfunction. Whether the correlation between these phenomena and burn size is linear was not determined, but we clearly observed these pathophysiologic changes with increased burn size.
Large burns cause a marked increase in inflammation and catecholamines, which leads to an increased metabolic rate. In the present study, we found that smaller burn injuries demonstrated decreased catecholamine and stress hormone levels that were associated with decreased hypermetabolism and catabolism. Smaller burns had significantly decreased and shortened predicted REE, body weight loss, and net muscle protein balance when compared with the larger burns. Further indicators that smaller burns are less hypermetabolic than larger burns are decreased acute stress hormones and increased anabolic hormones. Catecholamines, cytokines and proinflammatory mediators are known to block, and therefore decrease, endogenous anabolic agents via cellular mediators [
24,
25].
We showed in the present study that patients with large burns demonstrate a different cytokine expression profile compared with patients with smaller burn injuries. Patients with burns over 80% TBSA had persistent and marked increased levels of IL-8, IL-6, monocyte chemoattractant protein-1 and TNF. All of these cytokines are proinflammatory and enhance catabolism and hypermetabolism via hyperinflammation. We therefore suggest that these high levels of proinflammatory cell mediator trigger and enhance the hypermetabolic response with increased stress hormones and protein catabolism. On the other hand, lower inflammation and hypermetabolism was associated with higher endogenous anabolic hormone levels. Patients with smaller burns had lower inflammatory marker and stress hormones, which was associated with higher IGF-I, insulin like growth factor binding protein-3 and growth hormone levels. IGF-I was shown to exert anabolic effects on skeletal muscle protein synthesis, to attenuate the hepatic acute phase response, and to improve dermal and epidermal regeneration [
26‐
30]. Furthermore, decreased growth hormone and IGF-I levels lead to a deficit in transmembrane amino acid transport and a compromised immune system [
31‐
35].
The glucose kinetics in severely burned patients is almost always abnormal [
36,
37]. Glucose utilization in burned patients is almost entirely through inefficient anaerobic mechanisms, as characterized by increased lactate production, which accounts for increased glucose consumption [
22,
23,
38]. Glucose production, particularly from alanine, is elevated in almost all patients with severe burn [
5]. The increased gluconeogenesis from amino acids renders these amino acids unavailable for reincorporation into body protein. Nitrogen is excreted, primarily in urea, thus contributing to the progressive depletion of body protein stores. Plasma insulin levels usually remain normal or slightly elevated in burn patients [
39,
40]. The fact that the basal rate of glucose production is elevated despite normal or elevated plasma insulin levels indicates hepatic insulin resistance, since under normal conditions elevated serum insulin would lower the rate of glucose production [
38‐
40]. Furthermore, the plasma glucose concentration is frequently increased, which would normally directly inhibit glucose production [
41,
42]. As hyperglycemia is associated with increased mortality in critically ill patients [
43,
44] and worsens the outcome in severely burned patients [
41,
42], we determined serum insulin levels in our four burn groups. We found that insulin levels were significantly increased in the >80% TBSA burn group and were lower in the smaller burn groups. This indicates that with the increased severity of the burn injury insulin resistance increases and more insulin needs to be synthesized to maintain normoglycemia.
Another striking finding of this study was the cardiac function. Burn patients with burns of >80% TBSA demonstrated significantly decreased cardiac output and percentage predicted cardiac output. Furthermore, these patients had significantly decreased percentage predicted stroke volumes and cardiac index when compared with the other groups. There were no differences in the heart rate, the predicted heart rate, and the central venous pressure between groups. As the preload was not significantly different between groups, we therefore suggest that a severe burn over 80% TBSA causes a marked myocardial depression. A myocardial depression associated with a severe burn injury has been shown in several studies [
45‐
47]. These data demonstrate that myocardial depression plays an important role during the postburn response and that modulation of myocardial depression may improve clinical outcome. The hypothesis that myocardial dysfunction may be one of the main contributors to mortality in large burns was confirmed in a recent retrospective autopsy study [
10].
Conclusion
Based on our findings, we suggest that a burn injury involving more than 80% of the total body surface causes marked and prolonged inflammation, marked increases in hypermetabolism, catabolism and cardiac dysfunction, and, subsequently, higher incidences of infection, sepsis and death. Treatment should focus on several aspects of the pathophysiologic events postburn, such as treatment of the inflammatory response, insulin resistance, hypermetabolism, catabolism, and cardiac dysfunction.
Acknowledgements
This study was supported by the American Surgical Association Foundation, by the Shriners Hospitals for Children 8660, 8760, and 9145, by the National Institutes of Health R01-GM56687, T32 GM008256, and P50 GM60338, and by the National Institute on Disability and Rehabilitation Research H133A020102.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MGJ designed the study, gathered data, conducted the statistics and wrote the manuscript. RPM collected data, reviewed the statistical analysis and reviewed the manuscript. CCF performed experiments to obtain data, conducted the statistical analysis, and helped to write the manuscript. WBN helped to collect data and write the manuscript. GAK helped to obtain data, performed and established methods to obtain serum analyses, and reviewed the manuscript. GGG collected data, reviewed the statistical analysis and reviewed the manuscript. DNH gathered data, reviewed the analysis, and helped to write the manuscript.