SERUM NEOPTERIN AND PLASMA ENDOTOXINS AS PREDICTORS OF ENDOTOXAEMIA IN BURNED PATIENTS

Mabrouk A. R, Shetta A. M.

Department of Plastic Surgery' and Clinical Pathology, Faculty of Medicine, Ain Shams University, Egypt

SUMMARY. Serum neopterin, plasma endotoxins, ALT, AST, BUN, creatinine and TLC were determined in 35 burned patients on the fourth day after burn injury. The TBSA ranged from 10-75 % (mean, 34 ± 21 %). The results were compared with those of nineteen normal healthy controls and correlated with TSBA, sepsis, mortality, and hepatic and renal function tests. The results demonstrated higher serum neopterin and endotoxins, AST, ALT, BUN and creatinine in the patient groups than in controls (p < 0.001). Serum neopterin and plasma endotoxins were significantly increased and were related to the extent of the burn surface area (p < 0.001). They were significantly higher in the septic and deceased group than in the non-septic and survivors groups. These results suggest that thermal injury can lead to an elevation of serum neopterin. Endotoxin release in the circulation may be responsible for continuous induction of neopterin. In addition, plasma endotoxin and serum neopterin act as predictive factors for the occurrence of early sepsis with subsequent multiple organ failure and death.

Introduction

Burn patients often experience numerous devastating reactions to the burn injury. Major burns are followed by extensive impairment of the cellular and Immoral immunological systems. Subsequent complications such as infections and bacterial invasions are commonly reported. Even today, the commonest causes of death after burn injury are endotoxaernia with subsequent septicaemia, inhalation injury, severe shock, and multiple organ failure (MOF) (renal, hepatic, cardiac, pulmonary and gastrointestinal).
It is thus very important to monitor burn patients' defence mechanisms against infection and other complications. Monitoring provides information for the evaluation of the patients' condition and prognosis.
The determination of neopterin, a macrophage product, offers valuable insights into the activation of the T-cell system in various diseases, including endotoxaemia and septic complications.' Endotoxin, a component of grainnegative bacteria, is known to play an important role in the pathogenesis of sepsis with subsequent MOF in burn patients.' Although the main stimulus for the release of neopterin from macrophages is IFN-y, various studies have indicated that neopterin is released after stimulation of macrophages with other cytokines and endotoxin.
A better understanding of how neopterin behaves during the post-burn period, of its relation to endotoxin levels, and of the subsequent development of sepsis will clearly contribute to the reduction of the incidence of morbidity and mortality.
The aim of this work was to determine the level of neopterin and endotoxins during the post-burn period and to correlate these two parameters to total body surface area (TBSA), mortality and sepsis.

Patients and methods

This study was conducted on 35 burn patients (20 females and 15 males) admitted to the burn Unit of Ain Shams University Hospital (Egypt) over a period of one year (1996-97). The study was performed with consent given by the patients themselves or by members of the family. TBSA ranged from 10 to 75%, with a mean of 34 21%. The ages ranged from 16 to 55 yr (25.7 ± 9.5 yr).
All patients were initially resuscitated with the Parkland formula. The patency of the airways was checked to detect early inhalation injury, using bronchoscopy. Patients with suspected inhalation injury were placed on assisted ventilation. Early escharotomy was performed, when needed, in the resuscitation room with prompt application of topical silver sulphadiazine. The excision and grafting of deep partial- and full-thickness burns were performed as early as 48 h post-burn, the maximum body surface area excised each time being 10%. The intention was to reduce the load of burn toxin and to enhance the patients' immunity. Force feeding nutrition was provided by means of a nasogastric tube, using the Currie formula:

25 kcal x wt (kg) + 40 kcal x TBSA (%)
Proteins = 1 gin x kg body wt + 3 gin x TBSA

The patients were divided into three groups on the basis of TBSA percentage, as follows: group A, TBSA 10-20% (13 ± 2.7%), n = 10,); group B, TBSA 20-40% (25 ± 3.5%), n = 15; group C, TBSA over 40% (51 ± 11%), n = 10). Nineteen patients survived (54.3%) and sixteen died (45.7%): eight of septicaemia, five of MOF, and three of severe pulmonary inhalation injury.
Sepsis, on the evidence of serious bacterial infection with systemic inflammatory response, was defined in line with previous reports.` The diagnosis of sepsis was based on the patient's fulfilling at least two of the following three criteria:

• align="left">Fever (>39 'C) or hypothermia (<35.5 'C)
• Evidence of obvious infection or positive blood cultures of commonly accepted pathogens
• Leukocytes <6000 or >15000 mm

A group of nineteen healthy individuals matched for age and sex served as controls.
Sample collections were collected on day 4 post-injury. The blood samples were collected with an endotoxin-free syringe. The blood collected was divided into three sets of tubes. In the first set, the blood was left to clot and then centrifuged at 3000 rpm for 1 min. The sera were separated and used for determination of neopterin. All the sera were immediately stored at -70 'C until the time of the assay. The second set of tubes consisted of lyophilized ethylene diamine tetra-acetate (EDTA, 1.5 mg/pl) as an anticoagulant, the total leukocyte count being assayed immediately. In the third set of tubes, heparin was added, the blood was centrifuged, and plasma was separated and then stored at -70 'C until it was assayed for plasma endotoxins.
Determination of neopterin level. The serum neopterin level was measured with a commercially available enzymelinked assay kit (Diagnostic Merch [E. Merch, Post, Foch 449, Federal Republic Germany] based on the competitive binding principle).
Assessment ofplasma endotoxins. Plasma endotoxins were assayed qualitatively using a commercially available kit (EToxate, Sigma [PO box 14508, Saint Louis, Missouri 63178, USA]). The plasma samples were pre-treated using the method described by Harris et a]." To remove factors interfering with theLimulus amoebocyte lysate, the plasma was diluted 1/10 in endotoxin-free water and heated at 75 'C, after which it was ready to be assayed. Total proteins, alburnin, AST, ALT, BUN and creatinine were determined by Synchron CX-5.
Statistical analysis. The results were expressed as mean ± SD. The level of significance (p) was calculated by Student's t test. The correlation study was performed using Pearson's correlation coefficient (r). The Z test was used to study the incidence of positivity of plasma endotoxin.

Results

The results are summarized in Tables I-VI. Table I presents a statistical comparison between the patients and the control groups. Serum neopterin, plasma endotoxins, AST, ALT, BURN and creatinine were significantly increased in the patient group compared with the control group. Total proteins and alburnin were significantly decreased in the patient group compared with the control group.

Table II presents a statistical comparison of groups A, B and C with regard to the main parameters studied. Serum neopterin and endotoxins were significantly increased in group C compared with groups B and A and in group B compared with group A. ALT and AST were significantly increased in group C compared with groups A and B.
BURN was significantly decreased in group A compared with group C. There was no significant difference between the three groups as regards TLC, creatinine, alburnin and total proteins.

Table III presents a statistical comparison between septic and non-septic groups with regard to the main parameters studied. Serum neopterin and plasma endotoxins were significantly increased in the septic group compared with the non-septic group. ALT was significantly lower in the septic group than in the non-septic group.

Table IV presents a statistical comparison between the groups of survivors and deceased patients with regard to the main parameters studied. Serum neopterin and plasma endotoxins, ALT, AST and BUN were significantly increased in deceased patients compared with surviving patients.

Table V presents a correlation between the main parameters studied. Serum neopterin was significantly correlated with plasma endotoxins and TBSA. TBSA was significantly correlated with neopterin, endotoxins, BUN, ALT and AST.

Table VI presents a correlation study between serum neopterin and plasma endotoxins in septic and non-surviving groups. Serum neopterin was significantly correlated to endotoxin in the septic and non-surviving groups.

Discussion

In spite of modem therapeutic advances, severe sepsis remains the commonest cause of death in patients resuscitated after major thertnal injury. The inflammatory response to the burn injury leads to massive activation of the immune system, with the subsequent release of mediators. Measurement of neopterin is frequently used for monitoring diseases associated with cellular immune activation. However, little is known about whether neopterin serves as a valuable marker to differentiate between septic and non-septic patients and whether endotoxaemia could be involved in the increase of neopterin induction following the burn.
The inflammatory response to the burn injury is always experienced within the first two weeks. In our study, day 4 was chosen for sampling since it has been reported that neopterin increases significantly by that time in response to immunological stimuli. Endotoxins have also been proved to increase during the first week post-burn.
Our results showed that serum neopterin was significantly increased in the patient groups compared with the controls. This finding is consistent with the results of other researchers'and suggest that the burn injury causes a constant increase in neopterin. The increase in serum neopterin is due to various immunological stimulations following the burn injury.
The immune response to the burn injury depends on a symbiotic relationship between the monocyte phagocytic system and T-lymphocytes. In other words, lymphocytes play the music but macrophages call the tune." The cells, after being stimulated by the burn injury, orchestrate the immune response by the release of cytokines, thereby stimulating the macrophages that in turn produce neopterin and other cells (including B and NK cells)." These cytokines include mainly INF-y and other mediators such as TNF-y and 11-2.
Serum neopterin was significantly increased in group C compared with groups B and A and in group B compared with group A. In contrast to our data, Balogh et al.' and Yao et al.' found that the neopterin level was not significantly correlated with the extent of the burn surface area. 2,6 However, Leithauser et al." reported that serum neopterin was significantly correlated with the severity of the illness and with the onset of sepsis. The finding in our study that the extent of the burn injury was correlated with the level of neopterin could be explained by the fact that the immune response increased in relation to the increase in the percentage of the burn injury.
Endotoxins were also significantly increased in patient groups compared with control. This result was consistent with those reported by Dobke et al.," who found that the rise in endotoxins was evident immediately post-burn. The peak level occurred 7-12 h post-injury, probably owing to translocation from the gut or to the passage of endotoxins across the gut barrier. A further peak occurred on day 4, coinciding with colonization of the burn wound." Plasma endotoxins were significantly increased in group C compared with groups B and A. This finding confirmed those of Yao et al.,' who reported that the extent of burns and the outcome were correlated with the blood endotoxin concentration.
In this study, there was a positive significant correlation between the level of neopterin and the incidence of positivity of endotoxins in the burned group. This positive significant correlation was also reported by Yao et al.' Although it is known that the main stimulus for the production of neopterin from the macrophage is the INF-y," other stimuli for the macrophages have also been reported. These include other mediators such as TNF-), and IL-2, which exert activities regulating the generation of neopterin."It has also been reported that endotoxins were identified as a potential stimulus for sustained neopterin biosynthesis,' and in that report the patients with endotoxaemia presented much higher neopterin values than those without endotoxaemia. In vitro and in vivo studies have shown that endotoxins directly augment neopterin production from macrophages and that they also effectively induce neopterin biosynthesis under conditions in which the participation of INF-y is excluded! In addition, it is evident that marked neopterin release can be induced by as little as 100 pgmV of endotoxins.'Although we determined plasma endotoxins by the semiquantitative method, it was still demonstrated that the low levels of endotoxins induced high level of neopterin release.
There was a highly significant increase in both endotoxin and neopterin in the septic groups but not in the nonseptic groups. These results were consistent with those reported by Balogh et al.' and Grabosch et al." The increase in endotoxins and neopterin in septic rather than nonseptic patients as early as day 4 is not only as an indicator of sepsis but also a prognostic factor before sepsis actually occurs. The early rise of endotoxins in patients subjected to removal of the eschar stimulates the macrophages to continuously produce neopterin.` The increase in endotoxins in the treated group would be explained by other precipitating factors, such as the existence of a pre-existing disease that may also influence sepsis. Fungal infection, diabetes, cardiopulmonary disease, and the presence of full-thickness burn trauma are predictors of septic mortality.
In the present study, serum neopterin and plasma endotoxins were significantly increased in the deceased group compared with the surviving group. Grabosch et al." found that mean serum neopterin was higher in nonsurvivors than in survivors during the first two weeks post-burn and that an increase in neopterin was observed during the last five days before death. However, Yao et al.' and Balogh et al.' reported that there was no significant difference of serum neopterin levels between survivor and non-survivor groups, and concluded that serum neopterin per se does not appear to be a good marker for predicting mortality. Although their study failed to find a definite relationship between the increase of serum neopterin and mortality, plasma endotoxin concentrations were correlated with sepsis and death. The suggestion has been made that the evaluation of both neopterin and endotoxins may be more useful in the monitoring of the clinical course after burn trauma.
In conclusion, serum neopterin and plasma endotoxins are considered to be important parameters in immunoaffection and the modulation of infection in the post-burn period. Our data also suggest that the presence of a high neopterin level together with increased plasma endotoxins can be considered a prognostic factor for the occurrence of both sepsis and mortality. An understanding of these immunodefects is essential for the development of new therapeutic approaches in the post-burn injury.

RESUME. La néopterine sérique, les endotoxines plasmatiques, FALT, PAST, l'azote de l'urée sanguine (BUN), la créatinine et le TLC ont été déterminés dans 35 patients brûlés le quatrième jour après la brûlure. La surface brûlée variait entre 10 et 75% (moyenne, 34 ± 21%). Les résultats ont été comparés avec ceux de dix-neuf témoins sains et corrélés avec les résultats pour ce qui concerne la sepsis, la mortalité et la fonction hépatique et rénale. Les résultats démontraient un niveau élevé de la néopterine sérique et des endotoxines, de FAST, de FALT, de la BUN et de la créatinine dans les groupes des patients par rapport aux témoins (p < 0.001). La néoptérine sérique et les endotoxines plasmatiques étaient significativement élevées et corrélés avec l'extension de la surface brûlée (p < 0.001). Elles étaient augmentées significativement dans les patients non septiques décédés par rapport au groupe de patients non septiques et des survécus. Ces résultats font penser que la lésion thermique peut provoquer une élévation de la néopterine sérique. La libération de l'endotoxine dans la circulation pourrait être responsable de l'induction continuelle de la néoptérine. En outre, l'endotoxine plasmatique et la néoptérine sérique agissent comme des facteurs précurseurs de la vérification de la sepsis précoce suivie par l'insuffisance multiorganique et la mort.

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