The severity of an initial burn injury is critical for determining the treatment plan and prognosis of burn patients. Here, we measured serum neutrophil gelatinase-associated lipocalin (NGAL) levels to determine whether NGAL can be used as a biomarker for severity of burn injuries. A study of the demographic, clinical, and laboratory markers for various organ damage was performed at Bestian Burn Center (n = 10 healthy people, n = 31 patients). NGAL and organ damage marker levels were measured in 31 patients with severe burns within 2 - 3 days following their admission to the intensive care unit. Serum NGAL level of the expired patients was 788.5 (685.0 - 998.0) pg/mL, whereas that of the discharged patients was 421.2 (356.2 - 480.6) pg/mL, showing that the initial serum NGAL level can be used to estimate mortality. We also determined the correlation between serum NGAL level and the currently used severity markers (total body surface area burned and abbreviated burn severity index) and confirmed that serum NGAL level could be used as a severity marker. We also found that serum NGAL level was correlated with damage of organs such as the liver, kidney, heart, and respiratory organs in patients with severe burns.
In humans, following skin damage and infection, various types of reactions prevent tissue damage and activate the recovery process to restore organ functions. The acute-phase response begins at the injured sites where the circulatory plasma protein level changes when a soluble mediator is released, which in turn induces a metabolic and inflammatory response in organs [
The secretion of NGAL is activated by lactoferrin and vitamin B12, and its expression increases in epithelial cells in the inflammatory state. NGAL is secreted mainly from neutrophils as well as various organs including the kidney, liver, lung, and bronchus. NGAL has antibacterial effects, thus playing a critical role in early bacterial infection [
NGAL expression is low in the normal state but increases rapidly in an acute inflammatory response [
After acute injury in patients with severe burns, several pathophysiological changes occur following skin tissue damage by heat and acute systemic disease due to burn shock and production of inflammatory mediators. The prognosis of such burn patients is influenced by age, gender, injured area, size of burn injury, existence of other diseases, cause of injury, and presence of respiratory injuries. Therefore, accurate and objective evaluation of severity is critical to determine appropriate treatment for burn patients [
The severity of burn injuries can be evaluated by a number of methods including measurement of the Acute Physiology and Chronic Health Evaluation II (APACHE II) score, TBSA and ABSI. Although only the ABSI classifies burn patients on the basis of five variables, others are low as its clinical applicability [
We conducted an observational study in the emergency department and intensive care unit (ICU) of Bestian Medical Center (Seoul and Daejeon, Republic of Korea) between August 2012 and May 2014. This study was performed with standard of selection. All consecutive patients aged ≥18 years and <65 years with a flame burn involving ≥25% of the TBSA. Patients were admitted to the Bestian Medical Center within 12 hours of their injury, were included in the study.
Of 45 patients assessed, 31 patients were included in the study. The exclusion criteria were as follows: age < 18 or ≥65 years, burn size < 25% of the TBSA, other concomitant trauma, diabetes, and hypertension. Ten healthy subjects were included in the control group. Further, we evaluated the following parameters that could have influenced the mortality rate: age, sex, cause of burn, burn size, presence of inhalational injury, number of surgeries, length of stay in the ICU and hospital, and TBSA. We assessed serum NGAL & clinical levels in both patients and control subjects. The patient group was further divided into expired patients and discharged patients. The study protocol was approved by the Institutional Review Board of Bestian Hospital.
Initial clinical data were measured at medical-surgical ICU within 12 hours after the burn occurred. Additional blood samples for serum NGAL were collected every 2 days from the ICU patients and every 2 days from a general ward patients for the duration of the study. Blood samples were no longer collected after a patient left the hospital or died. Blood samples were collected and processed within 1 hour from the time of collection. We acquired serum samples from 10 healthy human subjects and 31 burn patients. The samples were centrifuged at 3000 rpm for 15 minutes (Hanil Sciences, Korea) and immediately stored at −80˚C until analysis.
Serum NGAL levels were measured using an ELISA kit (Bioporto, Denmark) with an assay range of 10 - 1000 pg/mL. Based on the ELISA results, samples were diluted 500-fold. Briefly, 100 μL of the sample was added to the antibody- coated microwell and incubated for 60 minutes at room temperature on a shaking platform. The plate was washed four times, after which 100 μL of biotinylated NGAL antibody was added. The plate was incubated again for 60 minutes at room temperature on a shaking platform, followed by washing four times again. Subsequently, 100 μL of 3, 3’, 5, 5’-tetramethylbenzidine substrate was dispensed into each microwell and incubated for 10 minutes at room temperature in the dark. The reaction was stopped by adding 100 μL of stop solution. Colorimetric analysis was performed using a Spectra Max Micro Plate Reader (Molecular Devices, CA) at 450 nm. For comparative purposes, serum NGAL levels were measured in anticoagulated plasma of apparently healthy donors. We evaluated the changes serum NGAL level for 35 days or until death or discharge from the hospital. Baseline clinical data included hemodynamic parameters, serum lactate levels, and heart, renal, and liver function.
The following characteristics of patients with severe burns in each group were analyzed: age, sex, and number of hospitalization days. In addition to the ABSI and percentage of TBSA burned, the following levels were measured in serum: aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin, blood urea nitrogen (BUN), creatinine, lactic dehydrogenase (LDH), creatinine phosphokinase, C-reactive protein (CRP), protein, myoglobin and amlyase. We examined a retrospective analysis of burn patients who had measurement of these factors within 48 hours of injury and healthy subjects center at the Bestian Medical Center.
The study was reviewed and approved by the Institutional Review Board of the Bestian Seoul Burn Center, Seoul, Korea.
Analysis of variance with Spearman correction, paired and unpaired χ2 tests, and Mann-Whitney tests were used for analysis. Data are expressed as mean ± standard deviation or standard error of the mean. Values of p < 0.05 were considered statistically significant.
All demographic data are presented in
Serum NGAL levels were significantly higher in the patient group (611 pg/mL) compared with the control group (174 pg/mL) (p < 0.001) and in the expired patients (788.5 pg/mL) compared with the discharged patients (421.2 pg/mL) (p < 0.001) (
Regarding hospital stay, the expired patients had a shorter stay than the discharged patients. Along with serum NGAL levels, the mean TBSA and AST, LDH, and myoglobin levels were significantly higher (p < 0.001) in the expired patients than in the discharged patients: TBSA, 66.5% vs. 30.0%; AST, 68 IU/L vs. 31 IU/L; LDH, 527 IU/L vs. 290IU/L; myoglobin, 311 ng/ml vs. 62 ng/ml.
Normal Range | Discharged (n = 17) | Expired (n = 14) | p | |
---|---|---|---|---|
Age, years | 44.9 ± 13.5 | 41.9 ± 16.6 | 0.58 | |
Male, N (%) | 14 (82. 4) | 13 (92. 9) | 0.607 | |
Duration of hospitalizationa, days | - | 61 (30, 75) | 7.5 (6.0, 12.0) | <0.001 |
Number of surgerya | - | 2 (1, 4) | 1 (1, 2) | 0.418 |
TBSAa | - | 30.0 (22.0, 44.0) | 66.5 (50.0, 78.0) | <0.001 |
ABSIa | - | 8 (6, 9) | 11 (9, 12) | 0.002 |
NGALa (pg/mL) | 157.1 | 421.2 (356.2, 480.6) | 788.5 (685.0, 998.0) | <0.001 |
BUN (mg/dL) | 8 ~ 20 | 14.2 ± 4.7 | 17.3 ± 7.7 | 0.176 |
Creatinine (mg/dL) | 0.8 ~ 1.2 | 0.88 ± 0.18 | 0.97 ± 0.26 | 0.3 |
ASTa (IU/L) | 8 ~ 38 | 31 (23, 37) | 68 (50, 75) | <0.001 |
ALTa (IU/L) | 4 ~ 44 | 22 (18, 29) | 28 (17, 47) | 0.203 |
GGTa (IU/L) | 16 ~ 73 | 22 (19, 78) | 44 (22, 86) | 0.506 |
Total bilirubina | 0.1 ~ 1.20 | 0.67 (0.50, 1.00) | 0.85 (0.50, 1.60) | 0.371 |
LDHa (IU/L) | 106 ~ 211 | 290 (214, 353) | 527 (391, 651) | <0.001 |
CPKa (IU/L) | 43~165 | 208.5 (168.0, 320.0) | 277.5 (230.0, 540.0) | 0.148 |
Proteina (mg/L) | 1.0 ~ 3.0 | 6.2 (6.0, 6.6) | 5.2 (3.6, 6.2) | 0.012 |
CRP (g/dl) | 5.8 ~ 8.1 | 61.5 (10, 98) | 89.7 (46, 95) | 0.234 |
Amylase (T_amylase)a (IU/L) | 43 ~ 116 | 50 (41, 74) | 42 (25, 53) | 0.347 |
pO2 (mmHg) | 80 ~ 100 | 128 (95.1, 165.3) | 194 (100.2, 208.4) | 0.418 |
Myoglobin (ng/ml) | 10 ~ 95 | 62 (46.5, 77.3) | 311 (156.2, 804.3) | 0.001 |
Values are expressed as mean ± SD or number (percentage). aValues are expressed as median (lower quartile-upper quartile). p value from Mann-Whitney U test.
Spearman correlation analysis revealed correlations between serum NGAL levels and each laboratory parameter was evaluated in all patients (
Serum NGAL levels in the expired patients remained consistently high for 5 - 35 days (until death). Serum NGAL levels in the survival group were initially high but decreased slightly within 14 days or increased temporarily but eventually decreased again (
A logistic regression analysis with odds ratios was performed using TBSA, ABSI, and serum NGAL levels with death as a dependent variable. TBSA, ABSI, and
Factors | All patients (n = 31) |
---|---|
Age | −0.115 (0.537) |
Duration of hospitalization, Day | −0.403 (0.025) |
Number of surgery | −0.017 (0.928) |
TBSA | 0.572 (0.001) |
ABSI | 0.503 (0.004) |
BUN | 0.280 (0.128) |
Creatinine | 0.188 (0.312) |
AST | 0.526 (0.002) |
ALT | 0.352 (0.052) |
GGT | 0.010 (0.957) |
Total bilirubin | 0.071 (0.703) |
LDH | 0.580 (<0.001) |
CPK | 0.200 (0.307) |
Protein | −0.348 (0.055) |
CRP | 0.331 (0.069 ) |
Amylase | −0.063 (0.743) |
pO2 | 0.204 (0.281) |
Myoglobin | 0.559 (0.016) |
Values are expressed as Spearman’s correlation coefficients (p value).
serum NGAL levels were statistically correlated (p < 0.05). As the TBSA and ABSI levels increased by 1 unit, the odds ratio increased 1.085 times and 1.884 times, respectively. In contrast, as serum NGAL increased by 10 units, the odds ratio increased 1.109 times (
To determine the correlation between serum NGAL levels and AKI, we comparatively analyzed factors associated with kidney damage. The BUN level remained within the normal range (8 - 25 mg/dL) in the discharged patients, but in expired patients, it eventually increased 10 days after the burn injury (
Odds ratio for death | |
---|---|
(95% confidence interval) | |
TBSAa | 1.085 (1.028 - 1.145) |
ABSIa | 1.884 (1.190 - 2.983) |
NGAL | 1.109 (1.038 - 1.185) |
(per 10 increase)a | |
NGAL | 1.092 (1.013 - 1.178) |
(per 10 increase)b |
aUnadjusted odds ratio. bAdjusted for age, TBSA, ABSI.
BUN levels increased later. During the early stage after a burn injury, the mean levels of BUN and creatinine showed no significant difference between the discharged patients and the expired patients (p value = 0.176, 0.300). Similar to serum NGAL levels, AST levels in the expired patients were higher than those in the discharged patients throughout the evaluation period (p value < 0.001). But ALT levels in both patients groups showed no significant difference.
The initial severity of the burn patients was evaluated within 48 hours of the burn by comparing the levels of NGAL and ABSI. In some cases, the ABSI was higher in discharged patients than in the expired patients. However, serum NGAL levels were mostly higher in the expired patients than in the discharged patients (Supplement 1).
A severe burn is a serious and complex injury that may result in disability and death. Thus, special methods for its diagnosis and treatment are required [
We analyzed the severity of burn injuries in ICU patients by measuring the TBSA, ABSI, and serum NGAL level. We classified the ICU patients into discharged patients, including those that survived, and expired patients. The results showed that TBSA, ABSI, and serum NGAL level were higher in the expired patients than in the discharged patients. ABSI is reportedly a more accurate and useful tool than TBSA, but given the advancements in therapeutic techniques, treatments, and technologies that have increased the survival rate of patients aged >60 years and with burns involving 30% of the TBSA, the score for sex and age need to be revised [
We additionally compared serum NGAL levels over time between the discharged patient and the expired patients. We found that as serum NGAL level decreased, the burn patients were moved to general wards or discharged, whereas if serum NGAL level remained high or increased, many patients died or stayed in the ICU for a long duration. Briefly, serum NGAL level allowed us to diagnose and provide a prognosis of burn patients to some extent.
On comparing the use of only TBSA and ABSI to determine the severity of burns in patients with respiratory damage, we noted that the severity was higher in patients with a high TBSA. However, serum NGAL level was higher in the patients with respiratory damage. Moreover, these patients died earlier than those without respiratory damage.
Severe burn injury damages several organs of the body [
To determine the correlation between NGAL expression and other organ dysfunction in patients with severe burns, we analyzed blood chemistry. Our results showed that the AST and LDH levels increased more significantly in the expired patients than in the discharged patients with severe burns. As such, muscle damage, as well as liver and heart damage, might occur in the early stage.
Serum NGAL level as an early predictor of AKI might reflect the severity of burns and could also be used as an indicator of inflammation in burned children [
NGAL is a major inflammatory protein [
The relevance of CRP and NGAL in inflammatory conditions has been reported [
In epithelial cells the level of NGAL increases by the inflammatory state. The secretion of NGAL is greatly increased in cases of damaged epithelial cells of the kidneys, large intestine, liver, and lungs [
There are several limitations in this study. Patients were included by convenience sampling and this study was performed with a relatively small number of patients. In addition, the study was only limited to adult patients with severe burn and therefore may not accurately reflect all burn patients. The small patients and limited sample tests may increase that the results are spurious. This report is a pilot study to help predict the severity of burns. Future studies with larger sample number sand better experimental strategies are needed to prove the value of NGAL as a good marker for predicting mortality.
Measurement of serum NGAL level together with ABSI and TBSA in the early stage of burns can help determine the condition of burn patients more accurately. In addition, the change in the NGAL level of burn patients during their hospital stay can appropriately represent the condition of such patients. Thus, we believe that serum NGAL level serves as a biomarker for burn patients within 72 hours of injury and can guide the provision of effective treatment for burn patients. Nonetheless, we may infer that NGAL expression is associated with the regeneration of damaged cells and tissues of organs such as the heart and liver, which should be further studied.
The authors do not have any financial relationship with any companies that may have financial interest in the information provided by this manuscript.
Lee, S., Lee, S., Choi, Y., Ahn, S.V., Yoon, C. and Lee, J. (2017) Correlation between Serum Neutrophil Gelatinase Associated Lipocalin and Burn Severity: A Pilot Study. Journal of Biosciences and Medicines, 5, 11-25. http://dx.doi.org/10.4236/jbm.2017.51002