0 1997 Elsevier

Bums Vol. 23r No. 3, pp. X8-224, 1997 Science Ltd for ISBI. AII rights reserved Irinted in Great Britain 0305-4179/97 $17.00+0.00

II: !%~05-41:?9(96)00124-6

volution and significance of circulating rocakitonin levels compared with IL-6, TNFot and

endotoxin

levels early after thermal injury

ervk Carsir?, Marcel Assicot2, Frkdkric FegeP, Olivier RoyI, Isabelle Iennacinol, erve Le Beverl, Pierre Ainaudl and Claude Bohuon2
Centre de Traitement des brtiles, H.I.A. Percy, BP 406,92141 Clamart Cedex, France and 2Departement de Biologic Clinique, Institut Gustave Roussy, 94805 Villejuif, France

To determine the evolution and significance of circulating procalcitonin (ProCT), lL-6, TNFcx and endotoxin levels early after thermal injuy, we performed a prospective, single unit, longitudinal study. Forty burn patients with total body surface area (TBSA) >30 per cent were studied, of whom 33 suffered an inhalation injury. Blood samples were taken on the day of admission, every 4 h during the first day and daily during the first week. All patients had increased ProCT and lL-6 levels without any proven infection. Endotoxin and TNFM levels remained very low or undetectable. ProCT and IL-6 levels correlated well with the severity of skin burn inju y (respectively, ~<0.006 and p < 0.028, using the non-parametric Kruska- Wallis test). ProCT levels are not associated with smoke inhalation. ProCT and IL6 are prognostic factors of mortality at the time of admission but less reliable than the clinical LtBS (unit burn standard} score. Endotoxin and TNFx were undetectable, suggesting that the problem of the early gut bacterial translocation remains to be proven. 0 1997 Hsevier Science Ltd for ISBI. Key wolrds: Bum inju y, smoke inhalation, cytokines lL-6, TNFa, endotoxin. procalcitonin,

Furthermore, the rise in IroCT in the absence of an increase of mature calcitonin has been described during bacterial1 or parasitic2 infections. The injection of endotoxin in healthy volunteers resulted in elevated serum IroCT levels3. Calcitonin-like immunoreactivity has been described as an early marker of the pulmonary impairment in the burn patient who sustains smoke inhalation and of the subsequently developing adult respiratory distress syndrome (ARDS)5. Therefore, it seemed important to include the serum ProCT levels together with IL6, TNFa and endotoxinemia levels in the serum during the first hours following severe burns, in patients with and without smoke inhalation injury, in addition to the usual biological investigations performed.

Materials
Patients

and methods

Burns,Vol. 23,No.3,218-2241997

Introductio
The two major causes of death in burn patients are respiratory failure, associated with a primary respiratory lesion, and infection. These are difficult to diagnose and potential markers are difficult to identify within the overall postburn inflammatory response. lrocalcitonin (IroCT) is a prohormone physiologically transformed into calcitonin by the m.edullary cells of the thyroid. However, the origin of this substance can be extrathyroidal. In our centre, high IroCT levels were found in a thyroidectomized burn patient, while calcitonin levels were undetectable.

From January 1993 to June 1994, thje usual biological monitoring of all burn patients admitted to the Burn Care Centre and presenting with a :3Oper cent TBSA (total body surface area) or more burn injury, was complemented by measuring blood procalcitonin, IL-6, TNFx and endotoxin levels. Blood collections were performed after informed consent from the patients or their families. The burned surface area was calculated according to Lund and Browders rule. The lesions which, on hospital admission, seemed to be third-degree burns were included in the UBS score (umt burn standard= percentage of burned body surface + 3 x thirddegree burned body surface area)6. We performed a bronchial fibroscopic exam of the patients within 24 h following admission. We diagnosed respiratory burn injury on the discovery of mucosal lesions and/or soot deposit. These clinical examinations and analysis of the circumstances of the

Carsin et a%:Evolution and significance of circulating procalcitonin levels -~ Table I. Characteristics of the population studied. Group 0, patients without inhalation injuries; Group 1, patients with indoctr inhalation injuries; Group 2, patients with outdoor inhalation injuries -Group Patients Age (years) UBS T5SA Number of deaths ~~ 0 Group 1 Group 2 7 (5M, 2F) 44&12 120&65 49*19 0 21 (13M, 8F) 42*15 193&79 59&18 7 12 (8M, 4F) 41*14 193k76 59&20 4 -

219

accident patient:

enabled

us to identify

three

groups

of

Patients without smoke inhalation injury (group 0); patients with smoke inhalation injuries after an accident which occurred indoors (e.g. home fire) (group I.); patients with endoscopic inhalation lesions after an accident which occurred outdoors (group 2).. The characteristics of these three groups are shown in Table I. Excision and grafting were performed on 29 of the 40 patients during the first week of hospitalization. Healthy volunteers served as a control group for IL-6 TNFg and IroCT (respectively, 10, 19 and IO). Methods Blood collection was carried out immediately upon adm.ission to the ward and then every 4 h until >the 24th hour (the reference hour being the hour of the accident). Next, a daily blood collection was carried out during the first week. The laboratory is located within the Care Unit, so samples were transported and processed within a short time ( < 1 h). Determination of ProCT. Blood samples were centrifuged at 15OOg for 10 min, and the sera were stored at --8X before analysis. ProCT was assayed by an ultrasensitive sandwich immunoluminometric assay (Brahms, Berlin, Germany). This determination uses two assays and two monoclonal antibodies: a first binding antibody to an epitope located on the katacalcin and a second tracing antibody in the mid-region of calcitonin. This assay, which does not detect mature calcitonin, has a sensitivity of approximately 10 pdml. The luminescence is measured usin.g a Berilux analyser (Behring@, Mannheim, Germany) against a standard range from 120 to 61000 pg/ml; samples exceeding the maximum value werle diluted before re-analysis. Determination of endotoxin. Endotoxin levels were measured during the first 3 days following the burn injury, except for 15 patients in whom the determination was continued until day 7. Blood was collected in apyrogenic sterile tubes (Kabi endotubes) and stomd in ice. After 2OOg centrifugation for 15 min at 4C we immediately obtained a plasma rich in platelets, aliquots of which were prepared under laminar air flow, transferred to sterile apyrogenic tubes and

istored at -8PC until use. The assay technique is an lend-point chromogenic method based on the activation of a limulus amoebocyte lysate (LAL) by endotoxin (Coatest endotoxinm, Biogenic, Montpellier, France). In order to avoid activation and the inhibiting effects of plasma on the LAL test, all samples were diluted (1:lO) in apyrogenic sterile water, then heated to 75C for 10 min. The samples were then coincubated for 30 mm at 37Y with the LAL; the chromogenic substrate was added and after 10 min the reaction was stopped with an aqueous solution of 50 per cent acetic acid. Absorbances were read in a plate-reader (Uniskan IP, Labsystems, Les Ulis, France). The absorbance of a control is substracted from these absorbances in order to adjust the samples intrinsic colour development. The endotoxin concentration is read on a standardization curve plotted after addition of endotoxin (Es&en&a cdi Olll:B4-1.2 endotoxin unit (EU)=lOO pg) to a pool of control plasmas. This mlethod is sensitive to 0.06 EU/ml of endotoxin In accordance with the manufacturer, calculation of recoveries of added endotoxin allowed the validation of these results (range of acceptable recoveries: 50-150 per cent). Determination of IL-6 and tumaur necrosis factor a. Interleukin-6 (IL-6) and tumour necrosis factor a (TNFa) serum levels were measured using a specific enzyme-linked immunosorbent assay (ELISA) designed as a double antibody sandwich assay (Immunotech, Marseille, France). The normal range of IL-6 and TNFa levels observed by these assays are < 10 and < 5 pg/ml, respectively. Blood cultures, lactic acid and PaOJFi02 ratio. Blood cultures were performed three times a day for each patient, using the BacteP system (Becton-Dickinson, Pant-de-Claix, France). Likewise, arterial lactic acid and IaO#iOZ were determined at the time of hospital admission, using enzymatic assay (Boehringer Mannheim, Meylan, France) and an analyser ABL520@ (Radiometer, Copenhagen, Denmark), respectively. Statistical analysis. The SPS package for Windows (Version 6.1.2) was used for analysis. We first verified the normality of the distribution of variables using the Shapiro-Wilk test. IL-6 and IroCT distribution were normal after logarithmic transformation. Therefore, statistical analysis was performed using the logarithm value of these variables For ProCT, IL6 and TNF@, initial values (time HO) were average values of the panel of health controls. The role of various factors upon the variation of IL-6, ProCT and TNF!x during ti.me since injury was tested using non-parametric analysis of variance for repeated measurements (Friedman test). If this was significant, the multiple range test was used (Bonferroni) to compare the different values. To take into account the UBS score in the analysis, we used ANOVA with the UBS score as a covariate.

220

Burrls:

Vol.

23, No.

3,1997

To compare the lroCT and IL-6 peaks, and the UBS score among survivors and non-survivors, we used the non-parametric Kruskal-Wallis (KW) test. We used the Spearman rank correlation test (Q) to test thiz link between continued variables. Study of endotoxin variation was monitored using a Yates x-squared test because a few measurements were performed on health controls; categories are defined according to the presence (level 0.06 EU/ml) or absence (level ~0.06 EU/ml) of endotoxin in the sample. The rate of 0.06 EU/ml is the sensitivity baseline of the kit used.

HO

b&H7

H8-HS H,2

1 18

I Hxl

H24

I D2

, D3

, D4

, D5

I D7

Time

post

burn

Serum levels of ProCT, IL-6 and TNF,z during the week following a burn injury

The mean values of serum ProCT, IL-6 and TNFx during the study period are shown in Figure 1, Mean serum ProCT and IL-6 levels increased during the first hours following the burn injury and rapidly stabilized. For IroCT, the increase was significant between HO and other postburn times and between H4 and H7 and other times (Bonferroni test p < 0.05). Similarly, the increase in IL-6 was significant between HO and other times (p < 0.05). In contrast to IL-6 and ProCT, mean TNFti serum levels after burn injury did not increase significantly during the first seven postburn days (Figure 2). Only oue of the 40 patients exhibited increased TNFLYlevels during the early 24 h, with a peak level of 140 pdml at 8 h.
ProCT #and IL-6 serum levels, respiratory UBS score burns and

1
HO H4-H7 H&W H,,? hi,6 H20 Time

II@
m+ post

i
D2 bum

i
D3

:
04

:
D5

1
D7

Figure 2. Post burn course of ProCT ant! IL-6 serum levels (means) in each of the three groups: grouP 0 (01, group 1 (oh group 2 (9

When the threle groups were compared, the IL-6 and ProCT concentrations increased following a similar curve; Ihowever, the levels were different (Figure 2). For ProCT, we observed a lower mean in group 0 than in groups 1 and 2 (KW test). For IL-6, we observed a higher mean in group 1 (burns, inhalation injury, closed space) than in groups 0 and 2 (KW test).

If we focused on the first 24 h, the same differences were found for IroCT and IL-6 among the different groups. If groups 1 and 2 were considered together (burn patients with inhalation injury), and the concentrations of ProCT and IL-6 were compared with group 0 (burn patients without inhalation injury), then higher levels are observed in patients with inhalation injury (KW test - p < 10d6 and p < 0.003) (T&e 11). Were these levels the results of inhalation injury or severity of burn injury? Introducing the UBS score as a covariate in a variance analysis showed that, in this model, ProCT and IL-6 did not change significantly between groups with or without smoke inhalation injury, but correlated with the UBS score (p < 0.006 and p < 0.028) (Figures 3 and 4). Clearly, IL-6 and ProCT were not reliable markers of inhalation injury in these studies.
Prognostic value of ProCT and IL-6 serum levels

Eleven patients died during hospitalization. The maximum values of ProCT and IL-6 measured within
Table II. Peak ProCT and IL-6 levels (means k SEM) within the first 24 h after burn injury in patients with and without inhalation injury
HO H4-H7 H&H9 HI2 18 Time H20 post H24 bum DZ D3 D4 I,5 D7

ProCT Without inhalation injury (n = 7) With inhalation injury (n = 33) Group Groups 1 and 0

kg/m/)

IL-6

bg/m0

2543kll60 32894&11462 2

1256 & 384 6057k2186

Serial values (means) of serum IroCT (A), IL-6 (0) and TNFx (0) after burn injury for the entire group of 40 patients.
Figure 1.

Carsin et al.: Evolution

and significance

of circulating

procalcitonin

levels

221

e e
l l l

ee

:
50 e

*
l

l *

0-I
100 1000 10000 100000

8
1000000

Pro,CT concentration
Figure 3. Relationship between ProCT level and UBS score.

(pglml)

Figwe

4. Relationship

between

IL-6 level and UBS score.

the first 24 h and the UBS score from survivors and non-survivors are shown in Fi@ue 5. As shown in Table 111, IroCT, IL-6 and UBS were prognostic of death, UBS being the best prognostic score, followed by IL-6 and ProCT. These three prognostic values were used in a logistic regression model using as parameters ProCT and IL-6 peak values within the 24 h following injury and UBS (divided into two groups: ~240 and >240). The patient with a UBS score of at least 24@ had 23 times greater risk of death than patients with lower UBS values. This model gave accurate predictions for 85 per cent of patients (Tubk IV). IroCT and IL-6 peaks correlated with UBS (respectively, p = 0.61; p < 10m6 and p = 0.47; p = 0.002).

l 0 0 e & iB 3 l 8 u l l

0
350

l e
l
e 300

le S?
l l

--J-O

ProCT
Figure 5. Ie,ak IroCT 72= 11). and IL-6 levels within 24 h of burn

IL-6

UBS
(0,

and UBS score in survivors

n = 29) and non-survivors

(0,

222

J3m-m: Vol. 23, No. 3,1997 or nonUBS 270 (100-200) 240(200-312) p<o,oo1

TableIII. Peak ProCT, IL-6 and UBS score (median 2575%) within the first 24 h after burn injury in survivor survivor patients The differences for IL-6, ProCT and UBS between groups were significant according to KW test
ProCT(pglml) Survivors (f~ = 21) Non-survivors (n = I?) 3400 7000 (750-18700) (2100-44100) p <0.05 IL-6 kdrnl)

KW test

1020(542-2100) 2800 (1030-35500) pco.01

ProCT,, IL-6, lactic acid serum levels and PaOJFiOz The maximum values of ProCT correlated with blood lactic acid levels measured at study entry (p =0.33; p =O.O4:) and did not correlate with the laOJFiOz levels (Q= -0.05; NS), which were used to identify pulmonary problems (Figure 6). By contrast,, peak IL-6 levels correlated with the l?aO#iOz (Q=: -0.42; p=O.O07) and unrelated with blood lactic acid (p =0,15; NS) which was used as an indicator of tissue hypoxia (Figure 7). lood endotoxin levels and haemocultures Endotoxin levels measured showed no significant difference to that of controls, except the Hl2 and Hl6 periods postinjury (p = 0.05 and p -c 0.001, respectively). At these times, more patients had detectable endotoxin levels, nevertheless remaining low. Endotoxin was undetectable in 60 per cent of blood samples (F~~LMT8). No sorrelation could be observed between endotoxin, IL-6 or ProCT levels. No bacteremia w,as observed during the first week.

IL-6 concentration

(pgiml)

Figure 7. Relationship of IL-6 level with blood lactic acid level (0) and with IaOJJ30~ level (D) in total population of patients.

Discussion
The study of serum procalcitonin levels during the early hours following severe burn injuries exhibits a rapid rise followed by a plateau. The peak value is related to burn severity and correlates with the UBS score. According to ONeilP and Skolnick5, increased
calcitonin-Iike immunoreactivity levels on hospital

Table JV. Predictive models of death using ProCT peaks wij:hin 24. h following injuv and UBS score -Mode/ Nonsurvivors 2 7

and IL-6

~~ Observatih -Survivors Non-survivors

Survivors 27 4

Percent identified 93.10 63.64

. . . .

. . On

o--n 100

* 1000

*...... ,oooo

'."*.'a ,00004

*.."1000000

ProCl

concentration

(pglml)

Figure 6. Relationship of ProCT level with blood lactic acid level (0) and with IaOJFiOz level (EI) in total population of

patients.

admission are a marker of pulmonary injury from smoke inhalation. We attempted to test this hypothesis on a series of 40 burned patients, distributed to groups classed according to endoscopic data and analysis of the circumstances of the accident. Groups 1 and 2 included patients who inhaled smoke in accidents which occurred outdoors or indoors, respectively (indoor inhalation caused the worse condition). Comparison of peak ProCT values between group 0 and groups 1 and 2 together shows a marked difference (T&e 11). One might therefore conclude that the ProCT peak and the presence of inhalation injuries are correlated but patients with respiratory impairments are those who, as a rule, present with the most severe skin burns. Therefore, it was important to determine the role of the skin burn, eval.uated according to the UBS score; our statistical analysis shows that the ProCT peak correlates with the UBS score but not with inhalation injuries. Is this early ProCT level a prognostic factor? As seen currently, every patient who died had smoke inhalation injuries. In fact, the IroCT peak has a prognostic value but is less significant than UBS score alone. It seemed important to compare this level with the arterial lactic acid level, measured on hospital admission, as this is evidence of cellular hypoxia,

Carsin et al.: Evolution

and significance

of circulating

procalcitonin

levels

223

HO

H4

H12 *

HI6 **

H20

H24

D2

D3

Time post burn Figure 8. Endotoxin concentrations (EU/ml) measured at each blood collection time. The area under the dotted line repre-

sents the ran;ge of concentrations observed in the control group.

regardless of the cause. We also compared the IroCT peak with the admission time IaOfiiOZ ratio as it is a marker of the pulmonary effector. We found no correlation between the l?aOfiiOZ ratio and the IroCT peak; in contrast, there was a clear correlation with blood lactic acid levels. The origin of the early increase of ProCT remains to be discovered. Our first hypothesis was that this increase was secondary to endotoxin secretion and gut bacterial translocation. Injection of endotoxin to healthy volunteers causes a rise in ProCT and triggers an inflammatory reaction along with increased circulating IL-6, TNFg and IL-1 levels3. Experimentally, in the burned animal there is a relationship between lowered mesenteric flowrate due to the burn and circulating endotoxin levels71E.A number of authorP found elevated circulating endotoxin levels in men with severe burn injuries. Our previous studies have also shown the existence of a correlation between infection and high IroCT levels at a later stage of the course of burn injuriesI. In 60 per cent of the patients of this study, we found no circulating endotoxin during the early stage of the burn injury and no infection was observed in any of the 40 patients. Such results are in agreement with those reported by Endo et a1.l. This does not rule out possible bacterial translocation but, as proposed by Moore et al.lZ, one may think that in patients presenting with polytrauma, the released bacteria are blocked in the mesenteric nodes where they are likely to induce macrophage activation. Hoch et al.13 propose that the binding of circulating endotoxin to the lipopolysaccharide binding proteins (LBP) may preclude its detection in the plasma but does not prevent it from effecting its stimulating action. In

the critical discussion following Munsters studylo, Mannick emphasizes the difficulty in determining the endotoxin owing to contamination sources, plasma activators and inhibitors. For the purpose of our observations, all tubes were sterile and apyrogenic, the samples were treated under laminar air immediately after collection and assays were validated by calculating overload recoveries. Nevertheless, if there was endotoxin production, one would expect to find high serum TNFu levels; we o,bserved this rise in only one patient and he had no detectable plasmatic endotoxin. This absence of endotoxin in our patients is perhaps explained by the fact that they all were transported to the hospital in medically equipped vehicles where they received early vascular filling. Therefore, the early inflammatory reaction observed in the patients with burn injuries seems unrelated to gut bacterial translocation but simply to skin tissue alteration14J5. It is now well known that keratinocytes are initiators of inflammati0r-P. The early rise in IroCT seems to be a marker of the mass of destroyed tissue since the peak value observed in the first 24 h is correlated with the UBS score. IL-6 levels provide reliable eviclence of this inflammatory reaction9,17,1*. IL-6 elevation occurs as rapidly as ProCT and they are statistically proven to be reliable markers of burn severityL*.

Conclusion
Serum procalcitonin and IL-6 levels rise quickly following severe burn injuries. Peak levels observed in the first 24 h are related to burn severity but do not correlate with smoke inhalation injuries. Systemic increases in procalcitonin do not seem to be related

224

Bums: Vol. 23, No. 3,1997 6 Sachs A, Watson J. Four years experience at a specialized burn center. Lancef 1969; 1: 718. 7 Herndon DN, Zeugler ST. Bacterial transiocation after thermal injury. Crif Cure Med 1993; 21: S50-S54. 8 Winchurch RA, Thupari JN, Munster AM. Endotoxin in burn patients. Surgery 1987; 102~ 808-812. 9 Guo Y, Dickerson C, Chrest FJ et al. Increased level of circulating interleukin 6 in burn patients. Clin lmrnunol lmmunopafhol 1990; 54: 361-371. 10 Munster AM, Smith-Meek M, Dickinson C et al. Translocation, incidental phenomenon or true pathology?. Ann Surg 1993; 218: 321-327. 11 Endo S, Inada K, Kikuchi M et al. Are plasma endotoxin levels related to burn size and prognosis? Btirrrs 1992; 18: 486-489. 12 Moore FA, Moore EE, Pogetti R et al. Gut bacterial translocation via the portal vein: a clinical perspective with major trauma. J Trauma 1991; 31: 629-638. 13 Hoch RC, Rodriguez R, Manning T et al. Effects of accidental trauma on cytokin and endotoxin production. Crif Care Med 1993; 21: 839-845. 14 Cavaillon JM et al. Cytokines et inflammation. In: Les cytokines. Paris: Masson S.A, 1993; p. 341. 15 Youn YK, Lalonde C, Demling R. Oxidants and the pathophysiology of burn and smoke inhalation injury. Free Radic Biol Med 1992; 12~ 409-415. 16 Barker JNWN, Mitra RS, Griffiths CEM et al. Keratinocytes as initiators of inflammation. Lancet 1991; 332 211-214. 17 Gueugniaud PY, Bertin-Maghit M, Joly MO et al. Role de linterleukine 6 dans la phase oedemateuse du bnXe grave. Presse Med 1993; 22: 735. 18 Ueyama M, Maruyama I, Osame M et al. Marked increase in plasma interleukin-6 in burn patients. J Lab Clip Med 1992; 120: 693-698.

TV septic processes. We have been unable to find a significant gut bacterial translocation or endotoxin release in the early hours following the thermal injury. The origin of procalcitonin remains unknown. However, there is a clear relationship with the systemic inflammatory response, the beginning of which should be local, in relation to the mass of damaged tissues.

Acknowledgements
We are grateful to Patrick Galaup, Fabrice Jaunault,, Ihilippe Verbeke and Olivier Gadal for their scien tific co:ntribution; to Mr Arvers (Centre de Recherches du Service de Sante des Armees) for the statistical. analysis; and to Mrs Felten (Hopital Saint Louis) for her technical contribution. The present work was carried out thanks to a Clinical Research grant (RC 93/13) of the Direction Centrake du Service de Sante des Armees.

References
1 Assicot M, Gendrel D, Carsin H et al. High serum procalcitonin. concentrations in patients with sepsis and infection. Lancef 1993; 341: 515-518. 2 Davis THE, Assicot M, Bohuon C et al. Serum procalcitonin concentrations in acute malaria. Trans R Sot Trap Med 1994; 88: 670-671. 3 Dandona I, Nix D, Wilson MF et al. Procalcitonin increase following endotoxin injection in normal subjects. I C& Endocrinol Metab 1994; 79: 1605. 4 ONeill WJ, Jordan MH, Lewis MS et al. Serum calcitonin may be a marker for inhalation injury in burns. J Burn Care Rehabil 1992; 13: 605-616. 5 Skolnick A. Calcitonin assay may help identify burn patients at risk for respiratory distress. JAMA 1990; 264: 565-566.

Paper accepted 26 September 1996.

Correspondence should be addressed to: H. Carsin, Centre de traitement des brules, H.1.A Percy, BP 406, 92141 Clamart Cedex, France.

46th Congresso Nazionale della Societa Italiana di Chirurgia Plasti.ca Ricostruttiva ed Estetica
30 September-3 October 1997
Venice, IMy

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