Catecholamines (CA) are hormones produced in the adrenal medulla and other cell types and released into the circulation in response to stress (1). Upon binding to their cognate receptors, they signal through different pathways to affect a variety of physiologic processes, from nerve transmission to energy metabolism and cardiac function. Epinephrine is a direct agonist of α1, α2, β1, β2 adrenergic receptors, norepinephrine binds to α1, α2, β1 adrenergic receptors, and dopamine binds to dopamine receptors D1 and D2 and with less affinity to α and β receptors. These receptors signal downstream to different classes of G protein-coupled molecules to affect intracellular Ca2+ or cyclic adenosine monophosphate concentration. The effect of CA in various disease states has been extensively studied, but mechanisms still remain unknown. There is an intensive ongoing effort to decipher the mechanism of CA action in individual tissues or cell types and to design effective treatments to modulate their effects in the body (1).
Burn-/trauma-related sympathetic activity likely contributes to the activation of stress cascades, leading to increased secretion of cytokines. Sympathetic activity may function as an upstream trigger for activation of p38 mitogen-activated protein kinase (MAPK), JNK, and nuclear factor-κB (NF-κB) pathways (2), leading to inflammation, immunosuppression, and organ dysfunction (2). Sympathetic activation after injury is further associated with impaired immune function by inhibiting CCL3 production and increasing CCL2 secretion. These chemokines have been shown to contribute to increased susceptibility to infection in severely burned patients (3, 4).
We hypothesize that CA plays a central role in the aftermath of severe burn. We are currently trying to decipher and identify what are the specific cellular and metabolic consequences of CA postburn.
Catecholamine actions in critically ill patients are an important opportunity for pharmacological manipulation, but the secretion profiles in pediatric burned patients are not well described. The aim of this large unicenter study is to evaluate the magnitude and extent of CA surge in pediatric burned patients when compared with the CA levels in healthy, nonburned children. This is the first study to elucidate the pattern and physiology of CA postburn in a large prospective design.
Patients admitted from 1996 to 2008 to our burn center, with informed consent, were enrolled in the study (n = 413). Twenty-four-hour urine collections were performed during acute hospitalization and at 3, 6, 9, 12, 18, and 24 months postburn. Urine was also collected from healthy nonburned volunteers (n = 12). All specimens with total volume less than 0.5 mL kg−1 body weight h−1 were excluded to avoid incomplete collection and all specimens with creatinine clearance below 40 to avoid patients on dialysis. During acute hospitalization, 24-h urine collection was collected via Foley catheters, starting at 6:00 am ± 2 h. The urine collection after discharge from the intensive care unit (3, 6, 9, 12, 18, 24 months) started at random times during the day. The study was approved by the institutional review board of the University of Texas Medical Branch, and informed consent was obtained from patients, parents, or legal guardians before enrollment.
Relevant demographic and clinical information was obtained from medical records. Urine samples were acidified to pH = 2 with hydrochloric acid and stored at 4°C to 8°C until analyzed. Epinephrine, norepinephrine, and dopamine were extracted using Bio-Rad urinary catecholamine kit (BioRad Laboratories, Hercules, Calif) according to manufacturer's instructions and analyzed on a Discovery HS F5 (567516-U; Sigma-Aldrich, St. Louis, Mo) column. Mobile phase A consisted of 25 mM potassium phosphate monobasic in water high-performance liquid chromatography grade, pH = 2.7, with phosphoric acid, and mobile phase B comprised methanol high-performance liquid chromatography, grade pH = 2.7, with phosphoric acid. A gradient 0% to 12% B over 20 min at 0.7 mL min−1 flow rate at room temperature achieved complete separation and good resolution between peaks. We used both UV detection at 280 nm and electrochemical detection (0.75 V; filter, 2 s; sensitivity, 10 nA/V) for a wider dynamic range of the method.
Heart rates were continuously monitored during acute hospitalization and later admissions for surgical interventions.
Cytokines were measured using Bio-Rad's human cytokine multiplex kits according to manufacturer's instructions.
Student's t-test and one-way ANOVA were used to analyze the data where appropriate. Significance was accepted at P < 0.05.
Four-hundred thirteen patients were enrolled in this study (284 boys and 129 girls), average total body surface area burn, 59% ± 17%; third-degree burn, 45% ± 25%; age distribution, 8 ± 5 years; 17 patients died during acute hospitalization. Results from the samples collected from 12 normal, healthy volunteers, 8 boys, 4 girls, 12 ± 3 years, were compared with the data from the burned patients.
One of the first clinical symptoms of sympathetic stimulation is tachycardia. As shown in Figure 1, predicted heart rates according to age during acute hospitalization are significantly increased, which leads to profound alterations of cardiac function.
The increase in the secretion of proinflammatory mediators is a hallmark in burns. As shown in Figure 2, there is similarity between expression profiles of inflammatory cytokines such as IL-6, IL-8, granulocyte-colony-stimulating factor, TNF-α, and CA secretion.
CA Secretion Profiles
In Figure 3 (A-C), the levels of urinary norepinephrine (A), epinephrine (B), and dopamine (C) are depicted over time. In burned patients, norepinephrine levels (A) are consistently and significantly elevated for up to 2 years when compared with the levels in normal, healthy volunteers (P < 0.05). Epinephrine levels (B) vary over time, and statistical significance is reached up to 60 days postburn. Dopamine levels (C) are consistently lower than in normal, healthy volunteers, and significance is reached up to 90 days postburn (P < 0.05).
Patients were stratified by burn size. The previous hypothesis that CA levels correlate with burn size held true in our patient population only for burns greater than 40% (Fig. 4). Patients with burns greater than 80% total body surface area burn have the highest and longest secretion of CA.
To further assess if sex affects CA, we divided our patient population into boys and girls. All three CA levels were significantly higher in boys compared with girls at multiple time points (Fig. 5; P < 0.05).
Our data imply that age has a big impact on CA levels after severe burn over time (Fig. 6). Norepinephrine and dopamine patterns of secretion show that there is a direct relationship between the levels and age, older children having higher CA levels. Epinephrine data did not reveal a pattern of secretion with regard to age.
In a subgroup of patients, we compared CA levels in nonsurvivors with matched survivors. We found significant differences for two time points (Fig. 7; P < 0.05). We could not identify a pattern of secretion for epinephrine, norepinephrine, or dopamine as the time of death approached; therefore, we think that the amount of CA released into the circulation does not directly impact survival, but rather the ability of each individual patient to respond to the effects of CA surge is responsible for mortality. Our group has shown previously that severe infection episodes are associated with increased CA levels (5).
In our institution, CA levels are measured in a 24-h urine collection. We think that urine CA levels are a better indicator of adrenal function than plasma levels. Plasma levels give a snapshot of CA secretion, and they cannot be used as a reliable biomarker of catecholaminergic system.
There are few large cohort studies to compare CA levels of burned children with normal volunteers and the time course of secretion profiles after injury. In most cases, the plasma CA is analyzed. These measurements are problematic because of epinephrine administration during skin grafting or burn surgery to reduce blood loss, and plasma levels reflect only a snapshot of CA secretion. A better indicator of hormone levels is the 24-h urine amount.
The knowledge of action of CA in severe trauma has increased tremendously in the past 10 years worldwide. A study by Smith et al. (6) in 1997 found that the magnitude of stress response to burn in children is closely related to the size of burn surface area. The pathophysiologic response to severe burn is mediated through chemical mediators such as CA that are interrelated with inflammatory responses such as increases in cytokine production (7). Differences between the stress response in children and in adults have been noted (8), suggesting that the management of burn-injured children has to be designed according to their particular needs.
Our finding that girls have lower CA levels than boys correlates with the lower hypermetabolic response in girls found in a previous study (9).
One of the big improvements in burn care over the past 15 to 20 years has been the use of CA intraoperatively to minimize blood loss during surgery (10, 11). Topical or subcutaneous administration of epinephrine during burn wound excision had been shown to have transient effects on cardiac function (12) because of its short lifetime.
Catecholamine has profound catabolic effects in the muscle. Decreases in lean body mass and muscle protein wasting are well documented in critically ill patients. Our group has shown that β-blockade is a useful tool in the arsenal for fighting postburn muscle proteolysis and to ameliorate the physical sequelae of severe burn (13, 14).
Gosain et al. (15) have shown that CA modulates the inflammatory and proliferative phases of wound healing in a temporally defined, cell-specific manner. Norepinephrine seems to have a protective role in defense against infection by recruiting innate immune cells and expediting wound closure. In another study, the group found that norepinephrine has an immunosuppressive effect on wound macrophage function that is tissue specific and seems to be mediated through adrenergic receptors and their downstream signaling pathway (15). This might provide an explanation for the reduced morbidity and mortality associated with β-blockade.
Catecholamine surge after severe illness has been involved in insulin resistance due to the effects on fat metabolism (16) and liver function (17). In a large study analyzing the insulin resistance postburn, we found a strong correlation between insulin resistance and increased CA levels (18). Patients had an oral glucose tolerance test performed at multiple time points up to 3 years post-initial injury, and at each return to clinic, 24-h urine collection was analyzed. There was a strong correlation between increased CA levels and insulin resistance, reflected especially in peripheral insulin sensitivity indices.
An unexpected association between CA levels and the severity of traumatic stress disorder (both acute stress disorder [ASD] and long-term posttraumatic stress disorder [PTSD]) has emerged in the past few years. Studies in acute and long-term survivors of multiple trauma, burn, or myocardial infarction have demonstrated a clear and vivid recall of different categories of traumatic memory such as nightmares, anxiety, respiratory distress, or pain with little or no factual recall of the events, and the number of traumatic memory recalls was proportional to the levels of CA (19). In our patient population, the incidence of ASD is approximately 8%, and the incidence of PTSD is approximately 7% (20-22). These values are lower than other institutions have reported and are a good indicator of the efforts by our psychiatrists and psychologists and the value the programs they are conducting for our patients. We are currently investigating the correlation between CA levels and incidence of ASD and/or PTSD.
The effects of septic episodes on the outcome of a critically ill patient are well documented. Our group has shown that septic episodes are accompanied by an enormous increase in CA levels (5).
β-Blockade treatment has been shown to be beneficial in the treatment of severe burns, resulting in decreased energy expenditure and muscle catabolism (23). Supportive therapy and pharmacological manipulations with inhibitors and antagonists of adrenergic receptors can ameliorate hypermetabolic response, immune deficiency, peripheral lipolysis, and bone metabolism (24).
Catecholamines play a key role in orchestrating the response to stress (1). Production and secretion of CA is crucial to handle emergency situations. When the exposure to stress stimuli is prolonged, adaptation of the organism becomes a big burden, increasing the susceptibility for a wide range of serious diseases. Catecholaminergic systems are responsive to activation of the hippocampal-pituitary-adrenal axis, and corticosteroid hormones may have a direct effect. Different signaling pathways and transcription factors are activated depending on the frequency and intensity of exposure to stressful stimuli. Most of the animal studies investigating these processes were performed in boys; however, there is evidence that girls respond differently to stress. It has been shown that estradiol can regulate the expression of genes involved in CA biosynthesis such as tyrosine hydroxylase or modulate their response to second messengers such as cyclic adenosine monophosphate.
Catecholamine surge may cause activation of transcription factors such as CREB and c-Fos, leading to induction of MAPK family: ERK1/2, JNK1/2/3, and p38 (25), thus contributing to the hyperinflammatory response after severe burn. Inflammatory cytokines are increased for a prolonged period of time after severe injury. These findings further strengthen our hypothesis that the inflammatory response and the humoral response after burn are linked. We think that the initial hormonal response after severe injury occurs within 15 min, followed by the inflammatory response, which occurs in 3 to 6 h (26). They are part of the fight-or-flight response. The mechanism remains to be determined, but we can hypothesize that stress response through G protein-coupled receptors and adrenergic receptors are linked to activation of NF-κB through MEK protein, which is a member of the MAPK family downstream from the adrenergic receptor and G protein-coupled receptor and in the cytoplasm forms a complex with NIK (inducible NF-κB kinase). The link between sympathetic nervous system and activation of stress-responsive pathways in cardiomyocytes was hypothesized by Ballard-Croft et al. (2) Their study aimed to determine the effects of α1-adrenergic receptor antagonists on the activation of stress response pathways.
SUMMARY AND CONCLUSIONS
In this study, we show that CA levels are increased for up to 2 years after severe burn. We found correlation between CA levels and burn size over time. Our data show that girls have lower CA levels over time, and older patients have higher levels over time. This is the first cohort study to examine the patterns of CA secretion over a 2-year period after burn. It gives clinicians a useful insight into the extent and magnitude of CA elevation to better design treatment strategies.
The authors thank Maricela Pantoja for support in analyzing the specimens, clinical research nurses for collecting the samples, and Eileen Figueroa and Steve Schuenke for expert support in editing the manuscript.
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